AROMATIC-CATIONIC PEPTIDE FORMULATIONS,

COMPOSITIONS AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Application No.

62/018,931 , filed on June 30, 2014 the content of which is incorporated herein by reference in its entirety.

FIELD OF THE TECHNOLOGY

[0002] The present technology relates to aromatic-cationic peptide pharmaceuticals where the active compounds include a plurality of amino acids and at least one peptide bond in the molecular structure, and to methods of providing pharmaceuticals of such peptide active compounds that are bioavailable when administered to subjects.

BACKGROUND

[0003] Peptide pharmaceuticals are frequently administered by injection or by nasal administration. However, injection and nasal administration are significantly less convenient, and involve more patient discomfort than, for example, oral administration.

[0004] Often this inconvenience or discomfort results in substantial patient noncompliance with a treatment regimen. Oral administration tends to be problematic, however, because peptide active compounds are very susceptible to degradation in the stomach and intestines. Thus, there is a need in the art for more effective and reproducible oral administration of peptide pharmaceuticals like insulin, salmon calcitonin and others discussed in more detail herein.

[0005] Proteolytic enzymes of both the stomach and intestines may degrade peptides, rendering them inactive before they can be absorbed into the bloodstream. Any amount of peptide that survives proteolytic degradation by proteases of the stomach (typically having acidic pH optima) is later confronted with proteases of the small intestine and enzymes secreted by the pancreas (typically having neutral to basic pH optima).

[0006] Specific difficulties arising from the oral administration of peptides involve the size of the molecule, and the charge distribution it carries. These physical properties may make it more difficult for the peptide to penetrate the mucus along intestinal walls or to cross the intestinal brush border membrane into the blood, and may result in limited bioavailability. [0007] Oral dosage forms which at least partially surmount many of the difficulties described above are disclosed and claimed in U.S. Pat. Nos. 5,912,014 and 6,086,918 to Stern et al, issued Jun. 15, 1999 and Jul. 11, 2000, respectively, which are incorporated herein by reference in their entireties. Both patents describe peptide dosage formulations which target release of the peptide to the intestine and which enhance bioavailability by administering the peptide in an oral dosage formulation which comprises, in addition to the peptide, at least one pharmaceutically acceptable pH-lowering agent and at least one absorption enhancer effective to promote bioavailability of the peptide. Moreover, the dosage formulation is coated with an enteric coating capable of conducting the peptide, the absorption enhancer and the pH-lowering agent through a subject's stomach, while protecting the peptide from degradation by stomach proteases. Thereafter, the coating dissolves and the peptide, absorption enhancer and pH lowering agent are released together into the intestine of the subject.

[0008] In certain instances, however, the condition to be treated by the oral peptide would benefit from more rapid remediation than that provided by the relatively slow dissolution of an enteric coating and related release of the active component(s) within the intestine. One particular example of a condition which benefits from such rapid remediation, involves the area of pain relief, where the speed with which relief is achieved is obviously an important, if not critical, factor to a patient. Furthermore, it is not always required that the aromatic- cationic peptide be transported all of the way through the stomach and into the intestine. That is, in the case of certain aromatic-cationic peptides, including but not limited to various pain-relievers, it may be most efficacious for absorption of the therapeutic peptide to occur prior to entry of the formulation into the intestine, e.g. , as the material passes down the esophagus or when it is within the patient's stomach. Under such circumstances, while oral bioavailability is still a factor to be considered, patients and/or clinicians may be willing to accept a limited reduction in bioavailability if such reduction is balanced by a corresponding increase in the speed of absorption, and thus of action, by the therapeutic peptide(s) contained within the formulation.

[0009] There has thus been a long-felt need for an oral peptide formulation which is capable of more rapid therapeutic action, e.g. , in contrast to the formulations described in the '014 and '918 patents discussed above, while still providing a desirable degree of

bioavailability. [0010] Normally, the plasma membrane of eukaryotic cells is impermeable to large peptides or proteins. However, certain hydrophobic amino acid sequences, variously called as ferry peptides or membrane translocating sequences, when fused to the N- or C-terminus of functional proteins, can act as membrane translocators, and mediate the transport of these proteins into living cells. This method of protein delivery into cells, while potentially very useful, has two main drawbacks. First, the protein cannot be targeted to any specific cell type. Therefore, once it is injected and enters the circulation, it will presumably enter all cell types in a non-specific, non-receptor mediated manner. This would cause a huge dilution effect, such that very high concentrations of the protein need to be injected in order to achieve an effective concentration in the target cell type. Second, the protein could be extremely toxic when it enters cells in non-target tissues. A third drawback is that the continued presence of the ferry peptide could make the protein very antigenic, and could also interfere with its biological activity. These above drawbacks apply whether the fusion was delivered by injection or nasal or oral route.

[0011] Nasal delivery is also frequently plagued by low bioavailability of the therapeutic peptide. Even where nasal delivery is possible, manufacturing costs can be undesirably high because of the large concentration of therapeutic peptide required to provide clinical efficacy in view of low bioavailability occasioned by the difficulty of peptides crossing the nasal mucosa.

[0012] Therapeutic peptides are often poorly absorbed by tissues, and are readily degraded by bodily fluids. For this reason, formulations were developed for the administration of peptide therapeutics via the nasal route. The nasal formulation was designed to be stored in a multi-dose container that was stable for an extended period of time and resisted bacterial contamination. The preservative in the formulation, benzalkonium chloride, was found to enhance the absorption of the peptide therapeutic. However, benzalkonium chloride was reported (P. Graf et al , Clin. Exp. Allergy 25 :395-400; 1995) to aggravate rhinitis medicamentosa in healthy volunteers who were given a decongestant nasal spray containing the preservative. It also had an adverse effect on nasal mucosa (H. Hallen et al, Clin. Exp. Allergy 25:401-405; 1995), Berg et al. (Laryngoscope 104: 1 153-1 158; 1994) disclose that respiratory mucosal tissue that was exposed in vitro underwent severe morphological alterations. Benzalkonium chloride also caused significant slowing of the mucocilary transport velocity in the ex vivo frog palate test (P.C. Braga et al , J. Pharm. Pharmacol. 44:938-940; 1992). SUMMARY

[0013] In one aspect, the present technology provides a composition including a therapeutically effective amount of an aromatic-cationic peptide and at least one counter ion, wherein the aromatic-cationic peptide and counter ion are encapsulated within a lipid particle.

[0014] In some embodiments, the lipid particle of the composition includes a phospholipid.

[0015] In some embodiments, the composition also includes one or more absorption enhancers. In some embodiments, the composition also includes one or more additional composition selected from the group consisting of a membrane translocator (MT), a membrane fluidizing agent, a peptide active ingredient, a second peptide, a lubricant, and combinations thereof.

[0016] In some embodiments, the aromatic-cationic peptide of the composition is D-Arg- 2'6'-Dmt-Lys-Phe-NH ₂ . In some embodiments, the aromatic-cationic peptide of the composition is Phe-D-Arg-Phe-Lys-NH ₂ .

[0017] In some embodiments, the composition also includes an enteric coating.

[0018] In one aspect, the present technology provides a process for producing a

pharmaceutical composition including combining to form a first composition: a

therapeutically effective amount of aromatic-cationic peptide, at least one counter ion, and a phospholipid; and suspending the first composition in a hydrophobic medium.

[0019] In some embodiments, the first composition further comprises a matrix forming polymer. In some embodiments, the first composition is in a solid phase. In some embodiments, the first composition is a suspension. In some embodiments, the first composition is an adsorbate. In some embodiments, the first composition also includes one or more absorption enhancers.

[0020] In some embodiments, the process also includes adding an enteric coating.

[0021] In some embodiments, the lipid or the phospholipid of the composition or the process includes a medium chain fatty acid.

[0022] In some embodiments, the aromatic-cationic peptide is D-Arg-2'6'-Dmt-Lys-Phe- Ν¾. In some embodiments, the aromatic-cationic peptide is Phe-D-Arg-Phe-Lys-NH2. [0023] In some embodiments, the counter ion is selected from the group consisting of tosylate, napsylate, docusate, sodium laurel sulfate (SLS), caprate, laurate, myristate, palmitate, stearate, and oleate.

[0024] In another aspect, the present technology provides a method for increasing the bioavailabilty or stability of an aromatic-cationic peptide comprising: (i) linking the aromatic-cationic peptide to at least one counter ion to generate a peptide-counter ion complex; and (ii) encapsulating the peptide-counter ion complex within a lipid particle, thereby increasing the bioavailabilty or stability of the aromatic-cationic peptide.

[0025] In some embodiments of the method, the counter ion is selected from the group consisting of tosylate, napsylate, docusate, sodium laurel sulfate (SLS), caprate, laurate, myristate, palmitate, stearate, and oleate.

[0026] In some embodiments of the method, the aromatic-cationic peptide is D-Arg-2'6'- Dmt-Lys-Phe-NH ₂ . In some embodiments, the aromatic-cationic peptide is Phe-D-Arg-Phe- Lys-NH ₂ .

[0027] In some embodiments of the method, the lipid particle comprises a phospholipid. In some embodiments of the method, the lipid particle or the phospholipid includes a medium chain fatty acid.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 is a graph showing the dissolution of 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ acetate salt in the absence (triangle) and presence (square) of 20 μΜ trypsin. D-Arg-2'6'- Dmt-Lys-Phe-NH ₂ Dose = 0.78 mgA/mL.

[0029] FIG. 2A is a graph showing the dissolution of 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ docusate in the absence (triange) and presence (square and diamond) of 20 μΜ trypsin. D- Arg-2'6'-Dmt-Lys-Phe-NH ₂ Dose = 0.70 mgA/mL.

[0030] FIG 2B is a graph showing the dissolution of 1 :6 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ docusate in the absence (triangle) and presence (square and diamond) of 20 μΜ trypsin. D- Arg-2'6'-Dmt-Lys-Phe-NH ₂ Dose = 0.73 mgA/mL.

[0031] FIG. 3A is a graph showing the dissolution of 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ napsylate in the absence (triangle) and presence (square and diamond) of 20 μΜ trypsin. D- Arg-2'6'-Dmt-Lys-Phe-NH ₂ Dose = 0.75 mgA/mL. [0032] FIG. 3B is a graph showing the dissolution of 1 :6 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ napsylate in the absence (triangle) and presence (square and diamond) of 20 μΜ trypsin. D- Arg-2'6'-Dmt-Lys-Phe-NH ₂ Dose = 0.77 mgA/mL.

[0033] FIG. 4A is a graph showing the dissolution of 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ oleate in the absence (triangle) and presence (square and diamond) of 20 μΜ trypsin. D- Arg-2'6'-Dmt-Lys-Phe-NH ₂ Dose = 0.39 mgA/mL.

[0034] FIG. 4B is a graph showing the dissolution of 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ stearate in the absence (triangle) and presence (square and diamond) of 20 μΜ trypsin. D- Arg-2'6'-Dmt-Lys-Phe-NH ₂ Dose = 0.39 mgA/mL.

[0035] FIG. 5A is a graph showing the degradation of 1 :6 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ docusate complex in the presence of 20 μΜ trypsin dosed from the Type I (square), Type II (diamond), or Type III (triangle) lipid formulation at 15 mgA/g the lipid formulation. D-Arg- 2'6'-Dmt-Lys-Phe-NH ₂ Dose = 0.75 mgA/mL.

[0036] FIG. 5B is a graph showing the degradation of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ acetate (square), 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ docusate complex (diamond), and 1 :6 D- Arg-2'6'-Dmt-Lys-Phe-NH ₂ docusate complex (triangle) in the presence of 20 μΜ trypsin dosed from the Type II lipid formulation at 15 mgA/g in the lipid formulation. D-Arg-2'6'- Dmt-Lys-Phe-NH ₂ Dose = 0.75 mgA/mL.

[0037] FIG. 6A is a graph showing the degradation of 1 :6 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ docusate complex in the presence of 20 μΜ trypsin dosed from the Type II lipid formulation after varying stability times and conditions. The initial (square) samples were pre-emulsified for 2 hours, while the other samples were pre-emulsified for 1 hour. D-Arg-2'6'-Dmt-Lys- Phe-NH ₂ Dose = 0.75 mgA/mL.

[0038] FIG. 6B is a graph showing the degradation of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ acetate in the presence of 20 μΜ trypsin dosed from the Type II lipid formulation (15mgA/g) after varying stability times and conditions. The initial (square) samples were pre-emulsified for 2 hours, while the other samples were pre-emulsified for 1 hour. D-Arg-2'6'-Dmt-Lys- Phe-NH ₂ Dose = 0.75 mgA/mL.

[0039] FIG. 7 shows some of the known intestinal proteases.

[0040] FIG. 8 exemplifies amino acid sequences, each of which can be used as a membrane translocator. [0041] FIG. 9 shows some plasma proteases as well as their target sequences.

DETAILED DESCRIPTION

Definitions

[0042] The present technology is described herein using several definitions, as set forth throughout the specification. The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this present technology belongs.

[0043] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0044] As used herein, "about" will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, "about" will mean up to plus or minus 10% of the particular term.

[0045] As will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to groups having 1 , 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to groups having 1 , 2, 3, 4, or 5 atoms, and so forth.

[0046] As used herein, the "administration" of an agent, drug, or peptide to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or

subcutaneously), or topically. Administration includes self-administration and the administration by another.

[0047] As used herein, the term "amino acid" includes naturally-occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally-occurring amino acids. Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally-occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups {e.g. , norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally-occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally- occurring amino acid. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0048] As used herein, the term "antigen" refers to a molecule or a portion of a molecule capable of stimulating an immune response, which is additionally capable of inducing an animal or human to produce antibody capable of binding to an epitope of that antigen.

[0049] As used herein, "bioactive molecule" refers to those compounds that have an effect on or elicit a response from living cells, tissues, or the organism as a whole. A non-limiting example of a bioactive molecule is D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ .

[0050] As used herein, "biological barrier" is meant to include biological membranes such as the plasma membrane as well as any biological structures sealed by tight junctions (or occluding junctions) such as the mucosal or vascular epithelia, (including, but not limited to, the gastrointestinal or respiratory epithelia), and the blood brain barrier. Moreover, those skilled in the art will recognize that translocation may occur across a biological barrier in a tissue containing cells such as epithelial cells or endothelial cells.

[0051] As used herein, "coupled" is meant to include all such specific interactions that result in two or more molecules showing a preference for one another relative to some third molecule, including any type of interaction enabling a physical association between, e.g., an aromatic-cationic peptide and a penetrating peptide.

[0052] As used herein, the term "effective amount" refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or disorder or one or more signs or symptoms associated with a disease or disorder. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compounds may be administered to a subject having one or more signs or symptoms of a disease or disorder. For example, a

"therapeutically effective amount" of the aromatic-cationic peptide is meant levels in which the physiological effects of a disease or condition are, at a minimum, ameliorated. A therapeutically effective amount can be given in one or more administrations. The amount of a compound which constitutes a therapeutically effective amount will vary depending on the compound, the disorder and its severity, and the general health, age, sex, body weight and tolerance to drugs of the subject to be treated, but can be determined routinely by one of ordinary skill in the art.

[0053] As used herein, "effective translocation" or "efficient translocation" refers to the introduction of the composition to a biological barrier that results in at least 5%, at least 10%, or at least 20% translocation of the composition (e.g., a composition comprising an aromatic- cationic peptide) across the biological barrier.

[0054] As used herein, "epitope" refers to the portion of any molecule capable of being recognized by and bound by a major histocompatibility complex ("MHC") molecule and recognized by a T cell or bound by an antibody.

[0055] As used herein, "glycosaminoglycan" refers to a polysaccharide that contains amino containing sugars.

[0056] As used herein, "impermeable molecules" are molecules that are unable to efficiently cross biological barriers, such as the cell membrane or tight junctions. In some embodiments, anionic impermeable molecules are polysaccharides, e.g., glycosaminoglycans, nucleic acids, or net negatively charged proteins. In some embodiments, cationic

impermeable molecules are net positively charged proteins.

[0057] As used herein, "isolated" or "purified" polypeptide or peptide refers to a polypeptide or peptide that is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the agent is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. For example, an isolated aromatic-cationic peptide would be free of materials that would interfere with diagnostic or therapeutic uses of the agent. Such interfering materials may include enzymes, hormones and other proteinaceous and nonproteinaceous solutes. [0058] As used herein, the term "lamination" shall have its conventional meaning as something which is composed of layers of firmly united material, but which involves little, if any, interaction between the layers.

[0059] As used herein, "membrane fluidizing agents" are defined as medium chain alcohols which have a carbon chain length of from 4 to 15 carbon atoms (e.g., including 5 to 15, 5 to 12, 6, 7, 8, 9, 10, or 1 1 carbon atoms). For example, a membrane fluidizing agent can be a linear (e.g., saturated or unsaturated), branched (e.g., saturated or unsaturated), cyclical (e.g. , saturated or unsaturated), or aromatic alcohol. Examples of suitable linear alcohols include, but are not limited to, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, and pentadecanol. Examples of branched alcohols include, but are not limited to, geraniol, farnesol, rhodinol, citronellol. An example of a cyclical alcohol includes, but is not limited to, menthol, terpineol, myrtenol, perillyl and alcohol. Examples of suitable aromatic alcohols include, but are not limited to, benzyl alcohol, 4-hydroxycinnamic acid, thymol, styrene glycol, and phenolic compounds.

Examples of phenolic compounds include, but are not limited to, phenol, m-cresol, and m- chlorocresol.

[0060] A protein's net charge is determined by two factors: 1) the total count of acidic amino acids vs. basic amino acids, and 2) the specific solvent pH surroundings, which expose positive or negative residues. As used herein, "net positively" or "net negatively" charged proteins are proteins that, under non-denaturing pH surroundings, have a net positive or net negative electric charge.

[0061] As used herein "parenteral" refers to injections given through some other route than the alimentary canal, such as subcutaneously, intramuscularly, intraorbitally (i.e., into the eye socket or behind the eyeball), intracapsularly, intraspinally, intrasternally, or intravenously.

[0062] As used herein, a "penetration composition" includes any composition of a water soluble composition immersed in a hydrophobic medium, that facilitates the effective translocation of a substance, e.g., aromatic-cationic peptide, across a biological barrier. In some embodiments, penetration compositions utilize at least one membrane fluidizing agent.

[0063] As used herein, "pharmaceutically acceptable salt" refers to a salt prepared from a base or an acid which is acceptable for administration to a patient, such as a mammal (e.g., salts having acceptable mammalian safety for a given dosage regime). [0064] As used herein, "pharmaceutically active agent" and "therapeutic agent" are used interchangeably to refer to a chemical material or compound, which, when administered to an organism, induces a detectable pharmacologic and/or physiologic effect.

[0065] As used herein, "polysaccharide" refers to a linear or branched polymer composed of covalently linked monosaccharides; glucose is the most common monosaccharide and there are normally at least eight monosaccharide units in a polysaccharide and usually many more. Polysaccharides have a general formula of Cx(I¾0)y where x is usually a large number between 200 and 2500. Considering that the repeating units in the polymer backbone are often six-carbon monosaccharides, the general formula can also be represented as (CgHio0 ₅ )n where there are normally between 40 and 3000 monosaccharide units in a polysaccharide.

[0066] As used herein, "polynucleotide" refers to any molecule composed of DNA nucleotides, RNA nucleotides or a combination of both types which comprises two or more of the bases guanidine, citosine, timidine, adenine, uracil or inosine, inter alia. A

polynucleotide may include natural nucleotides, chemically modified nucleotides and synthetic nucleotides, or chemical analogs thereof and may be single-stranded or double- stranded. The term includes "oligonucleotides" and encompasses "nucleic acids."

[0067] As used herein, "polypeptide" refers to a molecule composed of covalently linked amino acids and the term includes peptides, polypeptides, proteins and peptidomimetics. A peptidomimetic is a compound containing non-peptidic structural elements that is capable of mimicking the biological action(s) of a natural parent peptide. Some of the classical peptide characteristics such as enzymatically scissile peptidic bonds are normally not present in a peptidomimetic.

[0068] As used herein, "selectively translocating" refers to the relative translocation of the aromatic-cationic peptide as compared to the relative impermeability of other non-aromatic- cationic peptides such as bystander molecules (e.g., impermeable molecules other than the aromatic-cationic peptide itself).

[0069] As used herein, "small molecule" refers to a low molecular weight organic compound which may be synthetically produced or obtained from natural sources and typically has a molecular weight of less than 2000 Da, or less than 1000 Da or even less than 600 Da e.g., less than or about 550 Da or less than or about 500 Da or less than or about 400 Da; or about 400 Da to about 2000 Da; or about 400 Da to about 1700 Da. Examples of small molecules are ergotamine (molecular weight= 82 Da), fondaparinux (molecular weight=1727 Da), leuprolide (molecular weight= 1209 Da), vancomycin (molecular weight=1449 Da), gentamicin (molecular weight=478 Da) and doxorubicin (molecular weight=544 Da), and aliskiren hemi-fumarate (molecular weight=610 Da).

[0070] As used herein, "stabilizers of protein structure" refer to any compounds that can stabilize protein structure under aqueous or non-aqueous conditions, such as polycationic molecules, polyanionic molecules, and uncharged polymers. One example of a polycationic molecule that can function as a protein stabilizer is a polyamine such as spermine. Examples of polyanionic molecule that can function as protein stabilizers include, but are not limited to, phytic acid and sucrose octasulfate. Non-limiting examples of uncharged polymers that can function as protein stabilizers include polyvinylpyrrolidone and polyvinyl alcohol.

[0071] As used herein, the terms "treating" or "treatment" or "alleviation" refers to therapeutic treatment, wherein the object is to reduce or slow down (lessen) the targeted pathologic condition or disorder. A subject is successfully "treated" for a disease or condition if, after receiving a therapeutic amount of the aromatic-cationic peptides according to the methods described herein, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of the disease or condition.

[0072] As used herein, "prevention" or "preventing" of a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0073] It is also to be appreciated that the various modes of treatment or prevention of medical conditions as described are intended to mean "substantial," which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved. Treating a disease or condition, as used herein, also refers to treating any one or more of the conditions underlying the disease or condition to be treated.

[0074] As used herein, a "signal peptide" or "signal sequence" refers to a sequence of amino acids generally but not necessarily of a length of about 10 to about 50 or more amino acid residues, many (typically about 55-60%) residues of which are hydrophobic such that they have a hydrophobic, lipid-soluble portion.

[0075] As used herein, "water soluble composition" refers to compositions which can be solubilized in a hydrophilic or partially hydrophilic solvent. A hydrophilic or partially hydrophilic solvent may include water, or a non-aqueous medium such as mono-alcohols, di- alcohols, or tri-alcohols. Examples of suitable mono-alcohols include, but are not limited to, ethanol, propanol, isopropanol and butanol. An example of a di-alcohol includes, but is not limited to, propylene glycol. An example of a tri-alcohol includes, but is not limited to, glycerol.

Aromatic-cationic peptides

[0076] Aromatic-cationic peptides which may benefit from oral delivery in accordance with the present technology include aromatic-cationic peptides that are physiologically active and have a plurality of amino acids and at least one peptide bond in its molecular structure. The present formulations, by several mechanisms, suppress the degradation of the active ingredients (e.g., aromatic-cationic peptides) by protease that would otherwise tend to cleave one or more of the peptide bonds of the active ingredient. The molecular structure may further include other constituents or modifications. Both man-made and natural peptides can be orally delivered in accordance with the present technology.

[0077] In some aspects, the present technology provides an aromatic-cationic peptide or a pharmaceutically acceptable salt thereof such as acetate salt or trifluoroacetate salt. In some embodiments, the peptide comprises at least one net positive charge; a minimum of three amino acids; a maximum of about twenty amino acids; a relationship between the minimum number of net positive charges (p _m ) and the total number of amino acid residues (r) wherein 3p _m is the largest number that is less than or equal to r + 1; and a relationship between the minimum number of aromatic groups (a) and the total number of net positive charges (p _t ) wherein 2a is the largest number that is less than or equal to p _t + 1 , except that when a is 1 , p _t may also be 1. In some embodiments, one or more amino acids (e.g., 1, 2, 3, 4 or all) of the peptides are D amino acids.

[0078] In some embodiments, the peptide comprises the amino acid sequence Phe-D-Arg- Phe-Lys-NH2 or D-Arg-2'6'-Dmt-Lys-Phe-NH2. In some embodiments, the peptide comprises one or more peptides of Table A:

TABLE A

H-D-Arg(N ^Q: Me)-Dmt(NMe)-Lys(N ^a Me)-Plie(NMe)-NH ₂

D-Arg-Dmt-Lys-Dmt-Lys-Trp-NH ₂

D-Arg-Dmt-Lys-Dmt-Lys-Met-NH ₂

H-D-Arg^[CH ₂ -NH]Dmt-Lys-Phe-NH ₂

H-D-Arg-Dmt-LysΨ[CH ₂ -NH]Phe-NH2

D-Arg-2'6'Dmt-Lys-Phe-NH2

Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr -Trp- D-His-Trp-His-D-Lys-Asp

Dmt-D-Arg-Phe-(dns)Dap-NH ₂

Γ I Dmt-D-Arg-Phe-Lys-Ald-NH ₂ j

2',6'-dimethyltyrosine (2'6'-Dmt or Dmt)

2 ^f ,6'-dimethylphenylalanine (2'6'-Dmp or Dmp)

Cha = cyclohexyl alanine

[0079] In some embodiments, the aromatic-cationic peptide is de ined by Formula I.

wherein R ¹ and R ² are each independently selected from

(i) hydrogen;

(ii) linear or branched Ci-Ce alkyl; where m = l-3;

R ³ and R ⁴ are each independently selected from

(i) hydrogen;

(ii) linear or branched Cy-Ce alkyl;

(iii) Ci-C ₆ alkoxy;

(iv) amino;

(v) C1-C4 alkylamino;

(vi) Ci-C ₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where "halogen" encompasses chloro, fluoro, bromo, and iodo;

R ⁵ , R ⁶ , R ⁷ , R ⁸ , and R ⁹ are each independently selected from

(i) hydrogen;

(ii) linear or branched C ₁ -C6 alkyl;

(iii) Ci-C ₆ alkoxy;

(iv) amino;

(v) C ₁ -C ₄ alkylamino;

(vi) C1-C4 dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where "halogen" encompasses chloro, fluoro, bromo, and iodo; and n is an integer from 1 to 5.

6

[0080] In a particular embodiment, R ¹ and R ² are hydrogen; R ³ and R ⁴ are methyl; R ⁵ , R ⁽ R ⁷ , R ^s , and R ⁹ are all hydrogen; and n is 4. [0081] In some embodiments, the aromatic-cationic peptide is defined by Formula II:

wherein R ¹ and R ² are each independently selected from

(i) hydrogen;

(ii) linear or branched C1-C6 alkyl; where m = 1 -3;

R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are each independently selected from

(i) hydrogen;

(ii) linear or branched Ci-Ce alkyl;

(iii) Ci-C ₆ alkoxy;

(iv) amino;

(v) C1-C4 alkylamino;

(vi) Ci-C ₄ dialkylamino;

(vii) nitro;

(viii) hydroxyl;

(ix) halogen, where "halogen" encompasses chloro, fluoro, bromo, and iodo; and n is an integer from 1 to 5. [0082] In a particular embodiment, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , and R ¹² are all hydrogen; and n is 4. In another embodiment, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , and R ¹¹ are all hydrogen; R ⁸ and R ¹² are methyl; R ¹⁰ is hydroxyl; and n is 4.

[0083] In one embodiment, the aromatic-cationic peptides of the present technology have a core structural motif of alternating aromatic and cationic amino acids. For example, the peptide may be a tetrapeptide defined by any of Formulas III to VIII set forth below:

Aromatic - Cationic - - Aromatic - Cationic (Formula III)

Cationic - Aromatic - - Cationic - Aromatic (Formula IV)

Aromatic - Aromatic - Cationic - Cationic (Formula V)

Cationic - Cationic - Aromatic - Aromatic (Formula VI)

Aromatic - Cationic - - Cationic - Aromatic (Formula VII)

Cationic - Aromatic - - Aromatic - Cationic (Formula VIII)

wherein, Aromatic is a residue selected from the group consisting of: Phe (F), Tyr (Y), and Trp (W). In some embodiments, the Aromatic residue may be substituted with a saturated analog of an aromatic residue, e.g., Cyclohexylalanine (Cha). In some embodiments, Cationic is a residue selected from the group consisting of: Arg (R), Lys ( ), and His (H).

[0084] The peptides disclosed herein may be formulated as pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" means a salt prepared from a base or an acid which is acceptable for administration to a patient, such as a mammal (e.g. , salts having acceptable mammalian safety for a given dosage regime). However, it is understood that the salts are not required to be pharmaceutically acceptable salts, such as salts of intermediate compounds that are not intended for administration to a patient. Pharmaceutically acceptable salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. In addition, when a peptide contains both a basic moiety, such as an amine, pyridine or imidazole, and an acidic moiety such as a carboxylic acid or tetrazole, zwitterions may be formed and are included within the term "salt" as used herein. Salts derived from pharmaceutically acceptable inorganic bases include ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, and zinc salts, and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally-occurring amines and the like, such as arginine, betaine, caffeine, choline, Ν,Ν'-dibenzylethylenediamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N- ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperadine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like. Salts derived from pharmaceutically acceptable inorganic acids include salts of boric, carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Salts derived from

pharmaceutically acceptable organic acids include salts of aliphatic hydroxyl acids (e.g. , citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic

monocarboxylic acids (e.g. , acetic, butyric, formic, propionic and trifluoroacetic acids), amino acids (e.g., aspartic and glutamic acids), aromatic carboxylic acids (e.g., benzoic, p- chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylacetic acids), aromatic hydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic, 1 -hydroxynaphthalene-2- carboxylic and 3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic and succinic acids), glucoronic, mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids (e.g. , benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic, isethionic, methanesulfonic, naphthalenesulfomc, naphthalene-l,5-disulfonic, naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid, and the like. In some embodiments, the salt is an acetate salt. Additionally or alternatively, in other embodiments, the salt is a trifluoroacetate salt. In some embodiments, the salt is a tartrate salt.

[0085] In some embodiments, the pharmaceutically acceptable salt includes the peptides of Formulas I or II and a pharmaceutically acceptable acid. In some embodiments, the pharmaceutically acceptable acid includes l-hydroxy-2 -naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4- aminosalicylic acid, acetic acid, adipic acid, ascorbic acid (L), aspartic acid (L),

benzenesulfonic acid, benzoic acid, camphoric acid (+), camphor- 10-sulfonic acid (+), capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1 ,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid (D), gluconic acid (D), glucuronic acid (D), glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid (DL), lactobionic acid, lauric acid, maleic acid, malic acid (- L), malonic acid, mandelic acid (DL), methanesulfonic acid, naphthalene- 1 , 5 -disulfonic acid, naphthalene-2 -sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, pyroglutamic acid (- L), salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid (+ L), thiocyanic acid,

toluenesulfonic acid (p), and undecylenic acid. In some embodiments, the pharmaceutically acceptable acid is tartaric acid, where such embodiments of the pharmaceutically acceptable salt are referred to as a tartrate salt.

[0086] In some embodiments of the pharmaceutically acceptable salt, the peptide comprises the amino acid sequence 2'6'-Dmt-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe-Lys-NH2, or D-Arg- 2'6'-Dmt-Lys-Phe-NH ₂ . In some embodiments of the pharmaceutically acceptable salt, the eptide comprises one or more peptides of Table A:

2',6'-dimethyltyrosine (2'6'-Dmt or Dmt) 2',6'-dimethylphenylalanine (2'6'-Dmp or Dmp)

Cha = cyclohexyl alanine

[0087] In some embodiments of the pharmaceutically acceptable salt, the pharmaceutically acceptable salt is a tartrate salt and the peptide includes the amino acid sequence 2'6'-Dmt-D- Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe-Lys-NH ₂ , or D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ .

[0088] The aromatic-cationic peptides of the present technology disclosed herein may be synthesized by any of the methods well known in the art. Suitable methods for chemically synthesizing the protein include, for example, liquid phase and solid phase synthesis, and those methods described by Stuart and Young in Solid Phase Peptide Synthesis, Second Edition, Pierce Chemical Company (1984), and in Methods Enzymol., 289, Academic Press, Inc, New York (1997). Recombinant peptides may be generated using conventional techniques in molecular biology, protein biochemistry, cell biology, and microbiology, such as those described in Current Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed.

(1997); Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989); DNA Cloning: A Practical Approach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis, Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds. (1985); Transcription and Translation, Hames & Higgins, Eds. (1984); Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the series, Meth. Enzymol., (Academic Press, Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring Harbor Laboratory, NY, 1987); and Meth.

Enzymol, Vols. 154 and 155, Wu & Grossman, and Wu, Eds., respectively.

[0089] Additional peptide active compounds of the present technology include, but are not limited to, polypeptides such as insulin, vasopressin, salmon calcitonin, glucagon-like peptide 1, calcitonin, and analogs thereof. Other examples include, but are not limited to, calcitonin gene-related peptide, parathyroid hormone (full length or truncated, amidated or in the free acid form, further modified or not), luteinizing hormone-releasing factor, erythropoietin, tissue plasminogen activators, human growth hormone, adrenocorticototropin, various interleukins, enkephalin, DALDA derivatives such as dmt-DALDA and the like. Many others are known in the art. It is expected that any pharmaceutical compound having peptide bonds which would be subject to cleavage in the gastrointestinal tract would benefit from oral delivery in accordance with the present technology because of the reduction in such cleavage that is afforded by the present formulations. [0090] In some embodiments, the aromatic-cationic peptide includes the sequence 2'6'- Dmt-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe-Lys-NH ₂ , D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ , or combinations of two or more thereof. In some embodiments, the aromatic-cationic peptide is from about 0.02 to 0.2 percent by weight relative to the total weight of the overall pharmaceutical composition. Other aromatic-cationic peptides of the present technology may be present at higher or lower concentrations depending on desired target blood concentrations for the peptide and its bioavailability in the oral delivery system of the present technology.

[0091] Aromatic-cationic peptide precursors may be made by either chemical (e.g., using solution and solid phase chemical peptide synthesis) or recombinant synthesis methods known in the art. Precursors of e.g. , amidated aromatic-cationic peptides of the present technology may be made in like manner. In some embodiments, recombinant production is believed significantly more cost effective. In some embodiments, precursors are converted to active peptides by amidation reactions that are also known in the art. For example, enzymatic amidation is described in U.S. Pat. No. 4,708,934 and European Patent Publications 0 308 067 and 0 382 403. Recombinant production can be used for both the precursor and the enzyme that catalyzes the conversion of the precursor to the desired active form of the aromatic-cationic peptide. Such recombinant production is discussed in Biotechnology, Vol. 11 (1993) pp. 64-70, which further describes a conversion of a precursor to an amidated product. During amidation, a keto-acid such as an alpha-keto acid, or salt or ester thereof, wherein the alpha-keto acid has the molecular structure RC(0)C(0)OH, and wherein R is selected from the group consisting of aryl, a C ₁ -C ₄ hydrocarbon moiety, a halogenated or hydroxylated Ci-C ₄ hydrocarbon moiety, and a C1-C4 carboxylic acid, may be used in place of a catalase co-factor. Examples of these keto acids include, but are not limited to, ethyl pyruvate, pyruvic acid and salts thereof, methyl pyruvate, benzoyl formic acid and salts thereof, 2-ketobutyric acid and salts thereof, 3-methyl-2-oxobutanoic acid and salts thereof, and 2-keto glutaric acid and salts thereof.

[0092] In some embodiments, the production of the recombinant aromatic-cationic peptide may proceed, for example, by producing glycine-extended precursor in E. coli as a soluble fusion protein with glutathione-S-transferase. An a-amidating enzyme catalyzes conversion of precursors to active aromatic-cationic peptide. That enzyme is recombinantly produced, for example, in Chinese Hamster Ovary (CHO) cells as described in the Biotechnology article cited above. Other precursors to other amidated peptides may be produced in like manner. Peptides that do not require amidation or other additional functionalities may also be produced in like manner. Other peptide active agents are commercially available or may be produced by techniques known in the art.

Oral Delivery of Peptide Pharmaceutical Compositions

[0093] It has surprisingly been found that in some embodiments, administering the pharmaceutical formulations of this technology without an enteric coating increases the speed of peptide absorption (relative to corresponding enteric-coated formulation) without reducing bioavailability below practical levels. While some reduction in bioavailability does occur, this reduction is not expected to preclude effective medical treatment, or to unduly detract from the advantages of e.g., in the case of pain relief. In some embodiments, the present formulations permit more rapid absorption of the active aromatic-cationic peptides of the present technology or pharmaceutically acceptable salts thereof, such as acetate salt or trifluoroacetate salt, due to the reduction in the time necessary for the vehicle (e.g. , a capsule or tablet) to be dissolved and the active ingredients to be released. In some embodiments, the formulations also permit such release further upstream in the alimentary canal, e.g. , in the esophagus and/or stomach, instead of awaiting passage of the material into the intestine. See e.g., U.S. Patent Publication No. 2005/0282756 and U.S. Patent Publication No.

2007/0134279, herein incorporated by reference in their entireties.

[0094] In accordance with some embodiments of the present technology, subjects in need of treatment with aromatic-cationic peptide active ingredients are provided with a finished pharmaceutical product, for example in tablet form of an ordinary size in the pharmaceutical industry, formed of an oral pharmaceutical composition comprising one or more of such peptide active ingredients (at appropriate dosage). The finished pharmaceutical product may additionally be prepared, if desired, in (for example) capsule form. The dosages and frequency of administering the products are discussed in more detail below. Subjects who may benefit are any who suffer from disorders that respond favorably to increased levels of a peptide-containing compound. In some embodiments, oral formulations include a peptide and one or more of an absorption enhancer, a pH lowering agent, other optional agents and an enteric coating.

[0095] By way of example, but not by way of limitation, the oral peptide formulations described herein are useful in the treatment of disorders stemming from or related to mitochondrial permeability transition (MPT) and/or cellular oxidative damage. For example, oral peptide formulations of the aromatic-cationic peptides of the present technology Phe-D- Arg-Phe-Lys-NH ₂ and D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ , or pharmaceutically acceptable salts thereof, may be used to treat subjects suffering from vascular occlusion, kidney ischemia, tissue ischemia-rep erfusion injury, acute myocardial infarction, diseases or disorders of the eye, or neurological disorders such as Alzheimer's and Parkinson's diseases.

Pharmaceutically acceptable salts include, but are not limited to, e.g., acetate salt and trifluoroacetate salt.

[0096] Not wishing to be bound by theory, the pharmaceutical formulations described herein are believed to overcome a series of different and unrelated natural barriers to bioavailability. Various components of the pharmaceutical compositions act to overcome different barriers by mechanisms appropriate to each, and in some embodiments, result in synergistic effects on the bioavailability of a peptide active ingredient. As discussed below, inherent physical and chemical properties of peptides make certain absorption enhancers more effective than others in boosting its bioavailability.

[0097] In some embodiments, the aromatic-cationic peptide active compound of the present technology is contained within a formulation adopted for oral administration. In accordance with the present technology, in some embodiments, proteolytic degradation of the peptide by stomach proteases (most of which are active in the acid pH range) is reduced due to administration of the formulation to the patient on an empty stomach (although this is not required in order to achieve adequate results), while degradation by intestinal or pancreatic proteases (most of which are active in the neutral to basic pH range) is reduced due to the effect of the pH lowering agent in adjusting the pH of the intestinal environment to sub- optimal levels. In some embodiments, solubility enhancers are employed to aid passage of the aromatic-cationic peptide through the intestinal epithelial barrier.

[0098] The pH-lowering agent is believed to lower the local pH (where the active agent has been released) to levels below the optimal range for many intestinal proteases. This decrease in pH reduces the proteolytic activity of the intestinal proteases, thus affording protection to the peptide from potential degradation should the peptide be present within the intestine. The activity of these proteases is diminished by the temporarily acidic environment as discussed herein. For example, sufficient acid should be provided that local intestinal pH is lowered temporarily to 5.5 or below, 4.7 or below, or 3.5 or below. The sodium bicarbonate test described below (in the section captioned "the pH-Lowering Agent") is indicative of the required acid amount. In some embodiments, conditions of reduced pH typically persist for a time period sufficient to protect the aromatic-cationic peptide from proteolytic degradation until at least some of the aromatic-cationic peptide has had an opportunity to cross into the bloodstream. By way of example, but not by way of limitation, for salmon calcitonin, a 32 amino acid peptide, experiments have demonstrated T _max of 5-15 minutes for blood levels of salmon calcitonin when the active components are injected directly into the duodenum, ilium or colon. The absorption enhancers of the present formulations synergistically promote peptide absorption into the blood while conditions of reduced proteolytic activity prevail. In some embodiments, the mechanism by which the present formulations are believed to accomplish the goal of enhanced bioavailability is aided by having active components of the finished pharmaceutical product released together as simultaneously as possible.

[0099] The absorption enhancer, which may be a solubility enhancer and/or transport enhancer (as described in more detail below), aids transport of the aromatic-cationic peptide from the alimentary canal into the blood, and may promote the process so that it better occurs during the time period of reduced intestinal pH and reduced intestinal proteolytic activity. Many surface active agents may act as both solubility enhancers and transport (uptake) enhancers. Again without intending to be bound by theory, it is believed that enhancing solubility provides (1) a more simultaneous release of the active components of the present formulations into the aqueous portion of the alimentary tract, (2) better solubility of the peptide in, and transport through, a mucous layer such as that found along the intestinal walls. Once the aromatic-cationic peptide reaches, e.g., the intestinal walls, an absorption enhancer provides better transport through the brush border membrane of the intestine into the blood, via either transcellular or paracellular transport. As discussed in more detail below, many compounds may provide both functions. In those instances, embodiments utilizing both of these functions may do so by adding only one additional compound to the pharmaceutical composition. In other embodiments, separate absorption enhancers may provide the two functions separately. Additionally, many charged lipophilic species can interact with an aromatic-cationic peptide via charge and via hydrophobic interactions such that transport may be facilitated not just by the direct effect of the permeation enhancer on the absorbing membrane but also enabling the interaction of the peptide with the membrane either as a complex or single entity that cannot occur in the presence of peptide alone.

[0100] Each of the ingredients of the finished pharmaceutical product of the present technology is separately discussed below. In some embodiments, combinations of multiple pH-lowering agents, or multiple absorption and/or solubility enhancers can be used as well as using just a single pH-lowering agent and/or single enhancer. Some exemplary, non-limiting combinations are discussed below.

The pH-Lowering Agent and Protease Inhibitor

[0101] In embodiments of formulations (e.g., oral formulations) including a pH lowering agent, the total amount of the pH-lowering compound to be administered with each administration of aromatic-cationic peptide is typically an amount which, when released into the intestine for example, is sufficient to lower the local intestinal pH substantially below the pH optima for proteases found there. The quantity required will necessarily vary with several factors including the type of pH-lowering agent used (discussed below) and the equivalents of protons provided by a given pH-lowering agent. In practice, the amount required to provide good bioavailability is an amount which, when the pharmaceutical product of the present technology is added to a solution of 10 milliliters of 0.1 M sodium bicarbonate, lowers the pH of that sodium bicarbonate solution to no higher than 5.5, no higher than 4.7, or no higher than 3.5. Enough acid to lower pH, in the foregoing test, to about 2.8 has been used in some embodiments. In some embodiments, at least 50, 100, 200, 300 or at least 400 milligrams of the pH-lowering agent are used in the pharmaceutical composition of the present technology. The foregoing values relate to the total combined weight of all pH- lowering agents where two or more of such agents are used in combination. In some embodiments, the oral formulation should not include an amount of any base which, when released together with the pH-lowering compound, would prevent the pH of the above- described sodium bicarbonate test from dropping to 5.5 or below.

[0102] The pH-lowering agent of the present formulations may be any pharmaceutically acceptable compound that is not toxic in the gastrointestinal tract and is capable of either delivering hydrogen ions (a traditional acid) or of inducing higher hydrogen ion content from the local environment. It may also be any combination of such compounds. In some embodiments, at least one pH-lowering agent used in the present formulations has a p a no higher than 4.2, or no higher than 3.0. In some embodiments, the pH lowering agent has a solubility in water of at least 30 grams per 100 milliliters of water at room temperature.

[0103] Non-limiting examples of compounds that induce higher hydrogen ion content include aluminum chloride and zinc chloride. Pharmaceutically acceptable traditional acids include, but are not limited to acid salts of amino acids (e.g., amino acid hydrochlorides) or derivatives thereof. Examples of these are acid salts of acetylglutamic acid, alanine, arginine, asparagine, aspartic acid, betaine, carnitine, carnosine, citrulline, creatine, glutamic acid, glycine, histidine, hydroxylysine, hydroxyproline, hypotaurine, isoleucine, leucine, lysine, methylhistidine, norleucine, ornithine, phenylalanine, proline, sarcosine, serine, taurine, threonine, tryptophan, tyrosine and valine.

[0104] Other examples of useful pH-lowering compounds include dicarboxylic and tricarboxylic carboxylic acids. Acids such as acetylsalicylic, acetic, ascorbic, citric, fumaric, glucuronic, glutaric, glyceric, glycocolic, glyoxylic, isocitric, isovaleric, lactic, maleic, oxaloacetic, oxalosuccinic, propionic, pyruvic, succinic, tartaric, valeric, adipic, benzoic, phthalic, sorbic, edetic, benzenesulfonic, p-toluenenesulfonic, methansulfonic, boric, saccharinic,, and the like have been found useful.

[0105] Other useful pH-lowering agents that might not usually be called "acids" in the art, but which may nonetheless be useful in accordance with the present technology are phosphate esters (e.g., fructose 1 ,6 diphosphate, glucose 1,6 diphosphate, phosphoglyceric acid, and diphosphoglyceric acid). Carbopol® and polymers such as polycarbophil may also be used to lower pH. Additional pH-lowering agents include, but are not limited to, alginic acid and other naturally occurring or synthetic polysaccharide acids, such as the substituted carboyxmethyl celluloses, xanthan gum, polymethacrylic acid and substituted derivatives, cellulose glycollic acid, and pectins. Any combination of pH lowering agents may be used.

[0106] In one embodiment, the pH lowering agent achieves a pH level of between about 1 to about 6. In some embodiments, the pH lowering agent achieves a pH level of between about 1.5 to about 5.5, or between about 2 to about 5, or between about 2.5 to about 4.5, or between about 3 to about 4. One embodiment utilizes, as at least one of the pH-lowering agents in the finished pharmaceutical product, an acid selected from the group consisting of citric acid, tartaric acid and an acid salt of an amino acid.

[0107] When aromatic-cationic peptides of the present technology or a pharmaceutically acceptable salt thereof, such as acetate salt or trifluoroacetate salt, are the active agent, certain ratios of pH- lowering agent to peptide may prove especially effective. For example, in some embodiments, the weight ratio of pH-lowering agent to aromatic-cationic peptide exceed 200: 1 , 800: 1 , or 2000: 1. In some embodiments, the weight ratio of pH-lowering agent to aromatic-cationic peptide exceeds 40: 1, 400:1, or 4000: 1. [0108] An alternative or a supplement to the use of pH-lowering agents is the use of protease inhibitors, in particular inhibitors of intestinal proteases. FIG. 7 illustrates some of the known intestinal proteases.

[0109] In some embodiments, the protease inhibitors can be proteins or peptides. In some embodiments, the protease inhibitor are trypsin inhibitors (TI). Examples of TI include, but are not limited to, soy protein (e.g., SBTI, Kunitz inhibitor, or Glycine Max®) of 20.1 kDa, bovine pancreas of 6.5 kDa, aprotonin, chicken or turkey ova-mucoid TI of 28 kDA, partially hydrolysed gelatin fractions, epsilon-aminocaproic acid, and di-Sodium EDTA.

Absorption Enhancers

[0110] In some embodiments of the compositions disclosed herein absorption enhancers are present in a quantity that constitutes from 0.1 to 20.0 percent by weight, from about 0.5 to about 1 percent by weight, from about 1 to about 10 percent by weight, from about 3 to about 7 percent by weight, or from about 4 to about 6 percent by weight relative to the overall weight of the pharmaceutical composition. In some embodiments, absorption enhancers are surface active agents which act both as solubility enhancers and uptake enhancers.

Genetically speaking, "solubility enhancers" improve the ability of the components of the present formulations to be solubilized in either the aqueous environment into which they are originally released or into, for example, the lipophilic environment of the mucous layer lining the intestinal walls, or both. "Transport (uptake) enhancers" (which, in some embodiments, are frequently the same surface active agents used as solubility enhancers) are those which facilitate the ease by which aromatic-cationic peptides of the present technology cross the intestinal wall.

[0111] One or more absorption enhancers may perform one function only (e.g. , solubility), or one or more absorption enhancers may perform the other function only (e.g. , uptake), within the scope of the present technology. It is also possible to have a mixture of several compounds some of which provide improved solubility, some of which provide improved uptake and/or some of which perform both. Without intending to be bound by theory, it is believed that uptake enhancers may act by (1) increasing disorder of the hydrophobic region of the membrane exterior of cells, allowing for increased transcellular transport; or (2) leaching membrane proteins resulting in increased transcellular transport; or (3) widening pore radius between cells for increased paracellular transport. [0112] Surface active agents are believed to be useful both as solubility enhancers and as uptake enhancers. For example, detergents are useful in (1) solubilizing all of the active components quickly into the aqueous environment where they are originally released, (2) enhancing lipophilicity of the components of the present formulations, especially the aromatic-cationic peptide, aiding its passage into and through the intestinal mucus, (3) enhancing the ability of the normally polar aromatic-cationic peptide to cross the epithelial barrier of the brush border membrane; and (4) increasing transcellular or paracellular transport as described above.

[0113] In some embodiments, when surface active agents are used as the absorption enhancers, the surface active agents are free flowing powders for facilitating the mixing and loading of capsules during the manufacturing process. Because of inherent characteristics of specific peptides (e.g., their isoelectric point, molecular weight, amino acid composition, etc.) certain surface active agents will likely interact better with certain peptides. For example, some agents may undesirably interact with the charged portions of a peptide and prevent its absorption, thus undesirably resulting in decreased bioavailability. In some embodiments, when increasing the bioavailability of aromatic-cationic peptides of the present technology or other peptides, a surface active agent used as an absorption enhancer may include one or more of the following: (i) anionic surface active agents that are cholesterol derivatives (e.g., bile acids), (ii) cationic surface agents (e.g., acyl carnitines, phospholipids and the like), (iii) non-ionic surface active agents, and (iv) mixtures of anionic surface active agents (especially those having linear hydrocarbon regions) together with negative charge neutralizers.

Negative charge neutralizers include but are not limited to acyl carnitines, cetyl pyridinium chloride, and the like. In some embodiments, the absorption enhancer is soluble at acid pH, particularly in the 3.0 to 5.0 range.

[0114] In some embodiments, one combination useful with aromatic-cationic peptides of the present technology (or a pharmaceutically acceptable salt thereof, such as acetate salt or trifluoroacetate salt) mixes cationic surface active agents with anionic surface active agents that are cholesterol derivatives, and which are soluble at acid pH.

[0115] In some embodiments, an acid soluble bile acid is combined with a cationic surface active agent. In some embodiments, an acyl carnitine and sucrose ester are combined. In some embodiments, when a particular absorption enhancer is used alone, it comprises a cationic surface active agent. In some embodiments, acyl carnitines (e.g., lauroyl L- carnitine), phospholipids and bile acids are used as absorption enhancers, especially acyl carnitine. Anionic surfactants that are cholesterol derivatives are also used in some embodiments. In some embodiments, it is the intent to avoid interactions with the aromatic- cationic peptide that interfere with absorption of aromatic-cationic peptide into the blood.

[0116] In some embodiments, to reduce the likelihood of side effects, either biodegradable or reabsorbable detergents (e.g., biologically recyclable compounds such as bile acids, phospholipids, and/or acyl carnitines) are used as the absorption enhancers in the present technology. Acyl carnitines are useful in enhancing paracellular transport. In some embodiments, when a bile acid (or another anionic detergent lacking linear hydrocarbons) is used in combination with a cationic detergent, aromatic-cationic peptides of the present technology are better transported both to and through the intestinal wall.

[0117] Absorption enhancers include but are not limited to, e.g. , (a) salicylates such as sodium salicylate, 3-methoxysalicylate, 5-methoxysalicylate and homovanilate; (b) bile acids such as taurocholic, tauorodeoxycholic, deoxycholic, cholic, glycholic, lithocholate, chenodeoxycholic, ursodeoxycholic, ursocholic, dehydrocholic, fusidic, etc.; (c) non-ionic surfactants such as polyoxyethylene ethers (e.g. Brij 36T, Brij 52, Brij 56, Brij 76, Brij 96, Texaphor A6, Texaphor A14, Texaphor A60 etc.), p-t-octyl phenol polyoxyethylenes (Triton X-45, Triton X-100, Triton X-l 14, Triton X-305 etc.) nonylphenoxypoloxyethylenes (e.g., Igepal CO series), polyoxyethylene sorbitan esters (e.g.. Tween-20, Tween-80 etc.); (d) anionic surfactants such as dioctyl sodium sulfosuccinate; (e) lyso-phospholipids such as lysolecithin and lysophosphatidylethanolamine; (f) acylcarnitines, acylcholines and acyl amino acids such as lauroyl L-carnitine, myristoylcarnitine, palmitoylcarnitine,

lauroylcholine, myristoylcholine, palmitoylcholine, hexadecyllysine, N-acylphenylalanine, N-acylglycine etc. ; g) water soluble phospholipids such as diheptanoylphosphatidylcholine, dioctylphosphatidylcholine etc. ; (h) medium-chain glycerides which are mixtures of mono-, di- and triglycerides containing medium-chain-length fatty acids (caprylic, capric and lauric acids); (i) ethylene-diaminetetraacetic acid; (j) cationic surfactants such as cetylpyridinium chloride; (k) fatty acid derivatives of polyethylene glycol such as Labrasol, Labrafac, etc.; and (1) alkylsaccharides such as lauryl maltoside, lauroyl sucrose, myristoyl sucrose, palmitoyl sucrose, etc.

[0118] While not wishing to be bound by theory, in some embodiments, cationic ion exchange agents (e.g. , detergents) are included to provide solubility enhancement by another possible mechanism. In particular, cationic ion exchange agents may prevent the binding of aromatic-cationic peptides of the present technology or other therapeutic agents to mucus. Exemplary cationic ion exchange agents include protamine chloride or any other polycation.

Other Optional Agents

[0119] In some embodiments, a water-soluble barrier separates the pH-lowering agent from an acid resistant enteric coating. A conventional pharmaceutical capsule may, for example, be used for the purpose of providing this barrier. Many water soluble barriers are known in the art and include, but are not limited to, hydroxypropyl methylcellulose and conventional pharmaceutical gelatins.

[0120] In some embodiments, a second peptide (such as albumin, casein, soy protein, other animal or vegetable proteins and the like) is included to reduce non-specific adsorption (e.g., binding of peptide to the intestinal mucus barrier) thereby lowering the necessary

concentration of the aromatic-cationic peptide. When added, the second peptide, in some embodiments, is between about 0.1 percent to about 10.0 percent by weight relative to the weight of the overall pharmaceutical composition. In some embodiments, the second peptide is between about 1.0 percent to about 10.0 percent, or between about 2.0 percent to about 9.0 percent, or between about 3.0 percent to about 8.0 percent, or between about 4.0 percent to about 7.0 percent, or between about 5.0 percent to about 6.0 percent by weight relative to the weight of the overall pharmaceutical composition. In some embodiments, the second peptide is between about 0.1 percent to about 1.0 percent, or between about 0.2 percent to about 0.9 percent, or between about 0.3 percent to about 0.8 percent, or between about 0.4 percent to about 0.7 percent, or between about 0.5 percent to about 0.6 percent by weight relative to the weight of the overall pharmaceutical composition. This second peptide is typically not physiologically active and is typically not a food peptide such as soy bean peptide or the like. Without intending to be bound by theory, this second peptide may also increase

bioavailability by acting as a protease scavenger that desirably competes with the aromatic- cationic peptide for protease interaction. The second peptide may also aid the active compound's passage through the liver.

[0121] The pharmaceutical compositions of the present technology may optionally also include common pharmaceutical diluents, glycants, lubricants, gelatin capsules,

preservatives, colorants and the like in their usual known sizes and amounts. The Enteric Coating or Protective Vehicle

[0122] In some embodiments, aromatic-cationic peptide formulations include an enteric coating, a carrier or vehicle that protects the formulation from stomach proteases. Any carrier or vehicle that protects the aromatic-cationic peptide from stomach proteases and then dissolves so that the other ingredients of the composition may be released in the intestine is suitable, in some embodiments.

[0123] Many such enteric coatings are known in the art, and are useful in accordance with the present technology. Examples include cellulose acetate phthalate, hydroxypropyl methylethylcellulose succinate, hydroxypropyl methylcellulose phthalate,

carboxylmethylethylcellulose and methacrylic acid-methyl methacrylate copolymer. In some embodiments, aromatic-cationic peptides of the present technology, absorption enhancers such as solubility and/or uptake enhancer(s), and pH-lowering compound(s), are included in a sufficiently viscous protective syrup to permit protected passage of the components of the composition through the stomach.

[0124] Suitable enteric coatings for protecting the aromatic-cationic peptide from stomach proteases may be applied, for example, to capsules after the remaining components have been loaded within the capsule. In other embodiments, enteric coating is coated on the outside of a tablet or coated on the outer surface of particles of active components which are then pressed into tablet form, or loaded into a capsule, which is itself coated with an enteric coating.

[0125] In some embodiments, it is desirable that all components of a formulation of the present technology be released from the carrier or vehicle, and solubilized in the intestinal environment as simultaneously as possible. In some embodiments, the vehicle or carrier releases the active components in the small intestine where uptake enhancers that increase transcellular or paracellular transport are less likely to cause undesirable side effects than if the same uptake enhancers were later released in the colon. It is emphasized, however, that formulations of the present technology are believed effective in the colon as well as in the small intestine. Numerous vehicles or carriers, in addition to those discussed above, are known in the art. In some embodiments, it is desirable keep the amount of enteric coating low. In some embodiments, the enteric coating adds no more than 30% to the weight of the remainder of pharmaceutical composition (the "remainder" being the pharmaceutical composition exclusive of enteric coating itself). In some embodiments, the formulation includes less than 20%, e.g., from about 12% to about 20% to the weight of the uncoated composition. In some embodiments, the enteric coating should be sufficient to prevent breakdown of the pharmaceutical composition of the present technology in 0. IN HC1 for at least two hours, then capable of permitting complete release of all contents of the

pharmaceutical composition within thirty minutes after pH is increased to 6.3 in a dissolution bath in which the composition is rotating at 100 revolutions per minute.

Exemplary Weight Ratios and Illustrative Embodiments of Component Combinations

[0126] In some embodiments, the weight ratio of pH-lowering agent(s) to absorption enhancer(s) is 3: 1 to 20: 1 , 4: 1 to 12: 1 , or 5 : 1 to 10: 1. In some embodiments, the total weight of all pH-lowering agents and the total weight of all absorption enhancers in a given pharmaceutical composition is included in the foregoing ratios. For example, if a

pharmaceutical composition includes two pH-lowering agents and three absorption enhancers, the foregoing ratios will be computed on the total combined weight of both pH- lowering agents and the total combined weight of all three absorption enhancers.

[0127] In some embodiments, the pH-lowering agent, the aromatic-cationic peptide and the absorption enhancer (whether single compounds or a plurality of compounds in each category) are uniformly dispersed in the finished pharmaceutical product. In one

embodiment, the finished pharmaceutical product may be produced in the form of a laminate having two or more layers, wherein the aromatic-cationic peptide is contained within a first layer and the pH-lowering agent and absorption enhancer are contained within a second layer laminated with the first layer. In another embodiment, the composition of the product comprises granules that include a pharmaceutical binder having the aromatic-cationic peptide, the pH-lowering agent and the absorption enhancer uniformly dispersed within the binder. Granules may also include an acid core, surrounded by a uniform layer of organic acid, a layer of enhancer and a layer of peptide that is surrounded by an outer layer of organic acid. Granules may be prepared from an aqueous mixture including pharmaceutical binders such as polyvinyl pyrrolidone or hydroxypropyl methylcellulose, together with the pH- lowering agents, absorption enhancers and aromatic-cationic peptides of the present technology to be used in the present formulations.

Using Enzyme-cleavable Translocators

[0128] In accordance with some embodiments of the disclosed methods, patients in need of treatment with aromatic-cationic peptides of the present technology are provided with an oral pharmaceutical composition thereof. In some embodiments, the composition is in the form of a tablet or capsule form of an ordinary size in the pharmaceutical industry. The dosages and frequency of administering the products are discussed in more detail below. Patients who may benefit are any who suffer from disorders that respond favorably to increased levels of a peptide-containing compound.

[0129] In some embodiments of the formulations disclosed herein, aromatic-cationic peptides of the present technology display higher bioavailability when administered orally in accordance with the present compositions, formulations and/or methods compared to controls. In some embodiments of oral formulations disclosed herein, the bioavailability of aromatic-cationic peptides of the present technology when linked to a membrane translocator (MT) according to the methods disclosed herein, is significantly increased.

[0130] Without intending to be bound by theory, some pharmaceutical compositions of the present disclosure are believed to overcome a series of different and unrelated natural barriers to bioavailability. Various components of the pharmaceutical compositions act to overcome different barriers by mechanisms appropriate to each, and result, in some embodiments, in synergistic effects on the bioavailability of a peptide active ingredient.

[0131] In some embodiments, the aromatic-cationic peptide may be administered orally. In accordance with the methods, the presence of at least one MT, or at least two MTs, to enhance the membrane permeability of the fusion peptide across the lumen of the intestine and provide for improved bioavailability. In some embodiments, the MT link to the active peptide can be cleaved by an enzyme in the blood or the lymphatic system, thereby leaving the active peptide free to reach its target.

[0132] Also, in accordance with the compositions, formulations and methods disclosed herein, proteolytic degradation of the peptide and of the membrane translocator by stomach enzymes (most of which are active in the acid pH range) and intestinal or pancreatic proteases (most of which are active in the neutral to basic pH range) is reduced.

[0133] Again, without intending to be bound by theory, it appears that, in accordance with some embodiments of the present method, the peptide is transported through the stomach under the protection of an appropriate acid-resistant protective vehicle for substantially preventing contact between the aromatic-cationic peptide or other peptide and any stomach proteases capable of degrading it. Once the pharmaceutical composition passes through the stomach and enters the intestinal region where basic to neutral pH predominates, and where proteases tend to have basic to neutral pH optima, the enteric coating or other vehicle releases the peptide and acid or protease inhibitors (in close proximity to each other).

[0134] The acid is believed to lower the local intestinal pH, where the aromatic-cationic peptide has been released, to levels below the optimal range for many intestinal proteases and other intestinal enzymes. This decrease in pH reduces the proteolytic activity of the intestinal proteases, thus affording protection to the peptide and the membrane translocator from potential degradation. The activity of these proteases is diminished by the temporarily acidic environment provided by the composition. According to embodiments of methods disclosed herien, sufficient acid is provided that local intestinal pH is lowered temporarily to 5.5 or below, 4.7 or below, or 3.5 or below. The sodium bicarbonate test described herein (in the section captioned "the pH-Lowering Agent") is indicative of the required acid amount.

Conditions of reduced intestinal pH persist for a time period sufficient to protect the aromatic-cationic peptide and the membrane translocator from proteolytic degradation until at least some of the aromatic-cationic peptide has had an opportunity to cross the intestinal wall into the bloodstream. By way of example, for salmon calcitonin, experiments have demonstrated a Tmax of 5-15 minutes for blood levels of salmon calcitonin when the active components are injected directly into the duodenum, ileum or colon of rats. The

simultaneous release of protease inhibitor, permeation enhancer, and peptide is critical in order to provide the pH reduction locally and together with high concentrations of peptide and enhancer, which drives absorption.

[0135] Alternatively, protease inhibitors are believed to reduce the proteolytic activity of the intestinal proteases, thus affording protection to the peptide and the membrane translocator from premature potential degradation.

[0136] Compositions of the present technology can optionally contain absorption enhancers. The absorption enhancers of the disclosure, in some embodiments, synergistically promote peptide absorption into the blood while conditions of reduced proteolytic activity prevail.

[0137] The mechanism by which the method is believed to accomplish the goal of enhanced bioavailability is aided by having active components of the pharmaceutical composition released together as simultaneously as possible. According to some

embodiments of the methods disclosed herein, the volume of enteric coating is kept as low as possible consistent with providing protection from stomach proteases. Thus, enteric coating is less likely to interfere with peptide release, or with the release of other components in close time proximity with the peptide. In some embodiments, the enteric coating is less than about 30% to the weight of the remainder of pharmaceutical composition (i.e., the other components of the composition excluding enteric coating). In some embodiments, it is less than 20%. In some embodiments, the enteric coating adds between 10%> and 20% to the weight of the uncoated ingredients.

[0138] In some embodiments, the absorption enhancer which may be a solubility enhancer and/or transport enhancer (as described in more detail below) aids transport of the aromatic- cationic peptide from the intestine to the blood, and may promote the process so that it better occurs during the time period of reduced intestinal pH and reduced intestinal proteolytic activity. Many surface agents may act as both solubility enhancers and transport (uptake) enhancers. Again without intending to be bound by theory, it is believed that enhancing solubility provides (1) a more simultaneous release of the active components of the present methods into the aqueous portion of the intestine, (2) better solubility of the peptide in, and transport through, a mucous layer along the intestinal walls. Once the peptide active ingredient reaches the intestinal walls, an uptake enhancer is useful to provide better transport through the brush border membrane of the intestine into the blood, via either transcellular or paracellular transport. As discussed in more detail below, some compounds may provide both functions. In those instances, embodiments utilizing both of these functions may do so by adding only one additional compound to the pharmaceutical composition. In other embodiments, separate absorption enhancers may provide the two functions separately.

[0139] Formulations including combinations of multiple pH-lowering agents, multiple enhancers, multiple MTs and multiple peptide active agents can be used as well as using just a single pH-lowering agent and/or single enhancer.

[0140] In some embodiments, peptide active agent (e.g., D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ) is linked to an MT sequence to facilitate absorption from the intestine. The MT is typically protected from cleavage by proteases in the stomach and intestine before its absorption. However, once absorbed, the MT should be able to be at least partially removed by proteases to free up the active peptide.

[0141] The MT can comprise an amino acid sequence, such as a signal peptide or signal sequence. The hydrophobic portion is a common, major motif of the signal peptide, and it is often a central part of the signal peptide of protein secreted from cells. A signal peptide is a peptide capable of penetrating through the cell membrane to allow the export of cellular proteins. In some embodiments, the signal peptides are also "importation competents," e.g., are capable of penetrating through the cell membrane from outside the cell to the interior of the cell. The amino acid residues can be mutated and/or modified (i.e., to form mimetics) so long as the modifications do not affect the translocation-mediating function of the peptide. Thus the word "peptide" includes mimetics and the word "amino acid" includes modified amino acids, as used herein, unusual amino acids, and D-form amino acids. In some embodiments, the importation competent signal peptides have the function of mediating translocation across a cell membrane from outside the cell to the interior of the cell. In some embodiments, they may also retain their ability to allow the export of a protein from the cell into the external milieu. A putative signal peptide can easily be tested for this importation activity following the teachings provided herein, including testing for specificity for any selected cell type. FIG. 8 exemplifies amino acid sequences, each of which can be used as an MT.

[0142] The MT can also comprise fatty acids and/or bile acids. In some embodiments, such molecules, when used, are linked to the active peptide by an amino acid bridge which is subject to cleavage by proteases in the plasma. Alternatively, the MT can be linked to the active peptide by a non-peptidyl linkage, in which case the in vivo enzyme that cleaves the linkage may be an enzyme other than protease. In some embodiments, the amino acid bridge is a target for cleavage by at least one plasma protease. Plasma proteases as well as their target sequences are well known in the art. FIG. 9 illustrates some of these enzymes as well as their specific targets.

[0143] In some embodiments, the formulations disclosed herein, by several mechanisms, suppresses the degradation of the active ingredient linked to an MT by protease that would otherwise tend to cleave one or more of the peptide bonds of the active ingredient. In some embodiments, the molecular structure of the active ingredient (e.g. , aromatic-cationic peptide) may further include other substituents or modifications. For example, aromatic- cationic peptides of the present technology can be amidated at the C-terminus. Both synthetic and natural peptides can be orally delivered in accordance with the method.

Illustrative Peptide Active Agents

[0144] As described previously, peptide active compounds or active agents of the present disclosure include, but are not limited to, aromatic-cationic peptides of the present technology, such as, e.g. , D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ as well as polypeptides such as insulin, vasopressin, and calcitonin. Other examples include calcitonin gene-related peptide, parathyroid hormone, luteinizing hormone -releasing factor, erythropoietin, tissue

plasminogen activators, human growth hormone, adrenocorticototropin, various interleukins, enkephalin, glucagon-like peptide 1 , and all analogs thereof. In some embodiments, the peptide has the amino acid sequence Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu- Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Xaa-Xaa- Gly-Xaa-Xaa-Thr-Xaa, wherein amino acids 26, 27, 28, 29, and 31 can be any naturally occurring amino acid, and wherein amino acid 31 is optionally amidated. Many others are known in the art. It is expected that any pharmaceutical compound having peptide bonds which would be subject to cleavage in the gastrointestinal tract would benefit from oral delivery in accordance with the present methods because of the enhancement of absorption of such compounds from the intestine coupled with the reduction in such cleavage that is afforded by the present methods.

[0145] In some embodiments of the formulations including aromatic-cationic peptides of the present technology, the peptide may comprise from 0.02 to 0.2 percent by weight relative to the total weight of the overall pharmaceutical composition (exclusive of enteric coating). Other peptide peptides may be present at higher or lower concentrations depending on desired target blood concentrations for the active compound and its bioavailability in the oral delivery system of the methods.

[0146] Aromatic-cationic peptides of the present technology may be made by either chemical or recombinant syntheses known in the art. Precursors of other amidated peptides may be made in like manner. Recombinant production is believed to be significantly more cost effective. For example, enzymatic amidation is described in U.S. Pat. No. 4,708,934 and European Patent Publications 0 308 067 and 0 382 403. Recombinant production may be used for both the precursor and the enzyme that catalyzes the conversion of the precursor to the final product. Such recombinant production is discussed in Biotechnology, Vol. 1 1 (1993) pp. 64-70, which further describes a conversion of a precursor to an amidated product.

[0147] The linking of an MT to an active peptide ingredient may also be made by either chemical or recombinant syntheses known in the art. By "linking" as used herein is meant that the biologically active peptide is associated with the MT in such a manner that when the MT crosses the cell membrane, the active peptide is also imported across the cell membrane. Examples of such means of linking include (A) linking the MT to the active peptide by a peptide bond, i.e., the two peptides (the peptide part of the MT and the active peptide) can be synthesized contiguously; (B) linking the MT to the active peptide by a non-peptide covalent bond (such as conjugating a signal peptide to a protein with a cross-linking reagent); (C) chemical ligation methods can be employed to create a covalent bond between the carboxy- terminal amino acid of an MT such as a signal peptide and the active peptide.

[0148] Examples of method (A) are shown below wherein a peptide is synthesized, by standard means known in the art, (Merrifield, J. Am. Chem. Soc. 85:2149-2154, 1963; and Lin et al., Biochemistry 27:5640-5645, 1988) and contains, in linear order from the amino- terminal end, a signal peptide sequence (the MT), an amino acid sequence that can be cleaved by a plasma protease, and a biologically active amino acid sequence. Such a peptide could also be produced through recombinant DNA techniques, expressed from a recombinant construct encoding the above-described amino acids to create the peptide. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).

[0149] For method (B), either a peptide bond, as above, can be utilized or a non-peptide covalent bond can be used to link the MT with the biologically active peptide, polypeptide or protein. This non-peptide covalent bond can be formed by methods standard in the art, such as by conjugating the MT to the peptide, polypeptide or protein via a cross-linking reagent, for example, glutaraldehyde. Such methods are standard in the art. (Walter et al., Proc. Natl. Acad. Sci. USA 77:5197; 1980).

[0150] For method (C), standard chemical ligation methods, such as using chemical crosslinkers interacting with the carboxy-terminal amino acid of a signal peptide, can be utilized. Such methods are standard in the art (Goodfriend et al. , Science 143: 1344; 1964, which uses water-soluble carbodimide as a ligating reagent) and can readily be performed to link the carboxy terminal end of the signal peptide to any selected biologically active molecule.

Manufacturing Process and Formulations

[0151] In some embodiments, compositions of the present disclosure, e.g. , formulations including one or more aromatic-cationic peptides, and optionally, one or more of the compounds disclosed herein (e.g. , pH lowering agent, absorption enhancer, enteric coating, MT, etc.) are formulated and/or manufactured as follows. Micelles or Lipidic Emulsions

[0152] In some embodiments, one or more aromatic-cationic peptide, and optionally at least one absorption enhancer or other agents, are sequestered at a molecular level in solution or colloid state, rather than as a solid, by liposomes or other lipid complexes. For example, in some embodiments, the aromatic-cationic peptide and absorption enhancer are encapsulated in a lipid or lipid-polymer particle or nanoparticle (termed lipid emulsion or micelle). The interaction between the heavily positively charged cationic drug and negatively charged phospholipids or lipid anions {e.g., oleic acid) produce complexes that tend to propel the charge to the exterior in polar environments and the hydrophobic tails into the lipid core or bi-lipid membrane. This structure is able to protect against proteases, e.g., trypsin, from attacking the aromatic-cationic peptide, and optionally another pharmaceutical agent. Other additives can be used to stabilize and balance the dispersion or bi-lipid layer. Micelles and lipidic emulsion formulations can alleviate the need for protease inhibitors. In some embodiments, the lipids are selected from the group consisting of caprylic/capric triglyceride {e.g. , Miglyol 812N, Cremer, Hamburg, Germany), glyceryl monocaprate {e.g., Capmul MCM CIO, Abitec, Columbus, Ohio), and non-ionic surfactant {e.g., polysorbate 80 or Tween 80).

[0153] In one embodiment, the process for making a lipid complex {e.g., lipid emulsions and micelles) encapsulating one or more aromatic-cationic peptides, and optionally at least one absorption enhancer or other agents, includes dissolving the aromatic-cationic peptide in a polar solvent, {e.g. , 10 mM or 0.0817g peptide/10 ml); making a 10 mM solution of phospholipid in methanol; mixing the phospholipid solution and aromatic-cationic peptide solution; re-suspending the lipid complexes, spray drying or spray coating the composition onto an inert solid; drying the mixture; blending the dried solid with direct compression excipients; and forming tablet or alternatively encapsulating the dried solids.

[0154] Examples of a polar solvent include, but are limited to, methanol, acetone, isopropanol, ME , ethanol, butanol, n-propanol, benzyl alcohol, di-methyl acetamide (DMA), DMF, or a combination thereof.

[0155] In some embodiment, re-suspension of the lipid complexes is in polar solvents that are non-solvents for peptide but solvents for the lipids. Examples of re-suspension solvents include, but are not limited to, methanol, acetone-methylene chloride, cyclohexane, ether, chloroform, toluene, ethyl acetate, isopropyl acetate, cyclohexanone, or any combination thereof.

[0156] In some embodiments, one or more absorption enhancers are added in the re- suspension step. In some embodiments, the ratio of absorption enhancer to aromatic-cationic peptide is about 10: 1. In some embodiments, the ratio of with absorption enhancer to aromatic-cationic peptide is about 1 : 1 , or about 2 : 1 , or about 3 : 1 , or about 4 : 1 , or about 5: 1 , or about 6: 1, or about 7: 1 , or about 8: 1 , or about 9: 1.

[0157] In some embodiments, the re-suspension step includes adding one or more oils. Examples of oils include, but are not limited to, oleic acid, stearic acid, ethyl oleate, castor oil, mineral oil, long chain ester oils, such as vegetable oils. In some embodiments, the re- suspension step includes adding one or more of: (a) glyceryl monooleate; (b) a sterol, such as cholesterol; (c) bile acids, such as taurocholic, tauorodeoxycholic, deoxycholic, cholic, glycholic, lithocholate, chenodeoxycholic, ursodeoxycholic, ursocholic, dehydrocholic, fusidic; (d) non-ionic surfactants, such as polyoxyethylene ethers (e.g., Brij 36T, Brij 52, Brij 56, Brij 76, Brij 96, Texaphor A6, Texaphor A14, or Texaphor A60 ), p-t-octyl phenol polyoxyethylenes (e.g., Triton X-45, Triton X-100, Triton X-l 14, or Triton X-305), nonylphenoxypoloxyethylenes (e.g., Igepal CO series), polyoxyethylene sorbitan esters (e.g., Tween-20 or Tween-80); (e) anionic surfactants, such as dioctyl sodium sulfosuccinate; (f) lecithins, such as phosphatidyl choline, phosphatidyl inositol, lyso-phospho lipids (e.g., lysolecithin and lysophosphatidylethanolamine); (g) acylcarnitines, or acylcholines, or acyl amino acids, such as lauroyl L-carnitine, myristoylcarnitine, palmitoylcarnitine,

lauroylcholine, myristoylcholine, palmitoylcholine, hexadecyllysine, N-acylphenylalanine, or N-acylglycine; or (h) water soluble phospholipids, such as diheptanoylphosphatidylcholine or dioctylphosphatidylcholine.

[0158] In some embodiments, the re-suspension step includes adding suspended or dissolved 1-20% hydroxypropylmethylcellulose acetate succinate (HPMCAS),

hydroxypropyl methylcellulose (HPMC), hydroxyethylcellulose (HEC), cellulose acetate phthalate polymer, polyvinyl pyrrolidone (PVP), polyvinyl acetate (PVA), polyethylene glycols (4-6K), polymethacrylic acid and derivatives, pluronic (polaxomers F68), or a combination thereof. [0159] In some embodiments, the inert solid includes, but are not limited to, Avicel, lactose, calcium carbonate, fumed silicon dioxide, di-calcium phosphate (various grades), and mixture of excipients, modified starches (Sta-Rx), and maltodextrins.

[0160] In some embodiments, the 10 mM solution of phospholipids in methanol includes 1) DPG cardiolipin at 0.1275 mg/10 ml; 2) phosphatidyl glycerol-synthetic at 0.0801 mg/10 ml; 3) and phosphatidyl serine-synthetic at 0.081 mg/10 ml. Examples of phospholipids include, but are limited to, phosphatidyl inositol, phosphatidic acid, phosphatidic inositol or mixtures of the above with neutral or positively charged lechithins, cyclodextrins (e.g., HPMCD), and sulfobutylether CD beta -CD can also be used to modulate the lipid interactions.

[0161] In some embodiments, the mixing of phospholipid and aromatic-cationic peptide solutions includes the steps of: 1) mixing 2 ml of phospholipid solution and 1 ml of aromatic- cationic peptide into a 20 ml centrifuge or mixing tube; 2) adding 3 ml of methylene chloride solvent; 3) vortexing/mixing the mixture; 4) ultrasonicating for about 1 minute; 5) evaporating to a film on the mixing tube using gentle flow of nitrogen over the solution; 6) mixing 1 ml of phospholipid solution and 1 ml of aromatic-cationic peptide into the mixing tube; 7) repeating steps 2-5; 8) mixing 3 ml of phospholipid solution and 1 ml of aromatic- cationic peptide into the mixing tube; and repeating steps 2-5.

[0162] In another embodiment, the phospholipid -peptide mixtures are micro fluidized using high shear emulsifiers, which can assist in formation of multi-lamellar vesicles (liposomes) in the 100-200 nm range as an alternative to ultrasonification. The mixture is then evaporated in a rotovaporator under vacuum and/or nitrogen flow dry the film out completely.

[0163] In some embodiments, an enteric coat or polymeric coating is applied to the tablet or capsule. In one embodiment, the coating is between about 0.5% to about 20%, or between about 1% to about 18%, or between about 3% to about 1 %, or between about 6% to about 12%, or between about 8% to about 10% of total tablet weight. In another embodiment, the coating is supplemented with between about 1% to about 15%, or between about 3% to about 12%), or between about 4% to about 10%, or between about 6%> to about 8% by tablet weight with water soluble HPMC or similar polymer.

[0164] In some embodiments, the pharmaceutical compositions described herein include one or more aromatic-cationic peptide such as D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ , one or more phospholipid, and optionally at least one absorption enhancer, in contact or association with a substantially hydrophobic (lipophilic) medium. In some embodiments, the pharmaceutical composition includes an aromatic-cationic peptide, a phospholipid, and a matrix forming polymer, and optionally an absorption enhancer in contact or association with a substantially hydrophobic (lipophilic) medium. For example, the aromatic-cationic peptide and a medium chain fatty acid or derivative thereof may be coated, suspended, sprayed by or immersed in a substantially hydrophobic medium forming a suspension. In some embodiments, the aromatic-cationic peptide and the medium chain fatty acid or derivative thereof, are in a solid form within the hydrophobic medium forming a suspension. In some embodiments, the compositions of the present technology are not emulsions. In some embodiments, the compositions include oily suspensions and the amount of water in the compositions is very low. In some embodiments, the compositions incorporate octanoic acid (e.g., about 60-80%), which, in some embodiments, is a suspension at the concentration of solids exemplified, but in some embodiments, at a lower concentration of solids (below the saturation threshold) a solution is obtained. In some embodiments, the suspension may be a liquid suspension incorporating solid material, or a semi-solid suspension incorporating solid material (an ointment).

[0165] In some embodiments, the compositions described herein comprise a suspension which comprises an admixture of a hydrophobic medium and a solid form wherein the solid form comprises a therapeutically effective amount of an aromatic-cationic peptide and at least one salt of a medium chain fatty acid. In some embodiments, the phospholipid is present in the composition at an amount of about 10% or more by weight. In some embodiments, the solid form may comprise a particle (e.g., consist essentially of particles, or consist of particles). The solid particle may be produced by lyophilization or by granulation or by spray-drying or by other means. In some embodiments, for example after milling, 90% (v/v) of the particles are below 500 microns, and 50% (v/v) of the particles are below 45 microns. In another embodiment, for example after milling, about 10% (v/v) of the particles are above about 50-250 microns, and about 50% (v/v) of the particles are above about 40-50 microns.

[0166] The phospholipid may generally facilitate or enhance permeability and/or absorption of the aromatic-cationic peptide. In some embodiments, the phospholipid may generally facilitate or enhance permeability and/or absorption of the aromatic-cationic peptide and an added absorption enhancer. In some embodiments, a matrix forming polymer serves to enhance permeability. In some embodiments, the phospholipids include derivatives of phospholipids. In some embodiments, the aromatic-cationic peptide, the phospholipid, and/or the matrix forming polymer, and optionally the absorption enhancer are in solid form. For example, the solid form may be a solid particle such as a lyophilized particle, a granulated particle, a pellet or a micro-sphere. In some embodiments, the aromatic-cationic peptide, the phospholipid, and/or the matrix forming polymer are all in the same solid form, e.g., all in the same particle. In other embodiments, the aromatic-cationic peptide, the phospholipid, and/or the matrix forming polymer may each be in a different solid form, e.g., each in a distinct particle. In some embodiments, the compositions described herein are substantially free of any membrane fluidizing agents. For example, in some embodiments, the compositions include no membrane fluidizing agents. In some embodiments, compositions may include for example less than 1% or less than 0.5% or less than 0.1% by weight of membrane fluidizing agents.

[0167] Unlike emulsions, where water is an essential constituent of the formulation, in some embodiments of the compositions described herein, a solid form such as a particle containing the aromatic-cationic peptide is provided. In some embodiments, the solid form is then associated with the hydrophobic (lipophilic) medium. In some embodiments, the amount of water in the compositions is less than about 3% by weight, usually less than about 2% or about 1% or less by weight.

Medium Chain Fatty Acid Salt

[0168] In some embodiments, the compositions described herein include one or more aromatic-cationic peptide, such as, e.g., D-Arg-2'6'-Dmt-Lys-Phe-NH2, and a phospholipid, wherein the phospholipid is a medium chain fatty acid, and wherein the phospholipid is included in a solid form. For example, in some embodiments, the salt of the medium chain fatty acid is in the form of a particle such as a solid particle. In some embodiments, the particle may be characterized as a granulated particle. In at least some embodiments, the solid form may generally result from a spray drying or evaporation process. In some embodiments, the salt of the medium chain fatty acid is in the same particle as the aromatic- cationic peptide. For example, the aromatic-cationic peptide and the salt of the medium chain fatty acid can be prepared together by first preparing a solution such as an aqueous solution comprising both the aromatic-cationic peptide and the salt of the medium chain fatty acid and co-lyophilizing the solution to provide a solid form or particle that comprises both the aromatic-cationic peptide and the salt of the medium chain fatty acid (and other ingredients). As described above, the resulting solid particles are associated with a hydrophobic medium. For example, the solid particles may be suspended or immersed in a hydrophobic medium.

[0169] In some embodiments of the compositions described herein the medium chain fatty acid salt and/or a matrix forming polymer (see below) may be in the same particle or in a different particle than that of the aromatic-cationic peptide. In some embodiments, it is anticipated that bioavailability of the aromatic-cationic peptide will be lower if the medium chain fatty acid is in a different particle than the aromatic-cationic peptide e.g., there will be improved bioavailability if the medium chain fatty acid salt and the aromatic-cationic peptide are together {e.g., dried together) after solubilization in the hydrophilic fraction. In some embodiments, the medium chain fatty acid salt, the aromatic-cationic peptide, and/or the matrix forming polymer are dried, after solubilization, together in the hydrophilic fraction then they are all in the same particle in the final powder.

[0170] Medium chain fatty acid salts include those having a carbon chain length of from about 6 to about 14 carbon atoms. Examples of fatty acid salts are sodium hexanoate, sodium heptanoate, sodium octanoate (also termed sodium caprylate), sodium nonanoate, sodium decanoate, sodium undecanoate, sodium dodecanoate, sodium tridecanoate, and sodium tetradecanoate. In some embodiments, the medium chain fatty acid salt contains a cation selected from the group consisting of potassium, lithium, ammonium and other monovalent cations e.g., the medium chain fatty acid salt is selected from lithium octanoate or potassium octanoate or arginine octanoate or other monovalent salts of the medium chain fatty acids. It was found that raising the amount of medium chain fatty acid salt increased the

bioavailability of the resulting formulation. In some embodiments, raising the amount of medium chain fatty acid salt, for example, sodium octanoate, above 10% to a range between about 12% to about 15% increased the bioavailability of the aromatic-cationic peptides in the pharmaceutical compositions described herein. In some embodiments, the medium chain fatty acid salt is present at between about 1% to about 5%, or between about 5% to about 10%), or between about 10% to about 15%, or between about 15% to about 20%, or between about 20% to about 25%, or between about 25% to about 30%, or between about 30% to about 35%, or between about 35% to about 40%, or between about 40% to about 45%, or between about 45% to about 50% by weight of the bulk pharmaceutical composition.

[0171] In general, the amount of medium chain fatty acid salt in the compositions described herein may be from about 0.01% up to about 50% by weight of the bulk pharmaceutical composition. For example, the medium chain fatty acid salt may be present at an amount of between about 10% to about 50%, or at an amount of between about 10%> to about 20% or between about 10 to about 15% or between about 15% to about 20%>, or between about 1 1% to about 40% by weight. In some embodiments, the amount of medium chain fatty acid salt in the compositions described herein may be, for example, from between about 1 1% to about 28%), or between about 12% to about 13%, or between about 13% to about 14%, or between about 14% to about 15%>, or between about 15%> to about 16%>, or between about 16% to about 17%, or between about 17% to about 18%, or between about 18% to about 19%, or between about 19% to about 20%, or between about 20% to about 21%, or between about 21 ) to about 22%, or between about 22% to about 23%, or between about 23% to about 24%), or between about 24% to about 25%, or between about 25% to about 26 %, or between about 26% to about 27%), or between about 21% to about 28%) by weight of the bulk pharmaceutical composition.

[0172] In other embodiments the medium chain fatty acid salt may be present at an amount of at least about 1 1%, at least about 12%, at least about 13%, at least about 14%, at least about 15% at least about 16%, at least about 17%), at least about 18%, at least about 19%, at least about 20%, at least about 21 %, at least about 22%, at least about 23%, at least about 24%o, at least about 25%, at least about 26%, at least about 27% or at least about 28% by weight of the bulk pharmaceutical composition.

[0173] In another embodiments the medium chain fatty acid salt (sodium, potassium, lithium or ammonium salt or a mixture thereof) is present between about 12% to about 21 % by weight of the bulk pharmaceutical composition, for example, between about 1 1 % to about 18%), or between about 1 1% to about 17%, or between about 12% to about 16%, or between about 12% to about 15%>, or between about 13% to about 16%, or between about 13% to about 15%, or between about 14% to about 16%), or between about 14% to about 15%, or between about 15%) to about 16%), or, for example, about 15% or about 16%).

[0174] In another embodiment, the medium chain fatty acid salt (having a carbon chain length of from between 6 to about 14 carbon atoms; in some embodiments, 8, 9 or 10 carbon atoms) is present at between about 12% to about 21 % by weight of the bulk pharmaceutical composition, for example, between about 1 1 % to about 18%), or between about 1 1% to about 17%), or between about 12% to about 16%, or between about 12% to about 15%, or between about 13% to about 16%>, or between about 13% to about 15%, or between about 14% to about 16%, or between about 14% to about 15%, or between about 15% to about 16%, or, for example, at about 15% or about 16%. [0175] In some embodiments, the medium chain fatty acid salt (for example salts of octanoic acid, salts of suberic acid, salts of geranic acid) is present at between about 12% to about 21% by weight of the bulk pharmaceutical composition, for example, between about 11 ) to about 18%o, or between about 11% to about 17%, or between about 12% to about 16%), or between about 12% to about 15%, or between about 13% to about 16%, or between about 13% to about 15%>, or between about or 14% to about 16%, or between about 14% to about 15%, or , or between about 15% to about 16%> or most for example about \5% or about 16%). In certain embodiments the medium chain fatty acid salt is present in the solid powder at an amount between about 50% to about 90%, for example, at an amount between about 70% to about 80%.

[0176] Some embodiments of the present technology comprise a composition comprising a suspension which consists essentially of an admixture of a hydrophobic medium and a solid form wherein the solid form comprises a therapeutically effective amount of the aromatic- cationic peptide and at least one salt of a medium chain fatty acid and/or a matrix forming polymer, and wherein the medium chain fatty acid salt is not a sodium salt. The salt may be the salt of another cation, e.g., lithium, potassium or ammonium; an ammonium salt.

[0177] In some embodiments of the compositions described herein the salt of the fatty acid is sodium octanoate and the hydrophobic medium is glyceryl tricaprylate or castor oil; in some embodiments the composition further comprises glyceryl monooleate and sorbitan monopalmitate or glyceryl monocaprylate and glyceryl tricaprylate and

polyoxyethylenesorbitan monooleate. In some embodiments the composition further comprises glyceryl tributyrate or lecithin or ethylisovalerate or a combination thereof and at least one stabilizer. In some embodiments the composition includes an absorption enhancer. In some embodiments, the absorption enhancer is octreotide, insulin, growth hormone, parathyroid hormone, or analogs thereof (e.g., parathyroid hormone amino acids 1-34 termed teriparatide, interferon-alfa (IFN-a)), a low molecular weight heparin, leuprolide, fondaparinux, somatostatin and analogs (agonists) thereof including peptidomimetics, exenatide, terlipressin, vancomycin or gentamicin inter alia, cholecytokinin or analogs thereof, cholecytokinin-8 (CCK-8) or analogs thereof, calcitonin or aliskiren or salts of these the absorption enhancers. In another embodiment the composition further comprises a bile salt. Examples of bile salts are sodium taurocholate, sodium deoxycholate, sodium glycocholate, sodium chenodeoxycolate, sodium cholate, sodium lithocholate, in some sodium taurocholate. Hydrophilic Fraction

[0178] In embodiments of the present technology, the above compounds, including the aromatic-cationic peptide such as D-Arg-2'6'-Dmt-Lys-Phe-NH2, the phospholipid, optional an absorption enhancer, and/or the matrix forming polymer (or substitute) are solubilized in an aqueous medium and then dried to produce a powder. The drying process may be achieved, for example, by lyophilization or granulation or by spray-drying or by other means. In some embodiments, the powder obtained is termed the "hydrophilic fraction". In some embodiments, in the hydrophilic fraction, water is normally present at an amount of less than 6% drying, and the water in the final bulk composition comprises residual water from the hydrophilic fraction. In some embodiments, the amount of solid form in the hydrophilic fraction of the formulations of the present technology is normally from about 0.5% to about 50% of the formulation (w/w). In certain aspects of the present technology, the amount of solid form is from about 17% to about 40%.

[0179] Lyophilization may be carried out as described herein and by methods known in the art e.g., as described in Lyophilization: Introduction and Basic Principles, Thomas Jennings, published by Interpharm/CRC Press Ltd (1999, 2002). The lyophilizate may optionally be milled {e.g., below 150 micron) or ground in a mortar. In some embodoiments, during industrial production the lyophilizate is, for example, milled before mixing of the hydrophilic fraction and the hydrophobic medium in order to produce batch-to-batch reproducibility.

[0180] Granulation may be carried out as described herein and by methods known in the art e.g., as described in Granulation, Salman et al , eds, Elsevier (2006) and in Handbook of Pharmaceutical Granulation Technology, 2nd edition, Dilip M. Parikh, ed., (2005).

[0181] Various binders may be used in the granulation process such as celluloses (including micro crystalline celluloses), lactoses (e.g., lactose monohydrate), dextroses, starch and mannitol and other binders as described in the previous two references.

Adsorbates

[0182] In some embodiments, the aromatic-cationic peptide is formulated in an adsorbate before being contacted or associated with a substantially hydrophobic (lipophilic) medium. An adsorbate includes the aromatic-cationic peptide and a substrate, and optionally an absorption enhancer. In some embodiments, the adsorbate is amorphous, meaning that the peptide is not crystalline as indicated by any conventional method, such as by powder X-ray diffraction (PXRD) analysis. The peptide in the adsorbate is substantially amorphous, meaning that the amount of the peptide in amorphous form is about 70%-90%. In some embodiments, the peptide in the adsorbate is in a completely amorphous form.

[0183] In some embodiments, the adsorbate also includes a high surface area substrate. The substrate may be any material that is inert, meaning that the substrate does not adversely interact with the peptide to an unacceptably high degree and which is pharmaceutically acceptable. The substrate also has a high surface area, meaning that the substrate has a

2 2 2 2 2 surface area of about 20 m /g to 180 m /g, or about 40 m /g to 160 m /g, or about 60 m /g to

2 2 2 2 2

140 m /g, or about 80 m /g to 120 m /g, or about 90 m /g to 110 m /g. The surface area of the substrate may be measured using standard procedures.

[0184] In some embodiments, the higher the surface area of the substrate, the higher the peptide-to-substrate ratio that can be achieved and still maintain high concentration- enhancements and improved physical stability. Thus, effective substrates can have surface areas of up to 200 m ² /g, up to 400 m ² /g and up to 600 m ² /g or more.

[0185] In some embodiments, the substrate may also be in the form of small particles ranging in size of from about 5 nm to about 1 μηι, preferably ranging in size from about 5 nm to about 100 nm. These particles may in turn form agglomerates ranging in size from 10 nm to 100 μιη.

[0186] In some embodiments the substrate is also insoluble in the process environment used to form the adsorbate. That is, where the adsorbate is formed by solvent processing, the substrate does not dissolve in the solvent. Where the adsorbate is formed by a melt or thermal process, the substrate has a sufficiently high melting point that it does not melt.

[0187] Exemplary materials which are suitable for the substrate include inorganic oxides, such as Si0 ₂ , Ti0 ₂ , Zn0 ₂ , ZnO, A1 ₂ 0 ₃ , MgAl Silicate, Ca Silicate, A10H ₂ , zeolites, and other inorganic molecular sieves; water insoluble polymers, such as cross-linked cellulose acetate phthalate, cross-linked hydroxypropyl methyl cellulose acetate succinate, cross-linked polyvinyl pyrrolidinone, (also known as cross povidone) microcrystalline cellulose, polyethylene/polyvinyl alcohol copolymer, polyethylene polyvinyl pyrrolidone copolymer, cross-linked carboxymethyl cellulose, sodium starch glycolate, cross-linked polystyrene divinyl benzene; and activated carbons, including those made by carbonization of polymers such as polyimides, polyacrylonitrile, phenolic resins, cellulose acetate, regenerated cellulose, and rayon. [0188] The surface of the substrate may be modified with various substituents to achieve particular interactions of the peptide with the substrate. For example, the substrate may have a hydrophobic or hydrophilic surface. By varying the terminating groups of substituents attached to the substrate, the interaction between the peptide and substrate may be influenced. For example, where the peptide is hydrophobic, it may be desired to select a substrate having hydrophobic substituents to improve the binding of the peptide to the substrate.

[0189] Generally, the interaction of peptide with the substrate should be sufficiently high such that mobility of the aromatic-cationic peptide in the peptide/substrate adsorbate is sufficiently decreased such that the composition has improved stability. However, the peptide/substrate interaction should be sufficiently low such that the peptide can readily desorb from the adsorbate when it is introduced to a use environment, resulting in a high concentration of peptide in solution. The adsorbates are formed so as to form a thin layer of amorphous peptide on the surface of the substrate. A "thin layer" is a layer that ranges in average thickness from less than one peptide molecule to as many as 10 molecules. When the peptide/substrate interaction is large and the average peptide layer thickness, based on the ratio of the mass of peptide-to-substrate surface area, is about the dimensions of one molecule, the peptide layer is generally termed a "monolayer."

Matrix Forming Polymer

[0190] In certain embodiments the composition of the present technology comprises a suspension which comprises an admixture of a hydrophobic medium and a solid form wherein the solid form comprises a therapeutically effective amount of the aromatic-cationic peptide such as D-Arg-2'6'-Dmt-Lys-Phe-NH2, a phospholipid and a matrix forming polymer, and wherein the matrix forming polymer is present in the composition at an amount of about 3% or more by weight. In some embodiments the composition comprises a suspension which consists essentially of an admixture of a hydrophobic medium and a solid form wherein the solid form comprises a therapeutically effective amount of an aromatic- cationic peptide, at least one salt of a phospholipid and a matrix forming polymer, and wherein the matrix forming polymer is present in the composition at an amount about 0.5% to about 10% by weight, or at an amount of about 1% to about 10% by weight, or at an amount of about 3% or more by weight. In some embodiments, the matrix forming polymer is present at an amount of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19% or about 20% or more, by weight.

[0191] In some embodiments, the matrix forming polymer includes, but is not limited to, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), dextran, alginate salt, hyaluronate salt or polyacrylic acid salt or a combination thereof. In certain some embodiments the matrix forming polymer is polyvinylpyrrolidone (PVP), Carbopol polymer or polyvinyl alcohol (PVA), ionic polysaccharides (for example alginic acid and alginates) or neutral

polysaccharides (for example dextran and HPMC), polyacrylic acid and poly methacrylic acid derivatives and high molecular weight organic alcohols (for example polyvinyl alcohol), or a combination thereof. In some embodiments the polyvinylpyrrolidone is present in the composition at an amount of about 2% to about 20% by weight, for example at an amount of about 3%) to about 18% by weight, or at an amount of about 5% to about 15% by weight, or at an amount of about 10% by weight. In certain some embodiments the polyvinylpyrrolidone is PVP- 12 and/or has a molecular weight of about 3000.

[0192] In certain some embodiments, the matrix forming polymer is polyvinylpyrrolidone (PVP), and the polyvinylpyrrolidone is present in the composition at an amount of about 0.5% to about 20% by weight or about 1% to about 18%, for example, at an amount of about 3% to about 18% by weight. In some embodiments, the matrix forming polymer is PVP at an amount of about 5% to about 1 % by weight, and in some embodiments, at an amount of about 10% by weight. In certain some embodiments the polyvinylpyrrolidone is PVP- 12 and/or has a molecular weight of about 3000.

[0193] Other matrix forming polymers are believed to have a similar effects. Instead of PVP in the formulation, a range of matrix forming polymers can be substituted e.g., carbomers (Carbopol® polymers Lubrizol, 29400 Lakeland Blvd, Wickliffe, OH.) or alginate or hyaluronate or polyacrylic acid sodium salt; glucosamine or glucose was also substituted. The matrix forming polymers which produce a higher or similar bioavailability in the formulations of the present technology as PVP include but are not limited to Carbopol® polymer and PVA (polyvinyl alcohol); glucose may give results similar to PVP. Carbopol® polymers are polymers of acrylic acid cross-linked e.g., with polyalkenyl ethers or divinyl glycol. In some embodiments, Carbopol® 934P may give higher bioavailability. Carbopol® 934P is a high molecular weight polymer of acrylic acid crosslinked with allyl ethers of sucrose. PVA is a water-soluble synthetic polymer of vinyl alcohol monomers. [0194] In some embodiments, replacing PVP-12 in the formulation, by e.g., Carbopol® 934P or by PVA or by some of the other matrix forming polymers, may reduce the total amount of matrix forming polymer in the particle phase (i.e., the solid form) of the formulation (the hydrophilic fraction) and thus in some embodiments, may bestow the ability to load more API into the formulation, which may be desirable in order to achieve desired blood levels or reduce capsule size and number.

[0195] Other matrix forming polymers include, but are not limited to, cross-linked PVP (cross-povidones); linear polyacrylic acid polymers including polymethacrylic acid polymers; cross-linked polyacrylic acid polymers (carbomers); amino-polysaccharides (e.g., chitosans), S -containing polymers (thiomers) and combinations thereof.

[0196] Carbomer is a generic name for cross-linked polymers of acrylic acid; carbomers may be homopolymers of acrylic acid, cross-linked with, for example, an allyl ether pentaerythritol, or allyl ether of sucrose or allyl ether of propylene or allyl sucrose or other sugars or allyl pentaerythritol or a polyalkenyl ether or divinyl glycol.

[0197] In some embodiments of the present technology, the matrix forming polymer is a cross-linked acrylic acid polymer (also termed carbomer). Carbopol® polymers are examples of cross-linked polymers of acrylic acid. The viscosity of the cross-linked acrylic acid polymer is about 2000-80000 cP, for example 4000-65000, most for example 25000- 45000 cP; the viscosity is measured in cP, 0.5% solution at pH 7.5. In some embodiments of the present technology, the cross-linked acrylic acid polymer is an allyl sucrose-linked carbomer, of viscosity about 29000 to about 40000, e.g., Carbopol® 934P. In some embodiments, the cross-linked acrylic acid polymers are present in the composition at an amount between about 0.01% to about 0.1%, or between about 0.1% to about 1.0%, or between about 1% to about 5%, or between about 5% to about 10% by weight.

[0198] In another embodiment of the present technology, the matrix forming polymer is polyvinyl alcohol of molecular weight 10000-60000 Da, for example 20000-30000 Da. In some embodiments the polyvinyl alcohol is polyvinyl alcohol of molecular weight of about 27000 Da, and may be present in the composition at an amount of about 0.1% to about 6% by weight, for example at an amount of about 0.5% to about 4% by weight, e.g., at an amount of about at an amount of about 1%, about 2%, or about 3% by weight.

[0199] Glucose and/or other sugars and/or mannitol may be substituted in certain embodiments instead of a matrix forming polymer. Hydrophobic (Lipophilic) Medium

[0200] As described herein, the water soluble composition (e.g., particle including at least one aromatic-cationic peptide, such as, e.g., D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ) in some embodiments, is generally suspended in a hydrophobic medium. Without wishing to be bound by theory, the hydrophobic medium improves the selective translocation of the aromatic-cationic peptide across a biological barrier (e.g., a membrane) in the composition. This capability can be assessed utilizing the "innocent bystander" assay, whereby an impermeable molecule is administered concomitantly to the composition by the same route of administration, and no translocation of the impermeable molecule can be detected. Such an assay utilizing insulin as the impermeable molecule may be tested.

[0201] As described herein, in the compositions of the present technology the aromatic- cationic peptides, such as D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ and the phospholipid salt are in contact or association with a hydrophobic (lipophilic) medium. For example, one or both may be coated, suspended, immersed or otherwise in association with a hydrophobic (lipophilic) medium. Suitable hydrophobic mediums can contain, for example, aliphatic, cyclic or aromatic molecules. Non-limiting examples of a suitable aliphatic hydrophobic medium include, but are not limited to, mineral oil (e.g., paraffin), fatty acid monoglycerides, diglycerides, triglycerides, ethers, esters, and combinations thereof. Non-limiting examples of a suitable fatty acid are octanoic acid, decanoic acid and dodecanoic acid, also C7 and C9 fatty acids and di-acidic acids such as sebacic acid and suberic acid, and derivatives thereof. Non-limiting examples of triglycerides include, but are not limited to, long chain

triglycerides, medium chain triglycerides, and short chain triglycerides. For example, the long chain triglyceride can be castor oil or coconut oil or olive oil, and the short chain triglyceride can be glyceryl tributyrate and the medium chain triglyceride can be glyceryl tricaprylate. Monoglycerides are considered to be surfactants and are described below. Non- limiting exemplary esters include ethyl isovalerate and butyl acetate. Non-limiting examples of a suitable cyclic hydrophobic medium include, but are not limited to, terpenoids, cholesterol, cholesterol derivatives (e.g., cholesterol sulfate), and cholesterol esters of fatty acids. A non-limiting example of an aromatic hydrophobic medium includes benzyl benzoate.

[0202] In some embodiments of the compositions described herein, it is desirable that the hydrophobic medium include a plurality of hydrophobic molecules. In some embodiments the hydrophobic medium also includes one or more surfactants. Exemplary surfactants include phospholipids such as lecithin or a block copolymer such as pluronic F-68 In some embodiments, compositions including a surfactant in the hydrophobic medium, comprises less than about 20% by weight of surfactant in the hydrophobic medium. In some embodiments, the hydrophobic medium generally comprises from about 30% to about 90%> by weight of the composition. In some embodiments, the hydrophobic medium comprises about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% or higher by weight of the composition.

[0203] In some embodiments of the compositions described herein, the hydrophobic medium also includes one or more adhesive polymers such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose (HPMC), or poly(acrylate) derivative Carbopol™ 934P (C934P). Such adhesive polymers may assist in the consolidation of the formulation and/or help its adherence to mucosal surfaces.

Biological barrier penetrating compositions using water soluble compositions in

hydrophobic media

[0204] In some embodiments, the present technology provides compositions for penetration that specifically target various tissues, for example, those containing epithelial and endothelial cells, for the delivery of drugs and other the aromatic-cationic peptides across a biological barrier. Existing transport systems known in the art are typically too limited to be of general application, and because they can be inefficient, they can alter the biological properties of the active substance, compromise the target cell, irreversibly destroy the biological barrier and/or pose too high of a risk to be used in human subjects. Embodiments of the present technology include compositions containing an aromatic-cationic peptide such as D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ in a water soluble composition. This complex can be optionally lyophilized and then immersed in a hydrophobic medium. The immersion of the water soluble composition containing the aromatic-cationic peptide, or a lyophilizate thereof, in the hydrophobic medium results in an a unique association between the aromatic-cationic peptide and the penetration enhancing compounds, thereby enabling the aromatic-cationic peptide to efficiently translocate across a biological barrier. In some embodiments, the compositions of the present technology will be defined by their efficiency, and translocation of at least 5% (but for example 10%, 20%, 30%, 40%,50%, 60%,70%, 80% or more) of the aromatic-cationic peptide across an epithelial barrier is achieved. In some embodiments, translocation of at least about 2 times {e.g., 3 times, 5 times, 10 times, 20 times, 50 times, or 100 times) the amount of aromatic-cationic peptide is achieved, than the amount of translocation of the aromatic-cationic peptide when formulated in an aqueous medium. In some embodiments, this efficiency will be greater than that of other compositions known in the art, which typically achieve translocation of only about 1-3% of an aromatic-cationic peptide.

[0205] In some embodiments, the compositions of the present technology selectively allow the translocation of an aromatic-cationic peptide across the biological barrier. The hydrophobic medium serves as a shield, thereby preventing neighboring molecules, such as proteins, toxins, and other "bystander" molecules, from co-translocating through the biological barrier with the aromatic-cationic peptide.

[0206] Commonly used microemulsions are thermodynamically stable dispersions of one liquid phase into another, that involve a combination of at least three components-oil, water, and a surfactant. Both water-in-oil (w/o) and oil-in- water (o/w) microemulsions have been proposed to enhance the oral bioavailability of drugs. They offer improved drug

solubilization and protection against enzymatic hydrolysis, as well as the potential for enhanced absorption afforded by surfactant-induced membrane permeability changes. For example, the oral release and bioactivity of insulin in water-in-oil microemulsions is described by Watnasirichaikul et al, J. Pharm. Pharm., 54:473-480 (2002).

[0207] As described above, in some embodiments, the penetration compositions of the present technology contain aromatic-cationic peptides such as D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ in a water soluble composition immersed in a hydrophobic medium, which facilitates the effective translocation of the aromatic-cationic peptide across a biological barrier. Unlike emulsions, where water is an essential constituent of the formulation, the water soluble composition, according to the present technology, can be dissolved either in water or in a non-aqueous medium such as, for example, mono-alcohols, di-alcohols, or tri-alcohols. Moreover, the water soluble composition according to the present technology can be totally evaporated, e.g., via lyophilization, prior to suspension in the hydrophobic medium. In some embodiments, the water soluble composition is totally evaporated, via lyophilization to provide a particle containing the aromatic-cationic peptide, which is, then suspended in the hydrophobic medium. The compositions also include a membrane fluidizing agent. The membrane fluidizing agent is contained within the hydrophobic medium. [0208] Additionally, unlike the water-in-oil (w/o) and oil-in-water (o/w) microemulsions, where the use of a surface active agent is obligatory, in some embodiments, the penetration compositions of this present technology provide an oral delivery system whereby the addition of a surface active agent is optional. In some embodiments, the compositions contain less than about 1-30% by weight of a surface active agent (e.g., less than about 20% less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, or is substantially free of surfactant).

Water Soluble Composition

[0209] In some embodiments, the water soluble composition is suspended within a hydrophobic region, which contains a membrane fluidizing agent. In some embodiments, the water soluble composition is a particle (e.g., a lyophilized particle) comprising one or more aromatic-cationic peptide, such as, e.g., D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ . In some

embodiments, the particles are from between about 10 nanometers and about 10 micrometers in diameter (e.g., from about 100 nanometers to about 1 micrometer in diameter). In some embodiments, the water soluble composition includes the aromatic-cationic peptide, and in some embodiments can include one or more additional agents, for example a stabilizer (e.g., a protein stabilizer), a surface active agent, one or more counter ions, a protective agent, or a viscosity adjusting agent.

[0210] In some embodiments, the water soluble composition can include a stabilizer (e.g., a stabilizer of protein structure). Stabilizers of protein structure are compounds that stabilize protein structure under aqueous or non-aqueous conditions and/or can reduce or prevent aggregation of the aromatic-cationic peptide. In some embodiments, an absorption enhancer can be added, for example during a drying process such as lyophilization or the processing step.

[0211] Currently, the delivery of aromatic-cationic peptides, and optionally an absorption enhancer (e.g., the delivery of insulin, erythropoietin, or heparin to the blood stream), requires invasive techniques such as intravenous or intramuscular injections. One advantage of the compositions of this present technology is that they can deliver such aromatic-cationic peptides across biological barriers through non-invasive administration, including, for example oral, buccal, nasal, rectal, inhalation, insufflation, transdermal, or depository. In addition, a further advantage of the compositions of the present technology is that they might be able to cross the blood-brain barrier, thereby delivering aromatic-cationic peptides to the central nervous system (CNS).

[0212] Compositions of this present technology facilitate the effective passage,

translocation, or penetration of a substance (e.g., an aromatic-cationic peptide such as D-Arg- 2'6'-Dmt-Lys-Phe-NH2) across a biological barrier, e.g., through or between cells sealed by tight junctions. Translocation may be detected and quantified by any method known to those skilled in the art, including using imaging compounds such as radioactive tagging and/or fluorescent probes or dyes incorporated into a hydrophobic composition in conjunction with a paracytosis assay as described in, for example, Schilfgaarde, et al., Infect, and Immun., 68(8):4616-23 (2000). In some embodiments, a paracytosis assay is performed by: a) incubating a cell layer with a composition described by this present technology; b) making cross sections of the cell layers; and c) detecting the presence of the aromatic-cationic peptides, or any other component of the compositions of this present technology. The detection step may be carried out by incubating the fixed cell sections with labeled antibodies directed to a component of the compositions of this present technology, followed by detection of an immunological reaction between the component and the labeled antibody.

Alternatively, the peptide may be labeled using a radioactive label, or a fluorescent label, or a dye in order to directly detect the presence of the peptide. Further, a bioassay can be used to monitor the peptide translocation. For example, using a bioactive molecules such as erythropoietin, included in a penetration composition, the increase in hemoglobin or hematocrit can be measured. Similarly, by using a bioactive molecule such as insulin coupled with the aromatic-cationic peptide composition, the drop in blood glucose level can be measured

Membrane Fluidizing Agent

[0213] Additionally or alternatively, in some embodiments, the compositions of this present technology comprising aromatic-cationic peptides such as D-Arg-2'6'-Dmt-Lys-Phe-NH2, employ membrane fluidizing agents. For example, a membrane fluidizing agent may be a linear, branched, cyclical or aromatic alcohol. Non-limiting examples of suitable linear alcohols include, but are not limited to, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, and dodecanol. Non- limiting examples of branched alcohols include geraniol and farnesol. An example of a cyclical alcohol includes menthol. Examples of suitable aromatic alcohols can include benzyl alcohol, 4-hydroxycinnamic acid, and phenolic compounds. Examples of phenolic compounds can include phenol, m-cresol, and m-chlorocresol.

[0214] In some some embodiments, membrane fluidizing agents are medium chain alcohols which have a carbon chain length of from 4 to 15 carbon atoms (e.g., including 5 to 1 , 5 to 12, 6, 7, 8, 9, 10, or 1 1 carbon atoms). For example, a membrane fluidizing agent may be a linear (e.g., saturated or unsaturated), branched (e.g., saturated or unsaturated), cyclical (e.g., saturated or unsaturated), or aromatic alcohol. Examples of suitable linear alcohols include, but are not limited to, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, and pentadecanol. In some some

embodiments, the membrane fluidizing agent includes 1-ocatanol Non- limiting examples of branched alcohols include geraniol, rhodinol, citronellol, and farnesol. In some some embodiments, the membrane fluidizing agent includes geraniol. Exemplary cyclical alcohol includes menthol, terineol, myrtenol, perilly alcohol. Examples of suitable aromatic alcohols can include benzyl alcohol, 4-hydroxycinnamic acid, thymol, styrene glycol, and phenolic compounds. Examples of phenolic compounds can include phenol, m-cresol, and m- chlorocresol.

[0215] In some embodiments, the composition includes from about 1% to about 5% by weight of membrane fluidizing agent (e.g., from between about 5% to about 40% by weight of a membrane fluidizing agent or combinations thereof).

[0216] As described above, membrane fluidizing agents increase the fluidity and decrease the order of lipids in biological membranes. This alteration of membrane dynamics may be detected by the decrease in the steady state anisotropy of fluorescent membrane probes, such as l,6-diphenyl-l ,3,5-hexatriene. Normal alcohols, or n-alkanols, are known membrane fluidizing agents. Due to their amphipathic properties, they partition the membrane lipid bilayer with their hydroxyl moiety near the phospholipids polar headgroups, and their aliphatic chains intercalated among the fatty acyl chains of the phospholipids. Alkanols of increasing chain length penetrate the bilayer to increasing depths, and thus affect bilayer order and dynamics to a different extent. See Zavoico et ah , Biochim. Biophys Acta, 812:299-312 (1985).

Polymeric counter ions

[0217] In some embodiments, one or more counter ions are combined with one or more aromatic-cationic peptide in the formulations of the present technology. Counter ions according to this present technology include anionic or cationic amphipathic molecules, i. e. , those having both polar and nonpolar domains, or both hydrophilic and hydrophobic properties. Anionic or cationic counter ions of this present technology are ions that are negatively (anionic) or positively (cationic) charged and can include a hydrophobic moiety. Under appropriate conditions, anionic or cationic counter ions can establish electrostatic interactions with cationic or anionic impermeable molecules, respectively. The formation of such a complex can cause charge neutralization, thereby creating a new uncharged entity, with further hydrophobic properties in case of an inherent hydrophobicity of the counter ion.

[0218] Suitable anionic counter ions are ions with negatively charged residues such as carboxylate, sulfonate or phosphonate anions, and can further contain a hydrophobic moiety. Examples of such anionic counter ions include sodium dodecyl sulphate, dioctyl

sulfosuccinate (docusate), actetate, tosylate, napsylate, caprate, laurate, myristate, palmitate, stearate, oleate, sodium lauryl sulfate (SLS), and other anionic compounds derived from organic acids. In one embodiment, the anionic counter ion is sulfobutyl ether cyclodextrin, which is available as a sodium salt and can interact with the cationic peptide electrostatically and hydrophobically. Anionic counter ions have the potential to protect the aromatic-cationic peptide from protease attack via charge neutralization and hydrophobic interaction (e.g., micellar solubilization or hydrophobic complex formation). Other cyclodextrins can also be used, but these lack the anionic charge (e.g. , HPMCD, beta-DD, gamma-CD). Other examples of anionic counter ions include, but are not limited to, negatively charged, long chain hydrophobic or aromatic sulfate, phosphate, carbamate ester, and carbonyl acid.

[0219] In some embodiments, the molar ratio of aromatic-cationic peptide to the anionic counter ion, e.g., sodium lauryl sulfate, is about 1 : 10, about 1 :9, about 1 :8, about 1 :7, about 1 :6, about 1 :5, about 1 :4, about 1 :3, about 1 :2, or about 1 : 1. In some embodiments, the molar ratio of aromatic-cationic peptide to the anionic counter ion is about 1 :6.

[0220] Exemplary suitable cationic counter ions include quaternary amine derivatives, such as benzalkonium derivatives or other quaternary amines, which can be substituted by hydrophobic residues. In general, quaternary amines contemplated by the present technology have the structure: 1-R1-2-R2-3-R3-4-R4-N, wherein Rl, 2, 3, or 4 are alkyl or aryl derivatives. Further, quaternary amines can be ionic liquid forming cations, such as imidazolium derivatives, pyridinium derivatives, phosphonium compounds or

tetraalkylammonium compounds. [0221] Ionic liquids are salts composed of cations such as imidazolium ions, pyridinium ions and anions, such as BF ₄ and PFg, and are liquid at relatively low temperatures. Ionic liquids are characteristically in liquid state over extended temperature ranges, and have high ionic conductivity. Other favorable characteristic properties of the ionic liquids include non- flammability, high thermal stability, relatively low viscosity, and essentially no vapor pressure. When an ionic liquid is used as a reaction solvent, the solute is solvated by ions only, thus creating a totally different environment from that when water or ordinary organic solvents are used. This enables high selectivity, applications of which are steadily expanding. Some examples are in the Friedel-Crafts reaction, Diels-Alder reaction, metal catalyzed asymmetric synthesis and others. Furthermore, some ionic liquids have low solubility in water and low polar organic solvents, enabling their recovery after reaction product is extracted with organic solvents. Ionic liquids are also used electrochemically, due to their high ion-conductivity, for example as electrolytes of rechargeable batteries.

[0222] For example, imidazolium derivatives have the general structure of 1-R1-3-R2- imidazolium where Rl and R2 can be linear or branched alkyls with 1 to 12 carbons. Such imidazolium derivatives can be further substituted for example by halogens or an alkyl group. Specific imidazolium derivatives include, but are not limited to, l-ethyl-3- methylimidazolium, l-butyl-3-methylimidazolium, l-hexyl-3-methylimidazolium, 1-methyl- 3-octylimidazolium, l-methyl-3-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-im idazolium, 1,3-dimethylimidazolium, and l,2-dimethyl-3-propylimidazolium.

[0223] Pyridinium derivatives have the general structure of 1-R1-3-R2 -pyridinium where Rl is a linear or branched alkyl with 1 to 12 carbons, and R2 is H or a linear or branched alkyl with 1 to 12 carbons. Such pyridinium derivatives can be further substituted for example by halogens or an alkyl group. Pyridinium derivatives include, but are not limited to, 3 -methyl- 1 -propylpyridinium, l-butyl-3-methylpyridinium, and l-butyl-4- methylpyridinium.

Enhancement of Bioavailability

[0224] In some embodiments, enhancement of bioavailability may be achieved with one or more classes of enhancers selected from fatty acids, sugar esters of fatty acids, acyl carnitines and citrates. Some embodiments use combinations thereof, except that acyl carnitines and fatty acids are not used together because of undesirable interaction between them. Molecular structures regarding each class is discussed below. Fatty Acids

[0225] Without intending to be bound by theory, it is believed that the fatty acids interact with peptides to desirably enhance their ability to penetrate cell membranes, thus enhancing transcellular transport. The hydrophobic region of fatty acids is believed important to this function, and should desirably include as many consecutive carbon atoms as possible, consistent with water solubility, at least 8 consecutive carbon atoms, or 10-14 carbon atoms. Illustrative fatty acids include but are not limited to lauric acid and oleic acid.

Sugar Esters of Fatty Acids

[0226] Without intending to be bound by theory, it is believed that the sugar esters of fatty acids may interact with cells in a manner that could alter their shape, increase pore size, and thereby desirably increase paracellular transport. They may also provide benefit in transcellular transport. When fatty acids and sugar esters of fatty acids are used in combination, bioavailability may be especially enhanced by the combination of enhanced transcellular and enhanced paracellular transport. Like the fatty acids, the hydrophobic region may also include at least 8 consecutive carbon atoms, especially 10-14 carbon atoms. The sugar moiety may aid water solubility. Illustrative sugar esters of fatty acids include but are not limited to sucrose laurate, glucose laurate and fructose laurate. When used, concentration of sugar esters of fatty acids may be between 0.1 and 10.0 mg/ml, or between 0.5 and 5.0 mg/ml.

Acyl Carnitines

[0227] Acyl carnitines are believed to enhance bioavailability, and in some embodiments are combined with a sugar ester of a fatty acid. Illustrative acyl carnitines include but are not limited to lauroyl-1- carnitine and myristoyl carnitine. When used, concentration of acyl carnitine may be between 0.1 and 10.0 mg/ml, or between 0.5 and 5.0 mg/ml.

Citrates

[0228] In some embodiments, citrate -type bioavailability enhancing agents selected from the group consisting of citric acid, citric acid salt and mixtures thereof are used in

combination with one or more of the other enhancers discussed herein. Without intending to be bound by theory, it is believed that citrate-type enhancing agents may increase paracellular transport. In some embodiments, the concentration of all such citrate-type enhancing agents will be no lower than 5 mM and no higher than 50 mM, or in the range of 10-25 mM.

Without intending to be bound by theory, it is believed that shelf stability may be undesirably reduced at higher citrate concentrations due to interaction of citrate with the active peptide at the amino terminus of the peptide, or at lysyl side chains.

Indications for Use

[0229] The aromatic-cationic peptide formulations of the present technology (e.g., those including 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe-Lys-NH ₂ , or D-Arg- 2',6'-Dmt-Lys-Phe-NH ₂ ) are useful in treating any disease or condition that is associated with, for example, MPT. Such diseases and conditions include, but are not limited to, ischemia and/or reperfusion of a tissue or organ, hypoxia, diseases and conditions of the eye, myocardial infarction and any of a number of neurodegenerative diseases. Mammals in need of treatment or prevention of MPT are those mammals suffering from these diseases or conditions.

[0230] Ischemia in a tissue or organ of a mammal is a multifaceted pathological condition which is caused by oxygen deprivation (hypoxia) and/or glucose (e.g., substrate) deprivation. Oxygen and/or glucose deprivation in cells of a tissue or organ leads to a reduction or total loss of energy generating capacity and consequent loss of function of active ion transport across the cell membranes. Oxygen and/or glucose deprivation also leads to pathological changes in other cell membranes, including permeability transition in the mitochondrial membranes. In addition other molecules, such as apoptotic proteins normally

compartmentalized within the mitochondria, may leak out into the cytoplasm and cause apoptotic cell death. Profound ischemia can lead to necrotic cell death.

[0231] Ischemia or hypoxia in a particular tissue or organ may be caused by a loss or severe reduction in blood supply to the tissue or organ. The loss or severe reduction in blood supply may, for example, be due to thromboembolic stroke, coronary atherosclerosis, or peripheral vascular disease. The tissue affected by ischemia or hypoxia is typically muscle, such as cardiac, skeletal, or smooth muscle.

[0232] The organ affected by ischemia or hypoxia may be any organ that is subject to ischemia or hypoxia. Examples of organs affected by ischemia or hypoxia include brain, heart, kidney, and prostate. For instance, cardiac muscle ischemia or hypoxia is commonly caused by atherosclerotic or thrombotic blockages which lead to the reduction or loss of oxygen delivery to the cardiac tissues by the cardiac arterial and capillary blood supply. Such cardiac ischemia or hypoxia may cause pain and necrosis of the affected cardiac muscle, and ultimately may lead to cardiac failure.

[0233] Ischemia or hypoxia in skeletal muscle or smooth muscle may arise from similar causes. For example, ischemia or hypoxia in intestinal smooth muscle or skeletal muscle of the limbs may also be caused by atherosclerotic or thrombotic blockages.

[0234] Reperfusion is the restoration of blood flow to any organ or tissue in which the flow of blood is decreased or blocked. For example, blood flow can be restored to any organ or tissue affected by ischemia or hypoxia. The restoration of blood flow (reperfusion) can occur by any method known to those in the art. For instance, reperfusion of ischemic cardiac tissues may arise from angioplasty, coronary artery bypass graft, or the use of thrombolytic drugs.

[0235] The compositions (e.g., formulations of the present technology comprising an aromatic-cationic peptide such as 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) can also be used in the treatment or prophylaxis of neurodegenerative diseases associated with MPT. Neurodegenerative diseases associated with MPT include, for instance, Parkinson's disease, Alzheimer's disease, Huntington's disease and Amyotrophic Lateral Sclerosis (ALS, also known as Lou Gehrig's disease). The compositions of the present disclosure can be used to delay the onset or slow the progression of these and other neurodegenerative diseases associated with MPT. In some embodiments, the compositions of the present disclosure are useful in the treatment of humans suffering from the early stages of neurodegenerative diseases associated with MPT and in humans predisposed to these diseases.

[0236] The aromatic-cationic peptide formulations of the present disclosure may also be used in preserving an organ of a mammal prior to transplantation. For example, a removed organ can be susceptible to MPT due to lack of blood flow. Therefore, the oral formulation of the peptides can be administered to a subject prior to organ removal, for example, and used to prevent MPT in the removed organ.

[0237] The formulations of the present disclosure may also be administered to a mammal taking a different drug to treat a condition or disease. For example, if a side effect of the drug includes MPT, mammals taking such drugs would greatly benefit from the oral formulations of aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Pfie-NH ₂ ) of the present technology disclosed herein.

[0238] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) for the treatment or prevention of peripheral neuropathy or the symptoms of peripheral neuropathy. In some embodiments, the peripheral neuropathy is drug-induced peripheral neuropathy. In some embodiments, the peripheral neuropathy is induced by a chemotherapeutic agent. In some embodiments, the

chemotherapeutic agent is a vinca alkaloid. In some embodiments, the vinca alkaloid is vincristine. In some embodiments, the symptoms of peripheral neuropathy include hyperalgesia.

[0239] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) for the treatment or prevention of hyperalgesia. In some embodiments, the hyperalgesia is drug-induced. In some embodiments, the hyperalgesia is induced by a chemotherapeutic agent. In some embodiments, the

chemotherapeutic agent is a vinca alkaloid. In some embodiments, the vinca alkaloid is vincristine.

[0240] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to reduce CD36 expression in post-ischemic brain in a subject in need thereof.

[0241] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to reduce CD36 expression in renal tubular cells after unilateral ureteral obstruction (UUO) in a subject in need thereof.

[0242] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-N]¾, or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to reduce lipid peroxidation in a kidney after UUO.

[0243] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH ₂ ) to reduce tubular cell apoptosis in an obstructed kidney after UUO.

[0244] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH ₂ ) to reduce macrophage infiltration in an obstructed kidney induced by UUO.

[0245] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH ₂ ) to reduce interstitial fibrosis in an obstructed kidney after UUO.

[0246] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH ₂ ) to reduce infarct volume and hemispheric swelling in a subject suffering from acute cerebral ischemia.

[0247] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH ₂ ) to reduce the decrease in reduced glutathione (GSH) in post-ischemic brain in a subject in need thereof.

[0248] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH ₂ ) to reduce up-regulation of CD36 expression in cold storage of isolated hearts.

[0249] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH ₂ ) to reduce lipid peroxidation in cardiac tissue (e.g., heart) subjected to warm reperfusion after prolonged cold ischemia.

[0250] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2', 6 '-Dmt-Lys-Phe-NH ₂ ) to abolish endothelial apoptosis in cardiac tissue (e.g., heart) subjected to warm reperfusion after prolonged cold ischemia. [0251] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to preserve coronary flow in cardiac tissue (e.g., heart) subjected to warm reperfusion after prolonged cold ischemia.

[0252] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-N]¾, or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to prevent damage to renal proximal tubules in diabetic subjects.

[0253] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to prevent renal tubular epithelial cell apoptosis in diabetic subjects.

[0254] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to inhibit mitochondrial swelling and cytochrome c release.

[0255] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to protect myocardial contractile force during ischemia-reperfusion in cardiac tissue.

[0256] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH2, or D-Arg-2',6'-Dmt-Lys-Phe-NH2) that administered with a cardioplegic solution, which will enhance contractile function after prolonged ischemia in isolated perfused cardiac tissue (e.g., heart).

[0257] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to reduce spontaneous generation of hydrogen peroxide by mitochondria in certain stress or disease states.

[0258] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to inhibit spontaneous production of hydrogen peroxide in mitochondria and hydrogen peroxide production stimulated by antimycin.

[0259] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to decrease intracellular ROS (reactive oxygen species) and increase survival in cells of a subject in need thereof, e.g., a subject suffering from a disease or condition characterized by mitochondrial dysfunction.

[0260] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to prevent loss of cell viability in subjects suffering from a disease or condition characterized by mitochondrial dysfunction.

[0261] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to decrease the percent of cells showing increased caspase activity in a subject in need thereof, e.g., a subject suffering from a disease or condition characterized by mitochondrial dysfunction.

[0262] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH2, or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to reduce the rate of ROS accumulation in a subject in need thereof, e.g., a subject suffering from a disease or condition characterized by mitochondrial dysfunction.

[0263] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to inhibit lipid peroxidation in a subject in need thereof, e.g., a subject suffering from a disease or condition characterized by mitochondrial dysfunction.

[0264] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to prevent mitochondrial depolarization and ROS accumulation in a subject in need thereof, e.g., a subject suffering from a disease or condition characterized by mitochondrial dysfunction. [0265] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to prevent apoptosis in a subject in need thereof, e.g., a subject suffering from a disease or condition characterized by mitochondrial dysfunction.

[0266] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-N]¾, or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to significantly improve coronary flow in cardiac tissue (e.g., heart) subjected to warm reperfusion after prolonged (e.g., 18 hours) cold ischemia.

[0267] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH2, or D-Arg-2',6'-Dmt-Lys-Phe-NH2) to prevent apoptosis in endothelial cells and myocytes in cardiac tissue (e.g., heart) subjected to warm reperfusion after prolonged (e.g., 18 hours) cold ischemia.

[0268] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to improve survival of pancreatic cells in a subject in need thereof.

[0269] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to reduce apoptosis and increase viability in islet cells of pancreas in subjects in need thereof.

[0270] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH2, or D-Arg-2',6'-Dmt-Lys-Phe-NH2) to reduce oxidative damage in pancreatic islet cells in subjects in need thereof.

[0271] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-N]¾, or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to protect dopaminergic cells against MPP+ toxicity in subjects in need thereof. [0272] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to prevent loss of dopaminergic neurons in subject in need thereof.

[0273] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to increase striatal dopamine, DOPAC (3,4- dihydroxyphenylacetic acid) and HVA (homovanillic acid) levels in subjects in need thereof.

[0274] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to reduce oxidative damage in a mammal in need thereof. Mammals in need of reducing oxidative damage are those mammals suffering from a disease, condition or treatment associated with oxidative damage. Typically, the oxidative damage is caused by free radicals, such as reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). Examples of ROS and RNS include hydroxyl radical (HO ), superoxide anion radical (02 ^' -), nitric oxide ( O ^' ), hydrogen peroxide (H ₂ 0 ₂ ), hypochlorous acid (HOCI), and peroxynitrite anion (ONOO-).

[0275] In some embodiments, a mammal in need thereof may be a mammal undergoing a treatment associated with oxidative damage. For example, the mammal may be undergoing reperfusion. "Reperfusion" refers to the restoration of blood flow to any organ or tissue in which the flow of blood is decreased or blocked. The restoration of blood flow during reperfusion leads to respiratory burst and formation of free radicals.

[0276] In some embodiments, a mammal in need thereof is a mammal suffering from a disease or condition associated with oxidative damage. The oxidative damage can occur in any cell, tissue or organ of the mammal. Examples of cells, tissues or organs affected by oxidative damage include, but are not limited to, endothelial cells, epithelial cells, nervous system cells, skin, heart, lung, kidney, eye and liver. For example, lipid peroxidation and an inflammatory process are associated with oxidative damage for a disease or condition.

[0277] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) for use in reducing oxidative damage associated with a neurodegenerative disease or condition. The neurodegenerative disease can affect any cell, tissue or organ of the central and peripheral nervous system. Examples of such cells, tissues and organs include, the brain, spinal cord, neurons, ganglia, Schwann cells, astrocytes, oligodendrocytes and microglia.

[0278] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to effect oxidation state of muscle tissue.

[0279] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to effect oxidation state of muscle tissue in lean and obese human subjects.

[0280] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH ₂ , Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to effect insulin resistance in muscle tissue.

[0281] In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe-Lys-NH2, Phe-D-Arg-Phe- Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to treat, prevent or ameliorate a disease or condition comprising a neurological or neurodegenerative disease or condition, ischemia, reperfusion, hypoxia, atherosclerosis, ureteral obstruction, diabetes, complications of diabetes, arthritis, liver damage, insulin resistance, diabetic nephropathy, acute renal injury, chronic renal injury, acute or chronic renal injury due to exposure to nephrotoxic agents and/or radiocontrast dyes, hypertension, metabolic syndrome, an ophthalmic disease or condition such as dry eye, diabetic retinopathy, cataracts, retinitis pigmentosa, glaucoma, macular degeneration, choroidal neovascularization, retinal degeneration, oxygen-induced retinopathy, cardiomyopathy, ischemic heart disease, heart failure, hypertensive

cardiomyopathy, vessel occlusion, vessel occlusion injury, myocardial infarction, coronary artery disease, oxidative damage. In some embodiments, the present technology provides formulations comprising aromatic-cationic peptides (e.g., 2',6'-dimethyl-Tyr-D-Arg-Phe- Lys-NH ₂ , Phe-D-Arg-Phe-Lys-NH ₂ , or D-Arg-2',6'-Dmt-Lys-Phe-NH ₂ ) to treat, prevent or ameliorate macular degeneration, and/or the signs or symptoms of macular degeneration, wet or dry. In some embodiments, the macular degeneration is age related macular degeneration. Bioavailability and Delivery of Peptides

[0282] Aromatic-cationic peptides of the present technology may be chosen as an active ingredient for treatment of medical conditions and diseases as recited herein. Orally, nasally, inhaled, ophthalmic, topically, transdermally, intrathecally and parenterally (e.g.,

intravenously) administered aromatic-cationic peptide will be effective against medical conditions or diseases such as those described herein. By way of example, but not by way of limitation, delivery to the lungs via aerosolized e.g., lipid emulsion or micelle or suspension peptide formulations provides rapid absorption into the cardiovascular system and rapid uptake into the heart and lungs. In some embodiments, the same formulations (e.g., a liquid preparation) can be used for multiple modes of delivery, e.g. , nasal, intravenous, orally and inhalation (e.g., aerosolized liquid preparation).

[0283] Serum levels may be measured by HPLC or mass spectroscopy, according to methods known in the art. The attending physician may monitor patient response, aromatic- cationic peptide blood levels, or surrogate markers of disease, especially during the initial phase of treatment. The physician may then alter the dosage somewhat to account for individual patient metabolism and response.

[0284] The bioavailability achievable in accordance with the present methods permits, for example, delivery of aromatic-cationic peptide into the blood while using between 10-200 micrograms, or between 200-500 micrograms, or between 500-1000 micrograms, or between 1000- 5000 micrograms, or between 5000- 10,000 micrograms, or between 10,000- 20,000 micrograms, or between 20,000- 30,000 micrograms, or between 30,000- 40,000 micrograms, or between 40,000- 50,000 micrograms of orally, ophthalmiclly, nasally, or inhaled administered aromatic-cationic peptides of the present technology. In some embodiments, the aromatic-peptide compositions of the present technology is give parenterally for example via intravenous (IV) infusion. In some embodiments, in IV infusion, aromatic-peptide compositions of the present technology are delivered at between 10-200 micrograms, or between 200-500 micrograms, or between 500-1000 micrograms, or between 1000- 5000 micrograms, or between 5000-10,000 micrograms, or between 10,000- 15,000 micrograms.

[0285] In some embodiments, a single dosage (e.g., a capsule, tablet or liquid formulation for oral, inhalation, ocular, parenteral or nasal administration) can be used at each

administration because a single dosage can provide simultaneous release of the peptide, pH- lowering agent and absorption enhancers. In some embodiments, an acid is able to reduce undesirable proteolytic attack on the peptide when the acid is released in close time proximity to release of the peptide. Near simultaneous release is, in some embodiments, achieved by administering all components of the methods as a single pill or capsule. However, the methods also include, for example, dividing the required amount of acid and enhancers, when used, among e.g., two or more capsules, tablets or doses (e.g., for nasal, inhalation or parenteral administration) which may be administered together such that they together provide the necessary amount of all ingredients.

[0286] Both man-made and natural peptides can be delivered orally, nasally, by inhalation, ophthalmic administration and/or parenterally, e.g., intravenously, in accordance with the methods and compositions disclosed herein.

[0287] The peptides for use in the methods may be in free form or in pharmaceutically acceptable salt or complex form, e.g. , in pharmaceutically acceptable acid addition salt form. Such salts and complexes are known and tend to possess an equivalent degree of activity and tolerability to the free forms. Suitable acid addition salt forms for use in accordance with the methods include for example the hydrochlorides and acetates.

Oral Delivery

[0288] In some embodiments, a single tablet capsule or liquid oral dosage is used at each administration because a single dose of the product provides simultaneous release of the aromatic-cationic peptide of the present technology, pH-lowering agent and absorption enhancers. In some embodiments, the acid is best able to reduce undesirable proteolytic attack on the polypeptide when the acid is released in close time proximity to release of the polypeptide. Near simultaneous release, in some embodiments, can thus be achieved by administering all components of the present formulations as a single tablet or capsule or liquid oral dosage. However, the present technology also includes, for example, dividing the required amount of acid and enhancers among two or more tablets or capsules or liquid dosages which may be administered together such that they together provide the necessary amount of all ingredients. The term "pharmaceutical composition," as used herein includes a complete dosage appropriate to a particular administration to a human patient regardless of how it is subdivided so long as it is for substantially simultaneous administration.

[0289] For certain indications, a first oral pharmaceutical composition is administered in a capsule or tablet or liquid formulation which does not contain a protective acid stable vehicle, such that the components will be relatively rapidly released in the stomach and thus be available for immediate pain relief, e.g. , within about 10-20 minutes. Subsequently, additional capsules or tablets formulated according to the methods with a protective vehicle may then be administered, resulting in bioavailability in the intestine of the active ingredient after the longer time interval that is required for gastric emptying, i. e. , typically around two hours.

[0290] In some embodiments, a sufficient amount of the aromatic-cationic peptide is included in the oral formulation of the composition to achieve a serum level ( . e. , Cmax) of the aromatic-cationic peptide is from 200 μg ml to 20 ng/ml, or from 200 μg ml to 2 ng/ml. Dosage levels of the aromatic-cationic peptide for achieving the above serum levels may range from 100 μg to 10 mg, or from 100 μg to 1 mg. With respect to the dosages recommended herein, however, the attending clinician should monitor the subject's response and adjust the dosage accordingly. Moreover, except where otherwise stated, the dosage of the aromatic-cationic peptide of the present technology is identical for both therapeutic and prophylactic purposes. The dosage for each aromatic-cationic peptide discussed herein is the same, regardless of the disease being treated (or prevented).

[0291] Except where otherwise noted, or where apparent from context, dosages herein refer to weight of aromatic-cationic peptide unaffected by pharmaceutical excipients, diluents, carriers or other ingredients, although such additional ingredients are desirably included.

Nasal Delivery

[0292] The above described aromatic-cationic peptide compositions may be applied in accordance with the methods to the nasal mucosa, e.g., either in drop or in spray form. The compositions of the present disclosure may of course also include additional ingredients, in particular components belonging to the class of conventional pharmaceutically applicable surfactants.

[0293] In some embodiments, the aromatic-cationic compositions of the present disclosure are formulated as liquid pharmaceutical compositions, and in some embodiments, contain a pharmaceutically acceptable diluent or carrier suitable for application to the nasal mucosa. Aqueous saline may be used for example.

[0294] In some embodiments, the compositions of the present disclosure are formulated so as to permit administration via the nasal route. For this purpose they may also contain, e.g., minimum amounts of any additional ingredients or excipients desired, for example, additional preservatives or, e.g., ciliary stimulants such as caffeine. [0295] Generally for nasal administration a mildly acid pH will be used. In some embodiments, the compositions of the present disclosure have a pH of from about 3.0 to 6.5. In some embodiments the compositions have a pH of from about 3 to 5, about 3.5 to about 3.9 or about 3.7. In some embodiments, adjustment of the pH is achieved by addition of an appropriate acid, such as hydrochloric acid.

[0296] In some embodiments, the compositions of the present disclosure also possess an appropriate isotonicity and viscosity. In some embodiments, the compositions have an osmotic pressure of from about 260 to about 380 mOsm/liter. In some embodiments, the viscosity for the nasal spray is less than 0.98 cP. In some embodiments, the osmotic pressure is from 250 to 350 mOsm/liter.

[0297] Compositions in accordance with the present disclosure, e.g., those comprising aromatic-cationic peptides, may also comprise a conventional surfactant, such as a non-ionic surfactant. When a surfactant is employed, the amount present in the compositions will vary depending on the particular surfactant chosen, the particular mode of administration (e.g., drop or spray) and the effect desired. In general, however, the amount present will be of the order of from about 0.1 mg/ml to about 10 mg/ml, about 0.5 mg/ml to 5 mg/ml, or about 1 mg/ml. In some embodiments, the use of surface active agents generally in relation to the nasal application of aromatic-cationic peptides of the present technology, may increase absorption via the nasal mucosa and hence improve obtained bioavailability rates.

[0298] In some embodiments, a pharmaceutically acceptable preservative is included. Many are known in the art, and have been used in the past in connection with aqueous nasal pharmaceuticals. For example, benzyl alcohol or phenylethyl alcohol or a mixture thereof may be employed. In one embodiment, 0.2% phenylethyl alcohol and 0.5% benzyl alcohol are used in combination.

[0299] The amount of peptide to be administered, and hence the amount of active ingredient in the composition will, of course, depend on the particular peptide chosen, the condition to be treated, the desired frequency of administration and the effect desired.

[0300] The quantity of the total composition administered at each nasal application suitably comprises from about 0.05 to 0.15 ml, typically about 0.1 ml.

[0301] For the purposes of nasal administration, in some embodiments, the compositions is provided in a container provided with means enabling application of the contained composition to the nasal mucosa, e.g., put up in a nasal applicator device. Suitable applicators are known in the art and include those adapted for administration of liquid compositions to the nasal mucosa in drop or spray form. Because dosing should be as accurately controlled as possible, use of spray applicators for which the administered quantity is susceptible to precise regulation will generally be preferred. Suitable administrators include, e.g., atomizing devices, pump-atomizers and aerosol dispensers. In the latter case, the applicator will contain a composition in accordance with the methods together with a propellant medium suitable for use in a nasal applicator. The atomizing device will be provided with an appropriate spray adaptor allowing delivery of the contained composition to the nasal mucosa. Such devices are well known in the art.

[0302] The container, e.g., nasal applicator, may contain sufficient composition for a single nasal dosing or for the supply of several sequential dosages, e.g., over a period of days or weeks. Quantities of individual dosages supplied may be as hereinbefore defined.

[0303] In accordance with the present methods it has now been surprisingly found that pharmaceutical compositions can be obtained comprising aromatic-cationic peptide as an active ingredient which meet the high standards of stability and bioavailability required for nasal application and which are, for example, eminently suitable for use in multiple dose nasal spray applicators, i.e., applicators capable of delivering a series of individual dosages over, e.g., period of several days or weeks, by the use of citric acid or a salt thereof in concentrations ranging from about 10 to about 50 mM as a buffering agent.

[0304] Surprisingly, it has also been found that use of citric acid or a salt thereof at increasing concentrations confers, in some embodiments, beneficial advantages in relation to the nasal absorption characteristics of aromatic-cationic peptide containing compositions and hence enhance aromatic-cationic peptide bioavailability levels consequential to nasal application. In addition, it has also been found, in some embodiments, that the use of citric acid or a salt thereof in concentrations ranging from about 10 to about 50 mM increase the stability of aromatic-cationic peptide compositions while at the same time higher concentrations of citric acid or salt thereof do not have the same stabilizing effect.

[0305] The aromatic-cationic peptide for use in the present methods may be in free form or in pharmaceutically acceptable salt or complex form, e.g. , in pharmaceutically acceptable acid addition salt form. Such salts and complexes are known and possess an equivalent degree of activity and tolerability to the free forms. Suitable acid addition salt forms for use in accordance with the methods include for example the hydrochlorides and acetates. In some embodiments, the aromatic-cationic peptide comprises D-Arg-2'6'-Dmt-Lys-Phe-NH2. In some embodiments, the peptide is provided as a salt, such as an acetate or trifluoroacetate salt.

[0306] The above defined compositions may be applied in accordance with the methods to the nasal mucosa, e.g., either in drop or in spray form. As hereinafter described however, they may be applied in spray form, i.e. , in the form of finely divided droplets.

[0307] In some embodiments, the liquid pharmaceutical compositions of the present methods contain a pharmaceutically acceptable diluent or carrier suitable for application to the nasal mucosa, such as aqueous saline.

[0308] For the aromatic-cationic nasal formulations disclosed herein, bioavailability, as determined in terms of blood-plasma concentration following nasal administration in accordance with the teachings of the present methods are anticipated to be surprisingly high.

[0309] For nasal administration in accordance with the present methods, in some embodiments, treatment may suitably comprise administration of dosages at a frequency of from about once daily to about three times daily. Dosages may be administered in a single application, i. e. , treatment will comprise administration of single nasal dosages of aromatic- cationic peptide of the present technology. Alternatively such dosages may be split over a series of 2 to 4 applications taken at intervals during the day. The total composition quantity administered at each nasal application will vary according to the condition being treated, the particular peptide being administered, and the characteristics of the subject.

[0310] The container, e.g., nasal applicator, in some embodiments, may contain sufficient composition for a single nasal dosing or for the supply of several sequential dosages, e.g., over a period of days or weeks. Quantities of individual dosages supplied may be as hereinbefore defined. The stability of the compositions may be determined in conventional manner. In some embodiments, the aromatic-cationic peptide content of the compositions will degrade less than 50 % in 15 days at 50°C as determined by standard analytical tests.

Combination Therapy with an Aromatic-Cationic Peptide and Other Therapeutic Agents

[0311] In some embodiments, the aromatic-cationic peptides may be combined with one or more additional agents for the prevention or treatment of a disease or condition. In some embodiments, a synergistic therapeutic effect is produced. A "synergistic therapeutic effect" refers to a greater-than-additive therapeutic effect which is produced by a combination of two therapeutic agents (e.g., an aromatic-cationic peptide and another agent), and which exceeds that which would otherwise result from individual administration of either therapeutic agent alone. Therefore, lower doses of one or both of the therapeutic agents may be used in treating or preventing a disease or condition, resulting in increased therapeutic efficacy and decreased side-effects.

[0312] In some embodiments, the multiple therapeutic agents (e.g., an aromatic-cationic peptide and another agent) may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between the multiple doses may vary from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents.

EXAMPLES

[0313] The formulations described herein are further illustrated by the following examples. The examples are intended to be illustrative only and are not to be construed as limiting in any way. The examples are intended to show trends relating to the formulations described herein and are not intended to limit the scope of composition or function of the formulations.

Example 1 : Effects of pH on the Bioavailability of Aromatic-cationic Peptides of the Present Technology

[0314] This example will demonstrate the effect of pH on the bioavailability of formulations comprising the aromatic-cationic peptides of the present technology, such as Phe-D-Arg-Phe-Lys- NH ₂ and D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ .

[0315] Female Wistar rats (250-275 g) (n=3 for each formulation) will be anesthetized with ketamine and xylazine prior to the insertion of a cannula in the carotid artery. The cannula will be fitted to a three way valve through which blood will be sampled and replaced with physiological saline. A midline incision will be made in the abdominal cavity and 0.5 ml of formulation will be injected directly into the exposed duodenum. The pH of the formulation will be adjusted by mixing citric acid and sodium citrate of equimolar concentrations. Blood (0.5 ml) will be collected before administration of the formulation and at 5, 15, 30, 60, and 120 minutes after the administration. Blood samples will be centrifuged for 10 minutes at 2600 x g and the resulting plasma supernatant will be stored at -20°C. The concentration of aromatic-cationic peptide in plasma will be determined by reverse phase HPLC

chromatography and/or mass spectroscopy (MS). One of skill in the art will understand that the aromatic-cationic peptides described herein may be analyzed by a number of HPLC methods, including reverse phase HPLC, such as those described in Aguilar, HPLC of Peptides and Proteins: Methods and Protocols, Humana Press, New Jersey (2004). Likewise, one of skill in the art will understand that the aromatic-cationic peptides described herein may be analyzed by a number of MS methods, such as those described in Sparkman, Mass Spectroscopy Desk Reference, Pittsburgh: Global View Pub (2000).

[0316] The absolute bioavailability or aromatic-cationic peptide (i.e., relative to an intravenous dose of aromatic-cationic peptide) will be calculated from the area under the curve obtained from plots of the plasma concentration of aromatic-cationic peptide as a function of time.

[0317] Anticipated trends in the effects of buffer pH on the bioavailability of aromatic- cationic peptide are shown in Table 1. It is anticipated that when the pH of the buffer is reduced from 5.0 (illustrative formulation I) to 4.0 (illustrative formulation II) the absolute bioavailability of aromatic-cationic peptide will increase as much as five-fold. It is expected that reduction of the buffer pH to 3.0 (illustrative formulation III) will increase the absolute bioavailability of the peptide as much as 32-fold compared to that achieved with buffer of pH 5.0. It is expected that reduction of the buffer pH to 2.0 (illustrative formulation IV) will result in very little additional increase in absolute bioavailability of the peptide. It is anticipated that a substantial increase in the absolute bioavailability of aromatic-cationic peptide will occur when the buffer pH is reduced from 5.0 to 3.0.

Example 2: Effects of Citric Acid on the Bioavailability of Aromatic-cationic Peptides of the

Present Technology

[0318] This example will demonstrate the effect of citric acid on the bioavailability of the aromatic-cationic peptides of the present technology, such as Phe-D-Arg-Phe-Lys- N¾ and D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ .

[0319] Formulations consisting of a fixed amount of taurodeoxycholic acid and two different amounts of citric acid will be prepared in a total volume of 0.5 ml. Mannitol will be included in the formulations as a marker to measure paracellular transport. The formulations will be administered to female Wistar rats as described in Example 1. Blood samples will be collected and bioavailability measured as described in Example 1.

[0320] Anticipated trends in the effect of citric acid on the bioavailability of aromatic- cationic peptide are shown in Table 2. It is anticipated that a relatively higher citric acid concentration will result in increased bioavailability of aromatic-cationic peptides of the present technology compared to a lower citric acid concentration. For example, illustrative formulation II is anticipated to increase the bioavailability of aromatic-cationic peptide by as much as 10-fold over that achieved with illustrative formulation I. In the presence of a fixed amount of taurodeoxycholic acid, the bioavailability of aromatic-cationic peptides of the present technology it is anticipated to increase when the amount of citric acid in the formulation is increased only 5 fold.

Example 3 : Effects of Absorption Enhancers on the Bioavailability of Aromatic-cationic Peptides of the Present Technology

[0321] This example will demonstrate the effect of absorption enhancers on the bioavailability of the aromatic-cationic peptides of the present technology, such as Phe-D- Arg-Phe-Lys- NH ₂ and D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ .

[0322] Formulations consisting of citric acid, aromatic-cationic peptide, and various classes of enhancers will be prepared in a total volume of 0.5 ml. Mannitol will be included in formulation V as a marker to measure paracellular transport. The formulations will be administered to female Wistar rats as described in Example 1. Blood samples will be collected and bioavailability measured as described in Example 1. Anticipated trends in the effect of enhancers on the bioavailability of aromatic-cationic peptide are shown in Table 3. It is anticipated that formulations including an enhancer will result in increased

bioavailability of aromatic-cationic peptide relative to formulations lacking an enhancer. The inclusion of a water soluble phospholipid (illustrative formulation VII) is expected to increase the bioavailability of aromatic-cationic peptide by as much as four- fold. The most effective enhancer is anticipated to be the sugar ester class (illustrative formulation V) in which the aromatic-cationic peptide bioavailability may be increased as much as eight- fold. The use of a mixture of bile acid and a cationic detergent (illustrative formulation III), a non-ionic detergent (illustrative formulation IV), or an acylcarnitine (illustrative formulation VI) are expected to increase the bioavailability of aromatic-cationic peptide as much as eight-fold compared to that achieved with illustrative formulation I. Variations in the bioavailability of aromatic-cationic peptide administered with various classes of enhancers are expected to be minor compared to variations observed when the peptide is formulated with citric acid only and no enhancer.

Taurodeoxycholic acid (5 mg)

III Aromatic-cationic peptide (0.1 mg)

Citric acid (77 mg) 8x 7x Cetylpyridinium chloride (5 mg)

IV Aromatic-cationic peptide (0.1 mg)

Citric acid (48 mg) 3x 5x Tween -20 (5 mg)

V Aromatic-cationic peptide (0.1 mg)

Citric acid (48 mg) Sucrose ester- 15 (5

8x 8x mg)

Mannitol (22 mg)

VI Aromatic-cationic peptide (0.1 mg)

Citric acid (48 mg) 8x 8x Lauroyl L-carnitine chloride (5 mg)

VII Aromatic-cationic peptide (0.1 mg)

Citric acid (48 mg) 4x 4x Diheptanoylphosphatidylcholine (5 mg)

^Relative to values obtained for formulation I

Example 4: Effect of Lauroyl L-carnitine on the Bioavailability of Aromatic-cationic

Peptides of the Present Technology

[0323] This example will demonstrate the effect of lauroyl L-carnitine on the

bioavailability of the aromatic-cationic peptides of the present technology, such as Phe-D- Arg-Phe-Lys- NH ₂ and D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ .

[0324] Formulations consisting of lauroyl L-carnitine, aromatic-cationic peptide of the present technology, and various other compounds will be prepared in a total volume of 0.5 ml. The formulations will be administered to female Wistar rats as described in Example 1. Blood samples will be collected and bioavailability measured as described in Example 1.

[0325] Anticipated trends in the effect of lauroyl L-carnitine on the bioavailability of aromatic-cationic peptide are shown in Table 4. It is anticipated that administration of aromatic-cationic peptide the absence of citric acid or any enhancer (illustrative formulation I) will result in reduced absolute bioavailability of peptide compared to formulations that include citric acid or an enhancer. It is anticipated that the inclusion of 5 mg lauroyl L- carnitine chloride (illustrative formulation II) will increase the bioavailability or aromatic- cationic peptide by approximately two-fold relative to illustrative formulation I. It is anticipated that the inclusion of lauroyl L-carnitine together with citric acid (illustrative formulation III), will increase the bioavailability of aromatic-cationic peptide by as much as 50-fold. It is anticipated that a five-fold reduction in the amount of lauroyl L-carnitine, but not citric acid (illustrative formulation IV), will not significantly reduce the bioavailability of aromatic-cationic peptide compared to that achieved with illustrative formulation III. It is expected that the inclusion of 5 mg diheptanoylphosphatidylcholine together with citric acid and lauroyl L-carnitine (illustrative formulation V) will increase the bioavailability of peptide by as much as 67-fold over that achieved with illustrative formulation I. The substitution of 25 mg bovine serum albumin for citric acid (illustrative formulation VI) is anticipated to increase the bioavailability of aromatic-cationic peptide compared to that achieved with illustrative formulation I (unformulated peptide), but to a lesser extent than illustrative formulations I-V. It is expected that these results will show the synergistic effects of pH- lowering agents (e.g. citric acid) and an enhancers (e.g. lauroyl L-carnitine) on the bioavailability of aromatic-cationic peptide.

Example 5 : Effect of Illustrative Formulations on the Absorption of Aromatic-Cationic Peptides of the Present Technology

[0326] This example will demonstrate the effect of illustrative formulations on absorption of the aromatic-cationic peptides of the present technology, such as Phe-D-Arg-Phe-Lys- NH ₂ and D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ . [0327] Modified vascular access ports will be surgically implanted into the duodenum, ileum and colon of male beagle dogs. The septum/reservoir bodies of the ports will be implanted under the skin and will be used as sites for the administration of aromatic-cationic peptide formulations. Before and after the administration of aromatic-cationic peptide formulations into conscious dogs, the ports will be flushed with 2 ml of a mock formulation lacking aromatic-cationic peptide. Blood (2 ml) will be collected through angiocatheter tubes in the leg vein at 30, 15, and 0 minutes before administration of aromatic-cationic peptide, and at 5, 10, 20, 30, 40, 50, 60, and every 15 minutes thereafter for 2 hours after

administration. Blood samples will be centrifuged for 10 minutes at 2600 g and the resulting plasma supernatant will be stored at -20° C. The concentration of aromatic-cationic peptide in plasma will be determined by a reverse-phase HPLC. The absolute bioavailability (i.e. relative to an intravenous dose of aromatic-cationic peptide) will be calculated from the areas under the curve obtained from plots of the plasma concentration as a function of time.

[0328] Anticipated trends in the effect of illustrative formulations on the bioavailability of aromatic-cationic peptide are shown in Table 5. It is anticipated that the absolute

bioavailability of aromatic-cationic peptide administered alone (illustrative formulation I) will be low compared to formulations that include taurodeoxycholic acid and/or citric acid. It is anticipated that including citric acid in the formulation (illustrative formulation II) will increase the bioavailability of the peptide by as much as 25-fold. It is anticipated that further including taurodeoxycholic acid in the formulation (illustrative formulation III) will increase the bioavailability of the peptide by as much as 50-fold.

Example 6: Effect of Citric Acid and Lauroyl L-carnitine on the Bioavailability of

Vasopressin, Aromatic-Cationic Peptides of the Present Technology, and Insulin

[0329] This example will demonstrate the effect of citric acid lauroyl L-carnitine on the bioavailability of the aromatic-cationic peptides of the present technology, such as Phe-D- Arg-Phe-Lys- NH ₂ and D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ , vasopressin, and insulin.

[0330] Formulations consisting of either [Arg ⁸ ]-vasopressin, aromatic-cationic peptide, or human insulin together with specified additives will be prepared in a total volume of 0.5 ml. The formulations will be administered to female Wistar rats as described in Example 1. Blood samples will be collected and bioavailability measured as described in Example 1.

[0331] Anticipated trends in the effect of lauroyl L-carnitine and citric acid on the bioavailability of vasopressin, aromatic-cationic peptide, and insulin are shown in Table 6. It is expected that the bioavailability of [Arg ⁸ ]-vasopressin formulated with citric acid

(illustrative formulation V-II) will be as much as 20-fold higher than that of unformulated [Arg ⁸ ] -vasopressin (illustrative formulation V-I). It is expected that the bioavailability of aromatic-cationic peptide formulated with citric acid and lauroyl L-carnitine (illustrative formulation ACP-II) will be as much as 50-fold higher than that of unformulated peptide (illustrative formulation ACP-I). It is expected that the bioavailability of insulin formulated with citric acid and lauroyl L-carnitine (illustrative formulation HI-II) will be as much as 1 1- fold higher than that of unformulated insulin (illustrative formulation HI-I). These results are anticipated to demonstrate that the bioavailability of unformulated therapeutic peptides is substantially lower than peptides formulated with an organic acid, such as citric acid, and an enhancer such as lauroyl L-carnitine.

e at ve to va ues o ta ne or ormu at on n eac group

Example 7: Effect of Enteric Coating on Absorption of formulations comprising Aromatic- Cationic Peptides of the Present Technology

[0332] This example will demonstrate the effect of enteric coating on absorption of illustrative formulations of the aromatic-cationic peptides of the present technology, such as Phe-D-Arg-Phe-Lys- NH ₂ and D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ .

[0333] Size 00 UPMC (hydroxypropylmethyl cellulose) capsules will each be filled with a powdered blend consisting of citric acid, lauroyl L-carnitine, and aromatic-cationic peptide. Half the capsules will be coated with an enteric coating solution of EUDRAGIT L30D-55 (a methacrylic acid co-polymer with methacrylic acid methyl ester, ROUM Tech Inc., Maidan, Mass.), and the remaining capsules will not be coated. The coating process will correspond to that taught in U.S. Pat. No. 6,086,918 at col. 11, line 50 to col. 12, line 11. The average capsule content for the enteric coated and non-enteric coated capsules is shown in Table 7.

[0334] Eight fasted dogs will be orally administered one uncoated capsule at week one. At week three, each subject will be orally administered one enteric-coated capsule. After each administration, samples of blood will be taken at 15 minute intervals from an indwelling catheter for up to 4 hours. The blood samples will be centrifuged and the resulting plasma supernatants will be stored frozen at -20° C. The plasma samples will be analyzed for aromatic-cationic peptides of the present technology by reverse phase HPLC chromatography and/or mass spectroscopy (MS). The maximum plasma concentration of aromatic-cationic peptides of the present technology will be normalized to a 1 mg dose.

[0335] Anticipated trends in the effect of enteric coating on the bioavailability of aromatic- cationic peptides of the present technology is shown in Table 7. It is anticipated that aromatic-cationic peptides of the present technology will be detected in plasma from dogs orally administered enteric coated as well as uncoated capsules. It is anticipated that maximal plasma concentrations will be about three-fold higher following administration of enteric- coated capsules as compared to non-coated capsules. It is anticipated that maximum plasma concentrations will be achieved within 30 minutes after administration of uncoated capsules, and 90 minutes after administration of enteric-coated capsules.

[0336] It is anticipated that these results will demonstrate that a therapeutically effective amount of an aromatic-cationic peptide is absorbed from non-coated capsules at a faster rate than from enteric-coated capsules, and that higher plasma concentrations will be achieved with coated capsules than with non-coated capsules. Faster rates of absorption may be advantageous, especially in the case of peptides wherein speed is more important than overall bioavailability (e.g., inhibition of MPT). There can also be an advantage in production efficiency when the enteric coating step is not required.

Example 8: Effects of Illustrative Formulations on Absorption of Aromatic-Cationic Peptide From Non-enteric Coated Capsules

[0337] This example will demonstrate the effect of illustrative formulations on absorption of the aromatic-cationic peptides of the present technology, such as Phe-D-Arg-Phe-Lys- NH ₂ and D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ .

[0338] Size 00 UPMC (hydroxypropylmethyl cellulose) capsules will each be filled with a powdered blend consisting of the indicated amount of citric acid, lauroyl L-carnitine, sucrose and aromatic-cationic peptide. The average capsule content for the capsules is shown in Table 8. Each week, eight fasted dogs will each be orally administered one uncoated capsule. After each administration, samples of blood will be taken at 15 minute intervals from an indwelling catheter for up to 4 hours. The blood samples will be centrifuged and the resulting plasma supernatants will be stored frozen at -20° C. The plasma samples will be subsequently analyzed for aromatic-cationic peptide by reverse phase HPLC chromatography and/or mass spectroscopy (MS).

[0339] Anticipated trends in the effect of illustrative formulations on absorption of aromatic-cationic peptide from non-enteric coated capsules are shown in Table 8. It is anticipated that administration of aromatic-cationic peptide alone (illustrative formulation I) will result in a relatively lower plasma concentration than formulations including citric acid and/or lauroyl L-carnitine. It is expected that administration of peptide together with lauroyl L-carnitine (illustrative formulation II), citric acid (illustrative formulation III), or both (illustrative formulation IV) will result in as much as 50-fold, 230-fold, and 270-fold higher plasma concentrations than achieved with unformulated peptide, respectively. These results are anticipated to demonstrate the importance of including both an acid and an absorption enhancer in aromatic-cationic peptide formulations.

Example 9: Effects of Illustrative Formulations Absorption of the Aromatic-Cationic Peptides of the Present Technology, such as Phe-D-Arg-Phe-Lys- NFL and D-Arg-2 ^f 6'-Dmt-Lys-Phe-

NH?

[0340] Size 00 UPMC capsules will each be filled with a powdered blend consisting of at least 500 mg citric acid, 50 mg lauroyl L-carnitine and 1.0 mg of the aromatic-cationic peptide Phe-D-Arg-Phe-Lys- NH2 or D-Arg-2'6'-Dmt-Lys-Phe-NFL; , or a pharmaceutically acceptable salt thereof, such as acetate or trifluoroacetate salt. Each week, eight fasted dogs will be orally administered one uncoated capsule. After each administration, samples of blood will be taken at 15 minute intervals from an indwelling catheter for up to 4 hours. The blood samples will be centrifuged and the resulting plasma supernatants will be stored frozen at - 20° C. The plasma samples will subsequently be analyzed for Phe-D-Arg-Phe-Lys- NH2 or D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ as described in Example 7. [0341] Anticipated trends in the effect of illustrative formulations on absorption of the aromatic-cationic peptides of the present technology Phe-D-Arg-Phe-Lys- NH ₂ or D-Arg- 2'6'-Dmt-Lys-Phe-NH2 from non-enteric coated capsules are shown in Table 9. It is anticipated that administration of the peptide alone (illustrative formulation I) will result in a relatively lower plasma concentration than formulations including citric acid and/or lauroyl L-carnitine. It is expected that administration of peptides together with lauroyl L-carnitine (illustrative formulation II), citric acid (illustrative formulation III), or both (illustrative formulation IV) will result in as much as 50-fold, 230-fold, and 270-fold higher plasma concentrations than achieved with unformulated peptide, respectively. These results are anticipated to demonstrate the importance of including both an acid and an absorption enhancer in Phe-D-Arg-Phe-Lys- NH ₂ and D-Arg-2 ^, 6'-Dmt-Lys-Phe-NH ₂ peptide formulations.

[0342] Although the present method has been described in relation to particular embodiments thereof, many other variations and modifications and other uses will become apparent to those skilled in the art. The present method therefore is not limited by the specific technology herein, but only by the claims.

Example 10: Effects of Enteric Coating on the Bioavailability of Aromatic-Cationic Peptides of the Present Technology.

[0343] This example will demonstrate the effect of illustrative formulations on absorption of the aromatic-cationic peptides of the present technology, such as Phe-D-Arg-Phe-Lys- NH ₂ and D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ . [0344] Administration of aromatic-cationic peptide in the oral formulation described herein provides unexpected improvements in bioavailability of the subject peptide.

[0345] With regard to a first series of tests, i.e. , on rats, the improved effect will be demonstrated by comparing the curves for formulated aromatic-cationic peptide vs.

unformulated aromatic-cationic peptide. Six anesthetized rats will be given 0.7 ml aromatic- cationic peptide (1.6 mg/ml) with a syringe through a 27 gauge needle into the duodenum. This injection procedure will be followed due to the technical difficulty inherent in preparing capsules which can be swallowed by small animals the size of a rat. The intraduodanal injection, therefore, will mimic the release of the components of an enteric-coated capsule formulation which would pass through the esophagus and stomach and release its contents in the duodenum. Three of the rats will be given unformulated aromatic-cationic peptide in which there are no additional components (z. e. , other than the aromatic-cationic peptide), while the other three rats will be given formulated aromatic-cationic peptide which include, in addition to the aromatic-cationic peptide, 0.5M citric acid and lauroyl L-carnitine (10 mg/ml). Samples of blood will be taken from the carotid artery through an indwelling catheter before and 5, 15, 30, 60 and 120 minutes after the administration of the respective formulations (i.e., formulated and unformulated).

[0346] The blood samples will be centrifuged and the resulting plasma supernatants will be stored frozen at -20° C. The plasma samples will be subsequently analyzed for aromatic- cationic peptide by high-performance liquid chromatography (HPLC) through a 50 x 4.6 mm polysulfoethyl-aspartamid- e column with a mobile phase of 15.4 mM potassium phosphate (pH 3), 210 mM sodium chloride, and 25% acrylonitrile at a flow rate of 1.5 ml/min. Peptide will be detected with an ultraviolet (UV) detector set at a wavelength of 210 nm. The results are expected to show that aromatic-cationic peptide is virtually undetectable in rats given unformulated aromatic-cationic peptide, whereas as much as 8 μg ml of aromatic-cationic peptide is predicted to be detectable in rats given aromatic-cationic peptide formulated in citric acid and lauroyl L-carnitine. These results are expected to demonstrate that formulating aromatic-cationic peptide in an oral formulation according to the present methods increases the Cmax and AUC compared to the unformulated peptide.

[0347] A second series of tests will be carried out, as noted above, using beagle dogs. The improved bioavailability of orally administered aromatic-cationic peptide will be demonstrated in this second series of tests by comparing the curves for (1) non-enteric coated salmon calcitonin (sCT) and (2) non-enteric coated aromatic-cationic peptide with the curves for (3) enteric coated sCT and (4) enteric coated aromatic-cationic peptide. In the experiments, size 00 HPLC capsules will be filled with 758 mg of a powdered blend consisting of citric acid (643 mg), lauroyl L-carnitine (66 mg), talc (33 mg), salmon calcitonin (sCT) (13 mg) and aromatic-cationic peptide (2.4 mg). Half of the capsules will be coated with an enteric coating solution of L30D-55, while the remaining 50% of the capsules will not be coated. Four fasted dogs will be each given 1 uncoated capsule, and 2 weeks later they will be each given an enteric coated capsule. After administration of each capsule, samples of blood will be taken at 15 minute intervals from an indwelling catheter for up to 4 hours. The blood samples will be centrifuged and the resulting plasma supernatants will be stored frozen at -20°C. The plasma samples will be subsequently analyzed for sCT by a direct ELISA, and for aromatic-cationic peptide by HPLC-mass spectrometry performed as set forth in Wan, H. and Desiderio, D., Quantitation of dmt-DALDA in ovine plasma by online liquid chromatography/quadrapole time-of-flight mass spectrometry, Rapid

Communications in Mass Spectrometry, 2003; 17, 538-546, the contents of which are incorporated herein by reference.

[0348] The results will be summarized as plasma peptide concentration normalized to a 1 mg dose as a function of time relative to the average Tmax, (i.e., the time at which the maximum amount of peptide is detected). The results are expected to indicate that both peptides, i.e., sCT and aromatic-cationic peptide, are detected in dogs given uncoated or enteric coated capsules. It is expected that nearly three times as much aromatic-cationic peptide as sCT will be detected in dogs given uncoated capsules; whereas, nearly equal amounts of both peptides will be detected in dogs given enteric coated capsules. It is expected that nearly four times as much aromatic-cationic peptide will be detected in the plasma of dogs given enteric coated capsules than those given non-coated capsules. It is expected that nearly eight times as much sCT will be detected in the plasma of dogs given enteric coated capsules than non-coated capsules. The maximum concentration of aromatic-cationic peptide and sCT in dogs given uncoated capsules is expected to be seen 30 minutes after their administration, whereas the maximum concentration of these materials when given in coated capsules is expected to be seen 105 minutes after their administration, due to the additional time necessary for the oral formulation to pass through the stomach while remaining protected from the proteolytic enzymes therein. These results are expected to demonstrate that coating the capsules with an enteric polymer such that the capsule does not release its contents until reaching the small intestine, significantly enhances peptide absorption. [0349] The Cmax and AUC values for both sCT and aromatic-cationic peptide are expected to be significantly enhanced when the peptides are administered in enteric coated capsules versus in non enteric-coated capsules. The Cmax of enteric coated aromatic-cationic peptide is expected to be 4-fold higher than that of non enteric coated aromatic-cationic peptide. The bioavailability of both enteric coated and non-coated aromatic-cationic peptide is expected to be better than that of sCT. It would be expected that the bioavailability of a molecule such as aromatic-cationic peptide, which is positively charged and hydrophilic, would be extremely poor. The data is expected to indicate that when the aromatic-cationic peptide is

administered in combination with the ingredients of the present composition, either with or without an enteric coating, the bioavailability is increased to the point where it is superior to that of sCT, a molecule that has previously been shown to be highly bioavailable when formulated according to the present methods.

Example 11 : Effect of OmPA-MT3 on the Absorption of Aromatic-Cationic Peptides of the Present Technology from Rat Duodenum

[0350] The following example will demonstrate the effect of OmPA-MT3 on the absorption of aromatic-cationic peptides of the present technology, such as Phe-D-Arg-Phe-Lys- Ν¾ and D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ from rat duodenum. Female Sprague-Dawley rats (250- 275 g) (n = 4 for each peptide) are anesthetized with ketamine and xylazine prior to the insertion of a cannula in the carotid artery. The cannula is fitted to a three way valve through which blood is sampled and replaced with physiological saline containing heparin. A midline incision is made in the abdominal cavity, and 0.45 ml of either aromatic-cationic peptide (10 mg/ml) or OmpA-MT3-aromatic-cationinc peptide (10 mg/ml) in 0.5M citric acid is injected directly into the duodenum. Blood (0.5 ml) is collected before and at 5, 15, 30, 45 and 60 minutes after administration of the peptides. The blood is centrifuged, and the concentration (.+/-. SEM [standard error of the mean]) of aromatic-cationic peptide or OmpA-MT3- aromatic-cationinc peptide in the plasma supernatant is determined by a competitive enzyme immunoassay (EIA). Peak plasma concentration (Cmax) is determined by inspection. The absolute bioavailability of each peptide (relative to an intravenous dose of aromatic-cationic- peptide is calculated from plots of the plasma concentration of each peptide as a function to time.

[0351] It is predicted that the maximum concentration of aromatic-cationic-peptide in the blood will be reached between 30 and 60 minutes after their administration. The Cmax of OmpA-MT3-aromatic-cationinc peptide is expected to be more than 25 fold greater than that of aromatic-cationic peptide. The bioavailability of OmpA-MT3-aromatic-cationinc peptide is expected to be more than 20 times greater than that of aromatic-cationic peptide. These results will indicate that attaching OmpA-MT3 to aromatic-cationic peptide significantly enhances peptide absorption through the intestinal wall.

Example 12: Effect of the HIV TAT Protein Transduction Domain as an MT on the

Absorption of Aromatic-Cationic Peptides of the Present Technology from Dog Duodenum

[0352] Two formulations are used to test the efficacy of MT3 -aromatic-cationic peptide fusion. The first formulation (Fl) is prepared by blending 13g citric acid, 1.3g lauroyl L- carnitine, 0.65g talc and 0.03g aromatic-cationic peptide with a mortar and pestle. The other formulation (F2) is prepared by blending the same mixture except that s aromatic-cationic peptide is replaced with an equivalent amount of MT3 -aromatic-cationic peptide. Both blends are used to fill size 00 gelatin capsules, and the capsules are coated with Eudragit L30D-55. The resulting enteric-coated capsules contain approximately 1 to 2 mg of either aromatic- cationic peptide (Fl) or MT3-aromatic-cationic peptide (F2) per capsule. Fasted dogs (n=8) are administered F 1 by mouth and blood samples are collected in heparinized tubes at t=- 10 min, 0 min, and every 15 min thereafter for 240 minutes. The blood samples are centrifuged, and the resulting plasma stored at -20°C. for further analysis. After a 1 week washout period, the same dogs are given F2 by mouth, and the same protocol is followed.

[0353] The amount of aromatic-cationic peptide in plasma samples of dogs given either of the two formulations is HPLC using methods known in the art. Both formulations are expected to produce measurable amounts of aromatic-cationic peptide in the blood, the maximum concentration of aromatic-cationic peptide in the blood of dogs given Fl is expected to be in the range of 0.5 to 6.0 ng/ml, whereas the maximum concentration of aromatic-cationic peptide in dogs given F2 is expected to be at least 1 to 12 ng/ml.

[0354] The bioavailability of aromatic-cationic peptide in dogs given Fl is expected to be approximately 1%, whereas the bioavailability of aromatic-cationic peptide in dogs given F2 is expected to be at least 1.2%. The in vivo cleavage of MT from aromatic-cationic peptide in dogs given F2 is proven by applying samples of plasma from dogs given Fl and F2 to an HPLC column and collecting the effluent in plastic tubes. The solvent in the tubes is removed under vacuum and analyzed for the presence of aromatic-cationic peptide by HPLC. The in vivo cleavage of MT3-aromatic-cationic peptide is established by showing that the retention time of aromatic-cationic peptide in the plasma from dogs given F2 is the same as the retention time of aromatic-cationic peptide in the plasma of dogs given Fl .

Example 13 : Methods of Administering Aromatic-Cationic Peptides of the Present

Technology and Measurement of Plasma Concentration

[0355] The following example will demonstrate methods of administering aromatic- cationic peptides of the present technology, such as Phe-D-Arg-Phe-Lys- NH ₂ and D-Arg- 2'6'-Dmt-Lys-Phe-NH2, and measurement of plasma concentrations. Female Wistar rats, weighing between 225 and 250 g are anesthetized with a combination of ketamine and xyalzine, and a cannula is inserted into the carotid artery. The cannula is fitted to a three-way valve through which blood is sampled and replaced with physiological saline containing heparin. Formulated aromatic-cationic peptide (5 μg per 25 μΐ) is administered intranasally through a micropipette tip inserted 8 mm into the rat's nostril. For single-dose studies, 5 μg of aromatic-cationic peptide is administered. In multiple dose studies, aromatic-cationic peptide is administered four times in a volume of 25 μΐ each at 0, 30, 60 and 90 minutes for a total dose of 20 μg.

[0356] In single-dose studies, blood samples are collected prior to dosing and at 5, 15, 30, 60 and 120 minutes after dosing. In multiple-dose studies, blood samples are collected prior to dosing and at 30, 60, 90, 120 and 150 minutes after the administration of the first dose. Blood samples are always collected immediately before the administration of any additional doses.

[0357] Each sample (0.5 ml) of blood is collected into a heparinized 1 ml syringes and then transferred to chilled 1.5 ml polypropylene tubes containing 10 μΐ of heparin (500 U per ml). The tubes are centrifuged at approximately 3000 rpm for 20 minutes at 2-8°C. and the plasma supernatant is transferred to microcentrifuge tubes that are stored at -20°C. The

concentration of aromatic-cationic peptide in plasma is determined by HPLC using methods known in the art.

[0358] The values of Cmax are determined by inspection and the values for bioavailability (relative to an intravenous injection) are calculated from the areas under the curve that is obtained from plots of plasma aromatic-cationic peptide concentration as a function of time.

[0359] The following example will demonstrate the effect of the concentration of citric acid on the bioavailability and plasma concentration of nasally administered aromatic-cationic peptides of the present technology, such as Phe-D-Arg-Phe-Lys- H ₂ and D-Arg-2'6'-Dmt- Lys-Phe-NH ₂ .. Rats are administered intranasally as described previously 20 μΐ of aromatic- cationic peptide (200 μg/ml) in 0.85% sodium chloride, 0.1 % Tween 80, 0.2% phenylethyl alcohol, 0.5% benzyl alcohol and varying amounts of citric acid adjusted to pH 3.7 at t=0, 20, 60 and 90 minutes. Samples of blood are taken prior to the administration of aromatic- cationic peptide at these time points as well as at t=120 and 150 minutes. The resulting plasma samples are analyzed for aromatic-cationic peptide by HPLC. Maximum aromatic- cationic peptide levels are expected to be detected at t=120 minutes. It is expected that the bioavailability and peak concentration of aromatic-cationic peptide will be a function of the concentration of citric acid in the formulation. It is expected that relatively higher concentrations of citric acid in the formulations will result in higher levels of bioavailability and peak serum concentration as compared to control formulations lacking citric acid.

[0360] The following study will demonstrate the effect of different preservatives on the plasma concentration of nasally administered aromatic-cationic peptides of the present technology, such as Phe-D-Arg-Phe-Lys- N¾ and D-Arg-2'6'-Dmt-Lys-Phe-NH2. Rats are administered intranasally as described previously 20 μΐ of aromatic-cationic peptide (200 μg/ml) in 0.85% sodium chloride, 0.1% Tween 80 and a combination preservatives of either 0.2% phenylethyl alcohol and 0.5% benzyl alcohol or 0.27% methyl parabens and 0.04% proply parabens at t=0, 30, 60 and 90 minutes. It is expected that the bioavailability and peak concentration of aromatic-cationic peptide will not significantly affected by the addition of the different preservatives.

[0361] The following study will demonstrate the effect of the concentration of citric acid on the stability of aromatic-cationic peptide stored for varying periods at a temperature of 50°C. Nasal formulations containing aromatic-cationic peptide (200 μg/ml), 0.25% phenylethyl alcohol, 0.5% benzyl alcohol and 0.1% Tween 80 are adjusted to pH 3.7 with either HC1 or the indicated amount of buffered citric acid. The formulations are stored at 50°C in sealed glass containers for the indicated amount of time and analyzed for aromatic-cationic peptide by high performance liquid chromatography. It is expected that in the absence of citric acid, the amount aromatic-cationic peptide in the formulation will decrease steadily between 0 and 9 days, but that in presence of citric acid (10-50 mM) the rate of disappearance of aromatic- cationic peptide will decrease significantly. It is further expected that as the concentration of citric acid is further increased, the rate of aromatic-cationic peptide disappearance from vials stored at 50°C will increase in proportion to the amount of buffered citric acid in the formulation.

I l l [0362] Although the present method has been described in relation to particular embodiments thereof, many other variations and modifications and other uses will become apparent to those skilled in the art. The present method therefore is not limited by the specific disclosure herein, but only by the claims.

Example 14: Exemplary Formulation for Lipid Emulsion and Micelle Compositions

[0363] The following tables provide formulations for lipid emulsion and micelle compositions of the present technology. The tables are merely representative of formulations and should not be construed as limiting in any way. In addition to the exemplified compositions and formulations, the following compositions and formulations may also be used in any of the methods disclosed herein (e.g. , to treat a disease or condition, or be used in any of the disclosed test models).

Table 12. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate

NaOH Ethyl isovalerate

MgCl ₂ Glycerol monooleate

PVP-12 Lecithin

Sodium octanoate Span-40

Sodium caprate One or more of:

MC 400 Coconut oil

Water Castor oil

Adsorption enhancer (for example lauroyl L- Mineral oil

carnitine) Olive oil

If castor oil, optionally octanoic acid

Table 13. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate

NaOH Ethyl isovalerate

MgCl ₂ Glycerol monooleate

PVP-12 Lecithin

Sodium octanoate Span-40

Sodium caprate One or more of:

MC 400 Coconut oil

Water Castor oil

Adsorption enhancer (for example lauroyl L- carnitine)

Table 14. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Castor oil

NaOH Glyceryl tributyrate

MgCl ₂ Ethyl isovalerate

PVP-12 Glycerol monooleate

Sodium octanoate Lecithin

Sodium dodecanoate Span-40

MC 400 Coconut oil

Water Octanol

Adsorption enhancer (for example lauroyl L- Geranol

carnitine) Table 15. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate

MgCl ₂ Ethyl isovalerate

PVP-12 Glycerol monooleate

Potassium octanoate Lecithin

Lithium octanoate Span-40

Arginine octanoate Castor oil

MC400 water

Sodium caprate

MC 400

Water

Adsorption enhancer (for example lauroyl L- carnitine)

Table 16. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate

NaOH Ethyl isovalerate

MgCl ₂ Glycerol monooleate

PVP-12 Lecithin

Sodium octanoate Span-40

MC 400 Castor oil

Water

Adsorption enhancer (for example lauroyl L- Optionally, one or more of octanoic acid, carnitine) ricinoleic acid, ethyl octanoate.

Table 17. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate

PVP-12 Ethyl isovalerate

Sodium octanoate Glycerol monooleate

Water Lecithin

Adsorption enhancer (for example lauroyl L- Span-40

carnitine) Castor oil

Optionally, one or more of octanoic acid, ricinoleic acid, ethyl octanoate.

Table 18. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate

PVP-12 Ethyl isovalerate

MgCl ₂ Glycerol monooleate

Sodium octanoate Lecithin

MC 400 Span-40

Water Castor oil

Adsorption enhancer (for example lauroyl L- carnitine) Optionally, one or more of octanoic acid, ricinoleic acid , ethyl octanoate.

Table 21. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate PVP-12 Ethyl isovalerate

Sodium octanoate Glycerol monooleate Water Lecithin

Adsorption enhancer (for example lauroyl L- Span-40

carnitine) Castor oil

Tween 80

Table 22. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tricaprylate PVP-12 Glycerol monocaprylate

Sodium octanoate Castor oil

Water Tween 80

Adsorption enhancer (for example lauroyl L- carnitine)

Table 23. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate PVP-12 Ethyl isovalerate

Mono-Sodium Subernate Glycerol monooleate di-Sodium Subernate Lecithin

Water Span-40

Adsorption enhancer (for example lauroyl L- Castor oil

carnitine)

Table 24. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tricaprylate

NaOH Glycerol monocaprylate

PVP-12 Tween 80

Sodium geranate Castor oil

Water

Adsorption enhancer (for example lauroyl L- carnitine) Table 25. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate

Mannitol Ethyl isovalerate

MgCl ₂ Glycerol monooleate

Sodium octanoate Lecithin

MC 400 Span-40

Water Castor oil

Adsorption enhancer (for example lauroyl L- carnitine)

Table 26. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate Sodium octanoate Ethyl isovalerate PVP- 12/17/25 Glycerol monooleate Water Lecithin

Adsorption enhancer (for example lauroyl L- Span-40

carnitine) Castor oil

Table 27. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate Sodium octanoate Ethyl isovalerate PVP- 12 Glycerol monooleate Water Lecithin

Adsorption enhancer (for example lauroyl L- Span-40

carnitine) Castor oil

Table 28. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tricaprylate

Sodium octanoate Glycerol monocaprylate

PVP- 12 Tween 80

Dextran Castor oil

Water

Adsorption enhancer (for example lauroyl L- carnitine)

Table 29. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate

PVP- 12 Ethyl isovalerate

Sodium octanoate Glycerol monooleate

Sodium nonanoate Lecithin

Sodium decanoate Span-40

Water Castor oil

Adsorption enhancer (for example lauroyl L- carnitine) Table 30. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate

PVP-12 Ethyl isovalerate

NaOH Glycerol monooleate

Sodium octanoate Lecithin

Water Span-40

Adsorption enhancer (for example lauroyl L- Castor oil

carnitine) Tween 80

Glyceryl tricaprylate Glycerol monocaprylate

Table 31. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate PVP-12 Ethyl isovalerate

Sodium octanoate Glycerol monooleate Water Lecithin

Adsorption enhancer (for example lauroyl L- Span-40

carnitine) Castor oil

Tween 80

Glyceryl tricaprylate Glycerol monocaprylate

Table 32. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Castor oil

PVP-12 Tween 80

NaOH Glyceryl tricaprylate

Sodium octanoate Glycerol monocaprylate Water

Adsorption enhancer (for example lauroyl L- carnitine)

Table 34. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate

MgCl ₂ Ethyl isovalerate

PVP-12 Glycerol monooleate

Sodium octanoate Lecithin

MC 400 Span-40

Water Castor oil

Adsorption enhancer (for example lauroyl L- Tween 80

carnitine) Glyceryl tricaprylate

Glycerol monocaprylate

Table 35. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tricaprylate

PVP-12 Glycerol monocaprylate

PVA Tween 80

Glucose

Carbopol 934P

NaOH

Sodium octanoate

Water

Adsorption enhancer (for example lauroyl L- carnitine)

Table 36. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Tween 80

PVP-12 Glyceryl tricaprylate

NaOH Glycerol monocaprylate

Sodium octanoate

Sodium hyaluronate

Sodium alginate

Glucosamine

Polyacrilyc acid

Water

Adsorption enhancer (for example lauroyl L- carnitine)

Table 37. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tricaprylate

Carbopol 934P Glycerol monocaprylate

NaOH Tween 80

Sodium octanoate

Water

Adsorption enhancer (for example lauroyl L- carnitine) Table 38. Illustrative formulations

Hydrophilic Fraction Hydrophobic Medium

Aromatic-Cationic peptide Glyceryl tributyrate

PVP-12 Ethyl isovalerate

PVA Glycerol monooleate

Carbopol 934P Lecithin

NaOH Span-40

Sodium octanoate Castor oil

Water Tween 80

Adsorption enhancer (for example lauroyl L- Glyceryl tricaprylate

carnitine) Glycerol monocaprylate

Lutrol F-68

Octanoic acid

Example 15 : The use of Counter Anions in Compositions to Translocate Aromatic-cationic peptides Across an Epithelial Barrier

[0364] The composition is prepared by the lyophilization of (1) an aromatic-cationic peptide, e.g., D-Arg-2'6'-Dmt-Lys-Phe-NH2, (2) a counter anion, such as sodium dodecyl sulfate (SDS) or dioctyl sulfosuccinate (DSS),and (3) an absorption enhancer, for example lauroyl L-carnitine. Additional components of possible formulations are exemplified in Tables 41-46. Table 41. Additional constituents of the penetration compositions

N-methyl pirolidone (NMP)

Cremophor EL

Tricaprine

Pluronic F-68

Aprotinin

Solutol HS-15 (SHS)

N-acetyl cysteine (NAC)

Table 42. Additional constituents of the penetration compositions

N-methyl pirolidone (NMP)

Cremophor EL

Tricaprine

Pluronic F-68

Aprotinin

Solutol HS-15 (SHS)

N-acetyl cysteine (NAC)

Table 43. Additional constituents of the penetration compositions

NaOH

Acetic Acid

Sodium acetate

L-arginine

Pluronic F-68

Aprotinin

Solutol HS-15 (SHS)

N-acetyl cysteine (NAC)

Table 44. Additional constituents of the penetration compositions

Phytic acid

NaOH

Acetic Acid

Sodium acetate

L-arginine

Tricaprine

Pluronic F-68

Aprotinin

Solutol HS-15 (SHS)

N-acetyl cysteine (NAC)

Ethanol Table 45. Additional constituents of the penetration compositions

Acetate buffer

Arginine

Tricaprine

Pluronic F-68

Aprotinin

Solutol HS-15 (SHS)

N-acetyl cysteine (NAC)

Benzyl benzoate:Butanol 1 : 1

Table 46. Additional constituents of the penetration compositions

Tricaprine

Pluronic F-68

Aprotinin

Solutol HS-15 (SHS)

N-acetyl cysteine (NAC)

Benzyl benzoate:Butanol 1 : 1

[0365] The composition is then administered to test animals, e.g. , mice, in two forms: rectally or by injection into an intestinal loop. The experimental procedure involves male BALB/c mice, which are deprived of food, 18 hours prior to the experiment. For intra- intestinal injection the mice are then anesthetized and a 2 cm long incision is made along the center of the abdomen, through the skin and abdominal wall. An intestine loop is gently pulled out through the incision and placed on wet gauze beside the animal. The loop remains intact through the entire procedure and is kept wet during the whole time. The tested compound is injected into the loop, using a 26 G needle. For rectal administration the mice are anesthetized and the penetration composition is then rectally administered to the mice, 100 μΐ/mouse, using a plastic tip covered with a lubricant.

[0366] Penetration is assessed by direct measurement of aromatic-cationic peptide concentrations in the blood.

[0367] It is anticipated that peptide levels will be detected in both groups of mice administered the peptide composition, indicating D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ absorption from the intestine into the blood stream. In contrast, it is anticipated that control subjects will show no peptide in the blood stream.

[0368] Thus, this drug delivery system provides an efficient, safe and convenient route of administration. Example 16: Utilization of Compositions Disclosed herein to Translocate D-Arg-2'6'-Dmt-

Lys-Phe-NH? Across an Epithelial Barrier

[0369] A number of compositions disclosed herein are tested for their ability to cross an epithelial barrier. A peptide formulation will be administered to rats and/or pigs im, rectally or nasally. Blood samples are taken at various times after administration, and peptide levels determined by methods known in the art.

[0370] Formulation 1 : D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ will be dissolved with spermine, an absorption enhancer, for example lauroyl L-carnitine, and phytic acid in double distilled water ("DDW") containing NaOH. The solution is then lyophilized and suspended with sodium dodecanoate (SD), octanol and geraniol in a mixture of mineral oil, medium chain triglyceride (MCT) oil and castor oil.

[0371] Eight male SD rats, 175-200 g, are deprived of food, 18 hours prior to the experiment. The animals are divided into 2 groups, and anesthetized by a solution of 85% ketamine, 15% xylazine, 0.1 ml/100 g of body weight. Each preparation is administered either i.m. (100 ul/rat, containing 1.11 IU D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ) or rectally (100 μΐ/rat, containing 2.8 IU D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ). Rectal administration is done by gently inserting through the rectal orifice a plastic canule protected by a soft coating, to a depth of 2 cm. Blood samples are measured at various time intervals post administration, drawn from the tip of the tail.

[0372] It is anticipated that after the composition is administered rectally, the peptide will be found in the blood, indicating D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ absorption from the intestine into the blood stream.

[0373] Formulation 2: D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ will be dissolved with spermine, an absorption enhancer, for example lauroyl L-carnitine, and phytic acid in DDW containing NaOH. The solution will be lyophilized and suspended with sodium dodecanoate (SD), octanol and geraniol in a mixture of mineral oil, medium chain triglyceride (MCT) oil and castor oil.

[0374] Eight male SD rats, 175-200 g, are deprived of food, 18 hours prior to the experiment. The animals are divided into 2 groups, and anesthetized by a solution of 85% ketamine, 15% xylazine, 0.1 ml/100 g of body weight. Each preparation is administered either i.m. (100 ul/rat, containing 1.11 IU D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ) or rectally (100 ul/rat, containing 2.8 IU D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ). Rectal administration is done by gently inserting through the rectal orifice a plastic canule protected by a soft coating, to a depth of 2 cm. Blood levels of peptide are measured at various time intervals post administration, in samples drawn from the tip of the tail.

[0375] It is anticipated that after the composition is administered rectally, the peptide will be found in the blood, indicating D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ absorption from the intestine into the blood stream.

[0376] Formulation 3 : D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ is dissolved with spermine, an absorption enhancer, for example lauroyl L-carnitine, polyvinylpyrrolidone (PVP-40), sodium dodecanoate (SD) and methylcellulose (MC-400) in DDW containing NaOH. The solution is then lyophilized and suspended with octanol and geraniol in a mixture of medium chain triglyceride (MCT) oil and castor oil, further containing sorbitan monopalmitate (Span- 40).

[0377] Six female mini-pigs, 45-50 kg, are deprived of food, 18 hours prior to the experiment. The animals are divided into 2 groups, and anesthetized by a solution of 66% ketamine, 33% xylazine, 0.3 ml/kg of body weight. The superior vena cava is canulated transdermally to facilitate blood collection. Each preparation is administered either i.m. (0.22 IU/kg D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ) or rectally (1.1 IU/kg D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ). Rectal administration is done by gently inserting through the rectal orifice a plastic syringe, to a depth of 2 cm. Blood levels of the peptide will be measured at various time intervals post administration, to assess the levels in the serum.

[0378] It is anticipated that after the composition is administered rectally, serum D-Arg- 2'6'-Dmt-Lys-Phe-NH ₂ levels will be detected, indicating D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ absorption from the intestine into the blood stream.

[0379] Formulation 4: D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ is combined with spermine, an absorption enhancer, for example lauroyl L-carnitine, polyvinylpyrrolidone (PVP-40), and sodium dodecanoate (SD) in DDW containing NaOH, octanol and geraniol. The solution is then lyophilized and suspended with an additional amount of octanol and geraniol in a mixture of medium chain triglyceride (MCT) oil and castor oil further containing sorbitan monopalmitate (Span-40), methylcellulose (MC-400), and glyceryl monooleate (GMO).

[0380] Eight male SD rats, 175-200 g, are deprived of food, 18 hours prior to the experiment. The animals are divided into 2 groups, and anesthetized by a solution of 85 % ketamine, 15% xylazine, 0.1 ml/100 g of body weight. Each preparation is administered either i.m. (100 ul rat, containing 1.11 IU D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ) or rectally (100 υΐ/rat, containing 2.8 IU D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ). Rectal administration is done by gently inserting through the rectal orifice a plastic canule protected by a soft coating, to a depth of 2 cm. Blood samples are measured at various time intervals post administration, drawn from the tip of the tail.

[0381] It is anticipated that after the composition is administered rectally, the peptide will be found in the blood, indicating D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ absorption from the intestine into the blood stream.

[0382] Formulation 5 : D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ is dissolved with spermine, an absorption enhancer, for example lauroyl L-carnitine, and sodium dodecanoate in DDW containing NaOH. The solution is then lyophilized and suspended with octanol and geraniol in a mixture of medium chain triglyceride (MCT) oil and castor oil further containing sorbitan monopalmitate (Span-40), methylcellulose (MC-400), glyceryl monooleate, and pluronic (F-127).

[0383] Five male CB6/F1 mice, 9-10 wks, are divided into 2 groups, and anesthetized by a solution of 85% ketamine, 15% xylazine, 0.01 ml/10 g of body weight. Each preparation is administered either i.p. (100 ul/mouse, containing 0.2 mg D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ) or rectally (100 ul/mouse, containing 1 mg D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ). Rectal administration is done by gently inserting through the rectal orifice a plastic canule protected by a soft coating, to a depth of 1 cm. Blood samples at various time intervals post administration are drawn from the tip of the tail into a glass capillary.

[0384] It is anticipated that D-Arg-2'6'-Dmt-Lys-Phe-NH2 is absorbed from the intestine into the bloodstream.

[0385] Formulation 6: D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ is dissolved with spermine, polyvinylpyrrolidone (PVP-40, an absorption enhancer, for example lauroyl L-carnitine), and sodium dodecanoate (SD) in DDW containing NaOH. The solution is then lyophilized and suspended with octanol and geraniol in a mixture of medium chain triglyceride (MCT) oil and castor oil further containing sorbitan monopalmitate (Span-40), methylcellulose (MC- 400), and glyceryl monooleate (GMO).

[0386] Six male SD rats, 175-200 gr arere divided into 2 groups, and anesthetized by a solution of 85% ketamine, 15% xylazine, 0.1 ml/100 g of body weight. The external jugular veins are then exposed by removing the overlaying skin. The compositions are administered either nasally (25 ul/rat, containing 2.5 meg D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ) or rectally (50 ul/rat, containing 5 meg D-Arg-2'6'-Dmt-Lys-Phe-NH2). Nasal administration is done by smearing of the composition over the external nasal orifices. Rectal administration is done by gently inserting through the rectal orifice a plastic canule protected by a soft coating, to a depth of 2 cm. Blood samples are drawn from the jugular veins at various time intervals post administration. Serum was analyzed for detection of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ .

[0387] It is anticipated that both nasal and rectal administration of D-Arg-2'6'-Dmt-Lys- Phe- H ₂ will result in significant levels of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ in the blood stream, indicating D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ absorption from the intestine into the blood stream.

[0388] As a comparison, rectal administration of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ dissolved in phosphate buffered saline are performed in parallel, utilizing equivalent amounts of D- Arg-2'6'-Dmt-Lys-Phe-NH ₂ per rat. It is anticipated that the results will show low or D-Arg- 2'6'-Dmt-Lys-Phe-NH ₂ in the blood stream, and therefore no detected absorption from the intestine in the absence of the formulation.

[0389] Formulation 7: D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ is dissolved with spermine, an absorption enhancer, for example lauroyl L-carnitine), polyvinylpyrrolidone (PVP-40), sodium dodecanoate, and methylcellulose (MC-400) in DDW containing NaOH. The solution is then lyophilized and suspended with octanol and geraniol in a mixture of medium chain triglyceride (MCT) oil and castor oil further containing sorbitan monopalmitate (Span- 40). The control composition is prepared as described above, without the D-Arg-2'6'-Dmt- Lys-Phe-NH ₂ .

[0390] Six male SD rats, 175-200 g, are deprived of food, 18 hours prior to the experiment. The animals are divided into 3 groups, and each animal was given 200 mg glucose from a 50% glucose solution in water, by oral lavage. Ten minutes afterwards, each preparation is administered either i.p. (50 ul/rat, containing 25 meg D-Arg-2'6'-Dmt-Lys-Phe-NH2) or rectally (200 ul/rat, containing 100 meg D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ). Rectal

administration is done by gently inserting through the rectal orifice a plastic canule protected by a soft coating, to a depth of 2 cm. Blood levels of the peptide are measured at various time intervals post administration, in blood samples drawn from the tip of the tail. [0391] It is anticipated that rectally administered D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ will attenuate the rise in blood glucose and that the peptide will be detected in the blood stream. Likewise, parenterally administration of D-Arg-2'6'-Dmt-Lys-Phe-NH2, is anticipated to indicate absorption from the intestine into the blood stream.

[0392] Formulation 8: An exemplary composition used for mucosal delivery will contain a desired D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ , an absorption enhancer, for example lauroyl L- carnitine), and protein stabilizers, e.g. , spermine and phytic acid, which can be dissolved and then lyophilized together, along with additional components such as polyvinylpyrrolidone and a surface active agent, e.g. , Na dodecanoate, and then suspended with membrane fluidizing agents, e.g., octanol and geraniol, in a hydrophobic medium, e.g., a mixture of MCT oil or glyceryl tributyrate and castor oil. Additional possible components of the composition have been described. Such a composition can be administered nasally or orally to a subject in need of vaccination.

[0393] This method allows simple, efficient and rapid administration of a drug to a large populations in need thereof.

[0394] Formulation 9: D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ and an absorption enhancer, for example lauroyl L-carnitine), are dissolved with CaCl ₂ , polyvinylpyrrolidone (PVP-12), sodium dodecanoate (SD), sodium octanoate (SO) and silicon dioxide in DDW containing NaOH. The solution is then lyophilized and suspended with solution C (phosphatidyl choline (PC), sorbitan monopalmitate (Span-40), octanol and geraniol, and ethyl isovalerate, glyceryl monooleate (GMO) in a mixture of glyceryl tributyrate and castor oil).

[0395] Three male SD rats, 175-200g, are deprived of food, 18 hours prior to the experiment. Animals were anesthetized by a solution of 85% ketamine, 15% xylazine, 0.1 ml/ 100 g of body weight. The external jugular veins are then exposed by removing the overlaying skin. Each animal is given 50 μΐ of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ composition rectally (50 μΐ/rat, containing 25 meg D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ). Rectal administration is done by gently inserting through the rectal orifice a plastic canule protected by a soft coating, to a depth of 2 cm. Blood samples are drawn from the jugular veins at various time intervals post administration. Plasma is analyzed for D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ . It is anticipated that D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ will be present in the plasma samples. Example 17: Composition for Mucosal Delivery Using a Counter Ion

[0396] A composition for oral delivery containing D-Arg-2'6'-Dmt-Lys-Phe-NH2 encapsulated with a counter ion, i.e., sodium dodecyl sulfate (SDS) or dioctyl sulfosuccinate (DSS), and a hydrophobic agent, i.e., tricaprin is formulated as follows.

[0397] To prepare the counter ion complex of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ dissolve equimolar concentrations of the aromatic-cationic peptide and hydrophobic anion in a mutually acceptable solvent in which both are highly soluble and stable (e.g. , ethanol, acetone, isopropanol, or n-butanol ethyl acetate). The mixture is titrated until a haze or solubility limit has been reached. A polymer is dispersed, such as HMPCAS, PVP, or one of the substituted celluloses, between 5-10% by weight, into the solution in order to act as a solid support for the evaporated mixture. To provide a solid formulation, a fluid bed granulator is used to spray the solution at 25-35 °C onto lactose, Avicel, or Sta-Rx bed at 40- 50°C in order to provide a solid granulation when dry.

[0398] The solid formulation can be further coated with protective polymers (e.g., HPMC) and subsequently overgranulated or coated with a trypsin inhibitor, e.g., citric acid (2 to 5 : 1 ratio with drug) or other required excipient, such as an absorption enhancer (e.g. , 3-0- Lauroyl-L-Carnitine) at a 1 : 1 ration with drug. The particulates can be subsequently individually spray coated with an enteric coating polymer (e.g., Eudragit LlOO D-55) or can be co-compressed or encapsulated into tablets or capsules which can themselves be spray coated with the same enteric polymer.

[0399] Additional possible constituents of the pharmaceutical composition are exemplified in Tables 41-46. Such a composition can be administered to a subject in need thereof.

Example 18: Trypsin Resistant Lipophilic Salts of Aromatic-Cationic Peptides

[0400] This example shows that lipophilic salts of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ are resistant to trypsin digestion.

Methods and Materials Formation of Lipophilic Salt Complexes

[0401] Lipophilic salts of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ were prepared from D-Arg-2'6'- Dmt-Lys-Phe-NH ₂ acetate. D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ acetate and the free acid of counter ions were dissolved in methanol at a ratio of 1 :3 and 1 :6 (peptide: counter ion) and stirred overnight. Methanol and acetic acid were removed by rotary evaporation and a solid powder was isolated. The solid powder was then re-dissolved in methanol and dried by rotary evaporation a second time to remove remaining free acetic acid. Table 47 shows the lipophilic salts of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ that were prepared.

[0402] When docusate was used as the counter ion, sodium docusate was dissolved in tetrahydrofuran (THF) and acidified with one molar equivalent of HC1 using 1.25 M HC1 in methanol. The solution forms a cloudy suspension of precipitated NaCl and dissolved dioctyl sulfosuccinic acid. The cloudy suspension was filtered with a 0.45 micron nylon filter, resulting in a clear solution of dioctyl sulfosuccinic acid in THF. The THF is removed by rotary evaporation and then further under vacuum overnight. The isolated dioctyl sulfosuccinic acid is then dissolved in MeOH with D-Arg-2'6'-Dmt-Lys-Phe-NH2 acetate as described above.

Aqueous media

[0403] Phosphate buffered saline (PBS) is comprised of 80 mM NaCl, 46.7 mM KH ₂ P0 ₄ and 20 mM Na ₂ HP0 ₄ adjusted to pH 6.5.

[0404] Fasted state simulated intestinal fluid (FaSSIF) is comprised of PBS with 0.5% w/v SIF powder (sodium taurocholate and phospholipid in a 4 to 1 molar ratio).

Characterization Methods

[0405] Aqueous solubility of lipophilic salts of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ was determined using the HPLC after stirring excess salt complex in water at room temperature overnight. Sample solutions were filtered through a 0.45 μιτι nylon syringe filter. [0406] Octanol solubility for lipophilic salts of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ complexes was determined using the undissolved material remaining in the flask from the aqueous solubility measurement, which was dried overnight. 1 -Octanol was added until the salt complex was visually dissolved. 50 of the octanol solution was diluted with 450 of methanol in an HPLC vial prior to analysis by HPLC.

[0407] Octanol/Water partitioning (LogP or Kow) was determined using the shake flask method with 5 mL each of 1 -octanol and water in a 20 mL scintillation vial shaken overnight, the layers allowed to settle/separate for at least 2 hours, and then each layer analyzed individually using the HPLC method. The octanol layer was diluted as described above. Partitioning was initially determined using the aqueous solubility solutions, which generally had low concentrations and did not partition in the octanol layer as expected, which was confirmed the octanol solubility results. The caprate and laurate salts were retested by dissolving them in octanol and then adding water. The subsequent Kow determinations were made using the octanol solubility samples diluted to 5 mL before adding the 5 mL of water.

[0408] Dissolution and trypsin digestion kinetics of lipophilic salts of D-Arg-2'6'-Dmt- Lys-Phe-NH ₂ was investigated in FaSSIF as follows:

Prepared 40 mL FaSSIF and stored at 37°C while mixing for at least 1 hour

Transferred 25 mL FaSSIF to a separate container and dissolved 11.65 mg of trypsin (final trypsin concentration = 20 μΜ)

- Weighed lipophilic salts of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ at about 1.3 mM (in 10 mL) into three 20 mL vials

Pipetted 10 mL of FaSSIF without trypsin into one vial for a control

Pipette 10 mL of the 20 μΜ trypsin solution into the other two vials (i.e., Rep A and

Rep B)

Stored the vials at 37°C while stirring and sample 200 μΐ, at 0, 15, 30, 60, and 120 minutes into an HPLC vial with 200 μί of 1 % (v/v) formic acid in MeOH (For type I vehicles, THF was used instead of MeOH)

[0409] Trypsin digestion kinetics of lipophilic salts of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ pre- dissolved in micelle solutions was investigated as follows:

Prepared 50 mL micelle solution and stored at 37 °C while mixing for at least 1 hour Weighed D-Arg-2'6'-Dmt-Lys-Phe-NH2 at about 0.9 mgA/mL into the micelle solution and stored at 37 °C while mixing for at least 1 hour or until completely dissolved (API Solution)

Weighed 7.0 mg of trypsin into three 20 mL vials

In duplicate, sample 200 into an HPLC vial with 200 of 1% (v/v) formic acid in MeOH

Pipette 15 mL of the API solution into each of the 20 mL scintillation vials and stir at 37°C

At 5, 10, 15, and 20 minutes, transfer 1 mL to a 2 mL centrifuge tube and centrifuge at 12.8 Kg/min for 1 minute

Sample 200 of the supernatant into an HPLC vial with 200 μί of 1% (v/v) Formic acid in MeOH

Results

Characterization of Lipophilic Salts of D-Arg-2 '6' -Dmt-Lvs-Phe-NH?

[0410] Lipophilic salts of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ were tested for water solubility, octanol solubility, and octanol/water partitioning. These assays determined which counter ions resulted in complexes that were the most lipophilic, as indicated by low water solubility, high octanol solubility, and a high octanol/water partition coefficient. Lipophilic salt complexes will partition more preferentially into the oil phase of lipid formulations in the intestine, providing increased protection from trypsin digestion.

[0411] The octanol/water partition coefficient is defined as:

K _oW = [D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ Lipophilic Salt] _oci , _tota i

[D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ Lipophilic Salt]^, total wherein [D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ Lipophilic Salt] _0f¾tota i and [D-Arg-2' 6' -Dmt-Lys- Phe-NH ₂ Lipophilic Salt] _fl¾j total are the concentrations of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ in the octanol and aqueous phases, respectively, at the end of the shake flask partition coefficient measurement. The total drug concentration in each phase is given by:

[D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ] _{, total} =

[D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ ^K+ ] + [D-Arg-2 '6 '-Dmt-Lys-Phe-NH ₂ ^H+ Cr] +

[D-Arg-2 ' 6 '-Dmt-Lys-Phe-NH ₂ ^B+ CI ₂ ] + [D-Arg-2 ' 6 ' -Dmt-Lys-Phe-NH ₂ ³⁺ CI ₃ ^~ ] ; wherein x = oct or aq, CI = counter ion, and n = 1, 2, or 3 depending on the protonation state, predominantly n = 3 at physiological pH.

[0412] The effective logP represents some combination of the association constant between D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ and the counter ion and the logP of the resulting complexes at the concentration tested.

[0413] The acetate salt of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ is highly water soluble (205 mg/mL) while the free base form of D-Arg-2'6'-Dmt-Lys-Phe-NH2 has a solubility of 0.13 mg/mL. The free base form was prepared using a catch and release purification method with a polymer supported sulfonic acid ion exchange column. The resulting material was a clear oily liquid that was dissolved in water and measured by HPLC. The octanol solubility of the acetate salt is below LOD (LOD = Limit of Detection, estimated to be 10 ^"4 mg/mL). The partition coefficient of the free base is K^ _w = 0.01 (LogP = -2.2).

[0414] The partition coefficient of many of the lipophilic salt complexes is higher than the free base or acetate salt. The solubility and partition coefficients are summarized in Table 48.

[0415] Lipophilic salt D-Arg-2'6'-Dmt-Lys-Phe-NH2 complexes with docusate, SLS, oleate, laurate, stearate, and caprate had a significant effect on the partition coefficient (> lOOOx increase in Kow).

Stearate 1 :6 0.48 4.20 1.9

Oleate 1 :3 0.25 6.66 2.2

Oleate 1 :6 0.07 9.12 3.4

Trypsin Digestion

[0416] Lipophilic salts of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ were tested for dissolution rate and trypsin degradation in FaSSIF to identify the protection of D-Arg-2'6'-Dmt-Lys-Phe- NH ₂ from trypsin by decreasing the aqueous solubility (and increasing the solubility in bile salt micelles present in intestinal media).

[0417] D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ acetate dissolved rapidly in FaSSIF, but was simultaneously degraded rapidly in the presence of trypsin. FIG. 1. Greater than 90% of the D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ was dissolved at the first sampling time (< 2 minutes) in the absence of trypsin (FIG. 1, triangles). However, in the presence of trypsin, no D-Arg-2'6'- Dmt-Lys-Phe-NH ₂ is detected by the first sampling time (FIG. I , squares).

[0418] The D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ docusate complexes dissolved more slowly and less completely than the D-Arg-2'6'-Dmt-Lys-Phe-NH2 acetate salt, which indicates reduced solubility. The D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ docusate complexes were also digested much more slowly by trypsin (FIGs. 2A-B). The trypsin digestion was significantly reduced for the 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ docusate complex relative to the D-Arg-2'6'-Dmt-Lys- Phe- H ₂ acetate salt, while it is effectively eliminated in the case of the 1 :6 D-Arg-2'6'-Dmt- Lys-Phe-NH ₂ docusate complex.

[0419] Other lipophilic counter ions that reduced trypsin digestion of D-Arg-2'6'-Dmt-Lys- Phe-NH ₂ when compared to D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ acetate include, e.g. , napsylate (at 1 :3 and 1 :6), oleate (at 1 :3), and stearate (at 1 :3) (see FIGs. 3A, 3B, 4A, and 4B).

[0420] These results show that D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ complexed with lipophilic counter ions have greater resistance to trypsin digestion as compared to D-Arg-2'6'-Dmt- Lys-Phe-NH ₂ complexed with acetate. Accordingly, administration of aromatic-cationic peptides complexed with lipophilic counter ions can increase bioavailabilty of the aromatic- cationic peptides. As such, these results show that the compositions of the present technology are useful in methods for increasing the bioavailabilty of aromatic-cationic peptides. Example 19: Trypsin Resistant Lipid Formulations of Lipophilic Salts of Aromatic-Cationic Peptides

[0421] This example shows that lipophilic salts of D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ encapsulated in a lipid particle are resistant to trypsin digestion.

Methods and Materials

Preparation of Lipid Formulations

[0422] D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ docusate complexes were formulated into three different lipid particle formulations to assess the trypsin resistance of the lipid particles. The lipid formulations were comprised of long chain triglycerides, monoglycerides, and a non- ionic surfactant (see Table 49). The Type I lipid vehicle contains no surfactant and is not self-emulsifying. The Type II and III lipid vehicles contain surfactant and are self- emulsifying drug delivery systems (SEDDS).

[0423] D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ docusate complexes were dissolved in Type I, II, or III lipid vehicles at 15 or 30 mgA (mg Active/mL)(FIG. 5A). The saturated solubility of the 1 :3 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ docusate complex in the Type III lipid vehicle was 30 mgA/mL.

[0424] In the Type III lipid vehicle, the solubility of the 1 :3 and 1 :6 D-Arg-2'6'-Dmt-Lys- Phe-NH ₂ docusate complexes is 29 and 19 mg/mL, respectively. As a comparison, the solubility of the 1 :3 and 1 :6 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ SLS complexes was 9 and 5 mg/mL, respectively and in the Type II lipid vehicle, the solubility of the D-Arg-2'6'-Dmt- Lys-Phe-NEL acetate salt is at least 15 mg/mL.

Trypsin Digestion Lipid Formulations

[0425] Lipid formulations were investigated for protection from trypsin degradation. This assay was performed similarly to the test described above for the D-Arg-2'6'-Dmt-Lys-Phe- NH ₂ /lipophilic salt powders, but dosing was based on the lipid formulation. The test was either run by adding 10 mL FaSSIF (with or without trypsin) directly to the lipid formulation.

[0426] By way of example, for a 15 mgA/g lipid formulation, 10 mL FaSSIF (with or without trypsin) would be added to 0.5 g of lipid formulation to achieve a final concentration of 0.71 mgA/mL in the test.

Results

[0427] Type I and Type II lipid formulations of 1 :6 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ docusate complex led to higher in vitro area under the curve (AUC) in the presence of trypsin as compared to non- lipid formulation 1 :6 D-Arg-2 ' 6 '-Dmt-Lys-Phe-NH ₂ docusate complex (FIGs. 2B and 5A).

[0428] Three different salt complexes were tested for trypsin degradation using the Type II lipid vehicle. D-Arg-2 '6 '-Dmt-Lys-Phe-NH ₂ triacetate complex and the 1 :3 and 1 :6 D-Arg- 2'6'-Dmt-Lys-Phe-NH ₂ docusate complexes were tested for trypsin digestion (FIG. 5B). The total AUC in the presence of trypsin was higher in the case of the 1 :6 D-Arg-2 '6 '-Dmt-Lys- Phe-NH ₂ docusate complex as compared to 1 :3 D-Arg-2 '6 '-Dmt-Lys-Phe-NH ₂ docusate complex or for the D-Arg-2' 6' -Dmt-Lys-Phe-NH ₂ triacetate salt complex (FIG. 5B).

[0429] These results show that D-Arg-2' 6'-Dmt-Lys-Phe-NH ₂ complexed with lipophilic counter ions and formulated in a lipid formulation have a greater resistance to trypsin digestion as compared to D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ complexed with acetate in a lipid formulation. Accordingly, administration of aromatic-cationic peptides complexed with lipophilic counter ions in a lipid formulation can increase bioavailabilty of the aromatic- cationic peptides. As such, these results show that the compositions of the present technology are useful in methods for increasing the bioavailabilty of aromatic-cationic peptides.

Example 20: Stability of Lipid Formulations of Lipophilic Salts of Aromatic-Cationic Peptides

[0430] This example shows that lipid formulations of lipophilic salts of D-Arg-2 '6 '-Dmt- Lys-Phe-NH ₂ are more stable than lipid formulations of non- lipophilic salts of D-Arg-2 '6'- Dmt-Lys-Phe-NH ₂ . [0431] Stability of 1 :6 D-Arg-2' 6 '-Dmt-Lys-Phe-NH ₂ docusate complex and D-Arg-2 '6'- Dmt-Lys-Phe-NH2 triacetate complex in Type II lipid formulations was assessed at 5 days and 11 days after storage at 25°C .

[0432] The preparation of each lipid formulation was subdivided into test vials such that there were three vials for each time point ( . e. , 3 test vials for day 5 and 3 test vials for day 11) for the potency assay. The potency was determined in the absence of trypsin.

[0433] The potency and exposure in the absence of trypsin showed that at 25°C for 5 days and 11 days the 1 :6 D-Arg-2' 6' -Dmt-Lys-Phe-NH ₂ docusate complex Type II lipid formulation showed greater potency than the D-Arg-2' 6 '-Dmt-Lys-Phe-NH ₂ triacetate complex in Type II lipid formulation (Table 50).

[0434] The initial tests were performed with a 2 hour pre-emulsification while the remaining samples were tested with a 1 hour pre-emulsification.

[0435] The trypsin digestion of 1 :6 D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ docusate complex and D-Arg-2'6'-Dmt-Lys-Phe-NH2 triacetate complex, both in Type II lipid formulations stored at 25°C, shows that 1 :6 D-Arg-2 '6 '-Dmt-Lys-Phe-NH ₂ docusate complex Type II lipid formulations have a greater stability as compared to D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ triacetate complex in Type II lipid formulation (FIGs. 6A and 6B).

[0436] These results show that D-Arg-2' 6'-Dmt-Lys-Phe-NH ₂ when complexed with a lipophilic counter ion and encapsulated by lipids has a greater stability than lipid

encapsulated D-Arg-2'6'-Dmt-Lys-Phe-NH ₂ that is not complexed to a lipophilic counter ion. Accordingly, administration of aromatic-cationic peptides complexed with lipophilic counter ions in a lipid formulation can increase bioavailabilty of the aromatic-cationic peptides. As such, these results show that the compositions of the present technology are useful in methods for increasing the bioavailabilty of aromatic-cationic peptides. EQUIVALENTS

[0437] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present technology is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.