COCHLEATE COMPOSITIONS AND METHODS OF USE

Related Applications

This application is related and claims priority to U.S. Provisional Application Serial No. 61/149,646, filed February 3, 2009, the entire contents of which are incorporated herein by reference.

Government Support

Portions of the subject matter disclosed herein were supported by Federal Grant No. N01-AI-70043, awarded by the National Institutes of Health. The U.S. Government may have certain rights in the invention.

Background

Cochleate structures were first prepared by D. Papahadjopoulos as an intermediate in the preparation of large unilamellar vesicles (U.S. Pat. No. 4,078,052).

Cochleate compositions incorporating a variety of cargo moieties, methods of making and methods of using such cochleates have also been disclosed, e.g., in U.S. Pat. Nos.

5,840,707, 5,994,318, and 6,153,217, and International Application No. WO 04/091578.

Specifically, U.S. Pat. No. 5,840,707 discloses protein-cochleates and polynucleotide- cochleates. The entire contents of these patents are incorporated by this reference.

Summary of the Invention

Leishmaniasis, a parasitic infection, currently impacts over 12 million people every year, largely in economically disadvantaged areas. The World Health Organization estimates that 90% of visceral Leishmaniasis occurs in Bangladesh, India, Nepal, Sudan and Brazil, whereas 90% of cutaneous Leishmaniasis occurs in Afghanistan, Iran, Saudi Arabia, Syria, Peru and Brazil. Treatment for Leishmaniasis is often difficult to obtain due to cost and/or resources. For example, amphotericin B is an effective treatment for Leishmaniasis, however, it is typically delivered intravenously. This requires not only sterile water, needles and possibly a means for refrigeration and/or a hospital/clinic, but also a skilled person to administer the treatment. These resources often prevent treatment, e.g., in third world countries where such resources may be difficult or impossible to find. The present invention provides, at least in part, a means for oral administration of amphotericin B to treat a parasitic infection, such as Leishmaniasis. Such oral administration allows for a more accessible and inexpensive therapy, which in turn allows for the treatment of Leishmaniasis in a broader population. Accordingly, in some embodiments, the present invention provides methods for treating a parasitic infection comprising: administering a pharmaceutically effective amount of a composition comprising an amphotericin-B cochleate to a subject in need thereof, such that the parasitic infection is treated.

In some embodiments, the subject has Leishmaniasis, e.g., cutaneous or visceral Leishmaniasis. In some embodiments, the composition comprising an amphotericin-B cochleate is orally administered to the subject. In some embodiments, the composition comprising an amphotericin-B cochleate is administered daily for a treatment period of at least about 14 days. In some embodiments, the composition comprising an amphotericin-B cochleate is administered in a dosage of at least about 50mg of amphotericin B per kilogram of the subject's weight. In some embodiments, the composition comprising an amphotericin-B cochleate is administered within about 3-5 days of infection.

In some embodiments, the subject was exposed to at least one Leishmania parasite selected from the group consisting of Leishmania donovani, Leishmania infantum, Leishmania chagasi, Leishmania mexicana, Leishmania amazonensis, Leishmania venezuelensis, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) panamensis, and Leishmania (Viannia) peruviana. In some embodiments, the subject was exposed to Leishmania major, Leishmania chagasi or Leishmania donovani.

In some embodiments, the subject experiences reduced swelling at or near at least one infection site subsequent to administration of the composition comprising an amphotericin-B cochleate. In some embodiments, the subject experiences reduced parasite load at or near at least one infection site subsequent to administration of the composition comprising an amphotericin-B cochleate. In some embodiments, the subject experiences about a 20% reduction of parasite load. In some embodiments, the subject experiences reduced swelling within 4 days subsequent to administration of the composition comprising an amphotericin-B cochleate.

In some embodiments, the administration of the composition comprising an amphotericin-B cochleate results in an amastigote inhibition of between about 30% and about 40%.

In some embodiments, the composition administered includes about 20Og of amphotericin B, and administration of the composition exhibits at least one in vivo plasma profile selected from the group consisting of: a C _max of about 25 ng/mL to about 31 ng/mL; a T _max of about 7 hours to about 11 hours; an AUCo-i _nf of about 425 hr.ng/mL to about 475 hr.ng/mL; and an AUCo- ₂₄ of about 382 hr.ng/mL to about 432 hr.ng/mL. In some embodiments, the composition administered includes about 400g of amphotericin B, and administration of the composition exhibits at least one in vivo plasma profile selected from the group consisting of: a C _max of about 34 ng/mL to about 40 ng/mL; a T _max of about 9 hours to about 13.5 hours; an AUCo-i _nf of about 701 hr.ng/mL to about 751 hr.ng/mL; and an AUCo- ₂₄ of about 498 hr.ng/mL to about 548 hr.ng/mL. In some embodiments, the composition is a powder formulation.

Description of the Drawing

Figure 1 is a graph depicting the pharmacokinetics of exemplary cochleate compositions of the present invention.

Detailed Description of the Invention

Cochleates

In some embodiments, the present invention provides cochleates and cochleate compositions, e.g., for the treatment of Leishmaniasis. Cochleates and methods for making and using have been disclosed, e.g., in U.S. Patent Nos. 5,999,318 and 6,592,894. Cochleate delivery vehicles are stable lipid-cation precipitates that can be composed of simple, naturally occurring materials, e.g., phosphatidylserine, and calcium. Mixtures of naturally occurring molecules (e.g., soy lipids) and/or synthetic or modified lipids can be utilized.

As used herein, the terms "cochleate," "lipid precipitate" and "precipitate" are used interchangeably to refer to a lipid precipitate component that generally includes alternating cationic and lipid bilayer sheets with little or no internal aqueous space, typically stacked and/or rolled up, wherein the cationic sheet is comprised of one or more multivalent cations. Additionally, the term "encochleated" means associated with the cochleate structure. The cochleate structure provides protection from degradation for associated encochleated moieties. Divalent cation concentrations in vivo in serum and mucosal secretions are such that the cochleate structure is maintained. Hence, the majority of cochleate-associated molecules, e.g., cargo moieties such as amphotericin B, are present in the inner layers of a primarily solid, non-aqueous, stable, impermeable structure. Since the cochleate structure includes a series of solid layers, components within the interior of the cochleate structure remain substantially intact, even though the outer layers of the cochleate may be exposed to harsh environmental conditions or enzymes. The cochleate interior is primarily free of water and resistant to penetration by oxygen. Oxygen and water are primarily responsible for the decomposition and degradation of molecules which can lead to reduced shelf-life. Accordingly, encochleation can also impart extensive shelf-life stability to encochleated molecules.

With respect to storage, cochleates can be stored in cation-containing buffer, or lyophilized or otherwise converted to a powder, and stored at room temperature. If desired, the cochleates also can be reconstituted with liquid prior to administration. Cochleate preparations have been shown to be stable for more than two years at 4°C in a cation-containing buffer, and at least one year as a lyophilized powder at room temperature.

As used herein, the term "multivalent cation" refers to a divalent cation or higher valency cation, or any compound that has at least two positive charges, including mineral cations such as calcium, barium, zinc, iron and magnesium and other elements capable of forming ions or other structures having multiple positive charges capable of chelating and bridging negatively charged lipids. Additionally or alternatively, the multivalent cation can include other multivalent cationic compounds, e.g., cationic cargo moieties. The term "divalent metal cation," as used herein, refers to a metal having two positive charges.

In one embodiment, the cochleate comprises a negatively charged lipid component and a multivalent cation component. The lipid employed in the present invention may include one or more negatively charged lipids. As used herein, the term "negatively charged lipid" includes lipids having a head group bearing a formal negative charge in aqueous solution at an acidic, basic or physiological pH, and also includes lipids having a zwitterionic head group. In one embodiment, the lipid is a mixture of lipids, comprising at least 45%, at least 50%, at least 60%, at least 70% at least 80%, at least 90%, at least 95% or even at least 99% negatively charged lipid. All ranges and values between 40% and 100% negatively charged lipid are meant to be encompassed herein.

The negatively charged lipid can include soy-based lipids. In some embodiments, the lipid includes phospholipids, such as soy-based phospholipids. The negatively charged lipid can include phosphotidyl serine (PS), dioleoylphosphatidylserine (DOPS), phosphatidic acid (PA), phosphatidylinositol (PI), and/or phosphatidyl glycerol (PG) and or a mixture of one or more of these lipids with other lipids. Additionally or alternatively, the lipid can include phosphatidylcholine (PC), phosphatidylethanolamine (PE), diphosphotidylglycerol (DPG), dioleoyl phosphatidic acid (DOPA), distearoyl phosphatidyl serine (DSPS), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylgycerol (DPPG) and the like. In some embodiments, the negatively charged lipid includes phosphotidyl serine (PS). The lipids can be natural or synthetic. For example, the lipid can include esterified fatty acid acyl chains, or organic chains attached by non-ester linkages such as ether linkages (as described in U.S. Patent No. 5,956,159), disulfide linkages, and their analogs.

In one embodiment the lipid chains are from about 6 to about 26 carbon atoms, and the lipid chains can be saturated or unsaturated. Fatty acyl lipid chains useful in the present invention include, but are not limited to, n-tetradecanoic, n-hexadecanoic acid, n-octadecanoic acid, n-eicosanoic acid, n-docosanoic acid, n-tetracosanoic acid, n- hexacosanoic acid, cis-9-hexadecenoic acid, cis-9-octadecenoic acid, cis,cis-9,12- octadecedienoic acid, all-cis-9,12,15-octadecetrienoic acid, all-cis-5,8,11,14- eicosatetraenoic acid, all-cis-4,7,10,13,16,19-docosahexaenoic acid, 2,4,6, 8-tetramethyl decanoic acid, and lactobacillic acid, and the like.

The cochleates of the invention also can include non-negatively charged lipids

(e.g., positive and/or neutral lipids). The cochleates of the invention can further include additional compounds known to be used in lipid preparations, e.g., cholesterol and/or pegylated lipid. Pegylated lipid includes lipids covalently linked to polymers of polyethylene glycol (PEG). PEG's are conventionally classified by their molecular weight, thus PEG 6,000 MW, e.g., has a molecular weight of about 6000. Adding pegylated lipid generally will result in an increase of the amount of compound (e.g., peptide, nucleotide, and nutrient) that can be incorporated into the cochleate. An exemplary pegylated lipid is dipalmitoylphosphatidylehtanolamine (DPPE) bearing PEG

5,000 MW.

The cochleate compositions of the present invention can be provided in a variety of forms (e.g. powder, liquid, suspension) with or without additional components. Suitable forms and additives, excipients, carriers and the like are known in the art.

Leishmania

The present invention provides novel methods for treating parasitic infections, e.g., Leishmaniasis, using amphotericin B cochleates. That is, in some embodiments, cochleates of the present invention are useful in the treatment of Leishmaniasis.

Leishmaniasis, which is typically spread through the bite of certain species of sandflies, is caused by the parasite Leishmania. The primary host of the sandfly is the vertebrate, and Leishmania commonly infect hyraxes, canids, rodents, and humans. Leishmania currently affects 12 million people in 88 countries. Leishmania cells have two morphological forms: promastigote (with an anterior flagellum) in the insect host, and amastigote (without flagella) in the vertebrate host. Without wishing to be bound by any particular theory, it is believed that metacyclic promastigotes (the infective stage) are injected into the host by the sandfly during the bite. When it reaches the puncture wound, macrophages phagocytize the metacyclic promastigote and transform it into an amastigote. Amastigotes then multiply in infected cells and affect different tissues, depending in part on which Leishmania species is involved. These differing tissue specificities cause the differing clinical manifestations of the various forms of leishmaniasis. A cycle continues when sandflies bite infected host, thus ingesting macrophages infected with amastigotes.

Infections are regarded as cutaneous, mucocutaneous, or visceral. Cutaneous (localized and diffuse) infections appear as obvious skin reactions. Visceral infections are often recognized by fever, swelling of the liver and spleen, and anemia.

Accordingly, in some embodiments, the present invention provides methods for treating a parasitic infection. The method generally includes administering a pharmaceutically effective amount of a composition comprising an amphotericin-B cochleate to a subject in need thereof, such that the parasitic infection is treated. In some embodiments, the subject has Leishmaniasis, e.g., cutaneous Leishmaniasis or visceral Leishmaniasis.

In some embodiments, the present invention provides methods for treating Leishmaniasis which includes administering a pharmaceutically effective amount of a composition comprising an amphotericin-B cochleate to a subject in need thereof, such that the Leishmaniasis is treated. Insome embodiments, the present invention includes oral administration of composition comprising an amphotericin-B cochleate, e.g., oral administration of a powdered amphotericin-B cochleate composition.

In some embodiments, the cochleates of the present invention are useful in treating Leishmaniasis triggered by, for example, Leishmania donovani, Leishmania infantum, Leishmania chagasi, Leishmania mexicana, Leishmania amazonensis, Leishmania venezuelensis, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) panamensis, and Leishmania (Viannia) peruviana. Accordingly, in some embodiments, the present invention may be used to treat a subject who has been exposed to one or more of the above parasites. In some embodiments, the present invention is used to treat a subject who was exposed to Leishmania major. In some embodiments, the present invention is used to treat a subject who was exposed to Leishmania chagasi. In some embodiments, the present invention is used to treat a subject who was exposed to Leishmania donovani. In some embodiments, the amphotericin-B cochleate is orally administered to the subject. In some embodiments, the amphotericin-B cochleate is administered in a single dose. In some embodiments, the amphotericin-B cochleate is administered in multiple doses, e.g., two doses. In some embodiments, the amphotericin-B cochleate is administered daily for a treatment period of between 7 and 120 days. In some embodiments, the amphotericin-B cochleate is administered daily for a treatment period of about 7 days. In some embodiments, the amphotericin-B cochleate is administered daily for a treatment period of at least 14 days. In some embodiments, the amphotericin- B cochleate is administered daily for a treatment period of at least one month. In some embodiments, the amphotericin-B in the cochleate is administered in a dosage of at least about 10mg/kg, e.g., at least about 25mg/kg, e.g., at least about 50mg/kg. In some embodiments, the amphotericin-B in the cochleate is administered in a dosage of at least about 50mg/kg. In some embodiments, the amphotericin-B cochleate is administered in a bolus initial dose followed by one or more maintenance doses.

In some embodiments, the amphotericin-B cochleate is administered within a week of infection (e.g., infection with a Leishmania parasite). In some embodiments, the amphotericin-B cochleate is administered within 4 days of infection. In some embodiments, the amphotericin-B cochleate is administered within 3 days of infection.

In some embodiments, the subject experiences reduced swelling at or near at least one infection site subsequent to administration of the composition comprising an amphotericin-B cochleate. In some embodiments, the subject experiences a 5%, 10%, 20% 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in swelling at or near at least one infection site subsequent to administration of the composition comprising an amphotericin-B cochleate. In some embodiments, the subject experiences reduced parasite load at or near at least one infection site subsequent to administration of the composition comprising an amphotericin-B cochleate. In some embodiments, the subject experiences a 5%, 10%, 20% 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater reduction in parasite load at or near at least one infection site subsequent to administration of the composition comprising an amphotericin-B cochleate. In some embodiments, the subject experiences about a 20% reduction of parasite load.

In some embodiments, the subject experiences reduced swelling within 3 to 5 days following completion of the treatment period. In some embodiments, the subject experiences reduced swelling within 4 days following completion of the treatment period. In some embodiments, the subject experiences reduced parasite load within 3 to 5 days following completion of the treatment period. In some embodiments, the subject experiences reduced parasite load within 4 days following completion of the treatment period.

In some embodiments, the administration of amphotericin-B cochleates results in an amastigote inhibition of between about 10% and about 50%. As used herein, the term "amastigote inhibition" refers to the decrease in number of amastigotes in comparison to an untreated host or a host treated with a placebo. In some embodiments, the administration of amphotericin-B cochleates results in an amastigote inhibition of between about 20% and about 40%. In some embodiments, the administration of amphotericin-B cochleates results in an amastigote inhibition of between about 30% and about 40%. In some embodiments, the administration of amphotericin-B cochleates results in an amastigote inhibition of about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40%. All ranges and values between the ranges and values listed herein are meant to be encompassed by the present invention. In some embodiments, the administration of amphotericin-B cochleates results in an amastigote inhibition of about 38.07%. In some embodiments, the administration of amphotericin-B cochleates results in an amastigote inhibition of about 30.17%.

Pharmacokinetics The present invention is also based, at least in part, on the discovery that cochleates and cochleate compositions are able to provide a novel and improved pharmacokinetic profile of a cargo moiety {e.g., amphotericin B). Such pharmacokinetic profile may be advantageous, e.g., in the oral treatment of subjects {e.g., for fungal or parasitic infections). Accordingly, in some embodiments, the present invention provides for the treatment of an indication other than a parasitic infection {e.g., a fungal infection) using amphotericin B cochleates. In some embodiments, the present invention provides a method for treating a fungal or parasitic infection which includes administering to a human subject a cochleate composition comprising 10Og, 200g, 30Og, 400g, 50Og, 60Og, 70Og, 800g or 90Og of amphotericin B. In some embodiments, the present invention provides cochleate compositions that include amphotericin B {e.g., about 200g of amphotericin B), which composition exhibits upon oral administration to a subject (e.g., a human subject) at least one in vivo plasma profile selected from the group consisting of: a C _max of about 25 ng/mL to about 31 ng/mL; a T _max of about 7 hours to about 11 hours; an AUCo-i _nf of about 425 hr ng/mL to about 475 hr ng/mL; and an AUCo- ₂₄ of about 382 hr ng/mL to about 432 hr ng/mL. In one embodiment, the composition exhibits upon oral administration to a subject a C _max of about 28.11 ng/mL. In one embodiment, the composition exhibits upon oral administration to a subject a T _max of about 9 hours. In one embodiment, the composition exhibits upon oral administration to a subject an AUCo- _mf of about 449.8 hr ng/mL. In one embodiment, the composition exhibits upon oral administration to a subject an AUCo-24 of about 407.4 hr ng/mL.

In some embodiments, the present invention provides cochleate compositions that include amphotericin B (e.g., about 40Og of amphotericin B), which exhibits upon oral administration to a subject (e.g., a human subject) at least one in vivo plasma profile selected from the group consisting of: a C _max of about 34 ng/mL to about 40 ng/mL; a T _max of about 9 hours to about 13.5 hours; an AUCo-mf of about 701 hr ng/mL to about 751 hr ng/mL; and an AUCo-24 of about 498 hr ng/mL to about 548 hr ng/mL.

In one embodiment, the composition exhibits upon oral administration to a subject a C _max of about 37.09 ng/mL. In one embodiment, the composition exhibits upon oral administration to a subject a T _max of about 11.33333 hours. In one embodiment, the composition exhibits upon oral administration to a subject an AUCo-mf of about 726.0 hr ng/mL. In one embodiment, the composition exhibits upon oral administration to a subject an AUCo- ₂₄ of about 522.9 hr ng/mL.

In some embodiments, the present invention provides cochleate compositions that include amphotericin B (e.g., about 800g of amphotericin B), which exhibits upon oral administration to a subject (e.g., a human subject) at least one in vivo plasma profile selected from the group consisting of: a C _max of about 37 ng/mL to about 44 ng/mL; a T _max of about 10 hours to about 15 hours; an AUCo-i _nf of about 758 hrng/niL to about 808 hr ng/niL; and an AUCo- ₂₄ of about 600 hr ng/mL to about 650 hr ng/mL. In one embodiment, the composition exhibits upon oral administration to a subject a C _max of about 40.76 ng/mL. In one embodiment, the composition exhibits upon oral administration to a subject a T _max of about 12.66667 hours. In one embodiment, the composition exhibits upon oral administration to a subject an AUCo- _mf of about 783.3 hr ng/mL. In one embodiment, the composition exhibits upon oral administration to a subject an AUCo-24 of about 624.5 hr ng/mL.

In some embodiments, the cochleate compositions of the present invention are used in the treatment of a fungal infection. In other embodiments, the cochleate compositions of the present invention are used in the treatment of a parasitic infection. In some embodiments, the cochleate compositions of the present invention are used to reduce the toxicity of treatment with amphotericin B.

Methods of Treatment

In yet another aspect, the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) parasitic or fungal infections.

"Treatment", or "treating" as used herein, is defined as the application or administration of a therapeutic agent {e.g., antibiotics encochleated by cochleates of the invention) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward disease or disorder. "Treated," as used herein, refers to the disease or disorder being cured, healed, alleviated, relieved, altered, remedied, ameliorated improved or affected.

The terms "cure," "heal," "alleviate," "relieve," "alter," "remedy," "ameliorate," "improve" and "affect" are evaluated in terms of a suitable or appropriate control. A "suitable control" or "appropriate control" is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined prior to administration of a cargo moiety cochleate, as described herein. In another embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined in a subject, e.g., a control or normal subject exhibiting, for example, normal traits. In yet another embodiment, a "suitable control" or "appropriate control" is a predefined value, level, feature, characteristic, property, etc.

The methods of the present invention include methods of administering a cargo moiety (e.g., amphotericin B) to a host, wherein the cargo moiety is associated with a cochleate or cochleate composition of the invention. The cochleates and cochleate compositions of the present invention may be administered orally, nasally, topically, intravenously, transdermally, buccally, sublingually, rectally, vaginally or parenterally. In some embodiments, the cochleates and/or cochleate compositions of the present invention are administered orally.

The present invention provides a method for treating a subject that would benefit from administration of a composition of the present invention. Any therapeutic indication that would benefit from a cargo moiety, e.g., a substantially toxic cargo moiety, can be treated by the methods of the invention. The method includes the step of administering to the subject a composition of the invention, such that the disease or disorder is treated. The disease or disorder can be, e.g., inflammation, pain, infection, fungal infection, bacterial infection, viral infection, parasitic disorders, immune disorders, autoimmune disorders, cardiovascular disease, genetic disorders, metabolic disorders, degenerative disorders, cancer, proliferative disorders, rheumatoid arthritis, multiple myeloma or myelodisplastic syndromes.

The present invention provides a means for treating a variety of fungal infections, including, but not limited to, asthma, chronic rhinosinusitis, allergic fungal sinusitis, sinus mycetoma, non-invasive fungus induced mucositis, non-invasive fungus induced intestinal mucositis, chronic otitis media, chronic colitis, inflammatory bowel diseases, ulcerative colitis, Crohn's disease, candidemia, intraabdominal abscesses, peritonitis, pleural space infections, esophageal candidiasis and invasive aspergillosis. Exemplary fungi that can be treated using antifungal cochleates of the invention include, without limitation, Absidia, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus terreus, Aspergillus versicolor, Alternaria, Basidiobolus, Bipolaris, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida lypolytica, Candida parapsilosis, Candida tropicalis, Cladosporium, Conidiobolus, Cunninahamella, Curvularia, Dreschlera, Exserohilum, Fusarium, Malbranchia, Paecilonvces, Penicillium, Pseudallescheria, Rhizopus, Schizophylum, Sporothrix, Acremonium, Arachniotus citrinus, Aurobasidioum,

Beauveria, Chaetomium, Chryosporium, Epicoccum, Exophilia jeanselmei, Geotrichum, Oidiodendron, Phoma, Pithomyces, Rhinocladiella, Rhodoturula, Sagrahamala, Scolebasidium, Scopulariopsis, Ustilago, Trichoderma, and Zygomycete.

In some embodiments, cochleates of the present invention have the ability to reduce fungal colony forming units (CFU's) by at least 10%. More preferably, echinocandin cochleates can reduce CFU's by at least 25 % and even more preferably by 50%, 75%, 85%, 95%, ...100%. All individual values and ranges falling between these ranges and values are within the scope of the present invention. Reduction in colony forming units may be in vivo or in vitro. The host of the fungal infection can be a human or non-human animal.

The above methods can be employed in the absence of other treatment, or in combination with other treatments. Such treatments can be started prior to, concurrent with, or after the administration of the compositions of the instant invention. Accordingly, the methods of the invention can further include the step of administering a second treatment, such as for example, a second treatment for the disease or disorder or to ameliorate side effects of other treatments. Such second treatment can include, e.g., radiation, chemotherapy, transfusion, operations (e.g., excision to remove tumors), and gene therapy. Additionally or alternatively, further treatment can include administration of drugs to further treat the disease or to treat a side effect of the disease or other treatments (e.g., anti-nausea drugs).

The language "therapeutically effective amount" is that amount necessary or sufficient to produce the desired physiologic response. The effective amount may vary depending on such factors as the size and weight of the subject, or the particular compound. The effective amount may be determined through consideration of the toxicity and therapeutic efficacy of the compounds by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it may be expressed as the ratio LD _5o /ED ₅ o- Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to unaffected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any composition used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the EC ₅₀ (i.e., the concentration of the test composition that achieves a half-maximal response) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. Methods of treatment (prophylactic and therapeutic), including therapeutically effective amounts, are described, e.g., in WO 04/091572.

Methods of Forming Cochleates In one aspect, the invention provides methods for forming cochleates. Any known method can be used to form cochleates, including but not limited to those described in U.S. Patent Nos. 5,994,318 and 6,153,217, the entire disclosures of which are incorporated herein by this reference.

In one embodiment, the method generally includes introducing a cargo moiety to a lipid in the presence of a solvent, adding an aqueous solution to form liposomes, and precipitating to form a cochleate. In one embodiment, the method generally includes introducing a cargo moiety to a liposome in the presence of a solvent such that the cargo moiety associates with the liposome, and precipitating the liposome to form a cargo moiety-cochleate. In some embodiments, the method includes introducing a cargo moiety to a lipid in the presence of a pH-adjusted aqueous solution to form liposomes, and precipitating to form a cochleate. Without wishing to be bound by any particular theory, it is believed that the removal of solvents from the processing of cochleates may simplify the preparation of dried cochleate powders for reconstitution.

An antioxidant (e.g., Vitamin E) may also be employed in the methods of the present invention. It can be introduced with the cargo moiety or with the liposome. In some embodiments, it is incorporated into the liposomal suspension or a solution of the cargo moiety and solvent.

The liposome may be prepared by any known method of preparing liposomes. Thus, the liposomes may be prepared for example by solvent injection, lipid hydration, reverse evaporation, freeze drying by repeated freezing and thawing. The liposomes may be multilamellar (MLV) or unilamellar (ULV), including small unilamellar vesicles (SUV). The concentration of lipid in these liposomal solutions can be from about 0.1 mg/ml to 500 mg/ml. In some embodiments, the concentration of lipid is from about 0.5 mg/ml to about 50 mg/ml, e.g., from about 1 mg/ml to about 25 mg/ml.

The liposomes may be large unilamellar vesicles (LUV), stable plurilamellar vesicles (SPLV) or oligolamellar vesicles (OLV) prepared, e.g., by detergent removal using dialysis, column chromatography, bio beads SM-2, by reverse phase evaporation (REV), or by formation of intermediate size unilamellar vesicles by high pressure extrusion. Methods in Biochemical Analysis, 33:337 (1988). Liposomes made by all these and other methods known in the art can be used in practicing this invention.

The method is not limited by the method of forming cochleates. Any known method can be used to form cochleates from the liposomes of the invention (i.e., the liposomes associated with the cargo moiety). In one embodiment, the cochleate is formed by precipitation. The liposome can be precipitated with a multivalent cation to form a cargo moiety-cochleate. The multivalent cation can consist entirely or consist essentially of a cationic metal, including, but not limited to calcium, magnesium, barium, zinc, and/or iron. Additionally or alternatively, the multivalent cation can include other multivalent cationic compounds. Any suitable solvent can be employed in connection with the present invention. Solvents suitable for a given application can be readily identified by a person of skill in the art. In some embodiments, the solvent is an FDA acceptable solvent. The solvent can be an organic solvent or an inorganic solvent. In one embodiment, the solvent is a water miscible solvent. Suitable solvents include but are not limited to dimethylsulfoxide (DMSO), a methylpyrrolidone, N-methylpyrrolidone (NMP), acetonitrile, alcohols, e.g., ethanol (EtOH), dimethylformamide (DMF), tetrahydrofuran (THF), and combinations thereof. In general, the cargo moiety concentration within the solvent is between about 0.01 mg/ml and 200 mg/ml. In some embodiments, the cargo moiety concentration is between about 0.05 mg/ml and about 100 mg/ml, e.g., between about 0.1 mg/ml and 20 mg/ml.

The solvent can optionally be removed, e.g., before the formation of liposomes, at the liposome stage and/or after the cochleates are formed. Any known solvent removal method can be employed. For example, solvent may be removed from the liposomal suspension by tangential flow and/or filtration and/or dialysis, or from the cochleates by washing, filtration, centrifugation, and/or dialysis. The cochleates can be washed, e.g., with buffer or water, optimally with calcium or another cation.

Utilizing the methods of the invention a wide range of lipid to cargo moiety ratios can be achieved. Different ratios can have varying biological activity. The amount of cargo moiety incorporated into the cochleates can be varied as desired. The optimal lipid:cargo moiety ratio for a desired purpose can readily be determined without undue experimentation. The cochleates can be administered to the targeted host to ascertain the nature and tenor of the biologic response to the administered cochleates. It is evident that the optimized ratio for any one use may range from a high ratio to a low ratio to obtain maximal amount of cargo moiety in the cochleates. All ratios disclosed herein are w/w, unless otherwise indicated. In one embodiment, the ratio of lipid to cargo moiety is between about 10,000:1 and 1000:1. Ratios in this range may be suitable when it is desired to administer small amounts of the moiety, (e.g. , in the case of administration of radioactive agents or highly active, rare or expensive molecules). In another embodiment, the ratio is between about 8,000:1 and 4,000:1, e.g., about 6,000:1. Such a ratio may be suitable, e.g., in delivering porphyrins. In yet another embodiment, the ratio is between about 5,000: 1 and 50:1. In yet another embodiment, the ratio of the lipid to the cargo moiety is between about 20:1 and about 0.5:1. In another embodiment, the ratio of the lipid to the cargo moiety is between about 1 : 1 and about 10:1. Such a ratio may be suitable, e.g., for delivery of an antifungal agent. In yet another embodiment, the ratio of lipid to the cargo moiety is about 2:1, about 3:1, or between about 1.5:1 and 3.5:1. All individual values and ranges between about 0.25:1 and about 40,000:1 are within the scope of the invention. Further values also are within the scope of the invention. The cochleate formulations also can be prepared both with and without targeting molecules (e.g., fusogenic molecules, such as Sendai virus envelope polypeptides), to target specific cells and/or tissues. Formation of the cochleates of the invention in the above methods involves crystallization of multivalent cation with negatively charged lipids. It is evident, therefore, that all of the parameters that govern crystallization, e.g., temperature, lipid concentration, multivalent cation concentrations, rate of cation addition, pH and rate of mixing, can be utilized to regulate cochleate formation. In certain embodiments, ionic conditions can be created or adjusted to affect the efficiency of the association and/or the encochleation of the cargo moiety. For example, increasing the salt concentration in a liposomal suspension can render the environment less hospitable to a hydrophobic or amphipathic cargo moiety, thereby increasing liposome and cochleate loading efficiency. Ionic conditions can also affect the ultimate structure of the cochleate generated. High loads of a cargo moiety can also affect the highly ordered structure observed in cochleates formed, e.g., exclusively from calcium and PS. Additionally or alternatively, pH conditions can be created or adjusted to affect the loading and structure of the resulting cochleates. Such variations can readily be manipulated by the skilled practitioner using no more than the instant specification and routine experimentation. In addition, because a cochleate is highly thermodynamically stable, once a cochleate formulation method is developed for a given product, the end product can be made predictably and reliably.

In another aspect, the present invention generally is directed to methods of making cochleates that include an aggregation inhibitor. The aggregation inhibitor can be introduced prior to, during or after formation of cochleates. In some embodiments, aggregated cochleates may be disaggregated using alternative disaggregation methods, e.g., homogenization, and an aggregation inhibitor can be introduced in order to prevent reaggregation.

The method can include forming cochleates with any or all of the optional ingredients disclosed herein. For example, the cochleates can include additional cationic compounds, cargo moieties, non-negative lipids, and/or aggregation inhibitors.

Any of the methods described herein can be utilized to produce anywhere from about lmg to about 50Og of cochleates in one batch. A smaller batch may be preferred in a laboratory setting where characterization of cochleates is desired. On the other hand, larger batches may be preferred in a manufacturing setting where mass production is desired. In some embodiments, larger batches are at least 5Og, e.g., at least 75g.

Once a cochleate formulation is formed, cochleates may optionally be dried. In some embodiments, dried cochleates are formed using commercially available processes for creating a dry powder from a liquid suspension, including spray drying, fluid bed drying, lyophilization and the like. In some embodiments, spray drying is used to dry cochleate formulations. Without wishing to be bound by any particular theory, it is believed that such dried cochleate formulations may be advantageous, e.g., because they may have a longer shelf life and/or may contain no residual solvent.

Safety/Biocompatibility Cochleates readily can be prepared from safe, simple, well-defined, naturally occurring substances, e.g., PS and calcium. Mixtures of naturally occurring {e.g., soy lipids), synthetic lipids, and/or modified lipids can also be utilized. Phosphatidylserine is a natural component of all biological membranes, and is most concentrated in the brain. The phospholipids used can be produced synthetically, or prepared from natural sources. Soy PS is inexpensive, available in large quantities and suitable for use in humans. Clinical studies indicate that PS is safe and may play a role in the support of mental functions in the aging brain. Unlike many cationic lipids, cochleates (which are composed of anionic lipids) are non-inflammatory and biodegradable. The tolerance in vivo of mice to multiple administrations of cochleates by various routes, including intravenous, intraperitoneal, intranasal and oral, has been evaluated. Multiple administrations of high doses of cochleate formulations to the same animal show no toxicity, and do not result in either the development of an immune response to the cochleate matrix, or any side effects relating to the cochleate vehicle.

The cochleates of the invention not only protect the cargo moiety from the host (e.g., from decomposition by proteolytic enzymes in the digestive tract), but also protect the host from the cargo moiety (e.g., preventing damage to vital organs caused by toxic levels of certain cargo moieties). In addition, the cochleates of the invention allow for efficient delivery of the cargo moiety across the digestive tract and to cells, e.g., by fusion and/or cellular uptake. Thus, a lower dosage of cargo moiety can be administered to generate the same beneficial results as compared to conventional preparations, while minimizing the incidence of toxic side effects and/or buildup of cargo moiety in the digestive tract

The cochleates and cochleate compositions of the present invention can be administered to animals, including both human and non-human animals. It can be administered to animals, e.g., in animal feed or water. Accordingly, in some embodiments, the present invention is directed to reducing the toxicity of a substantially toxic cargo moiety in a subject, e.g., as compared to the unencochleated substantially toxic cargo moiety. The method generally includes encochleating the substantially toxic cargo moiety. In some embodiments, the present invention is directed to reducing the systemic toxicity of a substantially toxic cargo moiety. In some embodiments, the present invention provides methods of reducing the organ toxicity (e.g., renal toxicity) of a substantially toxic cargo moiety. Without wishing to be bound by any particular theory, it is believed that cochleate structures are taken up by macrophages, which reduces not only the acute toxicity of a cargo moiety, but also the systemic toxicity. Although other particulate items are taken up by macrophages, they do not necessarily exhibit low toxicity in animals. Cochleate compositions, e.g., amphotericin B cochleates, are shown to have reduced toxicity in in vivo studies for extended administration (see, e.g., Example 4 herein).

In some embodiments, the present invention is directed to reducing the toxicity of a substantially toxic cargo moiety, thus allowing substantially toxic cargo moieties to be administered to a subject. In some embodiments, an encochleated substantially toxic cargo moiety can be administered over a period of 1-10 weeks, e.g., daily for 7, 14, 21 or 28 days or more. In some embodiments, an encochleated substantially toxic cargo moiety can be administered over a period of greater than 10 weeks.

Without wishing to be bound by any particular theory, it is believed that activated phagocytes, including macrophages, neutrophils and dendritic cells, are professional particle scavengers. Foreign particles are rapidly cleared from the circulation by these cells and thus cochleates being foreign particles would be taken up by phagocytes. This phenomenon is seen with liposomes as well. If the surface of a liposome is not derivatized with special ligands, creating what has been called "stealth" liposomes, liposomes are rapidly cleared from the blood stream. Without wishing to be bound by any particular theory, it is also believed that cells generally have a natural lifespan before they begin to die. Macrophages are the "vacuum cleaners" that take up and destroy (clean up) the dead cell debris (a noninflammatory process). Therefore, macrophages can clean up debris without causing additional damage. One of the first signals of apoptosis is the appearance of phosphatidylserine on the external membranes of apoptotic cells. Macrophages have phosphatidylserine specific receptors, so the uptake of cochleates by macrophages may be mediated by specific receptors.

Once associated with a cell's membrane surface there are at least 2 possible mechanisms for drug release by cochleates. One is fusion of the cochleate membrane with the cell membrane. The cochleate lipid matrix becomes part of the cell membranes, and the drug is then free to interact with cellular components. A second is endocytosis of the intact cochleate into the cytoplasm of the cell. Extracellular fluid, including plasma, GI secretions and interstitial fluid, have a calcium concentration of 1.5-4 mM. This is sufficient calcium to maintain the cochleate structure. However, cells tend to aggressively maintain their cytoplasmic calcium concentration in the μM range. Thus, once within the low calcium environment of the cytoplasm the structure of the cochleate begins to loosen and the drug is released. These mechanisms are not mutually exclusive.

Cargo Moieties The cochleates of the present invention are associated or "loaded" with a cargo moiety (e.g., amphotericin B). A "cargo moiety" is a moiety to be encochleated, and generally does not refer to the lipid and ion employed to precipitate the cochleate. For example, in some embodiments, the cargo moiety is amphotericin B (e.g., where the cochleates are useful for the treatment of Leishmaniasis. Cargo moieties include any compounds having a property of biological interest, e.g., ones that have a role in the life processes of a living organism. A cargo moiety may be organic or inorganic, a monomer or a polymer, endogenous to a host organism or not, naturally occurring or synthesized in vitro and the like. In some embodiments, the cargo moiety is amphotericin B.

In some embodiments, the cochleates of the present invention are associated or "loaded" with a substantially toxic cargo moiety. A "substantially toxic cargo moiety" is a cargo moiety that is considered at least partially toxic at therapeutic doses. For example, in some embodiments, a substantially toxic cargo moiety is a cargo moiety that is classified as at least "Moderately Toxic" on the Hodge and Sterner Scale. In some embodiments, a substantially toxic cargo moiety is a cargo moiety that has an oral LD ₅₀ of less than about 100 mg/kg. In some embodiments, a substantially toxic cargo moiety is a cargo moiety that has an oral LD ₅₀ of less than about 50 mg/kg. In some embodiments, a substantially toxic cargo moiety is a cargo moiety that has an oral LD ₅₀ of less than about 25 mg/kg. In some embodiments, a substantially toxic cargo moiety is a cargo moiety that has an oral LD ₅₀ of less than about 10 mg/kg. In some embodiments, a substantially toxic cargo moiety is a cargo moiety that has an oral LD ₅₀ of less than about 5 mg/kg. In some embodiments, a substantially toxic cargo moiety is a cargo moiety that has an oral LD ₅₀ of less than about 1 mg/kg.

Substantially toxic cargo moieties include, but are not limited to amphotericin B, actinomycin D, aminopterin, benzedrine, busulfan, cisplatin, cyclophosphamide, daunomycin, melphalan, mytomycin C, myleran, uracil mustard and warfarin. Additional substantially toxic cargo moieties include p38 inhibitors. Suitable p38 inhibitors include, but are not limited to RO4402257 (Hoffmann-LaRoche), RO3201195 (Hoffmann-LaRoche), PH-797804 (Pfizer), AZD-6703 (AstraZeneca), GW681323 or SB-681323 (GlaxoSmithKline), VX-745 (Vertex Pharmaceuticals), VX-702 (Vertex Pharmaceuticals/Kissei), Scio-469 (Scios/Johnson & Johnson), Scio-323 (Scios/Johnson & Johnson), TAK-715 (Takeda Pharmaceuticals), PS540446 (Pharmacopeia/ Bristol- Myers Squibb), RWJ-67657 (Johnson & Johnson), BIRB-796 (Boehringer Ingelheim), KC706 (Kemia), ARRY-797 (Array BioPharma) and AMG-548 (Amgen). See, e.g., Roberts et al. Oncogene 26:3291-3310 (2007). The present invention provides, in some embodiments, novel means for reducing the toxicity of a cargo moiety, e.g., a substantially toxic cargo moiety, by encochleating the cargo moiety. Compositions made by such a method provide all the advantages of conventional cochleates, and additionally provide means for delivering a toxic cargo moiety to a subject in a non-toxic manner.

In some embodiments, an encochleated substantially toxic cargo moiety is administered to a subject for at least 7 days with no toxicity. In some embodiments, an encochleated substantially toxic cargo moiety is administered to a subject for at least 14, 21 or 28 days with no toxicity. In some embodiments, an encochleated substantially toxic cargo moiety is administered to a subject for at least 3 months with no toxicity.

In some embodiments, the compounds of the present invention also include one or more additional cargo moieties. Thus, examples include vitamins, minerals, nutrients, micronutrients, amino acids, toxins, microbicides, microbistats, co-factors, enzymes, polypeptides, polypeptide aggregates, polynucleotides, lipids, carbohydrates, nucleotides, starches, pigments, fatty acids, saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, flavorings, essential oils, extracts, hormones, cytokines, viruses, organelles, steroids and other multi-ring structures, saccharides, metals, metabolic poisons, antigens, imaging agents, porphyrins, tetrapyrrolic pigments, drugs and the like. The drug can be, but is not limited to, a protein, a small peptide, a bioactive polynucleotide, an antibiotic, an antiviral, an anesthetic, antipsychotic, an anti- infectious, an antifungal, an anticancer, an immunosuppressant, an immunostimulant, a steroidal anti-inflammatory, a non-steroidal anti-inflammatory, an antioxidant, an antidepressant which can be synthetically or naturally derived, a substance which supports or enhances mental function or inhibits mental deterioration, an anticonvulsant, an HIV protease inhibitor, a non-nucleophilic reverse transcriptase inhibitor, a cytokine, a tranquilizer, a mucolytic agent, a dilator, a vasoconstrictor, a decongestant, a leukotriene inhibitor, an anti-cholinergic, an anti-histamine, a cholesterol lipid metablolism modulating agent or a vasodilatory agent. The drug can also be any over the counter (non-prescription) medication. Specific cargo moieties are well known in the art and can be found, for example, in US Patent Application Publication No. 2005/0013854, the entire contents of which are hereby incorporated by this reference.

The cochleates of the invention can be prepared with a wide range of cargo moiety to lipid ratios. By way of example, the ratio of cargo moiety to lipid can be between about 20,000: 1 and about 0.5:1 by weight. In one embodiment the ratio is about 1:1 by weight. In others the ratio is about 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 50:1, 100: 1, 200: 1, or 400: 1 by weight. All individual ranges and values between 20,000: 1 and 0.5:1 are encompassed by the invention. Additional pharmacologically active agents may be delivered in combination with the primary active agents, e.g., the cochleates of this invention.

The cargo moiety can additionally be bound to a cochleate component or to a hydrophobic tail. In one embodiment, the cargo moiety is bound to the lipid cochleate component or the hydrophobic tail with a digestible, reducible, or otherwise reversible linker. The cargo moiety can be bound in a reversible manner (e.g., with a reducible or digestible linker) or a linker susceptible to target conditions (e.g., pH, temperature, ultrasonic energy and the like). This is particularly useful as the linker can be chosen such that it is readily digestible, e.g., by an enzyme, in the body generally or even in a target structure. Thus, e.g., a linker can be chosen such that it is degraded by an enzyme in the plasma, interstitial fluids, in a cell (e.g. a macrophage) or in an endosome, such that the protonized cargo moiety becomes detached and available in unbound form in these structures. In another embodiment, the reversible linker can be an electrostatic or other bond that is broken by a change in pH, e.g., in an organ or other structure in which the cochleate experiences a pH gradient. In another embodiment, the linker is reversed by a change in temperature, e.g., by exposure to body temperature.

Aggregation Inhibitors

In some embodiments, the cochleates of the present invention can optionally include one or more aggregation inhibitors. The term "aggregation inhibitor," as used herein, refers to an agent that inhibits aggregation of cochleates. The aggregation inhibitor typically is present at least on the surface of the cochleate, and may only be present on the surface of the cochleate (e.g., when the aggregation inhibitor is introduced after cochleate formation). The type and/or amount of aggregation inhibitor can be adjusted to obtain a desired cochleate size and/or distribution. Additionally or alternatively, aggregation inhibitor(s) can be used to stabilize cochleate size and/or size distribution such that aggregation of cochleates is minimized or eliminated. The terms "coat," "coated," "coating," and the like, unless otherwise indicated, refer to an agent (e.g. an aggregation inhibitor) present at least on the outer surfaces of a cochleate. Such agents may be associated with the bilayer by incorporation of at least part of the agent into the bilayer, and/or may be otherwise associated, e.g., by ionic attraction to the cation or hydrophobic or ionic attraction to the lipid. Suitable aggregation inhibitors that can be employed in accordance with the present invention, include but are not limited to at least one of the following: casein, K- casein, milk, milk products, albumin, serum albumin, bovine serum albumin, rabbit serum albumin, methylcellulose, ethylcellulose, propylcellulose, hydroxycellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinyl pyrrolidone, carboxymethyl cellulose, carboxyethyl cellulose, pullulan, polyvinyl alcohol, sodium alginate, polyethylene glycol, polyethylene oxide, xanthan gum, tragacanth gum, guar gum, acacia gum, arabic gum, polyacrylic acid, methylmethacrylate copolymer, carboxyvinyl polymer, amylose, high amylose starch, hydroxypropylated high amylose starch, dextrin, pectin, chitin, chitosan, levan, elsinan, collagen, gelatin, zein, gluten, carrageenan, carnauba wax, shellac, latex polymers, milk protein isolate, soy protein isolate, whey protein isolate and mixtures thereof. In some embodiments, the aggregation inhibitor includes casein. In some embodiments, the aggregation inhibitor includes methylcellulose.

More than one aggregation inhibitor may be employed in the compositions of the invention. For example, both milk and methylcellulose may be used as an aggregation inhibitor.

In one embodiment, the cochleate compositions of the invention include between about 10% and about 0.1% aggregation inhibitor. In some embodiments, the aggregation inhibitor comprises about 1% of the cochleate composition. A person of ordinary skill in the art will readily be able to determine the amount of aggregation inhibitor needed to form cochleates of the desired size with no more than routine experimentation . Cochleate Size and Distribution

The formation of cochleates can be envisioned as a crystallization event that occurs upon the interaction of charged lipids and oppositely charged multivalent cations. Modulating of the size of cochleate crystals formed, however, has proved difficult. In aqueous suspension, plain cochleates generally aggregate and upon long term storage form larger masses which can be several microns in size. Because of the association of the calcium with the lipid head group, the surfaces of cochleates have a hydrophobic character. When suspended in aqueous buffer, cochleate aggregation is a consequence of hydrophobic interactions, minimizing the amount of surface area exposed to water.

It has been discovered that aggregation can be inhibited and even reversed, and individual cochleate particles can be stabilized by changing the surface properties of the cochleates and thereby inhibiting cochleate-cochleate interaction. Aggregation can be inhibited by including in the liposome suspension a material that prevents liposome- liposome interaction at the time of calcium addition and thereafter. Alternatively, the aggregation inhibitor can be added after formation of cochleates. Additionally, the amount of aggregation inhibitor can be varied, thus allowing modulation of the size of the cochleates.

Accordingly, the present invention provides a cochleate composition comprising a plurality of cochleates and an aggregation inhibitor having a desired particle size distribution, and methods of making the same. The amount of aggregation inhibitor and/or time of addition can be varied to modulate and/or stabilize the size and/or size distribution of a cochleate composition.

In one embodiment, the aggregation inhibitor can be employed to achieve cochleates that are significantly smaller and have narrower particle size distributions than compositions without aggregation inhibitors. Such compositions are advantageous for several reasons including that they can allow for greater uptake by cells and rapid efficacy. Such a composition is suitable, e.g., when it is desired to rapidly and effectively deliver a cargo moiety {e.g., an antifungal or antibacterial agent against a fungal or bacterial infection). Moreover, cochleate size can have a targeting affect in that some cells may take up particles of a certain size more or less effectively. Size may also affect the manner in which cochleates interact with a cell (e.g., fusion events or uptake).

In another embodiment, the aggregation inhibitor can be employed in an amount to achieve cochleate compositions having a particle size relatively larger than that which can be achieved without or with other aggregation inhibitors (e.g., if more and/or a different aggregation inhibitor used). Such a composition can be useful, e.g., when delayed uptake and/or release of the cargo molecule is desired, or when targeted cells or organs more effectively take up cochleates in the relatively larger size range. Such compositions also may have sustained activity (relative to smaller cochleate compositions) because it can take longer for the cargo moiety to be released from a larger cochleate, e.g., if multiple fusion events are required.

In yet another embodiment, the amount and/or types of aggregation inhibitor can be chosen to manufacture a cochleate composition that has a wide particle size distribution such that the cargo moiety is released over a period of time because smaller cochleates are rapidly taken up initially followed by take up or fusion events with increasingly larger cochleates. In addition, size may not only affect what type of cells take up the cochleate, but also how the cochleates interact with certain cells, e.g., size may effect whether a cochleate is taken up by a cell or undergoes one or more fusion events with a cell. Cochleate compositions of the invention may have a mean diameter less than about 5, 4, 3, 2, or 1 micrometer. In some embodiments, the cochleate compositions of the present invention have a mean diameter less than about 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. All individual values between these values (880, 435, 350, etc.), are meant to be included and are within the scope of this invention. In some embodiments, the size distribution is narrow relative to that observed in standard cochleates (cochleates formed without aggregation inhibitors). In some embodiments, the cochleates have a size distribution of less than about 30, 20, 10, 5, 3 or 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. All individual values between these values (550 nm, 420 nm, 475 nm, etc.), are meant to be included and are within the scope of this invention. In another embodiment, a wider size distribution of cochleates is employed, e.g., about 10, 20, 50, 100, 200 ... 500 micrometers. All individual values within these ranges are meant to be included and are within the scope of this invention.

Additionally, the invention contemplates combination of cochleate populations with one or more cargo moieties, one or more size distributions, and one or more mean diameter, to achieve a desired release pattern, e.g., pulsed release, delayed release and/or timed release of different cargo moieties.

Delivery of Cargo Moieties

Many naturally occurring membrane fusion events involve the interaction of calcium with negatively charged phospholipids (e.g., PS and phosphatidylglycerol).

Calcium-induced perturbations of membranes containing negatively charged lipids, and the subsequent membrane fusion events, are important mechanisms in many natural membrane fusion processes. Therefore, cochleates can be envisioned as membrane fusion intermediates. Cochleates are useful for the delivery of a cargo moiety to cultured cells, tissues or organisms by a variety of administration routes. The term "delivery," as used herein, refers to any means of bringing or transporting a cargo moiety to a host, a food item, a formulation, a pharmaceutical composition, or any other system, wherein the cargo moiety maintains at least a portion of its activity. For example, the use of cochleates to deliver protein or peptide molecules as vaccines has been disclosed in U.S. Pat. No. 5,840,707, issued November 24, 1998. Similarly, polypeptide-cochleates are effective immunogens when administered to animals by intraperitoneal and intramuscular routes of immunization (G. Goodman-Snitkoff, et al., J. Immunol., Vol. 147, p.410 (1991); M. D. Miller, et al., J. Exp. Med., Vol. 176, p. 1739 (1992)). Further, cochleates are effective delivery vehicles for encapsulated proteins and/or DNA to animals and to cells in culture. For example, reconstituted Sendai or influenza virus glycoproteins are efficiently delivered in encochleated form (Mannino and Gould-Fogerite, Biotechniques 6(l):682-90 (1988); Gould-Fogerite et al., Gene 84:429 (1989); Miller et al., J. Exp. Med. 176: 1739 (1992)). The cochleates can be co-administered with a further agent. The second agent can be delivered in the same cochleate preparation, in a separate cochleate preparation mixed with the cochleate preparation of the invention, separately in another form (e.g., capsules or pills), or in a carrier with the cochleate preparation. The cochleates can further include one or more additional cargo moieties, such as other drugs, peptides, nucleotides (e.g., DNA and RNA), antigens, nutrients, flavors and/or proteins. In some embodiments, the amphotericin B cochleates of the present invention are co- administered with an additional anti-leishmanial treatment, such as B-miltefosine, paromomycin, and/or antimony treatments such as SbV.

The cochleates of the invention also can include a reporter molecule for use in in vitro diagnostic assays, which can be a fluorophore, radiolabel or imaging agent. The cochleates can include molecules that direct binding of the cochleate to a specific cellular target, or promotes selective entry into a particular cell type.

One advantage of the cochleates of the present invention is the stability of the composition. Cochleates can be administered by any route, e.g., mucosal or systemic, without concern. Cochleates can be administered orally or by instillation without concern, as well as by the more traditional routes, such as oral, intranasal, intraoculate, intrarectal, intravaginal, intrapulmonary, topical, subcutaneous, intradermal, intramuscular, intravenous, transdermal, systemic, intrathecal (into CSF), and the like. Direct application to mucosal surfaces is an attractive delivery means made possible with cochleates. Delivery can be effected by, e.g., a nasal spray or nasal bath or irrigation. Another advantage of the present invention is the ability to modulate cochleate size. Modulation of the size of cochleates and cochleate compositions changes the manner in which the cargo moiety is taken up by cells. For example, in general, small cochleates are taken up quickly and efficiently into cells, whereas larger cochleates are taken up more slowly, but tend to retain efficacy for a longer period of time. Also, in some cases small cochleates are more effective than large cochleates in certain cells, while in other cells large cochleates are more effective than small cochleates.

Cochleates and cochleate compositions can also be administered in food or beverage preparations. Such compositions can be introduced to the food or beverage compositions by the manufacturer (e.g., to supplement food with nutrients), or by the consumer (e.g., where the cochleate composition is sold separately as a food additive). For example, amphotericin B cochleates of the present invention may be added to normal food preparations (e.g., yogurt, etc.) such that sterile water is not necessary to reconstitute the cochleate formulation. Cochleates may be added at any stage into the preparation of food, as the cochleates are stable under extreme pressure and temperature conditions.

Another advantage of cochleates and cochleate compositions of the present invention is their ability to reduce a number of unwanted side effects. A number of drugs cause gastrointestinal distress and often high circulating blood levels lead to toxicity in a number of vital organs. In some embodiments, cochleates of the present invention may lower gastrointestinal distress, circulating blood levels or organ toxicity. For example, cochleates can be formulated for uptake by particular cells or organs. Conventionally, high levels of drugs are often administered intravenously to obtain moderate levels at the sites of infection in order to combat opportunistic infections. This can cause undesirable side effects, such as erythematous or urticarial reactions, flushing, tachycardia, and hypotension, generally non-permanent auditory impairment, ototoxicity associated with excessively high concentrations of the drug in plasma and less commonly, nephrotoxicity. By employing the cochleates of the present invention, toxicity levels can be lowered by decreasing the free drug in the circulating blood. Additionally, the cargo moiety can be delivered directly to the site of infection, which can lower or eliminate the incidence of gastrointestinal distress.

Cochleates of the present invention can be employed to avoid harmful side effects of drugs caused by their high concentration or presence in organs such as the kidneys, stomach or liver.

Pharmaceutical Compositions

The invention pertains to uses of the cochleate compositions of the invention for prophylactic and therapeutic treatments as described infra. Accordingly, the compounds of the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the compositions of the invention and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, wetting agents, emulsifiers and lubricants, antioxidants, water, antimicrobial agents, plasticizing agents, flavoring agents, surfactants, stabilizing agents, emulsifying agents, thickening agents, binding agents, coloring agents, sweeteners, fragrances, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.

Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which may be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of 100%, this amount will range from about 1% to about 99% of active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, gelcaps, crystalline substances, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, gel, partial liquid, spray, nebulae, mist, atomized vapor, aerosol, tincture, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) or as mouth washes and the like, each containing a predetermined amount of a composition of the present invention as an active ingredient. A composition of the present invention may also be administered as a bolus, electuary, or paste.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms for oral administration of the compounds of the invention may be formulated with water (e.g., sterile water or non-sterile water). In some embodiments, the present invention provides a cochleate composition which is dissolved or suspended in non-sterile water. In some embodiments, the present invention provides a cochleate composition which is dissolved or suspended in another edible carrier, e.g., juice, milk, yogurt, soup, etc.

The cochleates of the present invention can be administered to animals, including both human and non-human animals. They can be administered to animals, e.g., in animal feed or water. Methods for preparing pharmaceutical compositions containing the compositions of the present invention, including additional agents (e.g., wetting agents, emulsifiers and lubricants) used in such compositions, may be dependent upon the method of administration. Such method are known in the art, e.g., in WO 04/091572.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The pharmaceutical compositions can be included in a container along with one or more additional compounds or compositions and instructions for use. For example, the invention also provides for packaged pharmaceutical products containing two agents, each of which exerts a therapeutic effect when administered to a subject in need thereof. A pharmaceutical composition may also comprise a third agent, or even more agents yet, wherein the third (and fourth, etc.) agent can be another agent against the disorder, such as a cancer treatment (e.g., an anticancer drug and/or chemotherapy) or an HIV cocktail. In some cases, the individual agents may be packaged in separate containers for sale or delivery to the consumer. The agents of the invention may be supplied in a solution with an appropriate solvent or in a solvent- free form (e.g., lyophilized). Additional components may include acids, bases, buffering agents, inorganic salts, solvents, antioxidants, preservatives, or metal chelators. The additional kit components are present as pure compositions, or as aqueous or organic solutions that incorporate one or more additional kit components. Any or all of the kit components optionally further comprise buffers.

Practice of the invention will be more fully understood from the following examples, which are presented herein for illustration only and should not be construed as limiting in any way.

EXEMPLIFICATION

Example 1: Preparation of amphotericin B Cochleates (CAMB) and placebo cochleates

Amphotericin B cochleates (CAMB) were prepared with phospholipid to AmB ratio of 5:1 as follows. Amounts are provided in Table 1 for 35 and 87kg batches for preparation of the 2mg/mL intermediate formulation.

Liposomes are prepared by adding lecithin enriched with 50% phosphatidylserine (PS P50X) obtained from Lipoid (Ludwigshafen, Germany) to an EDTA solution and filtering the resultant mixture using a Millipore Opticap 5 μm filter. Amphotericin B (U.S. Pharmacopeia grade, obtained from Alpharma, Copenhagen, Denmark) was solubilized in dimethyl sulfoxide (DMSO) and vitamin E was added. The amphotericin B solution was then added to the liposomes at a pH of 8.85 through a spray nozzle, thus forming amphotericin B liposomes. Calcium chloride was then added through a spray nozzle to form cochleate structures containing 2 mg/mL amphotericin B.

Tangential flow filtration (TFF) using hollow fiber cartridges was then performed to concentrate the solution by about 2.5-fold. A six volume diafiltration with 2 mM CaCl ₂ was then performed to remove the DMSO solvent and other unbound excipients. Methylcellulose, methylparaben and propylparaben were then added and the solution was and adjusted to a final concentration of 5 mg/mL amphotericin B. The components are as listed below.

1 Starting batch size of 35 kg, theoretical ending batch size of 14 kg after TFF.

2 Starting batch size of 87.5 kg, theoretical ending batch size of 35 kg after TFF.

3 Total amount of calcium chloride used to make 2 mM CaCh for the TFF step.

The manufacturing process for the placebo was the same process as for the cochleate amphotericin B suspension drug product, except for the addition of β-carotene as a colorant in the final formulation and the absence of amphotericin B. Liposomes were prepared by adding PS P50X to an EDTA solution and filtering. Vitamin E was added to the DMSO and this DMSO solution was added to the liposomes. Calcium chloride was added to form cochleate structures.

Tangential flow filtration using hollow fiber cartridges was performed to remove the DMSO solvent and other unbound excipients, to buffer exchange into 2 mM CaCl ₂ , and to concentrate the suspension to 25 mg/mL phospholipid. The final formulation step was the addition of methylcellulose, methylparaben, propylparaben and β-carotene. The components are as listed below.

1 A 99% super refined soybean oil, 1% beta-carotene 20% solution

Example 2: Amphotericin B Cochleates for treatment of Leishmania major parasitic infections Materials

Female BALB/c mice weighing 18-20 g maintained under pathogen-free conditions were used herein.

Leishmania Parasites and Culture Conditions: L. major Friedlin V9 strain, grown as previously described, were used (see, e.g., Zhang, W. et al., 2003, J. Biol. Chem. 278: 35508-35515; and Zhang, W. and G. Matlashewski, 2004, MoI. Microbiol. 54: 1051-1062). Briefly, promastigotes were cultured at 27°C in M199 medium (pH 7.4) supplemented with 10% heat-inactivated fetal bovine serum, 40 mM Hepes (pH 7.4), 0.1 mM Adenine, 5 mg 1 1 Haemin, 1 mg 1 1 Biotin, 1 mg 1 1 Biopterine, 50 U ml 1 Penicillin and 50 μg ml 1 Streptomycin. Oral amphotericin B cochleate (CAMB) formulations with varying amphotericin

B concentrations as well as cochleate placebo were prepared as described in Example 1, immediately before the start of each drug trial. The CAMB formulations and cochleate placebo were allocated in tubes in the amount of daily required dose, stored at 4°C and protected from light. Methods

Infection of Mice: Stationary phase L. major promastigotes were harvested and washed once with phosphate buffered saline (PBS) and resuspended in PBS at a concentration of 2.5 x 108 ml ^-1 . 1 x 10 ⁷ L. major promastigotes in a volume of 40 μl were injected subcutaneously into the left rear footpad of each BALB/c mouse.

Oral administration of CAMB: Drug treatments in mice were started three days after Leishmania infection. Curved ball-tipped feeding needles (22 gauge) were used for oral dosing of CAMB formulas for BALB/c mice. A maximum of 200 μl of CAMB formulas or placebo were applied to each -20 g mouse. The tubes containing CAMB formulations and placebo were thoroughly vortexed before using and discarded after each use.

Footpad lesion monitoring and parasites quantification: Footpad (infection site) swelling measurements (weekly or every two days) were recorded using a metric caliper.

Parasite loads in the footpads of infected mice were determined by limiting dilution.

The number of viable parasites in the footpad was determined from the highest dilution that promastigotes could be grown out after 10 days of culture at 27°C.

Statistical analyses: The statistical difference between groups was determined by Student's t-test (for two groups) or by single factor Anova (for three or more groups). A P value of < 0.05 was considered significant. Five to ten mice were included in each experimental group.

Oral trial with high CAMB dose: A study was carried out with CAMB dosages including 25mg/ml and 50 mg/ml. In the study, Leishmania major (L major) infected BALB/c mice were divided into four groups (5 -10 mice per group). Mice were treated with oral CAMB at 25-50 mg/kg of body weight daily for a total of 14 days. Since the drug volume allowed to feed a 20g mouse is limited to 200 μl, 5 mg amphotericin B/ml in CAMB formula or 50 mg/kg for mice was the maximum dose the CAMB formula can achieve in this mouse model. Mice treated with 25 mg/kg CAMB still developed Leishmania lesions of similar size to lesions in mice receiving control placebo or saline treated mice. Mice treated with CAMB at 50 mg/kg had lesions of similar size to other mice before the CAMB treatment course was completed. However, beginning about 4 days after the completion of CAMB treatment course, the lesions of mice orally treated with CAMB at 50 mg/kg were observed to be smaller than these of control mice or mice treated with CAMB at 25 mg/kg (lesion size differences are statistically significant, with P<0.05). Two weeks after the completion of CAMB treatment course, four mice in each group were sacrificed for examination of footpad lesion parasite burden by limiting dilution. Mice treated with 50mg/kg oral CAMB had fewer parasites (about 20% less) in their footpad lesions than other groups of mice. Taken together, this data demonstrates that high oral CAMB dosage (50 mg/kg, the highest achievable dosage) showed inhibitory effects on footpad Leishmania lesion development in BALB/c mice.

Example 3: Amphotericin B Cochleates for treatment of Leishmania donovani parasitic infections Materials 8 -10 week old female BALB/c mice and RAG1.B6 mice, maintained under category 3 conditions were used herein.

Leishmania Parasites: The sodium stiboglucate sensitive Leishmania donovani (strain MHOM/ET/67/HU3) was used. L. donovani amastigotes were isolated from the spleen of infected donor mice, counted and a solution of 1 x 10 ⁸ amastigotes/ml in RPMI 1640 was prepared. Animals were infected intravenously (tail vein) with a 0.2 ml bolus (equivalent to 2.0 x 10 ⁷ amastigotes) on day 0. Infected mice were randomly assorted into 6 groups of five animals. On day 7 post-infection one mouse was sacrificed, liver smears taken, methanol fixed and Giemsa stained and infection assessed.

Oral amphotericin B cochleate (CAMB) formulations with varying amphotericin B concentrations as well as cochleate placebo were prepared as described in Example 1. Methods

Seven groups of 5 mice each were weighed before and after treatment and the % weight change noted. The 7 groups included: untreated control, cochleate placebo, 2 doses of CAMB, and 3 active controls. Mice were treated once a day for 5 days on days 7-11 of infection. On day 14 after infection, three days after the completion of treatment, livers were removed and weighed, smears were methanol fixed and Giemsa stained. Parasite burdens were determined microscopically (xlOO, oil immersion) by counting the number of amastigotes/500 liver cells.

The results were expressed as the mean number of amastigotes per liver cell x mg liver (Bradley and Kirkley 1977). The burden of drug treated groups was compared with that of the untreated group and the % inhibition calculated as reflected in Table 3 below. For the average level of infection, the standard drug treatment of sodium stibogluconate (GSK, UK) at 15 mgSbV/kg x5 gives about 50% reduction of liver parasite load.

Example 4: Rat and Dog Toxicity Studies of Amphotericin B Cochleates

A 28-day toxicity study was conducted to determine potential toxic effects, target organs of toxicity, and a no observable adverse effect level (NOAEL) in rats and dogs following daily oral dosing with CAMB.

Large scale GLP batches of CAMB and placebo (cochleate vehicle without AmB) were prepared for these 28 day studies as described in Example 1. CAMB was administered as single daily doses for 28 days by oral gavage to 10 Beagle dogs per group (5 males, 5 females) at doses of 15, 30, and 45 mg/kg and to 48 Sprague-Dawley rats per group (24 males, 24 females) at doses of 30, 45, and 90 mg/kg. In rats, 18 animals (9 males, 9 females) per dose group served as a satellite group for toxicokinetic samples (blood, urine, and fecal samples for drug analysis). Animals in the placebo groups received volume-matched to the highest dose of CAMB in both species. TK blood (Days 1 and 28), urine and feces (weeks 1 and 4), and tissue samples (at necropsy) were collected for AmB quantitation by validated LC-MS methods. Histopathology analysis and serum clinical chemistry tests were done on all animals. CAMB was well tolerated by rats and dogs at all doses with no mortalities or clinical abnormalities found in either species based on comparison with historical controls. Pharmacokinetic data in dogs for Day 1 were comparable to a previous 7-day study. The increase in maximum plasma AmB concentration (Cmax) in dogs was not dose-dependent after 28 days, similar to what was observed on Day 7 in the 7-day study. In dogs, the kidney had quantifiable concentrations of AmB at all doses while liver, lung, and spleen had AmB concentrations detectable at all 3 doses. In rats, quantifiable AmB concentrations were found in liver, lung, and kidney, but not in spleen. Additional analytical and histopathological analyses are underway in dog and rat.

Preliminary pharmacokinetic results for the dog study are listed in Table 4:

Preliminary results suggest a T _max in the dog study of about 8 hours. It also appeared that exposure and C _max increased with increasing dose, with less than proportional increase from 30 to 45 mg.

This study demonstrates that single daily oral doses of CAMB administered for 28 days is well tolerated in all dose groups in the rat and dog. The NOAEL for males and females was considered to be 45 mg/kg for dog and 90 mg/kg for rat. Quantifiable concentrations of AmB were measured in kidney of the dog and the rat as well as the liver and lung of the rat.

Example 5: Pharmacokinetic Study of Amphotericin B Cochleates

Amphotericin B cochleates (CAMB) were studied in a double-blind evaluation of the safety, tolerability, and pharmacokinetics following single-dose administration in healthy volunteers.

DOSE RATIONALE

Amphotericin B dosing cannot be determined by the standard pharmacologic approach of using a target serum concentration because the drug is concentrated in tissues, therefore effective doses of different formulations are associated with different concentrations.

In a mouse model of systemic candidiasis (Santangelo et al. (2000) Antimicrob Agents Chemother. v44(9):2356-60), oral (PO) CAMB 5 mg/kg/day was associated with 100% survival, which was superior to both deoxycholate amphotericin B (DAMB) 1 mg/kg/day intraperitoneal and liposomal amphotericin B lO mg/kg/day PO. In a mouse model of systemic aspergillosis (Delmas et al. (2002) Antimicrob Agents Chemother. v46(8):2704-7), CAMB 20 mg/kg/day and 40 mg/kg/day were associated with 70% survival and were superior to DAMB 4 mg/kg/day intraperitoneal.

The maximum dose that can be administered to animals is limited by volume. CAMB is formulated to provide 5 mg amphotericin B per mL of cochleate suspension. Animal toxicology studies have confirmed the feasibility of administering doses up to 45 mg/kg/day for up to 7 days.

Allosteric scaling of the maximal dog dose by body surface area to a maximal human dose (using a factor of 0.54, per Table 1 of the Food and Drug Administration (FDA) 2005 Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers (FDA. 2005) indicates a maximum dose in humans of 24 mg/kg/day. Assuming an average body weight of 70 kg yields a maximum dose of 1680 mg/day. The proposed 800 mg maximum dose in the current study therefore provides 2-fold cover from the no-effect dose in dogs.

Allosteric scaling of the maximal rat dose by body surface area to a maximal human dose (using a factor of 0.16, per Table 1 of the FDA Guidance 2005) indicates a maximum dose in humans of 7.2 mg/kg/day. Assuming an average body weight of 70 kg yields a maximum dose of 504 mg/day. The proposed 800 mg maximum dose in the current study therefore exceeds cover from the no-effect dose in rats by a factor of approximately 1.6-fold. However, the allosteric scaling does not account for the known plasma and tissue concentrations of amphotericin following CAMB administration in rats and dogs as compared with concentrations known to be acceptable in humans with this agent. In rats (study BDSI-CAMB-PK-4, Table 5), CAMB was dosed up to 30 mg/kg/day orally and DAMB was also given at 0.5 mg/kg IV.

In dogs (study BDSI-CAMB-PK3, Table 6), CAMB administration of doses up to 30 mg/kg/day for 7 days was associated with lower plasma concentrations and urine amphotericin excretion than intravenous DAMB at 0.5 mg/kg (Table 6). Tissue concentrations were not measured.

In a second dog study (TK-201), CAMB administration of doses up to 45 mg/kg/day for 7 days was associated with 2-fold higher amphotericin plasma concentrations than measured in the previous study and tissue amphotericin concentrations greater than 100 μg/gm in 3 tissues. DAMB was not given in this study. The observed concentration of amphotericin B is shown for the highest dose in Table 7.

Results indicate that oral availability in the uninfected rat and dog is low and most amphotericin presented in oral CAMB is excreted in feces. Therefore high doses of oral CAMB are expected to produce systemic exposure that is within concentrations that are generally recognized as safe.

In a meeting on 6 October 2006, the FDA concurred with initial human exposure limits of 45 mg/kg as being below the no observed adverse effect level and agreed that phase 1 studies could proceed at doses up to 800 mg. The purpose of this study is to evaluate the pharmacokinetic profile and relative bioavailability of amphotericin B, with cross-study comparison to other amphotericin formulations.

TRIAL OBJECTIVES AND PURPOSE

The objectives of this study were to: 1. Determine the pharmacokinetic profile of amphotericin B following an initial single oral dose of CAMB;

2. Estimate the relative bioavailability during the timeframe covered by blood sampling, with cross-study comparison to other amphotericin formulations; and

3. Assess the safety and tolerability of a single dose of CAMB administration. INVESTIGATIONAL PLAN

This was a single-dose, double-blind, dose-escalating, pharmacokinetic study in healthy males and females, designed to determine the safety, tolerability, and pharmacokinetic profile of amphotericin B and to estimate the relative bioavailability with cross-study comparison to other amphotericin formulations. A total of 48 subjects participated in this study, across 3 cohorts, 16 per treatment cohort. Within each treatment cohort, 12 subjects received active drug and 4 received placebo. Subjects checked into the clinic on Day 0 and were randomized to active treatment (200, 400, or 800 mg CAMB depending on cohort) or placebo (Table 8).

Blood samples for pharmacokinetic analysis were collected predose (0 hours), and postdose on Day 0 (1, 2, 4, 8, and 12 hours) and Day 1 (24 hours). Subjects checked out of the clinic when all scheduled procedures were completed. Additional blood samples were collected on an outpatient basis on Day 2 (48 hours), Day 3 (72 hours), Day 4 (96 hours), Days 8 and 9 (192 and 216 hours), and on Day 14 (336 hours).

Safety was evaluated by the monitoring of adverse events, concomitant medication, physical examination and medical history, clinical laboratory measurements, and vital sign measurements. Urine pregnancy test, ECG, drug and alcohol screen were also performed on various days of the treatment period. The Screening visit and the Day 0 visit may take place on the same day. Once an informed consent form has been signed, the following was completed:

1. Medical history and physical examination

2. Vital sign measurement (blood pressure, sitting heart rate, oral temperature, and respiratory rate) 3. Clinical laboratory testing

4. 12-lead electrocardiogram

5. Drug alcohol screen

On the day of drug administration (Day 0), the following was completed: 1. Check- in to the clinic (the evening prior)

2. Vital sign measurement (blood pressure, sitting heart rate, oral temperature, and respiratory rate)

3. Urine pregnancy test if applicable 4. Study drug administration (between 0600 and 0800)

5. Blood sample collection (predose and 1, 2, 4, 8, and 12 hours postdose) On clinic check-out (Day 1), the following was completed

1. Blood sample collection (24 hours)

2. Vital sign measurements (blood pressure, sitting heart rate, oral temperature, and respiratory rate)

3. Evaluation of AEs

4. Evaluation of concomitant medication

5. Check out of the clinic

On days 2-9, the following was completed 1. Blood sample collection (48, 72, 96, 192, and 216 hours)

2. Evaluation of AEs

3. Evaluation of concomitant medications On day 14, the following was completed

1. Physical examination and vital sign measurements, if indicated by occurrence of new medical event or adverse event after Screening

2. Clinical laboratory testing

3. Urine pregnancy test for females of childbearing potential

4. Blood sample collection (336 hours)

5. Evaluation of adverse events 6. Evaluation of concomitant medication

SELECTION AND WITHDRAWAL OF SUBJECTS

A subject was eligible for inclusion in this study if all of the following criteria apply:

1. 18 to 55 years of age 2. Within 20% of ideal body weight (Appendix 3) 3. Male or non-lactating female. Sexually active females of childbearing potential should either be surgically sterile (hysterectomy or tubal ligation), or should use a highly effective medically accepted contraceptive regimen.

4. Signed ICF (Appendix 2) obtained at Screening prior to any procedures being performed

A subject was not eligible for this study if 1 or more of the following applied:

1. Use of an investigational drug within 30 days preceding this study.

2. History of alcohol or drug abuse within the past 1 year.

3. History of hypersensitivity or intolerance to amphotericin B or to any cochleate product.

4. Use of a monoamine oxidase (MAO) inhibitor within 2 weeks preceding this study.

5. Subject is pregnant, actively trying to become pregnant, breast feeding or not using adequate contraceptive measures. 6. Current smokers.

The Investigator withdrew a subject upon request or upon occurrence of certain serious adverse events or intolerable adverse events. If a subject is discontinued due to an adverse event, the event was followed until it is resolved. In the event that a subject was withdrawn or did not complete the study for any reason, the Investigator made every effort to evaluate vital signs (blood pressure, sitting heart rate, oral temperature, and respiratory rate), perform a physical examination and perform a clinical adverse event assessment.

If a subject withdrew from the study at any time prior to completion of the specified treatment, either at the subject's request or the Investigator's discretion, the reason(s) for withdrawal were recorded by the Investigator. All subjects who withdrew from the study prematurely underwent all follow-up procedures and assessments, if possible. Subjects who withdrew from treatment because of a serious or non-serious adverse event were followed for at least 14 days after last dose of study drug.

One female subject, who became pregnant while enrolled in this study, was withdrawn immediately. TREATMENT OF SUBJECTS

CAMB was administered by the clinic staff at escalating doses of 200, 400, and 800 mg. Subjects were present in the clinic when study drug was administered. At the time of entry into the study, subjects were randomized to active CAMB or Placebo. The active dose was 200 mg in the first cohort, 400 mg in the second cohort, and 800 mg in the final cohort (see Table 8). The Placebo oral suspension was a cochleate vehicle suspension with no amphotericin B. Dose escalation was separated by at least 1 week. The CAMB dose was not escalated if 3 or more subjects in an active CAMB cohort experience one of the following: • a dose-related increase in serum creatinine of >50% above Baseline

• an absolute creatinine measure exceeding 1.2 mg/dL.

• a serum potassium <3 mmol/L.

After the subject was administered the study drug, 30 mL of an acceptable calcium containing liquid (defined below) was added to the dispensing cup to suspend the remaining study drug. The resuspended study drug was consumed by the subject as soon as possible. Acceptable calcium containing liquids include milk, orange juice fortified with calcium, water, if supplemented with an over the counter calcium supplement.

Subjects fasted for at least 10 hours before taking study drug and for 4 hours after. Subjects were allowed to drink acceptable calcium containing liquids, up to 1 hour prior to study drug administration and at least 2 hours after administration.

All concurrent therapies taken during the study were monitored throughout the study.

STUDY DRUG MATERIALS AND MANAGEMENT

CAMB was provided in 3 strengths: 200, 400, and 800 mg and was administered via oral suspension. A matching Placebo (cochleate vehicle suspension) was also provided. Study treatment was labeled with dose level and sequence.

The CAMB and cochleate vehicle suspension was packaged into Nalgene amber PETG 60 mL square bottles with HDPE closures and labeled as "Cochleate Vehicle Suspension" or "Cochleate Amphotericin B Suspension." At the study site, active drug or placebo was measured for each subject by unblended study personnel and doses were provided to the blinded study team, preserving blinding.

All study drug for this study was stored in a locked area. The area was also free of environmental extremes and with a limited access. Study drug was kept refrigerated at 2 to 8°C. The site maintained records of storage temperature and at end-of-study entered a copy of these records into the study documentation. Prior to dispensing, the study drug container was shaken, the required quantity poured into a glass cup, and the remainder of the study drug returned to storage. The measured dose was then allowed to warm to room temperature immediately prior to administration.

The volume administered determines the dose, so that each cohort received a different volume. The volume of placebo administered matched the volume of active drug (see Table 10).

Study drug was administered by drinking the liquid drug product. A small amount of water was then added to the dispensing cup to suspend remaining study drug, and was drunk by the subject.

PHARMACOKINETIC ASSESSMENT Blood samples for assessment of plasma concentrations of amphotericin B were collected predose (0 hour) and at 1, 2, 4, 8, 12, and 24 hours after administration of study drug while the subject was an inpatient and subsequently at the following time points on an outpatient basis: Day 2 (48 hours), Day 3 (72 hours), Day 4 (96 hours), Days 8 and 9 (192 and 216 hours), and Day 14 (336 hours). Blood samples were collected in a 3 mL K2-EDTA vacutainer tube for pharmacokinetic analysis at each collection time point. Samples were processed immediately and stored for shipping to the analytical laboratory at -7O°C, protected from light. Stability of -7O°C frozen samples was confirmed in rat plasma to 115 days. At the end of the study, samples were transferred to the bioanalytical laboratory for analyses of amphotericin B by a chromatographic method. The lower level of quantitation is approximately 20 ng/mL.

ASSESSMENT OF SAFETY

One of the objectives of this study is to assess the safety and tolerability of the drug; therefore, the Investigator was responsible for recording and reporting adverse events observed during and after study drug treatment. An adverse event is any reaction, side effect, or other untoward event experienced by a subject, regardless of relationship to study drug, including death. An adverse event may consist of a disease, an exacerbation of a pre-existing illness, or condition, a recurrence of an intermittent illness or condition, a set of related signs or symptoms, or a single sign or symptom. Serious and nonserious adverse events occurring after the consent form is signed, but prior to first dose of study drug will be collected as part of the medical history. In some embodiments, adverse events include any new medical occurrence not seen before initial study drug administration; a pre-existing medical condition which recurs with increased intensity or increased frequency subsequent to initial study drug administration; and a medical condition which is present at the time of initial study drug administration which exacerbates at any time following initial study drug administration. A pre-existing condition or signs or symptoms present at the time of initial study drug administration would not be considered an adverse event unless one of the other two indications listed above apply. In addition, signs or symptoms associated with a disease/condition being evaluated as part of assessments of study drug efficacy should ordinarily not be recorded as adverse events. An adverse drug reaction is any adverse event for which a causal relationship to study drug is at least a reasonable possibility, i.e., the relationship cannot be ruled out. A serious adverse event is an adverse event at any dose of study drug that results in death, is life-threatening, requires inpatient hospitalization or prolongation of existing hospitalization, results in a persistent or significant disability/incapacity, or is a congenital anomaly/birth defect. Additionally, important medical events that may not be immediately life-threatening or result in death or hospitalization may be considered a serious adverse event when, based upon appropriate medical judgment, they may jeopardize the subject or may require medical or surgical intervention to prevent one of the outcomes listed in the definition of serious adverse event above.

A "life-threatening" event is present when the subject was, in the view of the Investigator, at immediate risk of death from the event as it occurred. Note that this definition does not include an event which might have caused death, had it occurred in a more severe form.

Examples of serious adverse events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse.

Reports of therapeutic failure were not necessarily recorded or processed as adverse events. Complications of therapeutic failure, however, were recorded as adverse events.

The degree of "relatedness" of the adverse event to the study drug were assessed by the Investigator using the following scale:

• Not related indicates that the adverse event is definitely not related to the study drug.

• Unlikely related indicates that there are other, more likely causes and the study drug is not suspected as a cause. • Possibly related indicates that a direct cause and effect relationship between the study drug and the adverse event has not been demonstrated but there is a reasonable possibility that the adverse event was caused by the study drug.

• Probably related indicates that there probably is a direct cause and effect relationship between the study drug and the adverse event. It was the policy to consider "Probable" and "Possible" causality assessments as positive causality, i.e., related and to consider "Not" and "Unlikely" causality assessments as negative causality, i.e., not related.

At each visit, subjects had an opportunity to spontaneously mention any problems and the investigator inquired about adverse events (e.g., new medical problems and/or new medications). Each adverse event was evaluated for duration, intensity

(mild, moderate or severe), and relationship to study drug. Safety laboratory tests (such as hematology tests, serum chemistry tests, serology test and urinalysis tests) were obtained according to a study schedule for each subject, and were found to be in normal ranges.

RESULTS

CAMB was well-tolerated at doses of 200 and 400 mg. Adverse events are reported for the full cohort of 16 subjects. Gastrointestinal adverse events were seen in 6%, 38% and 56% of subjects in the 200, 400 and 800 mg cohorts respectively, and all were mild at doses of 200 and 400 mg. The most common adverse event was nausea of mild severity, seen in 6% and 19% of subjects in the 200 and 400 mg cohorts respectively. One subject became pregnant and underwent elective termination. There were no abnormalities in clinical laboratory testing of blood or urine.

This study demonstrates that CAMB is generally well tolerated in humans in single doses of 200 and 400 mg. CAMB appears to be a safe and feasible means of orally administering amphotericin B.

Pharmacokinetic results are as follows: For the 200mg cohort, see Table 11. For the 400mg cohort, see Table 12. For the 800mg cohort, see Table 13.

In each of Tables 11-13, the units of C _max are ng/mL, the units T _max of are hours; and the units of A plot of the average concentration versus time profile for the three cohorts can be seen in Figure 1.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.