LIPOSOMAL FORMULATIONS OF CATIONIC PEPTIDES AND USES

THEREOF

FIELD OF THE INVENTION

[001] The present invention pertains to the field of drug delivery and, in particular, to liposomal formulations of cationic peptide drugs.

BACKGROUND OF THE INVENTION

[002] Liposomes have been widely studied as drug delivery vehicles. Liposomal formulations of drugs that have been approved for use in the U.S., Europe and Japan include liposomal formulations of the chemotherapeutic, doxorubicin (for example, Doxil®, Caelyx® and Myocet®), liposomal formulations of the antifungal drug amphotericin B (for example, AmBisome®), liposomal vaccines against hepatitis A (Epaxal®) and influenza (Inflexal® V) and liposomal formulations of benzoporphyrin (Visudyne™).

[003] Liposomes are vesicular structures that consist of one or more concentric lipid bilayers that enclose an internal aqueous volume and are unique in their ability to accommodate a variety of therapeutic or diagnostic compounds, having significantly different physicochemical properties. Such compounds can localise to several locations in a liposome including the hydrophobic hydrocarbon core of the lipid bilayer(s), the polar surface of the liposome (which may be neutral or charged) and the internal aqueous space.

[004] The constituent lipids of the liposome can be selected to facilitate interaction with the drug cargo and/or with the target cell, making these vehicles extremely versatile drug delivery systems. For example, for gene delivery applications, a combination of neutral and cationic lipids is often employed to accommodate the polyanionic nature of DNA (see, for example, Balazs and Godbey, 201 1 , J. Drug Delivery, Vol. 201 1 ). Methods of stabilising low molecular weight diagnostic and therapeutic compounds through the use of cationic liposomes have been described (U.S. Patent Application No. 12/654,523; Publication No. 2010/0178243). [005] Anionic liposomes have also been used for delivery of therapeutic compounds. Incorporation of negatively charged lipids into liposomes carrying amphotericin B has been shown to stabilise the association of this drug with the liposomal membrane (see, Maurer, et al, 2001 , Expert Opin. Biol. Ther. , 1 (5): 1 -25). The use of anionic liposomes loaded with trehalose was shown to greatly increase the delivery of trehalose to the cytosol of red blood cells (Holovati, et al, 2008, Cell Preservation Technology, 6:207-218). The use of anionic liposomes has also been shown to improve loading efficiency of the chemo therapeutic mitoxantrone onto the liposomes as compared to the use of neutral liposomes, and to reduce the dose-limiting toxicity of this drug when administered intraperitoneally in a peritoneal carcinomatosis model (Chang, et al , 2010, Nanomedicine, 6(6):769-776).

[006] Anionic liposomes have also been proposed as a means to treat overdose by sequestering cationic drugs, such as the tricyclic antidepressants amitriptyline and nortriptyline (Howell and Chauhan, 2008, J. Colloid Interface Scl , 319:81-93; Howell and Chauhan, 2008, J. Colloid Interface Sci. , 324:61 -70), and the local anaesthetic bupivacaine (Howell and Chauhan, 2009, Anesth Analg. , 109:678-682), from the plasma.

[007] The entrapment of peptide drugs in another type of delivery vehicle - surfactant-based niosomes - has been shown to be enhanced by modifying the vesicular charge compositions, with the highest entrapment efficiencies for bovine serum albumin (BSA), bacitracin and insulin being achieved with neutral, anionic and cationic niosomes, respectively (Manosroi, et al, 2010, J. Microencapsulation, 27(3):272-280).

[008] Peptides are attracting increasing attention as drug candidates. Over 50 therapeutic peptides have received approval from at least one regulatory agency for various indications from HIV therapy to antimicrobials, and many more are in clinical or preclinical development (see Development Trends for Peptide Therapeutics: 2010 Report Summary, " Peptide Therapeutics Foundation, San Diego, CA). Insulin, vancomycin, oxytocin, cyclosporine, Fuzeon® (enfuvirtide) and Integrilin® (eptifibatide) are a few examples of approved peptide-based drugs. Peptide therapeutics have a number of advantages including their small size, specificity and lower side-effects than traditional small molecule drugs. Candidate peptide therapeutics can be quickly investigated for therapeutic potential. Peptide therapeutics, however, can have high manufacturing costs, short half-life, and limited in vivo bioavailability.

[009] International Patent Application Nos. PCT/CA06/00521 (Publication No. WO2006/108270) and PCT/CA06/01298 (Publication No. WO2007/016777) describe peptide- based inhibitors of protein kinases that are useful as therapeutics, for example, in the treatment of cancer.

[010] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY OF THE INVENTION

[011] An object of the present invention is to provide liposomal formulations of cationic peptides and uses thereof.

[012] In accordance with an aspect of the present invention, there is provided a liposomal formulation comprising a cationic peptide having a net positive charge at neutral pH associated with anionic liposomes comprising one or more anionic lipids and one or more neutral lipids and having a net negative charge at neutral pH, in which said one or more anionic lipids and said cationic peptide are present in amounts sufficient to provide a lipid:peptide charge ratio of greater than 1.0.

[013] In accordance with another aspect of the present invention, there is provided A method of preparing a liposomal formulation comprising a cationic peptide, said method comprising the steps of:

(a) providing a solution of a cationic peptide; (b) providing a mixture of one or more neutral lipids and one or more anionic lipids in which the anionic lipids comprise about 1 mol % and about 70 mol % of the mixture, and

(c) preparing liposomes associated with the cationic peptide from the solution of the cationic peptide and a sufficient amount of the mixture to provide an anionic lipid:peptide charge ratio between about 1.1 and about 30.0.

BRIEF DESCRIPTION OF THE DRAWINGS

[014] These and other features of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings.

[015] Figure 1 presents a schematic representation of the method of preparing liposomal formulations comprising egg phosphatidylcholine (EPC) and egg phosphatidylglycerol (EPG) in accordance with one embodiment of the invention.

[016] Figure 2 presents the results of assessment by size-exclusion chromatography of the association of a representative peptidic compound with liposomes in formulations containing (A) egg phosphatidylcholine (EPC) alone; (B) EPC:egg phosphatidylglycerol (EPG) at a ratio of 4:6; (C) EPC:EPG at a ratio of 6:4; (D) EPC:EPG at a ratio of 7:3; (E) EPC:EPG at a ratio of 8:2; (F) EPC:EPG at a ratio of 9: 1 .

[017] Figure 3 presents the results of assessment by size-exclusion chromatography of the association of a representative peptidic compound with liposomes in formulations containing EPC:EPG at a ratio of 7:3 in (A) water, and (B) 300mM sucrose-lOmM HEPES, pH7.0.

[018] Figure 4 presents the results of assessment by size-exclusion chromatography of the association of a representative peptidic compound with liposomes in formulations containing EPC:EPG at a ratio of 7:3 in differing amounts of total lipids (A) total lipid mass 29.4 mg (charge ratio 13.0: 1), (B) total lipid mass 23.5 mg (charge ratio 10.4: 1 ), (C) total lipid mass 17.6 mg (charge ratio 7.8: 1), (D) total lipid mass 1 1.8 mg (charge ratio 5.2: 1 ), and (E) total lipid mass 5.9 mg (charge ratio 2.6: 1 ). [019] Figure 5 presents the results of assessment by size-exclusion chromatography of the association of a representative peptidic compound with liposomes in formulations containing EPC:EPG at a ratio of 8:2 in differing amounts of total lipids (A) total lipid mass 29.4 mg (charge ratio 8.8: 1), (B) total lipid mass 23.5 mg (charge ratio 7.0:1), (C) total lipid mass 17.6 mg (charge ratio 5.3 : 1), (D) total lipid mass 1 1.8 mg (charge ratio 3.5:1), and (E) total lipid mass 5.9 mg (charge ratio 1.8 : 1).

[020] Figure 6 presents the results of assessment by size-exclusion chromatography of the association of a representative peptidic compound with liposomes in formulations containing EPC:EPG at a ratio of 9: 1 in differing amounts of total lipids (A) total lipid mass 29.4 mg (charge ratio 4.4:1 ), (B) total lipid mass 23.5 mg (charge ratio 3.5:1), (C) total lipid mass 17.6 mg (charge ratio 2.6:1), (D) total lipid mass 1 1.8 mg (charge ratio 1.8:1), and (E) total lipid mass 5.9 mg (charge ratio 0.9: 1 ).

[021] Figure 7 presents the results of assessment by size-exclusion chromatography of the association of a representative peptidic compound with liposomes in formulations containing EPC:EPG at a ratio of 7:3 and a charge ratio of lipids:peptide of 2.6 containing increasing amounts of total lipids and peptide, (A) lx concentration, (B) 2x concentration, (C) 5x concentration, (D) l Ox concentration, and (E) 20x concentration.

[022] Figure 8 presents the results of assessment by size-exclusion chromatography of the association of a representative peptidic compound with liposomes in formulations containing (A) dimyristoylphosphatidylcholine (DMPC):dimyristoylphosphatidylglcerol (DMPG) (7:3 mole ratio), and (B) EPC:EPG: l ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (PEG ₂₀ ooDSPE) (66.5:28.5 :5 mole ratio).

[023] Figure 9 presents a graph showing the effect of EPC:EPG (7:3) liposomal formulations of peptidic compound 1 [SEQ ID NO: l] on proliferation of A2780cp ovarian cancer cells in vitro.

[024] Figure 10 presents a graph showing the effect of EPC:EPG (7:3) liposomal formulations of peptidic compound 1 [SEQ ID NO:l ] on proliferation of OCC-1 ovarian cancer cells in vitro. [025] Figure 11 presents a graph showing the effect of EPC:EPG (7:3) liposomal formulations of peptidic compound 1 [SEQ ID NO: l ] on proliferation of TCCSUP bladder cancer cells in vitro.

[026] Figure 12 presents a graph showing the effect of EPC:EPG (7:3) liposomal formulations of peptidic compound 1 [SEQ ID NO:l] on proliferation of T24 bladder cancer cells in vitro.

[027] Figure 13 presents a graph showing the effect of EPC:EPG (7:3) liposomal formulations of peptidic compound 1 [SEQ ID NO:l ] on proliferation of T4 bladder cancer cells in vitro.

[028] Figure 14 presents a graph showing the effect of EPC:EPG (9: 1) liposomal formulations of peptidic compound 1 [SEQ ID NO: l] on proliferation of A2780cp ovarian cancer cells in vitro.

[029] Figure 15 presents a graph showing the effect of EPC:EPG (9: 1) liposomal formulations of peptidic compound 1 [SEQ ID NO:l] on proliferation of OCC-1 ovarian cancer cells in vitro.

[030] Figure 16 presents a graph showing the effect of EPC:EPG (9: 1) liposomal formulations of peptidic compound 1 [SEQ ID NO: l] on proliferation of TCCSUP bladder cancer cells in vitro.

[031] Figure 17 presents a graph showing the effect of EPC:EPG (9: 1) liposomal formulations of peptidic compound 1 [SEQ ID NO: l ] on proliferation of T24 bladder cancer cells in vitro.

[032] Figure 18 presents a graph showing the effect of EPC:EPG (9: 1 ) liposomal formulations of peptidic compound 1 [SEQ ID NO: l ] on proliferation of RT4 bladder cancer cells in vitro.

[033] Figure 19 presents a graph showing the effect of EPC:EPG:PEG ₂₀ ooDSPE (66.5 :28.5:5 mole ratio) liposomal formulations of peptidic compound 1 [SEQ ID NOT] on proliferation of A2780cp ovarian cancer cells in vitro.

[034] Figure 20 presents a graph showing the effect of EPC:EPG:PEG ₂₀ ooDSPE (66.5 :28.5:5 mole ratio) liposomal formulations of peptidic compound 1 [SEQ ID NOT ] on proliferation of OCC-1 ovarian cancer cells in vitro. [035] Figure 21 presents a graph showing the effect of EPC:EPG:PEG ₂ oooDSPE (66.5:28.5 :5 mole ratio) liposomal formulations of peptidic compound 1 [SEQ ID NO:l] on proliferation of TCCSUP bladder cancer cells in vitro.

[036] Figure 22 presents a graph showing the effect of EPC:EPG:PEG ₂ oooDSPE (66.5:28.5:5 mole ratio) liposomal formulations of peptidic compound 1 [SEQ ID NO: l] on proliferation of T24 bladder cancer cells in vitro.

[037] Figure 23 presents a graph showing the effect of EPC:EPG:PEG ₂₀ ooDSPE (66.5:28.5:5 mole ratio) liposomal formulations of peptidic compound 1 [SEQ ID NO:l] on proliferation of T4 bladder cancer cells in vitro.

[038] Figure 24 presents a graph showing the effect of DMPC:DMPG (7:3) liposomal formulations of peptidic compound 1 [SEQ ID NO:l] on proliferation of A2780cp ovarian cancer cells in vitro.

[039] Figure 25 presents a graph showing the effect of DMPC:DMPG (7:3) liposomal formulations of peptidic compound 1 [SEQ ID NO:l] on proliferation of OCC-1 ovarian cancer cells in vitro.

[040] Figure 26 presents a graph showing the effect of DMPC:DMPG (7:3) liposomal formulations of peptidic compound 1 [SEQ ID NO: l ] on proliferation of TCCSUP bladder cancer cells in vitro.

[041] Figure 27 presents a graph showing the effect of DMPC:DMPG (7:3) liposomal formulations of peptidic compound 1 [SEQ ID NO: l] on proliferation of T24 bladder cancer cells in vitro.

[042] Figure 28 presents a graph showing the effect of DMPC:DMPG (7:3) liposomal formulations of peptidic compound 1 [SEQ ID NO: l ] on proliferation of RT4 bladder cancer cells in vitro.

[043] Figure 29 presents a graph showing the ratio of pAKTl/AKT in free and liposomal formulated GAP-107B8.107 treated A2780cp cells. AKT and pAKTl were determined by ELISA on cells treated for 1 hour with peptide GAP-107B8.107or liposomal formulation GAP- 107B8.107. Ratios are expressed as normalized values against the untreated control (control). Experiments were done using duplicate samples in triplicate (n=6). Data was analyzed by One- WAY-ANOVA followed by Tukey test. Peptide treatment showed significance against the untreated control (P<0.05). Liposomal formulations were significantly different (P<0.05) against empty liposomes (data not shown).

[044] Figure 30 presents a graph showing the plasma concentration of GAP-107B8 in female rats after single Intravenous (IV) bolus treatment of GAP-107B8 liposomal formulation 107 or liposome-free formulations.

[045] Figure 31 presents a graph showing the plasma concentration of GAP-107B8 in female rats after single Intraperitoneal (IP) bolus treatment of GAP-107B8 liposomal formulation 107 or liposome-free formulations.

DETAILED DESCRIPTION OF THE INVENTION

[046] The present invention provides for liposomal formulations of cationic peptides. As demonstrated herein, cationic peptides can be effectively associated with liposomes by including one or more anionic lipids in the liposomes. Methods of producing liposomal formulations of cationic peptides are also provided.

[047] The liposomal formulations are useful, for example, for formulation of therapeutic or diagnostic peptides. In this context, the therapeutic or diagnostic peptide may itself be cationic, or it may be a neutral or anionic peptide that is rendered cationic by addition of an additional peptide domain. For example, many therapeutic and diagnostic peptides can be conjugated to a "protein translocation domain" or PTD to facilitate cell uptake. Most PTDs are strongly cationic and will confer an overall positive charge on the final peptide product.

[048] The liposomal formulations are suitable for formulation of cationic peptides having a range of positive charges, for example, net positive charges between about +1 and about +30. In one embodiment, liposomal formulations of highly charged cationic peptides are provided, for example, cationic peptides have a net negative charge of +5 or greater, +7 or greater, or +10 or greater.

[049] As demonstrated herein, the liposomal formulations can be prepared with relatively low proportions of anionic lipid in the formulation provided that the charge ratio of anionic lipid:peptide is maintained an greater than 1 .0.

Definitions

[050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[051] The term "cationic peptide," as used herein, refers to a peptide of 4 or more amino acids in length that has an overall net positive charge at neutral pH. For example, in the context of the invention, a cationic peptide may be between about 4 and about 200 amino acids in length and have a net positive charge of between about +1 and about +30.

[052] The term "liposome," as used herein, refers to a preparation of artificial membrane vesicles comprising lipid molecules that is useful as a delivery vector in vivo or in vitro and includes both unilamellar and multi-lamellar vesicles.

[053] The term "liposome-associated," as used herein, with reference to a cationic peptide refers to a cationic peptide that is encapsulated by, adhered to, embedded in, trapped between, mixed with or otherwise combined with one or more liposome.

[054] The term "mole percent" ("mol %"), as used herein, refers to a fraction of a concentration. For example, with respect to lipid concentrations of a liposome, mol % refers to the ratio (given in percent) of moles of a lipid to total lipid content (given in moles) in 1 liter. One liter of liposomes comprising 5 mmol of lipid A and 5 mmol of lipid B is 10 mM with respect to total lipid content, with lipid A contributing 50 mol % to the liposomes and lipid B contributing 50 mol %. This lipid composition can also be described as 10 mM lipid A:lipid B 5 :5. [055] The term "naturally-occurring," as used herein with reference to an object, such as a protein, peptide or amino acid, indicates that the object can be found in nature. For example, a protein, peptide or amino acid that is present in an organism or that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is considered to be naturally-occurring.

[056] The term "amino acid residue," as used herein, encompasses both naturally-occurring amino acids and non-naturally-occurring amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, D-amino acids (i.e. an amino acid of an opposite chirality to the naturally-occurring form), N- -methyl amino acids, C-a-methyl amino acids, β- methyl amino acids and D- or L- -amino acids. Other non-naturally occurring amino acids include, for example, β-alanine (β-Ala), norleucine (Nle), norvaline (Nva), homoarginine (Har), 4-aminobutyric acid (γ-Abu), 2-aminoisobutyric acid (Aib), 6-aminohexanoic acid (ε-Ahx), ornithine (orn), sarcosine, a-amino isobutyric acid, 3-aminopropionic acid, 2,3-diaminopropionic acid (2,3-diaP), D- or L-phenylglycine, D-(trifluoromethyl) -phenylalanine, and D-p- fluorophenylalanine.

[057] The term "retro-inverso sequence" or "retro-inverso peptide," as used herein, refers to a sequence of amino acids that has been altered with respect to a reference amino acid sequence in that the amino acid sequence has been reversed and all L-amino acids have been replaced with D-amino acids. Compared to the reference peptide, a retro-inverso peptide has a reversed backbone while retaining substantially the original spatial conformation of the side chains, resulting in an isomer with a topology that closely resembles the reference peptide.

[058] The terms "therapy" and "treatment," as used interchangeably herein, refer to an intervention performed with the intention of improving a subject's status. The improvement can be subjective or objective and is related to ameliorating the symptoms associated with, preventing the development of, or altering the pathology of a disease or disorder being treated. Thus, the terms therapy and treatment are used in the broadest sense, and include the prevention (prophylaxis), moderation, reduction, and curing of a disease or disorder at various stages. Preventing deterioration of a subject's status is also encompassed by the term. Subjects in need of therapy/treatment thus include those already having the disease or disorder as well as those prone to, or at risk of developing, the disease or disorder and those in whom the disease or disorder is to be prevented.

[059] The term "ameliorate" includes the arrest, prevention, decrease, or improvement in one or more the symptoms, signs, and features of the disease or disorder being treated, either temporarily or in the long-term.

[060] The term "subject" or "patient" as used herein refers to an animal in need of treatment.

[061] As used herein, the term "about" refers to an approximately +/-10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[062] Naturally-occurring amino acids are identified throughout by the conventional three-letter or one-letter abbreviations indicated below, which are as generally accepted in the peptide art and are recommended by the IUPAC-IUB commission in biochemical nomenclature:

Table 1. Amino Acid Codes

Name 3-letter I -letter Nam 3-letter I -letter

code code code eo e

Alanine Ala A Leucine Leu L

Arginine Arg R Lysine Lys K

Asparagine Asn N Methionine Met M

Aspartic Asp D Phenylalanine Phe F

Cysteine Cys C Proline Pro P

Glutamic acid Glu E Serine Ser S

Glutamine Gin Q Threonine Thr T

Glycine Gly G t ryptophan Trp w

Histidine His H Tyrosine Tyr Y

Isoleucine He I Valine Val V [063] The peptide sequences set out herein are written according to the generally accepted convention whereby the N-terminal amino acid is on the left and the C-tcrminal amino acid is on the right. By convention also, L-amino acids are represented by upper case letters and D-amino acids by lower case letters.

LIPOSOMAL FORMULATIONS AND METHODS OF PRODUCING SAME

[064] The liposomal formulations in accordance with the present invention comprise a cationic peptide associated with liposomes having a net negative charge at neutral pH ("anionic liposomes"). As demonstrated herein, effective association of cationic peptides with liposomes can be achieved by maintaining the charge ratio of lipid:peptide at greater than 1.0:1. Accordingly, the liposomal formulations in accordance with the present invention comprise anionic lipids and peptide in appropriate amounts to provide a lipid:peptide charge ratio of between about 1.1:1 and about 30.0:1.

[065] In certain embodiments, the liposomal formulations of the present invention have a lipid:peptide charge ratio of between about 1.1:1 and about 28.0:1, for example, between about 1.1:1 and about 26.0:1, between about 1.1:1 and about 24.0:1, between about 1.1:1 and about 22.0:1, or between about 1.1:1 and about 20.0:1. In some embodiments, the liposomal formulations of the present invention have a lipid : peptide charge ratio of between about 1.2:1 and about 30.0:1, for example, between about 1.3:1 and about 30.0:1, between about 1.4:1 and about 30.0:1, and between about 1.5:1 and about 30.0:1.

[066] In other embodiments, the liposomal formulations of the present invention have a lipid:peptide charge ratio of between about 1.1:1 and about 18.0:1, between about 1.1:1 and about 16.0:1, between about 1.1:1 and about 14.0:1, between about 1.1:1 and about 12.0:1, and between about 1.1:1 and about 10.0:1. In another embodiment, the liposomal formulations of the present invention have a lipid:peptide charge ratio of between about 1.1:1 and about 8.0:1, for example between about 1.1:1 and about 7.0:1, between about 1.2:1 and about 7.0:1, between about 1.2:1 and about 6.0:1, between about 1.3:1 and about 6.0:1, between about 1.4:1 and about 6.0:1, and between about 1.5:1 and about 6.0:1. [067] Methods of producing liposomal formulations of cationic peptides are also provided.

Liposomes

[068] The liposomes for use in the preparation of the liposomal formulations in accordance with the present invention comprise one or more anionic lipids and one or more neutral lipids. In certain embodiments, the liposomes may also comprise one or more cationic lipids provided that the liposome retains an overall net negative charge at neutral pH and an appropriate charge ratio with the peptide cargo.

[069] The lipids included in the liposomes may be naturally occurring lipids (for example, from sources such as egg, soy, brain or heart), modified natural lipids (such as hydrogenated or lyso- lipids), or synthetic lipids. In certain embodiments, one or more of the lipids included in the liposomes are naturally occurring lipids. In one embodiment, the liposomes comprise a mixture of naturally occurring and synthetic lipids.

[070] Suitable anionic lipids for inclusion in the liposomes include, but are not limited to, cardiolipin, 1 ,1 ',2,2'-tetramyristoyl cardiolipin, phosphatidylserine (PS), dimyristoyl phosphatidylserine (DMPS), dioleoyl phosphatidylserine (DOPS), dipalmitoyl phosphatidylserine (DPPS), phosphatidylglycerol (PG), dioleoyl phosphatidylglycerol (DOPG), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), phosphatidic acid (PA), dioleoyl phosphatidic acid (DOPA), dimyristoyl phosphatidic acid (DMPA), dipalmitoyl phosphatidic acid (DPP A), N-succinyl-dioleoyl phosphatidylethanolamine (N-succinyl-DOPE), N-glutaryl-dioleoyl phosphatidylethanolamine (N-glutaryl-DOPE), phosphatidylinositol (PI), and cholesteryl hemisuccinate (CHEMS). In one embodiment, the liposomes for use in the liposomal formulations comprise natural or synthetic phosphatidylglycerols as the anionic lipid component.

[071] Suitable neutral lipids for inclusion in the liposomes include, but are not limited to, cholesterol, phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), dilauroyl phosphatidylcholine (DLPC), distearoyl phosphatidylcholine (DSPC), phosphatidylethanolamine (PE), dioleoyl phosphatidylethanolamine (DOPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE) and distearoyl phosphatidylethanolamine (DSPE). In one embodiment, the liposomes for use in the liposomal formulations comprise natural phosphatidylcholine, synthetic phosphatidylcholine, cholesterol, or a combination thereof, as the neutral lipid component.

[072] Suitable cationic lipids for inclusion in the liposomes include, but are not limited to, N- [l -(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium methylsulfate (DOTAP), N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 3β-[Ν-(Ν',Ν'- dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and dioctadecylamidoglycylspermine (DOGS).

[073] One skilled in the art will understand that the ratio of anionic to neutral lipids used to prepare the liposomes will be dependent on a number of factors, for example, on the charge of the anionic lipid(s) being employed, on the desired charge ratio between the peptide cargo and the anionic lipid(s) (and thus also on the charge and amount of the peptide cargo), and on whether or not the liposome also comprises any cationic lipids or other charge-neutralizing groups. Appropriate ratios can be readily determined by the skilled worker taking these factors into consideration. The amount of anionic lipid(s) to include in the liposome can also be determined by assessing the zeta potential of the final liposome. In certain embodiments of the invention, therefore, the liposome comprises sufficient anionic lipid(s) to confer a negative zeta potential on the liposomes when measured in phosphate -buffered saline at room temperature.

[074] In general, the liposomes comprise 70 mol % or less of anionic lipids. In certain embodiments of the invention, the liposomes comprise 65 mol % or less, 60 mol % or less, 55 mol % or less, 50 mol % or less, and 45 mol % or less of anionic lipids. In one embodiment, the liposomes comprise anionic lipids in an amount of no more than 40 mol %.

[075] In certain embodiments of the invention, the amount of anionic lipids included in the liposomes is between about 1 mol % and about 70 mol %, for example between about 2 mol % and about 70 mol %, between about 3 mol % and about 70 mol %, between about 4 mol % and about 70 mol %, or between about 5 mol % and about 70 mol %. In some embodiments, the liposomes comprise anionic lipids in an amount between 1 mol % and about 60 mol %, for example between about 2 mol % and about 60 mol %, between about 3 mol % and about 60 mol %, between about 4 mol % and about 60 mol %, and between about 5 mol % and about 60 mol %. In another embodiment, the liposomes comprise anionic lipids in an amount between about 1 mol % and about 50 mol %, for example between about 2 mol % and about 50 mol %, between about 3 mol % and about 50 mol %, between about 4 mol % and about 50 mol %, or between about 5 mol % and about 50 mol %.

[076] In other embodiments, the liposomes comprise anionic lipids in an amount between about 1 mol % and about 40 mol %, between about 2 mol % and about 40 mol %, between about 3 mol % and about 40 mol %, between about 4 mol % and about 40 mol %, and between about 5 mol % and about 40 mol %. In some embodiments, the liposomes comprise anionic lipids in an amount between about 6 mol % and about 40 mol %, between about 6 mol % and about 35 mol %, between about 7 mol % and about 35 mol %, between about 8 mol % and about 35 mol %, between about 9 mol % and about 35 mol %, and between about 10 mol % and about 35 mol %. In other embodiments, the liposomes comprise anionic lipids in an amount between about 10 mol % and about 40 mol %, between about 10 mol % and about 35 mol %, and between about 10 mol % and about 30 mol %.

[077] The liposomes may further comprise one or more derivatized lipids, for example, polyethylene glycol (PEG)-derivatized lipids ("pegylated lipids"). As is known in the art, the inclusion of pegylated lipids can result in liposomes having a longer circulation half-life, which in turn can lead to accumulation of the liposomes at disease sites, such as tumours and sites of infection. In addition, as demonstrated herein, for liposomes which include lipids that result in the liposome exhibiting some toxicity, the inclusion of pegylated lipids in the liposomes results in a significant decrease in the toxicity of the liposomes thereby rendering them useful for in vivo use. Accordingly, in certain embodiments of the invention in which the selection of lipids for inclusion in the liposomes results in liposomes exhibiting some inherent toxicity, one or more pegylated lipids can be included in the liposomes in order to decrease their toxicity.

[078] Suitable pegylated lipids for inclusion in the liposomes are generally pegylated neutral lipids, although pegylated versions of charged lipids remain an alternative choice. The PEG moiety included in the pegylated lipid can have a MW of between about 350 and about 20,000. In certain embodiments, pegylated lipids comprising a PEG moiety having a MW between about 350 and about 5000 daltons, for example, between about 500 and about 5,000 daltons, are included in the liposomes. Examples of suitable pegylated lipids for inclusion in the liposomes include, but are not limited to, methoxy(PEG) derivatives of DMPE, such as mPEG ₃₅ oDMPE, mPEGssoDMPE, mPEGysoDMPE, mPEG _]0 ooDMPE, mPEG ₂₀ ooDMPE and mPEG ₅ oooDMPE (available from Avanti Polar Lipids, Inc., Alabama); methoxy(PEG) derivatives of DSPE, such as mPEG ₂ oooDSPE and mPEGsoooDSPE (NOF Corporation, Japan); methoxy(PEG) derivatives of DPPE, such as mPEG ₂ oooDPPE (NOF Corporation, Japan); methoxy(PEG) derivatives of DMPE, such as mPEGsoooDMPE (NOF Corporation, Japan), and poly(oxyethylene) cholesteryl ethers (PEG-chol) (NOF Corporation, Japan). Multi-arm PEG-derivatized DSPE (Sunbright DSPE-PTE020; MW 2,000) and comb-shaped PEG-derivatized DSPE (Sunbright DSPE- AM0530K; MW 20,000) available from NOF Corporation (Japan) are also contemplated for use in the liposomes. Also contemplated for use in the liposomes are PEG-Vitamin A and PEG- vitamin E.

[079] When pegylated lipids are included in the liposomes, they are typically included in an amount of about 10 mol % or less of total lipids. In one embodiment, pegylated lipids are included in the liposomes at about 9 mol % or less of the total lipids. In another embodiment, pegylated lipids are included in the liposomes at about 8 mol % or less. In other embodiments, pegylated lipids are included in the liposomes at about 7 mol % or less, about 6 mol % or less, and about 5 mol % or less of total lipids. In another embodiment, pegylated lipids are included in the liposomes in an amount between about 0.5 mol % and about 10 mol %, for example, between about 0.5 mol % and about 8 mol %, or between about 0.5 mol % and about 6 mol %.

[080] The inclusion of functionalized pegylated lipids in the liposomes is also contemplated. As is known in the art, pegylated lipids can be further modified to include one or more functional groups in order to provide additional functionality to the liposome. The functional group can be, for example, a targeting moiety or a detectable label. Examples of functional groups include, but are not limited to, biological ligands having binding affinity for a cell receptor, such as CD4, folate, insulin, LDL, vitamins, transferrin, asialoglycoprotein, selectins (for example, E, L, and P selectins), Flk-1 , Flk-2, FGF, EGF, integrins, HER2, and the like; antibodies and antibody fragments, such as F(ab')2, F(ab) ₂ , Fab', Fab, Fv fragments, scFv, small targeting peptides like RGD derivatized, potentially integrins, mannosylated PEGs and the like.

[081] Examples of suitable commercially available functionalized pegylated lipids for inclusion in the liposomes include, but are not limited to, DSPE-PEG ₂ ooofolate, DSPE-PEGsooofolate and DSPE-PEG2ooobiotin (Avanti Polar Lipids, Inc., Alabama).

[082] When functionalized pegylated lipids are included in the liposomes, they are typically included in amounts that are very small relative to the PEG lipid itself. When the liposomes include both pegylated and functionalized pegylated lipids, the total amount of the pegylated lipids (functionalized and non.functionalized) is within the amounts described above, for example, between about 0.01 mol % and about 10 mol %, between about 0.01 mol % and about 8 mol %, or _b etween _a bout 0.01 mol % and about 5 mol % or between 0.01 mol% - 3 mol%.

[083] In one embodiment of the present invention, the liposomes comprise one or more neutral lipids and one or more anionic lipids. In another embodiment, the liposomes comprise one or more neutral lipids and one anionic lip^ In another embodim _e nt, the liposomes co _mpr jSe one neutral lipid and one or more anionic lipids. In another embodiment, the liposomes comprise one neutral lipid and one anionic lipid. In another embodiment, the liposomes comprise two or more neutral lipids and one anionic lipid. In accordance with these embodiments, one or more of the neutral or anionic lipids may be pegylated.

[084] In certain embodiments, the liposomes comprise one or more neutral lipids and one or more anionic lipids, and at least one of the neutral lipids is a pegylated lipid.

Cationic Peptides

The liposomal formulations in accordance with the present invention comprise a cationic peptide, i.e. a peptide having a net positive charge at neutral pH. In certain embodiments, the formulations are particularly useful for peptides having multiple positive charges, for example, for peptides having a net positive charge at neutral pH of between +3 and about +30. The net positive charge may be intrinsic to the peptide, or the peptide may be a neutral or anionic peptide that is rendered cationic by addition of an additional peptide domain. For example, "protein translocation domains" or PTDs are often added to peptides to facilitate cell uptake. Most PTDs are strongly cationic and will confer an overall positive charge on the final peptide product. Examples of PTDs that are commonly used to facilitate cell uptake are provided in Table 2. Fragments (for example, of at least 5 amino acids in length), d-peptide analogs and retro-inverso forms of these sequences could also be used as PTDs.

Table 2: Cationic Peptides with Translocation Activity

[085] A wide variety of peptide drugs and diagnostics are known in the art which are suitable candidates for inclusion in the liposomal formulations (see for example, the Peptide Therapeutics Database maintained by the Peptide Therapeutics Foundation, and the DrugBank: Open Data Drug and Drug Target Database maintained by the University of Alberta, which currently lists 134 FDA-approved biotech (protein/peptide) drugs).

[086] Suitable peptides can be selected from known diagnostic or therapeutic peptides by one skilled in the art by identifying those peptides that are intrinsically cationic at neutral pH, or which can be modified (for example, by attachment of a PTD or the like) such that the peptide has an overall positive charge. The peptide preferably has a net positive charge at neutral pH of at least +3, for example between +3 and about +30. In one embodiment, the peptide for inclusion in the liposomal formulations has a net positive charge at neutral pH of between +4 and about +30. In another embodiment, the peptide for inclusion in the liposomal formulations has a net positive charge at neutral pH of between +5 and about +30. In other embodiments, the peptide for inclusion in the liposomal formulations has a net positive charge at neutral pH of between about +3 and about +25, between about +3 and about +20, between about +3 and about +15.

[087] In certain embodiments, the peptide for inclusion in the liposomal formulations is a highly charged cationic peptide, for example, a cationic peptide having a net positive charge at neutral pH of greater than +5, for example between +5 and about +30. In other embodiments, the peptide for inclusion in the liposomal formulations is a cationic peptide having a net positive charge at neutral pH of greater than +6, greater than +7, greater than +8, and greater than +9. In some embodiments, the peptide for inclusion in the liposomal formulations has a net positive charge at neutral pH of between +6 and about +30, for example, between +7 and about +30, between +8 and about +30, and between +9 and about +30. In one embodiment, the peptide for inclusion in the liposomal formulations is a cationic peptide having a net positive charge at neutral pH of greater than +10, for example between +10 and about +30.

[088] An example of a class of therapeutic peptides suitable for inclusion in the liposomal formulations in accordance with the present invention are cationic antimicrobial peptides. A large number of such peptides are known (see, for example, the Antimicrobial Peptide Database maintained by the Department of Pathology & Microbiology at the University of Nebraska Medical Center) and a number of cationic antimicrobial peptides are in clinical trials for various indications (see, Gordon and Romanowski, (2005), Curr Eye Res., 30(7):505-515).

[089] Additional examples of classes of peptide therapeutics for inclusion in the liposomal formulations are the peptidic compounds described in International Patent Application Nos. PCT/CA06/00521 (Publication No. WO2006/108270) and PC T/CA06/01298 (Publication No. WO2007/016777) (both herein expressly incorporated by reference in their entirety). These peptides are useful for the treatment of PKC-mediated diseases, including for the treatment of cancer. [090] In one embodiment, the peptide for inclusion in the liposomal formulations is a peptide of between about 12 and about 40 amino acid residues in length and having a sequence of general formula (I) or the retro-inverso form thereof (I-R):

N _x B _y (A/N) _x B _y N _y -J(M)-N _y B _z A _x B _y NyB _x - (linker) - (HB-HY) ₂ -HB2-HY (I)

HY-HB2-(HY-HB) ₂ - (linker) - B _x N _y B _y A _x B _z N _y -J(M)-N _y B _y (A/N) _x B _y N _x (I-R) wherein:

J is 1-2 Lys residues;

M is absent or is an ATP mimetic moiety attached to J via the side chain of one of the Lys residues;

each N is independently Ala, lie, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is independently 0-1 ;

each y is independently 0-2;

z = 0-l :

(linker) is 3 to 9 amino acid residues selected from the group of: glycine and alanine,;

each HY is 1 to 4 amino acid residues selected from the group of: Ala, Gly, He and Leu;

each HB is 1 to 3 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, and

HB2 is 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, and

wherein the sequences -N _y B _z A _x B _y N _y B _x and - B _x N _y B _y A _x B _z N _y are 2 or more amino acids in length. [091] In one embodiment, the peptide of general formula (I) has a sequence of general formula (II) or the retro-inverso form thereof (II-R):

B _y (A N) _x ByNy-J(M)-NyB _z A _x B _y N _y - (linker) - (HB-HY) ₂ -HB2-HY (II)

HY-HB2-(HY-HB) ₂ - (linker) - N _y B _y A _x B _z N _y -J(M)-N _y B _y (A/N) _x B _y (II-R) wherein:

J is 1-2 Lys residues;

M is absent or is an ATP mimetic moiety attached to J via the side chain of one of the Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2;

z = 0-l ;

(linker) is 3 to 9 amino acid residues selected from the group of: glycine, alanine, valine, leucine and isoleucine;

each HY is 1 to 4 amino acid residues selected from the group of: Ala, Gly, He and Leu;

each HB is 1 to 3 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, and

HB2 represents 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, and

wherein the sequences -N _y B _z A _x B _y N _y and - N _y B _y A _x B _z Ny are 2 or more amino acids in length. [092] In one embodiment, the peptide of general formula (I) has a sequence of general formula (III) or the retro-inverso form thereof (III-R):

(A/N) _x B _y N _y -J(M)-N _y A _x B _y N _y - (linker) - (HB-HY) ₂ -HB2-HY (III)

HY-HB2-(H Y-HB) ₂ - (linker) - N _y B _y A _x N _y -J(M)-N _y B _y (A N) _x (III-R) wherein:

J is 1-2 Lys residues;

M is absent or is an ATP mimetic moiety attached to J via the side chain of one of the Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys;

each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2;

(linker) is 3 to 9 Gly residues;

each HY is 1 to 4 amino acid residues selected from the group of: Ala, Gly, He and Leu;

each HB is 1 to 3 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser;

HB2 is 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser;

wherein the sequences -N _y A _x B _y N _y and - N _y B _y A _x N _y are 2 or more amino acids in length.

[093] In one embodiment, in general formulae (III) and (III-R):

J is Lys; M is absent or is an ATP mimetic moiety attached to J via the side chain of the Lys residue;

each N is independently He or Leu;

each B is independently Arg or Lys;

each A is independently Phe;

each x is 1 ;

each y is independently 0-2;

(linker) is 3 to 9 Gly residues;

each HY is 1 to 4 amino acid residues selected from the group of: Ala, Gly, He and Leu;

each HB is 1 to 2 amino acid residues selected from the group of: Asn, Asp, Gin and Lys, and

HB2 is 1 amino acid residue selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser.

[094] In one embodiment, the peptide of general formula (I) has a sequence of general formula (IV) or the retro-in verso form thereof (IV-R):

A _x B _y -J(M)-A _x B _y N _y - (linker) - (HB-HY) ₂ -HB2-HY (IV)

HY-HB2-(HY-HB) ₂ - (linker) - N _y B _y A _x -J(M)-B _y A _x (IV-R) wherein:

J is 1 -2 Lys residues;

M is absent or is an ATP mimetic moiety attached to J via the side chain of one of the Lys residues;

each N is independently Ala, He, Leu, Val or Gly;

each B is independently Arg or Lys; each A is independently Phe, His or Trp;

each x is 1 ;

each y is independently 0-2;

(linker) is 3 to 9 Gly residues;

each HY is 1 to 4 amino acid residues selected from the group of: Ala, Gly, He and Leu;

each HB is 1 to 3 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, and

HB2 is 1 or 2 amino acid residues selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser, and

wherein the sequences -A _x B _y N _y and - N _y B _y A _x are 2 or more amino acids in length. In one embodiment, in general formulae (IV) and (IV-R): J is Lys;

M is absent or is an ATP mimetic moiety attached to J via the side chain of the Lys residue;

each N is independently He or Leu;

each B is independently Arg or Lys;

each A is independently Phe;

each x is 1 ;

each y is independently 0-2;

(linker) is 3 to 9 Gly residues;

each HY is 1 to 4 amino acid residues selected from the group of: Ala, Gly, He and Leu; each HB is 1 to 2 amino acid residues selected from the group of: Asn, Asp, Gin and Lys, and

HB2 is 1 amino acid residue selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Ser.

[096] In one embodiment of the invention, the peptide of general formula (I) has a sequence of general formula (V) or the retro-inverso form thereof (V-R):

X-(linker)-Y (V)

[Y-R]-(linker)-[X-R] (V-R)

wherein:

X is an amino acid sequence comprising at least 5 consecutive residues of the sequence: FRRKFRL [SEQ ID NO: 12], wherein the Lys residue is optionally attached to an ATP mimetic moiety (M);

Y is an amino acid sequence comprising at least 8 consecutive residues of the sequence: KDAQNLIGISI [SEQ ID NO: 13];

[X-R] is an amino acid sequence comprising at least 5 consecutive residues of the sequence: lrfkrrf [SEQ ID NO:14], wherein the Lys residue is optionally attached to an ATP mimetic moiety (M);

[Y-R] is an amino acid sequence comprising at least 8 consecutive residues of the sequence: isigilnqadk [SEQ ID NO: 15], and

(linker) is 3 to 9 amino acid residues selected from the group of: glycine and alanine.

[097] In one embodiment, the peptide is a fragment of the peptide of general formula (I) having a length of about 5 to about 12 amino acids and a sequence of any one of general formulae (VI), (VI-R), (VII) or (VII-R):

N _x B _y (A/N) _x B _y N _y -J(M)-N _y B _z A _x B _y N _y B _x (VI) B _x N _y B _y A _x B _z N _y -J(M)-N _y B _y (A N) _x B _y N _x (VI-R) (HB-HY) ₂ -HB2-HY (VII) HY-HB2-(HY-HB) ₂ (VII-R) wherein:

J, M, N, B, A, HY, HB, HB2, x, y and z are as described above for general formula (I).

[098] In one embodiment, in general formulae (VI) and (VI-R): J is Lys;

M is absent or is an ATP mimetic moiety attached to J via the side chain of the Lys residue; each N is independently He or Leu; each B is independently Arg or Lys; each A is independently Phe; each x is 1 , and each y is independently 0-2.

[099] In one embodiment, in general formulae (VII) and (VII-R): each HY is 1 to 4 amino acid residues selected from the group of: Ala, Gly, He and Leu; each HB is 1 to 2 amino acid residues selected from the group of: Asn, Asp, Gin and Lys, and HB2 is 1 amino acid residue selected from the group of: Arg, Asn, Asp, Glu, Gin, Lys and Scr.

[0100] In one embodiment, a peptide fragment having a sequence of any one of general formulae (VI), (VI-R) (VII) and (VIT-R) is attached at the N- or C-terminus to a linker as defined in general formula (I).

[0101] In one embodiment of the present invention, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R) (V) and (V-R) is between about 12 and about 35 amino acid residues in length. In another embodiment, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R) (V) and (V-R) is between about 12 and about 30 amino acid residues in length. In other embodiments, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R) (V) and (V-R) is between about 12 and about 29 amino acid residues in length, between about 12 and about 28 amino acid residues in length, between about 12 and about 27 amino acid residues in length; between about 12 and about 26 amino acid residues in length and between about 12 and about 25 amino acid residues in length.

[0102] In certain embodiments of the present invention, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R) (V), (V-R), (VI), (VI-R) (VII) and (VII-R) comprises an ATP mimetic moiety (M). In the context of the present invention, an ATP mimetic is a compound that comprises adenine or a derivative of adenine. A "derivative of adenine," as used herein, refers to a compound that retains the heteroaromatic ring structure of adenine (shown below) but which may contain additional, fewer or different substituents attached to the ring structure and/or additional, fewer or different heteroatoms within the ring structure when compared to adenine.

[0103] The term "derivative of adenine" also encompasses molecules that are isosteric with adenine. In the context of the present invention, a molecule that is isosteric with adenine (an "adenine isostere") is a molecule that has a similarity of structure and spatial orientation to adenine and a resulting similarity of properties, in particular with respect to three-dimensional space-filling properties.

[0104] Suitable adenine derivatives are known in the art and include, but are not limited to, 1 - deazaadenine; 3-deazaadenine; 7-deazaadenine; 7-deaza-8-azaadenine; 1 -methyladenine; 2- aminoadenine; 2-propyl and other 2-alkyl derivatives of adenine; 2-aminopropyladenine; 8- amino, 8-aza, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines; 8-oxo-N ⁶ - methyladenine; N ⁶ -methyladenine; N ⁶ -isopentenyladenine; 2-aminopurine; 2,6-diaminopurine; 2-amino-6-chloropurine; 6-thio-2-aminopurine; hypoxanthine; inosine; xanthine; 8-aza derivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 7-deaza-8-aza derivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6- chloropurine, hypoxanthine, inosine and xanthine; 1-deaza derivatives of 2-aminopurine, 2,6- diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; 7-deaza derivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; and 3-deaza derivatives of 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and xanthine; and adenine isosteres, such as 4-methylindole.

[0105] Examples of ATP mimetics that can be incorporated into the peptides include adenine peptide nucleic acid (PNA) and PNAs that include an adenine derivative, such as those described above. In one embodiment of the invention, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI) and (VI-R) comprises an ATP mimetic moiety (M) that is an adenine PNA.

[0106] In certain embodiments of the present invention, in the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI) and (VI-R), the ATP mimetic moiety ( ) is absent.

[0107] In one embodiment of the present invention, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI), (VI-R), (VII) and (VII-R) further comprises a PTD. The PTD can be attached to the N- or C-terminus, or to the side chain of one of the constituent amino acids of the peptide, for example, to the side chain of a lysine, arginine, glutamate, aspartate, asparagine or glutamine residue. In those embodiments in which the peptide includes a ATP mimetic moiety, the PTD may be attached to the ATP mimetic. In one embodiment, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III- R), (IV), (IV-R), (V), (V-R), (VI), (VI-R), (VII) and (VII-R) comprises a PTD attached to the side chain of one of the constituent amino acids of the peptide. In another embodiment, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI), (VI-R), (VII) and (VII-R) comprises a lysine residue and a PTD attached to the side chain of the lysine residue.

[0108] In one embodiment of the present invention, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI), (VI-R), (VII) and (VII-R) further comprises a PTD having a sequence selected from the sequences provided in Table 2 or the retro-inverso form thereof or a fragment thereof. In another embodiment, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI), (VI-R), (VII) and (VII-R) further comprises a PTD having the sequence: KRRQRRKKR [SEQ ID NO:6] or rkkrrqrrk [SEQ ID NO: l 1 ] or a fragment of one of these sequences.

[0109] In one embodiment of the present invention, the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI), (VI-R), (VII) and (VII-R) comprises a modified N- and/or C-terminus. Examples of chemical substituent groups suitable for modifying the N-terminus and/or C-terminus of peptides are known in the art and include, but are not limited to, alkyl, alkenyl, alkynyl, amino, aryl, aralkyl, heteroalkyl, hydroxy, alkoxy, aralkyloxy, aryloxy, carboxy, acyl, aroyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamino, dialkylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, alkylthio, aralkylthio, arylthio, alkylene, and NZ]Z ₂ where Zi and Z2 are independently hydrogen, alkyl, aryl, or aralkyl, and the like. Blocking groups such as Fmoc (fluorenylmethyl-O-CO-), carbobenzoxy (benzyl-O-CO-), monomethoxysuccinyl, naphthyl-NH- CO-, acetylamino-caproyl and adamantyl-NH-CO-, can also be used. Other modifications contemplated by the present invention include C-terminal amidation, esterification, hydroxymethyl modification and O-modification (for example, C-terminal hydroxymethyl benzyl ether), as well as N-terminal modifications such as substituted amides, for example alkylamides and hydrazides.

[0110] In one embodiment of the present invention, in the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI), (VI-R), (VII) and

(VII-R), the N-terminus of the peptide is modified with an acyl group. Non-limiting examples of suitable acyl groups are benzoyl, acetyl, -butylacetyl, /j>-phenylbenzoyl, trifluoroacetyl, cyclohexylcarbonyl, phenylacetyl, 4-phenylbutanoyl, 3,3-diphenylpropanoyl, 4-biphenylacetyl, diphenylacetyl, 2-naphthylacetyl, 3-phenylbutanoyl, a-phenyl-or/^o-toluoyl, indole-3-acetyl, 3- indolepropanoyl, 3-indolebutanoyl, 4-(4-methoxyphenyl)butanoyl, and the like. In another embodiment, the N-terminus of the peptide is modified with an acetyl group. In another embodiment, in the peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (M-R), (IV), (IV-R), (V), (V-R), (VI), (VI-R), (VII) and (VII-R) the C-terminus of the peptide is modified with an amino group.

[0111] In one embodiment of the invention, the peptide for inclusion in the liposomal formulations is selected from the group of:

Compound 1 [SEQ ID NO: l] Compound 2

KDAQNLIGIS[-NH ₂

- rkkrrq rrk-Ac

Compound 3 [SEQ ID NO: 16] Compound 4 [SEQ ID NO: 17]

Compound 5 [SEQ ID NO: 18] Compound 6 [SEQ ID NO: 19]

Compound 7 [SEQ ID NO:20]

O O

Ac-FRR-H Ac— isigilnqadkggggggglrf— HN .

FRLGGGGGGGKDAQNLIGISI-NH2 rrf-NH,

(

/

HN— KRRQRRK R-Ac Ac— krrqrrkkr— HN )

Compound 8 [SEQ ID NO:21] Compound 9 O

Ac-FRR-HN

FRLGGGGGGGKDAQNLIGISI-NH ₂

Ac-FRRKFRLGGGGGGGKDAQNLIGISI-NH ₂

Compound 1 1 [SEQ ID NO :2]

Ac— rkkrrqrrk— HN

Compound 10 [SEQ ID NO:22]

Ac-KDAQNLIGISI-NH ₂ Ac-GGGGGGGKDAQNLIGISI-NH ₂

Compound 12 [SEQ ID NO:23] Compound 13 [SEQ ID NO:24]

Compound 14 [SEQ ID NO:25] Compound 15

[0112] In another embodiment of the invention, the peptide for inclusion in the liposomal formulations is selected from the group of:

.. rkkrrqrrk-Ac

Compound 1 [SEQ ID NO: l] Compound 2 Ac-F

Compound 3 [SEQ ID NO: 16] Compound 4 [SEQ ID NO: 17]

O

FRR-HN A _{FRLGGGGGGGKDAQNL|G|S|} . _N H ₂ Ac-isigilnqadkggggggglrf-

/

HN— KRRQRRKKR-Ac Ac— krrqrrkkr—

Compound 8 [SEQ ID NO:21] Compound 9

AC-FRRKFRLGGGGGGGKDAQNLIGISI-NH2 Compound 1 1 [SEQ ID O:2]

Ac— rkkrrqrrk— HN

Compound 10 [SEQ ID O:22]

PREPARATION OF LIPOSOMAL FORMULATIONS

[0113] The liposomal formulations can be prepared by standard techniques known in the art (see, for example, Gregoriadis, Liposome Technology, Volume I, 3 ^rd Edition (2006), Informa Healthcare, New York, NY).

[0114] For example, in some embodiments, the liposome formulations can be prepared by preparing a solution of the lipid components in a suitable organic solvent and a solution of the peptide in an appropriate aqueous solvent. The two solutions are then combined with stirring to provide multilamellar vesicles (MLVs). The MLVs can optionally be subjected to sonication or extrusion procedures in order to provide small, unilamellar vesicles (SUVs) or large, unilamellar vesicles (LUVs) of a desired size.

[0115] Sonication procedures generally produce SUVs with diameters in the range of about 15nm to about 50nm. Sonication procedures typically utilize a bath-type or probe-tip sonicator. Cup-horn sonicators may also be used to produce SUVs. The use of probe-tip sonicators, which deliver high energy input to the lipid suspension, requires careful monitoring of the lipid suspension to avoid overheating and any associated degradation. Sonication tips also tend to release titanium particles into the lipid suspension which must be removed by centrifugation prior to use of the resulting SUVs.

[0116] Extrusion procedures involve repeatedly passing the lipid suspension under pressure through one or a series of polycarbonate filters with a defined pore size to yield particles having a diameter near the pore size of the filter being used. Prior to extrusion, the MLVs may optionally be disrupted either by submitting them to several freeze-thaw cycles or by prefiltering the MLV suspension through a larger pore size filter. The extrusion should be carried out at a temperature above the gel-liquid crystal transition temperature (Tc or Tm) of the lipid with the highest Tc. The size of the large, unilamellar vesicles (LUVs) resulting from the extrusion can be controlled by selection of a filter having an appropriate pore size. For example, extrusion through filters with l OOnm pores typically yields LUVs with a mean diameter of about 120nm to about 140nm. Extrusion through filters with 80nm pores typically yields LUVs with a mean diameter of about 60nm to about 125nm.

[0117] Alternatively, lipsomes can be prepared by the method originally described in Bangham, J. Mol. Biol, (1965) 1 1 :238-252 ("the Bangham method"), which comprises hydration of a thin lipidic layer. For example, MLVs can be prepared by dissolving lipids in a suitable organic solvent (such as chloroform or chloroform-methanol mixtures), then evaporating the organic solvent to complete dryness under low pressure and at a temperature range of about 37-40°C, for example in a rotary evaporator. Following evaporation, a solution of the peptide in an appropriate hydrating medium is added to the dry lipid film. The temperature of the hydrating medium should be above the Tc of the lipid with the highest Tc before adding to the dry lipid film. Suitable hydration media include, but are not limited to, distilled water, buffer solutions, saline, and nonelectrolytes such as sugar solutions. Specific non-limiting examples of such solutions include 0.9% saline, 5% dextrose, and 10% sucrose, amongst others. The hydrated lipids are then agitated for a time and at a temperature range appropriate for the lipid composition resulting in the formation of MLVs. The MLVs can optionally be subjected to sonication or extrusion procedures as described above in order to provide SUVs or LUVs of a desired size.

[0118] The liposomes can also be prepared by the reverse-phase evaporation technique. In this method, a lipidic film is prepared from an organic solution of the constituent lipids through removal of the organic solvent. The system is purged with nitrogen and the lipids are re- dissolved in a second organic solution, for example, diethyl ether and/or isopropyl ether. An aqueous solution of the peptide is added to the re-dissolved lipids with sonication leading to the formation of water droplets in the organic solvent. Removal of the second organic solvent, for example by means of a rotary evaporator, provides a semi-solid gel. The gel is subjected to vigorous agitation to induce a phase reversal resulting in the formation of LUVs.

[0119] Another method that may be used in some embodiments involves the use of detergent to solubilize the lipid components. Basically, the lipid components are solubilized in an aqueous solution comprising a detergent and the peptide. Examples of detergents that may be used in this method include, for example, non-ionic surfactants (such as n-octyl-beta-D-glucopyranoside), anionic surfactants (such as dodecyl sulphate) and anionic surfactants (such as hexadecyltrimethylammonium bromide (CTAB)). The detergent is subsequently removed by dialysis or column chromatography to yield LUVs.

[0120] Suitable aqueous solvents for use in the above methods include, but are not limited to, water and buffer solutions. The aqueous solvent may further comprise a sugar, an alcohol, or a combination thereof, such as trehalose, maltose, sucrose, glucose, lactose, dextran, mannitol, sorbitol or the like. Various buffers are known in the art and can be used as an aqueous solvent. In one embodiment, the aqueous solution used in the preparation of the liposomes is a histidine or HEPES buffer. In other embodiments, the aqueous solution used in the preparation of the liposomes is a histidine or HEPES buffered sugar solution. [0121] Suitable organic solvents for use in the above methods include, but are not limited to, methanol, ethanol, propanol, isopropanol, ethylene glycol, tetrahydrofuran, chloroform, tert- butanol, diethylether, mixtures of these solvents, and mixtures of these solvents with water or an aqueous buffer. In one embodiment of the present invention, the liposomal formulations are prepared using ethanol or an ethanol/water mixture as an organic solvent. In one embodiment of, the ethanol to water ratio is 95 :5. In one embodiment, the ethanol to water ratio is up to 70:30.

[0122] As noted above, the liposomal formulations in accordance with the present invention comprise lipids and peptide in appropriate amounts to provide a lipid :peptide charge ratio of between about 1.1 and about 30.0.

[0123] In general, the liposomal formulations are prepared using a lipid:peptide mass ratio in the range of about 100:1 to about 10:1. In certain embodiments, the liposomal formulations are prepared using a lipid:peptide mass ratio in the range of about 100:1 to about 12: 1. In one embodiment, the liposomal formulations are prepared using a lipid:peptide mass ratio in the range of about 100: 1 to about 15: 1. In other embodiments, the liposomal formulations are prepared using a lipid:peptide mass ratio in the range of about 90: 1 to about 15: 1.

[0124] In certain embodiments, the formulations are prepared such that the liposomes have an average size between about 5 nm and about 5 μηι, for example, between about 5 nm and about 1 μιη, or between about 5 nm and about 500 nm. In another embodiment, the formulations are prepared to comprise liposomes having an average size between about 10 nm and about 500 nm. In other embodiments, the formulations are prepared to comprise liposomes having an average size between about 10 nm and about 400 nm, between about 15 nm and about 400 nm, and between about 25 nm and about 400 nm. In some embodiments, the formulations are prepared to comprise liposomes having an average size between about 15 nm and about 250 nm, and between about 25 nm and about 250 nm, between about 25 nm and about 200 nm, and between about 50 nm and about 200 nm. In other embodiments, the formulations are prepared to comprise liposomes having an average size between about 25 nm and about 150 nm, and between about 50 nm and about 150 nm. In certain embodiments, the formulations are prepared to comprise liposomes having an average size of 100 nm or less, for example, between about 25 nm and about 100 nm, between about 25 nm and about 90 nm, and between about 30 nm and about 90 nm.

[0125] The liposomal formulation may optionally be submitted to one or more additional purification steps and/or concentrated prior to use and/or for storage purposes. For example, the liposomal formulation can be submitted to dialysis procedures to remove any residual solvent, and/or concentrated by ultrafiltration or by lyophilization and reconstitution in a smaller amount of aqueous solvent. Other appropriate purification steps including cross-flow filtration and tangential filtration may be used. In addition, larger liposomes may be concentrated using centrifugation.

[0126] In one embodiment, the present invention provides for methods of producing effective liposomal formulations of cationic peptides. The method generally comprises the steps of:

(a) providing a solution of a cationic peptide;

(b) providing a mixture of one or more neutral lipids and one or more anionic lipids in which the anionic lipids comprise about 1 mol % and about 70 mol % of the mixture, and

(c) preparing liposomes associated with the cationic peptide from the solution of the cationic peptide and a sufficient amount of the mixture to provide an anionic lipid :peptide charge ratio between about 1.1 : 1 and about 30.0:1.

TESTING OF THE LIPOSOMAL FORMULATIONS

[0127] Various properties of the liposomal formulations can be tested as necessary by standard techniques. For example, association of the peptide with the anionic liposomes within the liposomal formulations can be tested, the size of the liposomes in the liposomal formulations and/or the zeta potential of the liposomes may be tested. Other properties of the formulations, including the relative therapeutic or diagnostic activity of the liposome-associated peptide compared to free peptide, may also be tested. [0128] The extent of association of peptide with the liposomes in the formulations may be tested, for example, by liquid chromatography techniques, including gravity chromatography, medium- and high-pressure liquid chromatography or the use of spin columns. For example, reverse- phase, size exclusion and/or ion-exchange chromatographic techniques may be used. Other techniques such as sometimes one uses centrifuging with a sucrose gradient, such that liposomes concentrate in sucrose layers of varying density may be used.

[0129] The size of the liposomes can be assessed using, for example, dynamic light scattering techniques with commercially available instrumentation (such as that available from Malvern Instruments, Malvern, UK).

[0130] The zeta potential of the liposomes comprised by the formulations may also be assessed if required. Zeta potential provides an indication of the overall charge the particle acquires from a particular medium and can be measured, for example, using laser Doppler velocimetry techniques with commercially available instrumentation (such as that available from Malvern Instruments, Malvern, UK).

[0131] The liposomal formulations can also be tested for their therapeutic or diagnostic properties either in vitro or in vivo by standard tests and appropriate animal models known in the art and compared to the properties of the unformulated peptide. The pharmacokinetics of the liposomal formulations may also be tested in vivo using standard techniques (see, for example, Current Protocols in Pharmacology, (1998 & updates), Enna and Williams (Ed.), J. Wiley & Sons, Hoboken, NJ).

PHARMACEUTICAL COMPOSITIONS

[0132] The present invention also provides for pharmaceutical compositions suitable for administration to a subject that comprise the liposomal formulations. The pharmaceutical compositions may comprise a sterile liposomal formulation alone, or they may comprise the liposomal formulation in combination with one or more pharmaceutically acceptable carriers, diluents or buffers. The pharmaceutical compositions may further comprise one or more stabilizers, preservatives, adjuvants or the like as is known in the pharmaceutical arts and described in standard texts, such as in "Remington: The Science and Practice of Pharmacy, ^' " Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000). In certain embodiments, the pharmaceutical compositions may comprise one or more additional active components, such as another therapeutic or diagnostic compound.

[0133] The pharmaceutical compositions can be formulated for administration by a variety of routes. For example, the compositions can be formulated for oral, topical, rectal, vaginal, non- parenteral or parenteral administration, or for administration by inhalation or spray. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intrathecal, intraperitoneal and intrasternal injection, implant, intravesical or infusion techniques. In certain embodiments, the pharmaceutical compositions can be formulated in unit dose or dosage form. Non-limiting examples of unit dose forms include dragees, tablets, ampoules, vials, suppositories and capsules.

USES OF THE LIPOSOMAL FORMULATIONS

[0134] In one embodiment, the present invention provides for liposomal formulations comprising therapeutic or diagnostic peptides and their use to treat or diagnose humans and/or animals. Accordingly, in certain embodiments the liposomal formulations have human medical uses and in certain embodiments, the liposomal formulations have veterinary uses.

[0135] One embodiment of the invention provides for liposomal formulations comprising a cationic peptide having anti-cancer activity. Non-limiting examples of anti-cancer peptides include peptides of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V- R), (VI), (VI-R), (VII) and (VII-R), as described above.

[0136] Accordingly, in one embodiment of the invention, there is provided a method of treating cancer comprising administering to a subject an effective amount of a liposomal formulation comprising a cationic peptide having anti-cancer activity, such as a peptide of any one of general formulae (I), (I-R), (II), (II-R), (III), (III-R), (IV), (IV-R), (V), (V-R), (VI), (VI-R), (VII) and (VII-R). In this context, treatment with a liposomal formulation of the invention may result in a reduction in the size of a tumour, the slowing or prevention of an increase in the size of a tumour, an increase in the disease-free survival time between the disappearance or removal of a tumour and its reappearance, prevention of an initial or subsequent occurrence of a tumour (e.g. metastasis), an increase in the time to progression, reduction of one or more adverse symptom associated with a tumour, or an increase in the overall survival time of a subject having cancer.

[0137] The liposomal formulation can be used to inhibit the growth and/or metastasis of a variety of tumours. Exemplary tumours include, but are not limited to, haematologic neoplasms, including leukaemias, myelomas and lymphomas; carcinomas, including adenocarcinomas and squamous cell carcinomas; melanomas and sarcomas. Carcinomas and sarcomas are also frequently referred to as "solid tumours." Examples of commonly occurring solid tumours include, but are not limited to, cancer of the brain, breast, cervix, colon, head and neck, kidney, lung, ovary, pancreas, prostate, stomach and uterus; non-small cell lung cancer,\; CNS cancer and colorectal cancer. Various forms of lymphoma also may result in the formation of a solid tumour and, therefore, are also often considered to be solid tumours.

[0138] One embodiment of the present invention provides for the use of the liposomal formulations comprising an anti-cancer peptide in the treatment of a solid tumour, single cell or metastasizing cancer or a melanoma. In one embodiment, the invention provides for the use of the liposomal formulations comprising an anti-cancer peptide in the treatment of a cancer of an organ in the abdominal cavity, such as, colon cancer, pancreatic cancer, stomach cancer, breast cancer, lymphoma, lung cancer or ovarian cancer.

[0139] One embodiment of the invention provides for liposomal formulations comprising a cationic peptide having anti-microbial activity, for example, anti-bacterial, anti-fungal and/or anti-viral activity. Liposomal formulations comprising anti-microbial peptides have utility in medical and veterinary contexts, as well as being suitable for inclusion in anti-microbial cleansers, polishes, paints, sprays, soaps, or detergents for cosmetic, personal care, household or industrial use.

[0140] Thus, in one embodiment, the present invention provides a method of inhibiting bacterial growth by contacting bacteria with an effective amount of a liposomal formulation of the invention comprising a cationic peptide having anti-bacterial activity. In certain embodiments, the cationic peptide may have broad spectrum anti-bacterial activity, in which case the liposomal formulations may be used against gram-positive or gram-negative bacteria, for example, Corynebacterium xerosis, Chlamydia neumonia, Chlamydia trachomatis, Enterobacter cloacae, Enterobacter faecalis, Enterococcus faecium, Escherichia coli, Escherichia coli 0157:H7, Haemophilus neumonia, Helicobacter pylori, Listeria monocytogenes, Moraxella catarrhalis, Neisseria gonorrhoae, Neisseria neumonia es, Pseudomonas aeruginosa, Pneumococci species, Salmonella neumon, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus aureus K147, Staphylococcus epidermidis, Staphylococcus typhimurium, Streptococcus mitis, Streptococcus neumonia, Streptococcus pyogenes, Vibrio neumon, Mycobacterium tuberculosis and other acid-fast staining bacteria {i.e. M. africanum, M. avium- intracellulare, M. neumonia, M. bovis, M. leprae, M. phlei), Bacillus anthracis and other endospore-forming rods and cocci. In certain embodiments, the liposomal formulations may be used against multidrug-resistant strains of bacteria, such as, methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant coagulase-negative staphylococci (MRCNS), penicillin- resistant Streptococcus pneumoniae, penicillin-resistant pneumococci and multidrug-resistant Enterococcus faecium.

[0141] Another embodiment of the invention provides for methods of treating a bacterial infection or bacterially-related disease in a subject by administering an effective amount of a liposomal formulation comprising a cationic peptide having anti-bacterial activity. Examples of bacterial infections include infections with one of the bacteria listed above. Examples of bacterially-related diseases include, for example, tuberculosis, meningitis, ulcers, septicaemia, bacteremia, cystic fibrosis, pneumonia, typhoid fever, bacterial conjunctivitis, gonorrhoea, impetigo, bacterial eye infections, bacterial ear infections, bacterial diarrhoea, cystitis, bacterial vaginitis, bacterial endocarditis, bacterial pericarditis, peliosis, superficial skin infections, toxic shock, food poisoning, hemolytic uremic syndrome, botulism, leprosy, gangrene, tetanus, lyme disease, plague, anthrax and chancroid.

[0142] In another embodiment, the present invention provides a method of inhibiting fungal growth by contacting a fungus with an effective amount of a liposomal formulation of the invention comprising a cationic peptide having anti-fungal activity. In certain embodiments, the liposomal formulations may be used against Histoplasma (e.g. H. capsulstum), Coccidioides, Blastomyces, Paracoccidioides, Cryptococcus (e.g. C. neoformans), Aspergillus (e.g. A. fumigatus, A. flaws, A. niger, A. nidulans, A. terreus, A. sydowi, A. flavatus, and A. glaucus), Zygomycetes (e.g. Basidiobolus, Conidiobolus, Rhizopus, Mucor, Absidia, Mortierella, Cunninghamella, and Saksenaea), Candida (e.g. C. albicans, C. tropicalis, C. parapsilosis, C. stellatoidea, C. krusei, C. parakrusei, C lusitaniae, C. pseudorropicalis, C. guilliermondi and C. glabratd), Cryptosporidium parvum, Sporothrix schenckii, Piedraia hortae, Trichosporon beigelii, Malassezia furfur, Phialophora verrucosa, Fonsecae pedrosoi, Madurella mycetomatis or Pneumocystis carinii.

[0143] Another embodiment of the invention provides for methods of treating a fungally-related disorder or disease in a subject by administering an effective amount of a liposomal formulation comprising a cationic peptide having anti-fungal activity. Examples of fungally-related disorders and diseases include, for example, Candidiasis; endemic mycoses (such as Histoplasmosis, Coccidioidomycosis, Blastomycosis, Paracoccidioidomycosis, Cryptococcosis, Aspergillosis, Mucormycosis, associated disseminated infections and progressive pulmonary disease; cryptococcal meningitis; narcotising patchy bronchopneumonia; haemorrhagic pulmonary infarction; rhinocerebral disease; neutropenia, black piedra; white piedra; tinea (versicolor, capitis, corporis); Pneumocystis pneumonia; chromoblastomycosis, and maduromycosis.

[0144] In certain embodiments in which the liposomal formulations comprising a cationic antimicrobial peptide are formulated for use in cosmetic, personal care, household and industrial products, the liposomal formulations may be foiinulated for application to surfaces to inhibit the growth of a microbial species thereon, for example, surfaces such as countertops, desks, chairs, laboratory benches, tables, floors, sinks, showers, toilets, bathtubs, bed stands, tools or equipment, doorknobs and windows. Alternatively, the liposomal formulations may be formulated for laundry applications, for example, for washing clothes, towels, sheets and other bedlinen, washcloths or other cleaning articles. In certain embodiments, the liposomal formulations are formulated for inclusion in personal care items, such as soaps, deodorants, shampoos, mouthwashes, toothpastes, and the like. Many compositions used in personal care applications are susceptible to microbial growth and it is thus desirable to incorporate into these compositions an effective anti-microbial material.

KITS

[0145] The present invention additionally provides for therapeutic or diagnostic packs or kits containing a liposomal formulation of the invention or a pharmaceutical composition comprising a liposomal formulation. Individual components of the kit would be packaged in separate containers and, associated with such containers, can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human or animal administration.

[0146] When the components of the kit are provided in one or more liquid solutions, the liquid solution can be an aqueous solution, for example a sterile aqueous solution. For in vivo use, the liposomal formulation may be formulated into a pharmaceutically acceptable syringeable formulation. In this case the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the animal, such as the lungs, injected into an animal, or even applied to and mixed with the other components of the kit.

[0147] The components of the kit may also be provided in dried or lyophilised forms. When reagents or components are provided as a dried form, rcconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means. Irrespective of the number or type of containers, the kits also may comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the composition within the body of an animal. Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.

[0148] To gain a better understanding of the invention described herein, the following examples are set forth. It will be understood that these examples are intended to describe illustrative embodiments of the invention and are not intended to limit the scope of the invention in any way. EXAMPLES

EXAMPLE 1: EPC/EPG Liposomal Peptide Formulations

[0149] Liposomal formulations of peptidic compound 1 [SEQ ID NO: l], shown below, using either egg phosphatidylcholine (EPC; neutral) alone, or a combination of EPC with egg phosphatidylglycerol (EPG; anionic) at ratios of 4:6, 6:4, 7:3, 8:2 and 9: 1 EPC:EPG, were prepared using the general method outlined schematically in Figure 1.

Compound 1 [SEQ ID NO: l ]

[0150] Vesicle size for each formulation was determined using a zetasizer. The results are summarized in Table 3.

Table 3: Charge Ratio and Vesicle Size for Various EPC:EPG Formulations of Compound 1

[SEQ ID NO:l]

Charge Ratio Vesicle Size

Formulation

(-ve : +ve ) nm (PDI*)

102.6

EPC 0

(0.141)

124.1

EPC:EPG (4:6) 26.0

(0.037)

106.4

EPC:EPG (6:4) 17.4

(0.144) Charge Ratio Vesicle Size

Formulation

(-ve : +ve ) nm (PDI*)

77.1

EPC:EPG (7:3) 13.0

(0.166)

82.7

EPC:EPG (8:2) 8.8

(0.129)

88.1

EPC:EPG (9: 1 ) 4.4

(0.180)

*PDI = polydispersity index

[0151] The amount of peptidic compound 1 [SEQ ID NO: l] associated with liposomes in each formulation was assessed by size exclusion chromatography as follows:

[0152] Columns of Sepharose CL-4B were poured into 2 ml Pasteur pipettes plugged with glass wool. The columns were washed through with ~10 ml H ₂ 0, then loaded with 0.5 ml of the formulation. The column was eluted with 0.5 ml aliquots of H ₂ 0 and each eluted fraction was collected separately.

[0153] A 200 μΐ aliquot of each collected fraction was combined with 200 μΐ H ₂ 0 + 500 μΐ 5% SDS in 0.1 M Na borate and incubated for 10 min. at 50°C. While vortexing, 100 μΐ 10 mM fluorescamine (1 1.1 mg/4 ml EtOH) was added to each tube, then 3x250 μΐ of each sample was transferred to wells of black microplate. Fluorescence for peptide analysis (Ex. 381 nm, Em 470 nm, cut-off filter 455 nm) and lipid analysis (Ex. 565 nm, Em. 590 nm, cut-off filter 570 nm) was then determined.

[0154] The results are shown in Figure 2 and show that ratios of EPC:EPG of 7:3, 8:2 and 9: 1 provided the best peptide-liposome associations.

EXAMPLE 2: Effect of Buffer on EPC/EPG Liposomal Peptide Formulations

[0155] The effect of using sucrose-HEPES buffer pH 7.0 on the interaction of peptidic compound 1 [SEQ ID NO: l ] with EPG in EPC/EPG (7:3 mole ratio) liposomes was investigated. A charge ratio (EPG/peptide) of 2.6, assuming 1 1 positive charges per peptide molecule, was used.

[0156] Liposomes were prepared as follows. 12.3 mg EPC (MW 760) was dissolved in 0.3 ml EtOH/H ₂ 0 (95/5, v/v) with heating. 5.5 mg EPG (MW 771) was dissolved in 0.3 ml EtOH/H ₂ 0 (95/5, v/v) with heating. Two samples containing 100 μΐ EPC solution, 100 μΐ EPG solution and 50 μΐ EtOH/H ₂ 0 were prepared providing 23.6 mg/ml lipid. To each sample, 5 μΐ 5 mg/ml rhodamine-phosphatidylethanolamine (RhPE) in EtOH was then added.

[0157] While vortexing, either 2.2 ml H ₂ 0 or 2.2 ml 300 mM sucrose-10 mM HEPES pH 7.0 was added to the sample to make multilamellar vesicles (MLVs). Vesicle size was determined using a zetasizer (Malvern, dual angle, in H ₂ 0 or sucrose as appropriate; 100 μΐ diluted to 1.0 ml).

[0158] While mixing, 48 μΐ of a 7.14 mg/ml solution of peptidic compound 1 [SEQ ID NO: l ] was added to each preparation of MLVs (2.4 ml total volume, 200 μg/ml crude peptide, 2.36 mg/ml lipid). Vesicle size was determined as above. 200 μΐ of each preparation was transferred to a glass test tube for peptide/lipid analysis.

[0159] Using a 10 ml extruder, each preparation was extruded at R.T. through 2x80 nm polycarbonate filters. Vesicle size was checked after 1 , 3, 5 passes (in H ₂ 0 or sucrose, as appropriate). 200 μΐ of each resulting formulation was transferred to a glass test tube for peptide/lipid analysis.

[0160] Using a 2 ml Pasteur pipettes plugged with glass wool, 2 columns of Sepharose CL-4B were prepared. One column was washed through with ~10 ml H ₂ 0, the other with -10 ml sucrose-HEPES. 0.5 ml of each formulation was loaded onto the column prepared in the corresponding solution (H ₂ 0 or sucrose-HEPES buffer) and eluted with 0.5 ml aliquots of H ₂ 0 or buffer as appropriate. Each eluted fraction was collected separately.

[0161] A 200 μΐ aliquot of each collected fraction or total sample (from after the extrusion step) was combined with 200 μΐ H ₂ 0 + 500 μΐ 5% SDS in 0.1 M Na borate and incubated for 10 min. at 50°C. While vortexing, 100 μΐ 10 mM fluorescamine (1 1.1 mg/4 ml EtOH) was added to each tube. 3x250 μΐ of each sample was then transferred to wells of black microplate and fluorescence for peptide analysis (Ex. 381 nm, Em 470 ran, cut-off filter 455 nm) and lipid analysis (Ex. 565 nm, Em. 590 nm, cut-off filter 570 nm) was determined.

[0162] The results are shown in Figure 3 and indicate that the sucrose-HEPES buffer does not affect the peptide-EPG ion pair.

[0163] Liposomal formulations prepared as described above but using phosphate-buffered saline (pH 7.4), phosphate-buffered sucrose (pH 7.4), histidine-buffered saline (pH 6.5) and histidine- buffered sucrose (pH 6.5) demonstrated differing degrees of solubility, with only the formulations in histidine-buffered sucrose (pH 6.5) providing a clear solution, fhe other buffers resulted in varying degrees of precipitation. As such, either histidine-buffered sucrose or HEPES (pH 7)-sucrose was used as the buffer in subsequent experiments.

EXAMPLE 3: Effect of Lipid Concentration on Peptide Association with EPC/EPG Liposomes

[0164] To investigate the effect of peptide and lipid concentrations on liposomal formulations of peptidic compound 1 [SEQ ID NO: l], liposomal formulations comprising EPCiEPG ratios of 7:3, 8:2 and 9: 1 were prepared by the general method outlined in Figure 1 with varying amounts of lipids as detailed in Table 4.

Table 4: Liposomal Formulations to Investigate Effect of Lipid Concentration

Formulation Lipid Mass (mg) Compound 1 [SEQ Charge Ratio

ID NO: l] Mass (mg)

EPCiEPG 7:3

Formulation A 29.4 0.3 13.0

Formulation B 23.5 0.3 10.4

Formulation C 17.6 0.3 7.8

Formulation D 1 1.8 0.3 5.2 Formulation Lipid Mass (mg) Compound 1 [SEQ Charge Ratio

ID NO:l] Mass (mg)

Formulation E 5.9 0.3 2.6

EPC:EPG 8:2

Formulation A 29.4 0.3 8.8

Formulation B 23.5 0.3 7.0

Formulation C 17.6 0.3 5.3

Formulation D 1 1.8 0.3 3.5

Formulation E 5.9 0.3 1.8

EPC:EPG 9:1

Formulation A 29.4 0.3 4.4

Formulation B 23.5 0.3 3.5

Formulation C 17.6 0.3 2.6

Formulation D 1 1.8 0.3 1.8

Formulation E 5.9 0.3 0.9

[0165] The liposomal formulations were assessed by size-exclusion chromatography as described in Example 2. The results are shown in Figure 4 (for the EPC:EPG 7:3 formulations), Figure 5 (for the EPC:EPG 8:2 formulations), and Figure 6 (for the EPC:EPG 9: 1 formulations).

[0166] The results show generally that lower charge ratio values provide better association of the peptide with the liposomes as demonstrated by improved recovery of peptide with lipid fractions (size exclusion studies). For the 7:3 and 8:2 formulations, optimal associations were seen at charge ratios of 2.6 and 1.8, respectively. The results for the 9: 1 formulation (see Figure 6) demonstrate the importance of retaining a net negative charge on the liposomes as at a charge ratio of 0.9, a two-phase elution profile was observed (see Figure 6E) with low overall peptide recovery in the range of ~ 30%.

EXAMPLE 4: Effect of Increased Lipid and Peptide Concentrations on EPC/EPG 7:3 Liposomal Peptide Formulations [0167] The effect of increasing lipid and peptide concentrations in a EPC:EPG 7:3 liposomal formulation in which the charge ratio of lipid:peptide is maintained at 2.6 was investigated. Lipid concentrations of 2.46 mg/ml (lx), 4.92 mg/ml (2x increase), 12.3 mg/ml (5x increase), and 24.6 mg/ml (lOx increase) were employed as described below. lx Formulation

[0168] 10.3 mg EPC (MW 760) and 4.5 mg EPG (MW 771) were dissolved in 0.6 ml EtOH/H ₂ 0 (95/5, v/v) with heating and 2 μΐ RhPE (5 mg/ml in EtOH) was added. 5.28 ml 300 mM sucrose- 10 mM HEPES pH 7.0 was combined with 120 μΐ 10 mg/ml solution of peptidic compound 1 [SEQ ID NO: l]. While mixing, the lipid mixture was then added to the diluted peptide solution to make MLVs (10% solvent, 2.47 mg/ml lipid, 200 μg/ml crude peptide). Vesicle size was determined (Malvern, dual angle, in sucrose; dilute 200 μΐ to 1.0 ml) and a 200 μΐ sample was transferred to glass test tube for peptide/lipid analysis.

[0169] Using a 10 ml extruder, the above lipid/peptide mixture was extruded at R.T. through 2x80 nm polycarbonate filters, 5 passes. Vesicle size was determined (Malvern, dual angle, in sucrose; dilute 200 μΐ to 1.0 ml) and a 200 μΐ sample was transferred to glass test tube for peptide/lipid analysis.

2x Formulation

[0170] 20.6 mg EPC (MW 760) and 8.9 mg EPG (MW 771 ) were dissolved in 0.6 ml EtOH/H ₂ 0 (95/5, v/v) with heating and 4 μΐ RhPE (5 mg/ml in EtOH) was added. 5.16 ml 300 mM sucrose- 10 mM HEPES pH 7.0 was combined with 240 μΐ 10 mg/ml solution of peptidic compound 1 [SEQ ID NO: l]. While mixing, the lipid mixture was then added to the diluted peptide solution to make MLVs (10% solvent, 4.92 mg/ml lipid, 400 μg ml crude peptide). Vesicle size was determined (Malvern, dual angle, in sucrose; dilute 100 μΐ to 1.0 ml) and a 100 μΐ sample was transferred to glass test tube for peptide/lipid analysis.

[0171] Using a 10 ml extruder, the above lipid/peptide mixture was extruded at R.T. through 2x80 nm polycarbonate filters, 5 passes. Vesicle size was determined (Malvern, dual angle, in sucrose; dilute 100 μΐ to 1.0 ml) and a 100 μΐ sample was transferred to glass test tube for peptide/lipid analysis.

5x Formulation

[0172] 25.7 mg EPC (MW 760) and 1 1.2 mg EPG (MW 771) were dissolved in 0.3 ml EtOH/H ₂ 0 (95/5, v/v) with heating and 5 μΐ RhPE (5 mg/ml in EtOH) was added. 2.4 ml 300 mM sucrose- 10 mM HEPES pH 7.0 was combined with 300 μΐ 10 mg/ml solution of peptidic compound 1 [SEQ ID NO:l]. While mixing, the lipid mixture was then added to the diluted peptide solution to make MLVs (10% solvent, 12.3 mg/ml lipid, 1.0 mg/ml crude peptide). Vesicle size was determined (Malvern, dual angle, in sucrose; dilute 40 μΐ to 1 .0 ml) and a 40 μΐ sample was transferred to glass test tube for peptide/lipid analysis.

[0173] Using a 10 ml extruder, the above lipid/peptide mixture was extruded at R.T. through 2x80 nm polycarbonate filters, 5 passes. Vesicle size was determined (Malvern, dual angle, in sucrose; dilute 40 μΐ to 1.0 ml) and a 40 μΐ sample was transferred to glass test tube for peptide/lipid analysis.

IQx Formulation

[0174] 51.4 mg EPC (MW 760) and 22.4 mg EPG (MW 771) were dissolved in 0.3 ml EtOH/H ₂ 0 (95/5, v/v) with heating and 10 μΐ RhPE (5 mg/ml in EtOH) was added. 2.1 ml 300 mM sucrose-10 mM HEPES pH 7.0 was combined with 600 μΐ 10 mg/ml solution of peptidic compound 1 [SEQ ID NO:l]. While mixing, the lipid mixture was then added to the diluted peptide solution to make MLVs (10% solvent, 24.6 mg/ml lipid, 2.0 mg/ml crude peptide). Vesicle size was determined (Malvern, dual angle, in sucrose; dilute 20 μΐ to 1 .0 ml) and a 20 μΐ sample was transferred to glass test tube for peptide/lipid analysis.

[0175] Using a 10 ml extruder, the above lipid/peptide mixture was extruded at R.T. through 2x80 nm polycarbonate filters, 5 passes. Vesicle size was determined (Malvern, dual angle, in sucrose; dilute 20 μΐ to 1.0 ml) and a 20 μΐ sample was transferred to glass test tube for peptide/lipid analysis. 20x Formulation

[0176] 51.4 mg EPC (MW 760) and 22.4 mg EPG (MW 771) were dissolved in 0.15 ml EtOH/H ₂ 0 (95/5, v/v) with heating andlO μΐ RhPE (5 mg/ml in EtOH) was added. 0.75 ml 300 mM sucrose- 10 mM HEPES pH 7.0 was combined with 600 μΐ 10 mg/ml solution of peptidic compound 1 [SEQ ID NO: l]. While mixing, the lipid mixture was then added to the diluted peptide solution to make MLVs (10% solvent, 49.2 mg/ml lipid, 4.0 mg/ml crude peptide). Vesicle size was determined (Malvern, dual angle, in sucrose; dilute 10 μΐ to 1.0 ml) and a 10 μΐ sample was transferred to glass test tube for peptide/lipid analysis.

[0177] Using a 10 ml extruder, the above lipid/peptide mixture was extruded at R.T. through 2x80 nm polycarbonate filters, 5 passes. Vesicle size was determined (Malvern, dual angle, in sucrose; dilute 10 μΐ to 1.0 ml) and a 10 μΐ sample was transferred to glass test tube for peptide/lipid analysis.

Sepharose CL-4B Separation

[0178] Using 2 ml Pasteur pipettes plugged with glass wool, columns of Sepharose CL-4B were poured (one for each formulation). Each column was washed through with ~10 ml sucrose- HEPES buffer. The lx formulation was used undiluted; for the 2x formulation, 500 μΐ was diluted to 1.0 ml; for the 5x formulation, 200 μΐ was diluted to 1.0 ml; for the lOx formulation, 100 μΐ was diluted to 1.0 ml, and for the 20x formulation, 50 μΐ was diluted to 1.0 ml, all with sucrose-HEPES buffer. 0.5 ml of each formulation was loaded per column and eluted with 0.5 ml aliquots of sucrose-HEPES buffer. Each eluted fraction was collected separately.

Peptide and Lipid Assays

[0179] Samples that were taken of MLVs and extruded LUVs were made up to 200 μΐ volume with sucrose-HEPES buffer. 200 μΐ H ₂ 0 and 0.5 ml 5% SDS-0.1 M Na borate was added to each diluted initial sample and each fraction collected from the Sepharose columns and the samples were incubated 10 min. at 50°C to dissolve the lipids. While vortexing, 100 μΐ 10 mM fluorescamine (1 1.1 mg/4 ml EtOH) was added to each sample and then 3x250 μΐ of each sample was transferred to wells of a black microplate. Fluorescence for peptide (Ex. 381 nm, Em 470 nm, cut-off filter 455 nm) and for lipid (Ex. 565 nm, Em. 590 nm, cut-off filter 570 nm) was determined.

Results

[0180] Vesicle sizes are provided in Table 5. Vesicle size of the LUVs was small for lx, 2x and 5x formulations (-60-70 nm), but tended to increase for the more concentrated formulations (-87 nm for lOx, and -95 nm for 2 Ox).

Table 5: Vesicle Size for Liposomal Formulations of Various Lipid and Peptide

Concentrations

Polydispersity index

[0181] The results of the size-exclusion chromatography are shown in Figure 7 and demonstrate good recovery of both peptide and lipid after extrusion, with a slight decrease at the highest peptide/lipid concentration. Recovery of peptide paralleled recovery of lipid in LUVs for all formulations. The Sepharose CL-4B separations showed similar patterns for all formulations, with peptide associated with lipid, and complete recovery of both peptide and lipid from the column.

EXAMPLE 5: DMPC/DMPG and EPC/EPG/PEG ₂₀ ooDSPE Liposomal Peptide Formulations

[0182] Dimyristoylphosphatidylcholine (DMPC) / Dimyristoylphosphatidylglcerol (DMPG) (7/3 mole ratio) and EPC/EPG/l ,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (PEG ₂₀ ooDSPE) (66.5/28.5/5 mole ratio) formulations of peptidic compound 1 [SEQ ID NO: l] at a charge ratio (PG/peptide) of 2.6 (200 crude peptide) were prepared as follows.

DMPC/DMPG/Peytide Formulation

[0183] 9.2 mg DMPC (MW 677.93) and 4.0 tng DMPG (MW 688.85) were dissolved in 0.48 ml EtOH/H ₂ 0 (95/5, v/v) with heating to 35°C and 2 μΐ RhPE (5 mg/ml in EtOH) was added. 5.4 ml 300 mM sucrose-10 mM HEPES pH 7.0 was combined with 120 μΐ 10 mg/ml solution of peptidic compound 1 [SEQ ID NO:l] and heated to 35°C. While mixing, the lipid mixture was then added to the diluted peptide solution to make MLVs (8% solvent, 2.20 mg/ml lipid, 200 μg/ml crude peptide). Vesicle size (Malvern, dual angle, in sucrose; dilute 200 μΐ to 1.0 ml) was determined and a 200 μΐ sample transferred to a glass test tube for peptide/lipid analysis.

[0184] Samples were extruded at 35°C through 2x80 nm polycarbonate filters and vesicle size determined as generally described in Example 4.

EP /EPC/PezvMiDSP K/P guide Formula/ion

[0185] 10.4 mg EPC (MW 760), 4.5 mg EPG (MW 771 ) and 2.9 mg Peg ₂₀ ooDSPE (MW 2810g) were dissolved in 0.48 ml EtOH/H ₂ 0 (95/5, v/v) with heating and 2 μΐ RhPE (5 mg/ml in EtOH) was added. 5.4 ml 300 mM sucrose-10 mM HEPES pH 7.0 was combined with 120 μΐ 10 mg/ml solution of peptidic compound 1 [SEQ ID NO: l]. While mixing, the lipid mixture was then added to the diluted peptide solution to make MLVs (8% solvent, 2.95 mg/ml lipid, 200 μg/ml crude peptide). Determine vesicle size (Malvern, dual angle, in sucrose; dilute 200 μΐ to 1.0 ml). Transfer 200 μΐ to glass test tube for peptide/lipid analysis. Vesicle size (Malvern, dual angle, in sucrose; dilute 200 μΐ to 1.0 ml) was determined and a 200 μΐ sample transferred to a glass test tube for peptide/lipid analysis.

[0186] Samples were extruded at 35°C through 2x80 nm polycarbonate filters and vesicle size determined as generally described in Example 4.

Sepharose CL-4B Separation [0187] Using 2 ml Pasteur pipettes plugged with glass wool, Sepharose CL-4B separation was carried out as generally described in Example 4.

Peptide and Lipid Assays

[0188] Amounts of peptide and lipid in the various samples were determined as generally described in Example 4.

Results

[0189] Vesicle size for the two liposomal formulations is shown in Table 6 and indicates that the EPC/EPG/PEG ₂ oooDSPE/peptide MLVs were very small and only slightly further reduced in size by extrusion. The MLVs and LUVs were larger for the DMPC/DMPG/peptide formulation and there was more reduction in vesicle size with extrusion.

Table 6: Vesicle Size for DMPC DMPG and EPC/EPG PEG ₂₀ ooDSPE Liposomal Peptide

Formulations

* Polydispersity index

[0190] The results of the size-exclusion chromatography are shown in Figure 8 and demonstrate that the peptide and lipid elution patterns paralleled one another, and essentially all the peptide and lipid were recovered from the column in the fractions collected. However, the EPC/EPG/PEG ₂ oooDSPE/peptide formulation appears to have lost -15% of both peptide and lipid during extrusion, while losses from the DMPC/DMPG/peptide formulation during extrusion were small.

EXAMPLE 6: EPC/EPG/DOTAP Liposomal Peptide Formulations [0191] -(2,3-Dioleoyloxy)]-N,N,N-trimethylammonium propane methyl sulfate (DOTAP) (7/3/4 mole ratio) formulations of peptidic compound 1 [SEQ ID NO: l] at a negative charge (EPG) to peptide ratio of 2.6 and excess positively charged lipid (DOTAP) relative to negatively charged lipid (EPG) were prepared by two different methods as follows.

Procedure A (Standard Protocol)

[0192] 10.3 mg EPC (MW 760), 4.5 mg EPG (MW 771) and 5.4 mg DOTAP (MW 698.55) were dissolved in 0.6 ml EtOH/H ₂ 0 (95/5, v/v) with heating and 2 μΐ RhPE (5 mg/ml in EtOH) was added. 5.28 ml 300 mM sucrose-10 mM HEPES pH 7.0 was combined with 120 μΐ 10 mg/ml solution of peptidic compound 1 [SEQ ID NO: l]. While mixing, the lipid mixture was then added to the diluted peptide solution to make MLVs (10% solvent, 3.4 mg/ml lipid, 200 g/ml crude peptide). Vesicle size (Malvern, dual angle, in sucrose; dilute 200 μΐ to 1.0 ml) was determined and a 200 μΐ sample transferred to a glass test tube for peptide/lipid analysis.

[0193] Samples were extruded through 2x80 nm polycarbonate filters and vesicle size determined as generally described in Example 4.

Sepharose CL-4B separation was carried out using 2 ml Pasteur pipettes plugged with glass wool, as generally described in Example 4. Amounts of peptide and lipid in the various samples were determined as generally described in Example 4.

Procedure B (Alternate Protocol)

[0194] 42.9 mg EPC (MW760) and 18.6 mg EPG (MW 771 ) were dissolved in 0.5 ml EtOH/H ₂ 0 (95/5, v/v) with heating and 10 μΐ RhPE (5 mg/ml in EtOH) was added. Using a 10 mg/ml solution of peptidic compound 1 [SEQ ID NO: l ] in 300 mM sucrose-10 mM HEPES pH 7.0, 0.5 ml peptide solution was combined with 4.0 ml 300 mM sucrose-10 mM HEPES pH 7.0. While vortexing, the lipid solution was added to the diluted peptide solution to make MLVs (10% solvent, 12.3 mg/ml lipid, 1.0 mg/ml crude peptide). Vesicle size (Malvern, dual angle, in sucrose; dilute 50 μΐ to 1.0 ml) was determined.

[0195] Samples were extruded through 2x80 nm polycarbonate filters and vesicle size determined as generally described in Example 4. [0196] 1 1.9 mg DOTAP (MW 698.55) was dissolved with heating in 0.5 ml EtOH/H ₂ 0. Aliquots of the DOTAP solution were then combined with the EPC/EPG/peptide formulation and with EtOH/H ₂ 0 such that the final solvent concentrations were increased from the initial 10% in the formulation to 20, 30, 40, and 50% as follows:

[0197] 20%: 50 μΐ DOTAP + 12.5 μΐ EtOH/H ₂ 0 (95/5) + 0.5 ml formulation

30%: 50 μΐ DOTAP + 93 μΐ EtOH/H ₂ 0 (95/5) + 0.5 ml formulation

40%: 50 μΐ DOTAP + 200 μΐ EtOH/H ₂ 0 (95/5) + 0.5 ml formulation

50%: 50 μΐ DOTAP + 350 μΐ EtOH/H ₂ 0 (95/5) + 0.5 ml formulation

[0198] An increased turbidity was observed in the samples with addition of DOTAP. The turbidity increased with increasing solvent concentration and may have been due to aggregation of liposomes and/or precipitation of peptide at higher solvent concentrations.

[0199] The samples were allowed to stand at R.T. for at least 30 min. before diluting into sucrose-HEPES buffer to decrease the solvent concentration below 10% (to a crude peptide concentration of -100 μg/ml) as follows:

[0200] 10%: 200 μΐ diluted to 2.0 ml (1 % solvent; 100 μg/ml crude peptide)

20%>: 225 μΐ diluted to 2.0 ml (2.25% solvent; 100 ig/m\ crude peptide)

30%: 250 μΐ diluted to 2.0 ml (3.75% solvent; 97 μg/ml crude peptide)

40%: 300 μΐ diluted to 2.0 ml (6% solvent; 100 μg/ml crude peptide)

50%: 350 μΐ diluted to 2.0 ml (8.75% solvent; 97 μg/ml crude peptide)

[0201] Corresponding control samples (without DOTAP) were prepared as follows:

[0202] 20%: 31 μΐ EtOH/H ₂ 0 (95/5) + 0.25 ml formulation

30%: 72 μΐ EtOH/H ₂ 0 (95/5) + 0.25 ml formulation

40%: 125 μΐ EtOH/H ₂ 0 (95/5) + 0.25 ml formulation

50%: 400 μΐ EtOH/H ₂ 0 (95/5) + 0.5 ml formulation [0203] Again, a slight increase in turbidity of samples was observed with increasing solvent concentration, suggesting that there is some precipitation of the peptide at the higher solvent concentrations, but significantly less turbidity than seen in the samples containing DOTAP.

[0204] The control samples were diluted into sucrose-HEPES buffer to decrease the solvent concentration to correlate to the corresponding sample containing DOTAP as follows:

[0205] 20%: 225 μΐ diluted to 2.0 ml (2.25% solvent; 100 crude peptide)

30%: 250 μΐ diluted to 2.0 ml (3.75% solvent; 97 μg ml crude peptide)

40%: 300 μΐ diluted to 2.0 ml (6% solvent; 100 μg/ml crude peptide)

50%: 350 μΐ diluted to 2.0 ml (8.75% solvent; 97 μg/ml crude peptide)

[0206] Vesicle size of the diluted samples was determined (NICOMP, in sucrose, without further dilution).

[0207] 500 μΐ of each diluted sample and control was transferred into microcentrifuge tubes, and centrifuged 5 min. at R.T., 14,000 rpm (~20,000xg). The supernatant was reserved. In the control samples, no pellet was seen in 20, 30, 40% solvent samples; and a very small pink pellet was seen in 50% solvent sample. In the DOTAP-containing samples, the supernatant was clearer than before centrifugation, no pellets were seen, but some lipid appeared to be floating on the surface of the supernatant.

[0208] 500 μΐ of each diluted sample was transferred into 100K MWCO Microcon centrifugal filtration devices, centrifuged 30 min. at 13,000xg and the filtrates reserved.

[0209] 400 μΐ of the initial diluted samples and controls, supernatants from centrifugation and filtrates from Microcons was combined with 500 μΐ 5% SDS-0.1 M Na borate and incubated 10 min. at 50°C to dissolve the lipid. While vortexing, 100 μΐ 10 mM fluorescamine (1 1.1 mg/4 ml EtOH) was added to each sample and control. 3x250 μΐ were transferred to the wells of a black microplate and peptide (Ex. 381 nm, Em. 470 nm, cut-off filter 455 nm) and lipid (Ex. 565 nm, Em. 590 nm, cut-off filter 570 nm) were determined. [0210] 500 μΐ of each diluted control was transferred into 100K MWCO Microcon centrifugal filtration devices, centrifuged 30 min. at 13,000xg and the filtrate reserved. 400 μΐ of the filtrates from the Microcons was combined with 500 μΐ 5% SDS-0.1 M Na borate. While vortexing, 100 μΐ 10 mM fluorescamine (1 1.1 mg/4 ml EtOH) was added to each control filtrate sample. 3x250 μΐ were transferred to the wells of a black microplate and peptide and lipid determined as above.

[0211] The results showed that preparation of EPC/EPG/DOTAP/peptide by solvent dilution into peptide solution resulted in a formulation that did not have peptide associated with the lipid. Preparation of EPC/EPG/DOTAP/peptide by addition of DOTAP (in solvent) to pre-formed EPC/EPG/peptide liposomes on the other hand resulted in displacement of the peptide from the liposomes. This latter result is significant as it demonstrates that the peptide can be readily released and bioavailable from the EPC/EPG liposomal formulations.

EXAMPLE 7: Inhibition of Cancer Cell Proliferation by Liposomal Peptide Formulations

[0212] The effect of EPC:EPG, EPC:EPG:PEG ₂₀₀₀ DSPE and DMPQDMPG liposomal formulations of peptidic compound 1 [SEQ ID NO:l] on the proliferation of human ovarian and bladder cancer cells in vitro was investigated.

Cultures

[0213] Five cells lines were used including two ovarian cancer cell lines gifted from Dr. Barbara Vanderhyden (Ottawa Hospital Research Institute, Ottawa, ON) and three human bladder cancer cell lines purchased from the American Type Culture Collections (ATCC, Rockville, MD). The two ovarian cancer cell lines were A2780cp (human ovarian carcinoma) and OCC-1 cells (ovarian clear cell carcinoma cells). The three bladder cancer cell lines tested were TCCSUP (transitional cell carcinoma from human urinary bladder; ATCC HTB-5), T24 (transitional cell carcinoma from human urinary bladder; ATCC HTB-4), and RT4 (transitional cell papilloma from human urinary bladder carcinoma; ATCC HTB-2). All cell lines were seeded at 4000 cells per well in 96-well plates (BD Falcon; 35307) in media supplemented with 5% FBS (HyClone SH30396.03) as follows: A2780cp in DMEM, OCC-1 in MEM alpha, and all bladder cell lines in RPMI (HyClone SH30243.01 , SH30265.01 , SH30027.01 respectively). Cells were allowed to adhere for 2 hours at 37°C in a water-saturated atmosphere with 5% C0 ₂ .

[0214] Sterile liposomal formulations of peptidic compound 1 [SEQ ID NO: l] prepared generally as described in Examples 1 to 5 were added to cultures to final peptide concentrations as shown in Table 8. As controls, empty liposomes were screened at equivalent concentrations with respect to lipid content (for empty liposomes) or unformulated peptide alone. Table 7 provides a description for each formulation of empty and loaded liposomes used, and Table 8 indicates final lipid content per sample as derived from final calculated peptide concentrations.

[0215] Cells were incubated for 24 and 72 hours at 37°C in a water-saturated atmosphere with 5% CO2, at which point media was removed by quick inversion of the plate, and plates were stored at -80°C until ready to be assayed.

Table 7: Liposomal Formulations Tested

Table 8: Peptide and Lipid Final Concentrations*

* Final l ipid content is calculated as lipid content of the stock solution divided by the dilution needed to obtain the desired final peptide content. The dilution required for the final peptide concentration target is calculated similarly from peptide content of loaded liposome stock solutions.

† Ovarian cancer cell lines only.

% Bladder cancer cell lines only.

Proliferation Assay [0216] Cell Proliferation was measured using the CyQUANT® GR cell proliferation assay kit (Invitrogen; C7026) according to the manufacturer's protocol, without the use of a standard curve. Briefly, CyQUANT lysis buffer and reagent were added to nuclease free ddH20 (Ambron; 9932) at 1 :20 and 1 :400 respectively. Care was taken to protect from light. Plates to be read were thawed at room temperature. The prepared reagent was added to each sample well (200 μΐ/well) and incubated (2-5min, RT, light protected). Three wells per plate contained prepared reagent only; these were considered background for all samples of the plate. Plates were read and sample fluorescence measured using a FLUOStar Galaxy Microplate Reader (BMG Technologies) with filters at 485nm excitation and 520 nm emission. Proliferation was determined by subtracting background from all samples. Untreated cells were considered to be 100% proliferation, and all results were normalized to untreated cells.

Statistics

[0217] All statistical significances were determined using SigmaPlot®. RESULTS

EPC:EPG Formulations

[0218] The results for the EPC:EPG (7:3) formulations are presented in Figure 9 (A2780cp ovarian cancer cells), Figure 10 (OCC-1 ovarian cancer cells), Figure 11 (TCCSUP bladder cancer cells), Figure 12 (T24 bladder cancer cells) and Figure 13 (RT4 bladder cancer cells).

[0219] The results for the EPC:EPG (9:1) formulations are presented in Figure 14 (A2780cp ovarian cancer cells), Figure 15 (OCC-1 ovarian cancer cells), Figure 16 (TCCSUP bladder cancer cells), Figure 17 (T24 bladder cancer cells) and Figure 18 (RT4 bladder cancer cells).

[0220] In each Figure, a summary of cell proliferation results is provided for each cell line and compared after 24 and 72 hours of exposure to varying concentrations of free peptide in buffer or liposomal formulated peptide over a similar concentration range. Included in each study are control groups of empty liposomes administered at near equivalent concentration position with respect to the corresponding peptide loaded liposome group and a control group of untreated cells. [0221] An analysis of the cell proliferation data obtained for liposome formulations derived from EPC:EPG lipids at a ratio of 7:3 and 9: 1 demonstrated that after 24 or 72 hours of exposure no significant inhibition of cell proliferation by peptide alone vs. untreated cells over the concentration ranges tested was found except in select cases for the highest concentrations of peptides examined.

[0222] For 7:3 liposome compositions significant improvement in potency can be found for peptide loaded liposomes vs, untreated cells after 24 hours with a lower limit of 1.56 uM (p < 0.05) for A2780cp and OCC-1 cell lines and 3.13 uM (p < 0.05) for T24 cells and 6.3 uM (p < 0.05) for SUPTCC and RT4 cell lines respectively.

[0223] Further improvement in potency can be found for peptide loaded liposomes vs, untreated cells after 72 hours with a lower limit of 0.78 uM (p < 0.05) for A2780cp cells and 3.13 uM (p < 0.05) for OCC-1 cell lines and 1.56 uM (p < 0.05) for SUPTCC and T24 cell lines and 3.1 uM (p < 0.001 ) for RT4 cells respectively.

[0224] For 9: 1 liposome compositions significant improvement in potency can be found for peptide loaded liposomes vs, untreated cells after 24 hours with a lower limit of 0.78 uM (p < 0.05) for A2780cp and OCC-1 cell lines and 1.56 uM (p < 0.05) for T24 cells, 6.3 uM (p < 0.05) for RT4 cells and 12.5 uM for SUPTCC (p < 0.05) cell lines respectively.

[0225] Further improvement in potency can be found for peptide loaded liposomes vs, untreated cells after 72 hours with a lower limit of 0.78 uM (p < 0.001) for A2780cp and OCC-1 cell lines and 1.56 uM (p < 0.001) for RT4 and T24 cell lines and 1.56 uM (p = 0.002) for SUPTCC cell lines respectively.

[0226] Further analysis of the cell proliferation data obtained for empty liposome formulations relative to peptide loaded liposomes confirms that significant differences in potency favouring peptide loaded liposomes can be found from liposomes comprised of EPC:EPG lipids at both ratios of 7:3 and 9: 1 , which is most evident after 72 hours of exposure.

[0227] Significant improvement in potency for peptide loaded liposomes vs, empty liposomes derived from 7:3 compositions was found at a lower limit of 1.56 uM (p < 0.05) for SUPTCC cells, at 3.13 uM (p = 0.004) for T24 cells and at 6.25 uM (p = 0.003) for RT4 cell lines respectively.

[0228] Significant improvement in potency was found for peptide loaded liposomes vs, empty liposomes derived from 9: 1 compositions with a lower limit of 0.78 uM (p < 0.001) for A2780cp cells and 3.13 uM (p < 0.05) for OCC-1 cell lines and 1.56 uM (p = 0.002) for SUPTCC and T24 cell lines, at 1.56 uM (p < 0.05) for RT4 cell lines respectively.

EPC:EPG:PEG _7M DSPE Formulations

[0229] The results for the EPC:EPG:PEG ₂₀₀ oDSPE (66.5:28.5 :5) formulations are presented in Figure 19 (A2780cp ovarian cancer cells). Figure 20 (OCC-1 ovarian cancer cells), Figure 21 (TCCSUP bladder cancer cells), Figure 22 (T24 bladder cancer cells) and Figure 23 (RT4 bladder cancer cells).

[0230] An analysis of the cell proliferation data obtained for liposome formulations derived from EPC:EPG:PEG ₂ oooDSPE lipids at a ratio of 66.5:28.5:5 demonstrates that after 24 or 72 hours of exposure no significant inhibition of cell proliferation by peptide vs. control untreated cells over the full concentration range tested was found.

[0231] Significant improvement in potency can be found for peptide loaded PEGylated liposomes vs, untreated cells after 24 hours with a lower limit of 0.78 uM (p < 0.05) for A2780cp and 1.56 uM for (p < 0.001) for OCC-1 cell lines and at 0.78 uM (p = 0.002) for T24 cells, 1.56 uM (p < 0.05) for SUPTCC and 3.13 uM (p = 0.001 ) for RT4 cell lines respectively.

[0232] Further improvement in potency can be found for peptide loaded PEGylated liposomes vs, untreated cells after 72 hours with a lower limit of 0.78 uM (p < 0.001 ) for A2780cp, OCC-1 , TCCSUP and T24 cell lines and 0.78 uM (p < 0.05) for for RT4 cells respectively.

[0233] Further analysis of the cell proliferation data obtained for empty liposome formulations relative to peptide loaded liposomes confirms that significant differences in potency favouring peptide loaded liposomes can be found from liposomes comprised of EPC:EPG:PEG ₂ oooDSPE lipids, which is most evident at both 24 and 72 hours of exposure. [0234] Significant improvement in potency for peptide loaded PEGylated liposomes vs, empty PEGylated liposomes after 24 hours was found at a lower limit of 1.56 uM (p < 0.05) for A2790cp and OCC-1 cell lines and at 1.56 uM (p < 0.001)) for T24 cells, 1.56 uM (p = 0.002) for SUPTCC cells and at 3.13 uM (p = 0.005) for RT4 cell lines respectively.

[0235] Significant improvement in potency was found for peptide loaded PEGylated liposomes vs, empty PEGylated liposomes with a lower limit of 0.78 uM (p < 0.001 ) for A2780cp, OCC-1 , TCCSUP and T24 cell lines and 1.56 uM (p = 0.001) for RT4 cell lines respectively.

DMPC-DMPG Formulations

[0236] The results for the DMPC:DMPG (7:3) formulations are presented in Figure 24 (A2780cp ovarian cancer cells), Figure 25 (OCC-1 ovarian cancer cells), Figure 26 (TCCSUP bladder cancer cells), Figure 27 (T24 bladder cancer cells) and Figure 28 (RT4 bladder cancer cells).

[0237] An analysis of the cell proliferation data obtained for liposome formulations derived from DMPC:DMPG lipids at a ratio of 7:3 demonstrates that after 24 or 72 hours of exposure no significant inhibition of cell proliferation by peptide vs. control untreated cells over the full concentration range tested was found.

[0238] Significant improvement in potency can be found for peptide loaded liposomes vs, untreated cells after 24 hours with a lower limit of 0.78 uM (p < 0.05) for A2780cp and 0.78 uM (p = 0.002) for OCC-1 cell lines and at 0.78 uM (p = 0.003) for T24 cells, 0.78 uM (p < 0.05) for RT4 and 6.3 uM (p = 0.005) for TCCSUP cell lines respectively.

[0239] Further improvement in potency can be found for peptide loaded liposomes vs, untreated cells after 72 hours with a lower limit of 0.78 uM (p < 0.001) for A2780cp, OCC-1 , RT4 and T24 cell lines and 0.78 uM (p = 0.006) for for RT4 cells respectively. [0240] Further analysis of the cell proliferation data obtained for empty liposome formulations relative to peptide loaded liposomes demonstrated differences in potency at both 24 or 72 hours of exposure

[0241J Significant improvement in potency for peptide loaded DMPC:DMPG liposomes vs, empty liposomes after 24 hours was found at a lower limit of 25 uM (p < 0.001) for A2790cp and 12.5 uM (p < 0.001) for OCC-1 cell lines and at 12.5 uM (p < 0.001) for SUPTCC, T24 and T4 cell lines respectively.

[0242] Significant improvement in potency for peptide loaded DMPC:DMPG liposomes vs, empty liposomes after 72 hours was found at a lower limit of 25 uM (p < 0.001) for OCC-1 cell line and at 3.13 uM (p < 0.05) for SUPTCC and 12.5 uM (p < 0.001) for RT4 cell lines respectively.

[0243] In summary all peptide loaded liposome compositions were found to significantly improve the potency of GAP-10B8.107 peptides relative to unformulated analogs in all cell liens examined.

[0244] The most effective liposome systems developed with respect to the potency of peptide loaded liposomes relative to empty liposome analogs were determined to be those comprised of EPC:EPG:PEGylated lipid components.

[0245] Peptide loaded liposomes derived from lipid components without PEGylated lipid as a component (EPC:EPG liposomes) proved to be effective over an intermediate concentration range relative to empty liposomes, whereas peptide loaded DMPC:DMPG liposomes relative to empty liposome analogs were found to be effective but at higher concentrations relative to the other lipid compositions examined.

EXAMPLE 8: Additional Liposomal Peptide Formulations

[0246] The following liposomal peptide formulations will be prepared following protocols similar to those outlined above in Examples 1 to 5 : [0247] Formulation A: EPC:EPG:PEG ₂₀ ooDSPE (6.5:3:0.5) with peptidic compound 1 [SEQ ID NO: l] at 4-5 mg/mL.

[0248] Formulation B: EPC:EPG:PEG ₂ oooDSPE:DSPE-PEG ₅ ooo-Folate (6.5:3:0.47:0.03) with peptidic compound 1 [SEQ ID NO:l] at 4-5 mg/mL.

[0249] Formulation C: EPC:EPG:PEG ₂₀ ooDSPE (6.5:3:0.5) with peptidic compound 9 at 4-5 mg/mL.

Compound 9

[0250] Formulation D: EPC:EPG (7:3) with peptidic compound 9.

[0251] Formulation E: EPC:EPG:PEG ₂ oooDSPE:DSPE-PEG ₅ ooo-Folate (6.5:3:0.47:0.03) with peptidic compound 9 at 4-5 mg/mL.

[0252] Substitution of mixed neutral lipids (EPC:cholesterol) in place of EPC alone in any of the above formulations, or those described in Examples 1 to 5, is also contemplated.

EXAMPLE 9: Treatment with GAP-107B8 peptide inhibits Akt phosphorylation.

[0253] Purpose: To compare the ratio of expressed pAKTl to total AKT in untreated control cells versus cells treated with unformulated GAP-107B8.107 peptide, as well as a PEGylated liposomal formulation of GAP-107B8.107 peptide at several concentrations.

Cell Plating and Treatment:

[0254] A2780cp ovarian cancer cells (passage 17-post thaw) were grown in DMEM (Hyclone) plus 5% fetal bovine serum and plated in 2mL of media in 6-well culture plates (BD Faclon) at a concentration of 348,000 cells/mL. Cells were allowed to adhere and grow for 23 hours in an incubator at 37°C and 5% C0 ₂ .

[0255] Free peptide GAP-107B8.107 stock was prepared at a concentration of ImM. PEGylated liposomal peptide stocks were prepared with GAP-107B8.107 concentration determined to be 3.19mg/mL.

[0256] Cells were treated with the 4 stocks mentioned above in duplicate wells. Two different volumes of each treatment were added, including: 12.5 μΙ, (6.25μΜ) and 50μΙ> (25μΜ) of free peptide (GAP-107B8.107) or PEGylated liposomes containing GAP-107B8.107 at Ι Ι . Ι μΙ, (3.13 μΜ) and 44.4μΙ. (12.5μΜ). All treatments were left on the cells for 1 hour in the incubator at 37°C and 5% C0 ₂ .

Cell Lysate Collection:

[0257] Treatment plates were removed from the incubator and the media was removed. Each well was washed 2 times with 2mL of phosphate buffered saline (PBS, Hyclone), after which 500μΙ, of ice cold lysis buffer (l OmM Tris pH 7.4, l OOmM NaCl, ImM EDTA, I mM EGTA, ImM NaF, 20mM Na P ₂ 0 ₇ , 2mM Na ₃ V0 ₄ , 1 % Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, ImM PMSF, and 250μ1_7ιηΕ Sigma protease inhibitor cocktail) was added and the plates were incubated on ice for 30 minutes. After visual verification under the microscope that the cells were detached, a pipette was then used to wash and scrape each well individually before transferring the lysed cells to microfuge tubes. The tubes were mixed by a vortex mixer before being spun in a centrifuge for 10 minutes at 13,000rpm and 4°C. Post centrifugation the cell lysates were aspirated off to fresh microfuge tubes and stored at -80°C.

BioRad® Protein Assay:

[0258] All cell lysates were diluted 1 :50 in Milli-Q® water for total protein measurement by the BioRad® protein assay. Samples were run in duplicate along-side a standard curve of BSA protein (3.75μg/mL to 30μg/mL) by adding 160μΙ. of sample or standard to wells of a 96-well microtiter plate, after which 40μΙ, of dye reagent (BioRad, 500-0006) was added and mixed. After 5 minutes the absorbance at 95nm was determined. The protein concentrations values were then used for normalization in the pAKTl and total AKT ELISAs. pAKT and Total AKT ELISA:

[0259] The concentration of pAKT l and total AKT was determined for each of the lysate samples through use of 2 ELISA kits supplied by Invitrogen (pAKTl - KHO0541, total AKT - KHO0101). All lysate samples were diluted based on the BioRad® protein assay to ensure the equal loading of 4μg of total protein for each sample onto the ELISA plates. After obtaining the concentrations of pAKTl and total AKT by ELISA, the ratio of pAKTl/AKT was determined for each sample.

Summary Conclusions:

[0260] An analysis of the data obtained by calculating the ratio pAKTl/AKT ratio obtained for lysates of A2780cp cells treated with GAP-107B8.107peptide either unformulated or associated with a PEGylated liposome after 1 hour of exposure indicates significant inhibition of AKT1 phosphorylation can be achieved. Differences relative to the control group for free unformulated GAP-107B8.107 peptide at 6.25 μΜ (21 %, p < 0.001) and 25 μ (37%, p < 0.001) exhibit a dose response whereas with PEGylated liposome formulations at 3.13 μΜ (67%, p < 0.001) and 12.5 μΜ (65%, p < 0.001 ) no dose response can be seen.

[0261] Statistically significant differences were also found between GAP-107B8.107 peptide at 6.25 μΜ and 25 μΜ (37.3%, p < 0.025) whereas no statistical differences were found between PEGylated liposome formulations at 3.13 μΜ and 12.5 μΜ respectively.

EXAMPLE 10: Intravenous Administration of unformulated GAP-107B8.107 peptide, as well as a PEGylated liposomal formulation of GAP-107B8.107

General method for IV and IP (liposome) and IV (free peptide)

[0262] In brief, rats were administered the test article intravenously (bolus injection) at an injection rate of 5 ml/kg/min. Dose given IV were 2.4 and 9.4 mg/Kg whereas doses given IP were 9.4 and 37.6 mg/Kg respectively. Dose of free peptide was 2.5 mg/Kg. On day 1 , each animal was weighed and received their respective formulations. Following administration, blood samples (up to 0.25 mL each) were collected at the following time points: one sample shortly before dose administration (i.e., predose), and then samples at 0.5, 1 , 2, 4, 8, 12, 24 and 72 hours after dose administration (IV and IP liposome study) or one sample shortly before dose administration (i.e., predose), one immediately after treatment (time 0) and then samples at 5, 15, 30, 45, 60, and 120 minutes after dose administration (IV free peptide) . After the collection of blood samples, blood was processed and the plasma was obtained. A qualified assay was developed which allowed for either free peptide or total peptide (liposomal associated) to be quantified in rat plasma. Plasma concentrations of GAP-107B8.107 were determined using an LC/MS/MS assay with an analytical range of 10-2500 ng/mL. Samples with concentrations higher than 2500 ng/mL were diluted appropriately.

[0263] Non-compartmental pharmacokinetic analysis were performed from individual plasma concentration values, these results are included in the present report. Pharmacokinetic analyses were performed on individual plasma concentrations at each time point. The maximum observed plasma concentration (C _max ) and time to C _ma , (T _max ), were observed values. Concentrations at t=0 were calculated by back extrapolation of plasma concentrations at the first two time points (0.5 and 1 hr post) following dose administration. The terminal elimination half-life (ti _/2 ) was calculated by dividing 0.693 by the K _e i (elimination rate constant). The area under the curve for the plasma concentration of GAP-107B8.107 versus the time curve from the first to the last measurable concentration (AUCo- _t ) was calculated by the linear trapezoidal method.

Summary and Conclusion.

[0264] Liposomal formulations are shown to significantly extend the half-life (8-26X) of the peptide GAP-107B8.107, increase the systemic exposure (>9X that found for free peptide) and reduce clearance rates (> 1 OX) when given by the IV or IP route. These findings indicate that liposomal formulations of peptide GAP-107B8.107 have increased plasma stability and exhibit prolonged circulation systemically. The bioavailability of Liposomal peptide given at similar doses (9.4 mg/Kg) was found to be less than 10% when given IP relative to the IV route. Further, when given IP the time to Tmax is delayed considerably indicating that systemic exposure of liposomal formulations given by this route is restricted, Increased local concentrations of drug formulations when given IP would be expected to reduce any potential toxicity risks for target organs accessible systemically. The therapeutic index of the drug would be expanded and these attributes would be useful for disease indications such as ovarian cancer (others) where tumours are found.

Table 9: Plasma concentration of GAP-107B8 in female rats after single Intravenous (IV) bolus treatment of GAP-107B8 liposomal formulation 107 or peptide.

Table 10: Plasma concentration of GAP-107B8 in female rats after single Intraperitonel (IP) bolus treatment of GAP-107B8 liposomal formulation 107 or peptide.

[0265] The disclosure of all patents, publications, including published patent applications, and database entries referenced in this specification are expressly incorporated by reference in their entirety to the same extent as if each such individual patent, publication, and database entry were expressly and individually indicated to be incorporated by reference. [0266] Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention. All such modifications as would be apparent to one skilled in the art are intended to be included within the scope of the following claims.