阅读说明：本技术 两性霉素b的固体口服调配物 (Solid oral formulations of amphotericin B ) 是由 彼得·海尼克 罗奇·泰伯特 艾美斯特·贝坦科尔特 于 2018-02-21 设计创作，主要内容包括：本公开描述了包括两性霉素B的固体剂型。本文还描述了治疗真菌感染和利什曼原虫(Lesishmania)感染的方法。(The present disclosure describes solid dosage forms comprising amphotericin B. Also described herein are methods of treating fungal infections and leishmania (leshmania) infections.)

1. A solid dosage form, comprising:

Amphotericin B coated on solid carrier and

At least one lipophilic component.

2. The solid dosage form according to claim 1, wherein the% w/w of amphotericin B in the solid dosage form is greater than the% w/w of the at least one lipophilic component.

3. The solid dosage form of claim 1 or 2, wherein the% w/w of amphotericin B ranges from about 20% to about 30% of the total weight of the solid dosage form.

4. The solid dosage form of any one of claims 1-3, wherein amphotericin B is present in an amount ranging from about 50mg to about 200 mg.

5. The solid dosage form of any one of claims 1-4, wherein amphotericin B is present in an amount of about 100 mg.

6. The solid dosage form of any one of claims 1-5, wherein the amphotericin B is present in an amount of about 150 mg.

7. The solid dosage form of any one of claims 1 to 6, wherein the at least one lipophilic component is selected from the group consisting of: polyethylene oxide-containing fatty acid esters, fatty acid glycerides, and combinations thereof.

8. The solid dosage form of any one of claims 1-7, wherein the solid carrier is a bead or a saccharide.

9. The solid dosage form of any one of claims 1-8, wherein the Cmax of amphotericin B is in the range of about 80% to about 125% of the Cmax of amphotericin B measured after oral administration of a liquid formulation having an equivalent dose of amphotericin B.

10. The solid dosage form of any one of claims 1-9, wherein the AUC0-24 of amphotericin B is in the range of about 80% to about 125% of the AUC0-24 of amphotericin B measured after oral administration of a liquid formulation having an equivalent dose of amphotericin B.

11. The solid dosage form of any one of claims 1-10, wherein the AUC0-48 of amphotericin B is in the range of about 80% to about 125% of the AUC0-48 of amphotericin B measured after oral administration of a liquid formulation having an equivalent dose of amphotericin B.

12. The solid dosage form of any one of claims 1-8, wherein the Tmax of amphotericin B is in the range of about 80% to about 125% of the Tmax of amphotericin B measured after oral administration of a liquid formulation having an equivalent dose of amphotericin B.

13. A capsule comprising the solid dosage form of any one of claims 1 to 12.

14. A method of treating leishmaniasis in a subject in need thereof, the method comprising administering an effective amount of a solid dosage form according to any one of claims 1 to 12.

Background

Amphotericin B is a potent antifungal agent and is the first choice drug for the treatment of severe systemic fungal infections and leishmania (leshmania) infections. However, amphotericin B has several adverse properties that severely hamper its use as a therapeutic agent. First, amphotericin B is insoluble in water. Second, amphotericin B cannot be absorbed in the gastrointestinal tract (GIT). Third, amphotericin B is unstable in the gastric acid environment. Each of these properties limits the bioavailability of amphotericin B.

To overcome the above-mentioned problems leading to limited bioavailability, amphotericin B is administered in the form of a liposome composition or as a colloidal dispersion. However, intravenous injection and infusion of amphotericin B have significant drawbacks. First, intravenous injection and infusion of amphotericin B have been associated with considerable side effects such as fever, chills, bone pain, nephrotoxicity and thrombophlebitis. Second, intravenous amphotericin B must be administered within 30-40 days, and thus this dosing regimen is expensive and may have low patient compliance. These disadvantages are particularly problematic in developing countries where leishmanial infections occur.

U.S.8,592,382 and U.S.8,673,866 describe liquid formulations for oral administration comprising amphotericin B and a mixture of fatty acid glycerides and polyethylene oxide-containing fatty acid esters. The fatty acid glycerides and polyethylene oxide-containing fatty acid esters are present in a significant excess (greater than 180:1) relative to amphotericin B, which is described as being critical to achieving bioavailability of amphotericin B in oral dosage forms. However, the large amount of oily component in these formulations may cause gastric discomfort such as nausea and diarrhea, which limits patient compliance (particularly because of the need for extended dosing regimens). In addition, administering such liquid suspensions is cumbersome and may result in under or over dosing due to dispensing errors, spillage and/or loss of residual formulation remaining in the dispensing device. Thus, there is a need to provide stable bioavailable dosage forms, ideally solid dosage forms, of amphotericin B that do not exhibit the limitations of known amphotericin B formulations.

The present disclosure provides solid dosage forms that overcome the limitations of conventional amphotericin B compositions.

Disclosure of Invention

In various embodiments, the present disclosure relates to solid dosage forms (e.g., solid or semi-solid dosage forms) comprising a lipophilic drug (e.g., amphotericin B). In embodiments, the solid dosage forms disclosed herein achieve bioavailability comparable to liquid formulations typically used for administering amphotericin B.

In some embodiments, the solid dosage form comprises amphotericin B and at least one lipophilic component coated on a solid carrier. In other embodiments, the% w/w of amphotericin B in the solid dosage form is greater than the% w/w of the at least one lipophilic component. In further embodiments, the% w/w of amphotericin B ranges from about 20% to about 30% of the total weight of the solid dosage form.

In some embodiments, amphotericin B is present in the solid dosage form in an amount ranging from about 50mg to about 200 mg. In other embodiments, amphotericin B is present in an amount of about 100 mg. In still other embodiments, wherein the amphotericin B is present in an amount of about 150 mg.

In some embodiments, the at least one lipophilic component is selected from the group consisting of: polyethylene oxide-containing fatty acid esters, fatty acid glycerides, and combinations thereof.

In some embodiments, the solid carrier is a bead or a saccharide. In other embodiments, the present disclosure provides a capsule comprising a solid dosage form as described herein.

In some embodiments, the present disclosure provides a method of treating leishmaniasis comprising administering an effective amount of a solid dosage form described herein.

Drawings

FIG. 1 illustrates the preparation of amphotericin B/Gelucire/Peceol/TPGS/powdered excipient formulations.

Figure 2 shows the thermogravimetric analysis (TGA) profile of amphotericin B (23%) for formulations 1-3.

FIG. 3 shows the dissolution profiles of amphotericin B for formulations 1-3.

Fig. 4 shows the dissolution profiles of amphotericin B in formulations 1A and 1B at T ═ 0/onset magnified compared to formulations 1, 2.

Figure 5 shows the dissolution profile of solid and semi-solid amphotericin B formulations in capsules in 0.5% SDS in water.

Fig. 6 shows the dissolution profile of solid and semi-solid amphotericin B formulations in capsules in FeSSIF at pH 5.8.

Figure 7 shows the dissolution profile of a 100mg capsule comprising a lipid-based formulation.

figure 8 shows the dissolution profile of amphotericin B granule formulation in capsules at T ═ 0 and under stable storage conditions.

Figure 9 shows the dissolution profile of amphotericin B lipid-based capsules of formulation 5A at T ═ 0 and under stable storage conditions.

Figure 10 shows the tissue concentration of amphotericin B measured in a canine model.

Fig. 11 shows plasma concentrations of amphotericin B measured for formulation 1a (a), formulation B (B), and conventional lipid formulation (C).

Figure 12 shows the individual plasma levels of amphotericin B after oral administration of 500mg amphotericin B in formulation 1A to dogs.

Figure 13 shows mean plasma levels of amphotericin B after oral administration of 500mg amphotericin B in formulation 1A to dogs.

Detailed Description

All publications, patents and patent applications (including any accompanying figures and appendices) herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application, figure or appendix was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

The term "pharmaceutically acceptable" means biologically or pharmacologically compatible for use in an animal or human, and may refer to use in animals and more particularly in humans that are approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia.

As used herein, the term "subject" includes any and all organisms and includes the term "patient". "subject" may refer to a human or any other animal.

The term "treating" means alleviating, delaying, reducing, reversing, ameliorating, or managing one or more of at least one symptom of a condition in a subject. The term "treating" may also mean one or more of arresting, delaying onset (i.e., a period prior to clinical manifestation of the condition), or reducing the risk of developing or worsening the condition.

As used herein, the term "about," when preceding a dose or dose range of a particular ingredient, refers to an amount or range that is slightly above and/or slightly below the recited amount or range, which does not significantly alter the therapeutic effect of the recited amount or range of the particular ingredient.

Any lipophilic therapeutic agent can be formulated using the solid dosage forms disclosed herein. For example, specific therapeutic agents that may be administered using the formulations and methods disclosed herein include tetracycline, doxycycline, oxytetracycline, chloramphenicol, erythromycin, acyclovir, idoxuridine, triamantadine, miconazole, ketoconazole, fluconazole, itraconazole, econazole, griseofulvin, amphotericin B, terbinafine, metronidazole benzoate, tinidazole, indomethacin, ibuprofen, piroxicam, diclofenac, disodium cromoglycate, nitroglycerin, isosorbide dinitrate, verapamil, nifedipine, diltiazem, digoxin, morphine, cyclosporine, buprenorphine, lidocaine, diazepam, nitrazepam, flurazepam, estazolam, flunitrazepam, triazolam, alprazepam, midazolam, temazepam, nitrazepam, brozolam, lorazepam, clobazapam, chlorazepam (lorazepam), lorazepam, doxazopam, doxepin, and, Oxazepam, buspirone, sumatriptan, ergotamine derivatives, cinnarizine, antihistamines, ondansetron, tropisetron, granisetron, metoclopramide, dithiol, vitamin K, paclitaxel, docetaxel, camptothecin, SN38, cisplatin, carboplatin, efavirenz, saquinavir, ritonavir, and clofazimine.

In a particular embodiment, the solid dosage form comprises amphotericin B. In further embodiments, the amphotericin B solid dosage forms of the present disclosure may further comprise a second therapeutic agent, such as any of those disclosed herein.

in embodiments, the bioavailability of amphotericin B in the solid dosage forms described herein is at least equivalent to conventional liquid formulations, such as those disclosed in u.s.8,592,382 and u.s.8,673,866, each of which is incorporated herein by reference in its entirety for all purposes. For example, the amphotericin B formulation disclosed in U.S.8,673,866 utilizes an isotropic mixture of lipophilic components (oil, surfactant, solvent and co-solvent/surfactant) in a weight ratio relative to amphotericin B of greater than about 189:1 to achieve a suitable level of bioavailability, which results in an oily formulation that causes gastric discomfort. Surprisingly and unexpectedly, the inventors of the present invention found that comparable levels of bioavailability can be achieved using solid dosage forms comprising a significantly reduced amount of lipophilic component without causing gastric discomfort.

In some embodiments, the solid dosage forms of the present disclosure provide bioavailability comparable to conventional liquid formulations mentioned above, wherein the ratio of amphotericin B relative to one or more lipophilic components of the formulation is greater, whereas conventional liquid amphotericin formulations employ a greater ratio of lipophilic components to amphotericin B to provide adequate bioavailability. In embodiments, the weight ratio of amphotericin B to lipophilic component of the solid compositions of the present disclosure ranges from about 100:1 to about 1:1, e.g., about 100:1, about 95:1, about 90:1, about 85:1, about 80:1, about 75:1, about 70:1, about 65:1, about 60:1, about 55:1, about 50:1, about 45:1, about 40:1, about 35:1, about 30:1, about 25:1, about 20:1, about 15:1, about 10:1, about 9.5:1, about 9:1, about 8.5:1, about 8:1, about 7.5:1, about 7:1, about 6.5:1, about 6:1, about 5.5:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2.5:1, about 2:1, about 5:1, about 1, and all ranges therebetween.

in other embodiments, the solid dosage forms of the present disclosure provide comparable bioavailability to the liquid formulations mentioned above, and have a smaller excess of lipophilic component relative to amphotericin B, as compared to conventional liquid formulations. In embodiments, the weight ratio of one or more lipophilic components (e.g., one, two, three, etc. lipophilic components) to amphotericin B in the solid dosage form of the present disclosure ranges from about 100:1 to about 1:1, for example, about 100:1, about 95:1, about 90:1, about 85:1, about 80:1, about 75:1, about 70:1, about 65:1, about 60:1, about 55:1, about 50:1, about 45:1, about 40:1, about 35:1, about 30:1, about 25:1, about 20:1, about 15:1, about 10:1, about 9.5:1, about 9:1, about 8.5:1, about 8:1, about 7.5:1, about 7:1, about 6.5:1, about 6:1, about 5.5:1, about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2.5:1, about 2:1, about 1.5:1, or about 1:1, including all ranges and subranges therebetween.

In alternative embodiments, the solid dosage form of the present disclosure comprises between about 10-30 wt.% of amphotoxin B and between about 1-10 (total) wt.% of one or more lipophilic components. For example, the weight% of amphotoxin B is about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%, including all ranges and subranges therebetween; and the total wt% of lipophilic component is about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10%, including all ranges and subranges therebetween.

In some embodiments, at least one lipophilic component is used in combination with a therapeutic agent (e.g., amphotericin B). In other embodiments, at least one lipophilic component is used to facilitate coating of the therapeutic agent onto the solid carrier. The lipophilic component may comprise any hydrophobic material in which the therapeutic agent (e.g., amphotericin B) may be dissolved or suspended and which is pharmaceutically acceptable. The lipophilic component used to solubilize the therapeutic agent may be selected based on the hydrophilic-lipophilic balance (HLB) of the therapeutic agent and the lipophilic component or the lipid and optional organic solvent to facilitate solubilization of amphotericin B in the lipophilic component. Suitable lipid materials for dissolving the therapeutic agent (e.g., amphotericin B) may have an HLB value equal to or otherwise sufficient to dissolve the therapeutic agent in an appropriate solvent. For example, the lipophilic component suitable for dissolving amphotericin B in ethanol may have an HLB of 14 or less (e.g., 13, 12, 11, or 10).

each lipophilic component in the compositions of the present disclosure may be selected from natural (human, animal or plant derived) or synthetic sources. The lipophilic component can be a liquid or a solid at room temperature, provided that the solid can melt upon heating and the lipophilic component after melting does not degrade or denature the therapeutic agent (e.g., amphotericin B). In some embodiments, at least one lipophilic component may be used to solubilize the therapeutic agent (e.g., a lipophilic drug, such as amphotericin B). In other embodiments, the lipophilic component may be selected to improve oral absorption of the therapeutic agent (amphotericin B). In further embodiments, the lipophilic component may be selected to improve the bioavailability of the therapeutic agent (amphotericin B). In still other embodiments, the lipophilic component may comprise a surfactant. In some such embodiments, the lipophilic component may be a nonionic surfactant. In even further embodiments, the lipophilic component is a lipophilic binder material that facilitates coating or adhering the therapeutic agent to the solid carrier.

In embodiments, a dosage form disclosed herein can comprise one lipophilic component or a mixture of two or more lipophilic components (e.g., a mixture of 3 lipophilic components, 4 lipophilic components, 5 lipophilic components, etc.). In embodiments where two lipophilic components are desired, the weight ratio of the first lipophilic component to the second lipophilic component ranges from about 99:1 to about 1:99, e.g., about 99:1, about 95:5, about 90:10, about 85:15, about 80:20, about 75:25, about 70:30, about 65:35, about 60:40, about 55:45, about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90, about 5:95, and about 1:99, including all ranges and subranges therebetween.

Non-limiting examples of lipophilic components that can be used in the solid dosage forms disclosed herein include pharmaceutically acceptable fats, fatty substances, oils, phospholipids, sterols, and waxes. Fat generally refers to esters of glycerol (e.g., mono-, di-, or tri-esters of glycerol and fatty acids). Suitable fats and fatty substances include, but are not limited to, fatty alcohols (such as lauryl, myristyl, stearyl, cetyl or cetostearyl alcohol, and the like), fatty acids and derivatives, including, but not limited to, fatty acid esters, fatty acid glycerides (mono-, di-and triglycerides), and hydrogenated fats. Fats may be solid or liquid at normal room temperature, depending on their structure and composition.

Suitable oils include pharmaceutically acceptable animals (e.g., fatty acid esters), minerals (e.g., paraffin oils), plants (e.g., vegetable oils), or synthetic hydrocarbons that are liquid at room temperature. Examples of pharmaceutically acceptable oils include, but are not limited to: mineral oils, such as paraffin oil; vegetable oils, such as castor oil, hydrogenated vegetable oil, sesame oil and peanut oil; and animal fats and oils such as triglycerides and butter. Partially hydrogenated vegetable oils are derived from natural products and generally comprise a glyceride mixture of C14-20 fatty acids (specifically, palmitic and stearic acids). Suitable examples of partially hydrogenated vegetable oils include partially hydrogenated cottonseed oil, soybean oil, corn oil, peanut oil, palm oil, sunflower seed oil, or mixtures thereof. Chemical equivalents of partially hydrogenated vegetable oils comprise synthetically produced C14-20 fatty acid glycerides having the same characteristics as the naturally derived products described above.

Suitable phospholipids include pharmaceutically acceptable vegetable phospholipids, animal phospholipids and synthetic phospholipids. Examples of pharmaceutically acceptable phospholipids include choline phosphatidylethanolamine and phosphatidylglycerol, such as, but not limited to, phosphatidylcholine, 1, 2-dioleoyl phosphatidylcholine, 1, 2-dimyristoyl phosphatidylcholine, 1, 2-dioleoyl phosphatidylserine, 1, 2-distearoyl phosphatidylglycerol, 1, 2-dipalmitoyl phosphatidylcholine, 1, 2-distearoyl phosphatidylglycerol, egg phosphatidylcholine, egg phosphatidylglycerol, soybean phosphatidylcholine, glycerophosphorylcholine, hydrogenated soybean phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, N- (carbonyl-methoxypolyethylene glycol 2000) -1, 2-distearoyl phosphatidylethanolamine sodium salt, sodium phosphatidylethanolamine, and combinations thereof, Muramyl tripeptide phosphatidylethanolamine, 1-palmitoyl-2-linoleoyl phosphatidylcholine, 1-palmitoyl-2-linoleoyl phosphatidylglycerol, 1-palmitoyl-2-oleoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylglycerol, polyenylphosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidyl glycerol, 1-stearoyl-2-linoleoyl phosphatidyl choline, 1-stearoyl-2-linoleoyl phosphatidyl glycerol, sphingomyelin, 1-stearoyl-2-oleoyl phosphatidyl choline, 1-stearoyl-2-oleoyl phosphatidyl glycerol, and the like.

Suitable waxes include animal waxes, vegetable waxes, mineral waxes, and petroleum waxes. Examples of waxes include, but are not limited to, glyceryl behenate, glyceryl monostearate, stearic acid, palmitic acid, lauric acid, carnauba wax, cetyl alcohol, glyceryl stearate, beeswax, paraffin wax, ozokerite (ozokerite), candelilla wax, cetyl alcohol, stearyl alcohol, spermaceti, carnauba wax, bayberry wax, montan wax, ceresin, and microcrystalline wax.

In particular embodiments, lipophilic components suitable for use in the solid dosage forms disclosed herein comprise fatty acid glycerides, polyethylene oxide-containing fatty acid esters, and combinations thereof.

In particular embodiments, amphotericin B formulations of the present disclosure comprise one or more fatty acid glycerides. As used herein, the term "fatty acid glyceride" refers to an ester formed between glycerol and one or more fatty acids comprising mono-, di-, and tri-esters (i.e., glycerides). Suitable fatty acids include saturated and unsaturated fatty acids having from eight (8) to twenty-two (22) carbon atoms (i.e., C8-C22 fatty acids). In certain embodiments, suitable fatty acids comprise C12-C18 fatty acids. The fatty acid glycerides that may be used in the formulation may be provided by commercially available sources. Representative sources of fatty acid glycerides are mixtures of mono-, di-and tri-esters (commonly known as "glycerol oleate" or "glycerol monooleate") commercially available as (Gattefosse, Saint Priest Cedex, france). In some embodiments, when used as a source of fatty acid glycerides in the formulation, the fatty acid glycerides comprise from about 32% to about 52% by weight fatty acid monoglycerides, from about 30% to about 50% by weight fatty acid diglycerides, and from about 5% to about 20% by weight fatty acid triglycerides. The fatty acid glycerides include greater than about 60% by weight oleic acid (C18:1) monoglycerides, diglycerides, and triglycerides. Other fatty acid glycerides include palmitic (C16) ester (less than about 12%), stearic (C18) ester (less than about 6%), linoleic (C18:2) ester (less than about 35%), linolenic (C18:3) ester (less than about 2%), arachidic (C20) ester (less than about 2%), and arachidic (C20:1) ester (less than about 2%). Free glycerol (typically about 1%) may also be included. In one embodiment, the fatty acid glycerides comprise about 44% by weight fatty acid monoglycerides, about 45% by weight fatty acid diglycerides, and about 9% by weight fatty acid triglycerides, and the fatty acid glycerides comprise about 75% by weight oleic acid (C18:1) monoglycerides, diglycerides, and triglycerides. Other fatty acid glycerides include palmitic (C16) ester (about 4%), stearic (CI5) ester (about 2%), linoleic (CIs:2) ester (about 12%), linolenic (C18:3) ester (less than about 1%), arachidic (C20) ester (less than about 1%), and arachidic (C20:1) ester (less than about 1%).

In embodiments, the fatty acid glyceride may be the only lipid in the amphotericin B formulation. In other embodiments, the formulation may comprise a mixture of fatty acid glycerides, such as any of those disclosed herein. In still other embodiments, one or more fatty acid glycerides may be used in combination with other lipophilic components as described herein, such as one or more polyethylene oxide-containing fatty acid esters as described herein.

In some embodiments, amphotericin B formulations described herein include at least one polyethylene oxide-containing lipophilic component, such as a fatty acid ester. As used herein, the term "polyethylene oxide-containing fatty acid ester" refers to a fatty acid ester comprising a polyethylene oxide group (i.e., a polyethylene glycol group) covalently coupled to a fatty acid through an ester bond. The polyethylene oxide-containing fatty acid esters comprise mono-and di-fatty acid esters of polyethylene glycol. Suitable polyethylene oxide-containing fatty acid esters are derived from fatty acids comprising saturated and unsaturated fatty acids having from eight (8) to twenty-two (22) carbon atoms (i.e., polyethylene oxide esters of C8-C22 fatty acids). In certain embodiments, suitable polyethylene oxide-containing fatty acid esters are derived from fatty acids comprising saturated and unsaturated fatty acids having twelve (12) to twenty-eight (18) carbon atoms (i.e., polyethylene oxide esters of C12-C18 fatty acids). Representative polyethylene oxide-containing fatty acid esters include saturated C8-C22 fatty acid esters. In certain embodiments, suitable polyethylene oxide-containing fatty acid esters comprise saturated C12-C18 fatty acids.

The molecular weight of the polyethylene oxide groups of the polyethylene oxide-containing fatty acid esters can be varied to optimize the solubility of the therapeutic agent (e.g., amphotericin B) in the formulation. A representative average molecular weight of the polyethylene oxide groups may be from about 350 to about 2000. In one embodiment, the polyethylene oxide group has an average molecular weight of about 1500.

In some embodiments, when the amphotericin B formulation comprises polyethylene oxide-containing fatty acids in the lipophilic component, the lipophilic component may comprise only one type of polyethylene oxide-containing fatty acid. In other embodiments, the polyethylene oxide-containing fatty acids in the lipophilic component can comprise a mixture of polyethylene oxide-containing fatty acid esters (mono and di fatty acid esters of polyethylene glycol).

Polyethylene oxide-containing fatty acid esters useful in the formulations of the present disclosure may be provided by commercially available sources. Representative polyethylene oxide-containing fatty acid esters (mixtures of mono-and diesters) are commercially available under the name Saint Priest Cedex, france. Suitable polyethylene oxide-containing fatty acid esters include 44/14, 50/13, 53/10, and 48/16. The numbers in these names refer to the melting point and the hydrophilic/lipophilic balance (HLB) of these materials, respectively. 44/14, 50/13, 53/10 and 48/16 are mixtures of (a) mono-, di-and tri-esters of glycerol (glycerol esters) and (b) mono-and di-esters of polyethylene glycol (polyethylene glycol). GELUCIRE may also comprise free polyethylene glycol (e.g., PEG 1500).

Lauric acid (C12) is the major fatty acid component of the glycerides and polyglycol esters in 44/14. 44/14 is referred to as a mixture of glycerol dilaurate (lauric diester and glycerol) and PEG dilaurate (lauric diester and polyethylene glycol) and is commonly referred to as PEG-32 glyceryl laurate (Glyphosate Inc.) lauroyl polyethylene glycol-32 glycerol ester EP or lauroyl polyoxylglyceride USP/NF. 44/14 was produced from the reaction of hydrogenated palm kernel oil with polyethylene glycol (average molecular weight 1500). 44/14 contains about 20% monoglycerides, diglycerides, and triglycerides; about 72% mono-and di-fatty acid esters of polyethylene glycol 1500; and about 8% polyethylene glycol 1500.

44/14 contains lauric acid (C12) ester (30 to 50%), myristic acid (C14) ester (5 to 25%), palmitic acid (C16) ester (4 to 25%), stearic acid (C18) ester (5 to 35%), caprylic acid (C8) ester (less than 15%) and capric acid (C10) ester (less than 12%). 44/14 may also contain free glycerol (typically less than about 1%). In a representative formulation, 44/14 included lauric acid (C12) ester (about 47%), myristic acid (C14) ester (about 18%), palmitic acid (C16) ester (about 10%), stearic acid (C18) ester (about 11%), caprylic acid (C8) ester (about 8%), and capric acid (C10) ester (about 12%).

Palmitic acid (C16) (40-50%) and stearic acid (C18) (48-58%) are the major fatty acid components of glycerol and polyethylene glycol esters in 50/13. 50/13 are known as PEG-32 glyceryl palmitostearate (Jiafa lion company), stearyl polyglycolyglyceride EP or stearyl polyoxyglyceride USP/NF. 50/13 contains palmitic (C16) ester (40 to 50%), stearic (C18) ester (48 to 58%) (stearate and palmitate > about 90%), lauric (C12) ester (less than 5%), myristic (C14) ester (less than 5%), caprylic (C8) ester (less than 3%) and capric (C10) ester (less than 3%). 50/13 may also contain free glycerol (typically less than about 1%). In a representative formulation, 50/13 included palmitic (C16) ester (about 43%), stearic (CIS) ester (about 54%) (stearate and palmitate about 97%), lauric (C12) ester (less than 1%), myristic (C14) ester (about 1%), caprylic (C8) ester (less than 1%), and capric (C10) ester (less than 1%). Stearic acid (C18) is the major fatty acid component of the glycerides and polyethylene glycol esters in 53/10. 53/10 is called PEG-32 glyceryl stearate (Jiafa Shi Co.).

In one embodiment, the polyethylene oxide-containing fatty acid ester is a laurate, palmitate, or stearate (i.e., monolaurate and dilaurate, monopalmitate and dipalmitate, and monostearate and distearate of polyethylene glycol). Mixtures of these esters may also be used.

In some embodiments, the solid dosage form comprises at least one fatty acid glyceride and at least one polyethylene oxide-containing fatty acid ester. In such embodiments, the ratio of the at least one fatty acid glyceride to the at least one polyethylene oxide-containing fatty acid ester ranges from about 90:10 to about 10:90, including about 90:10, about 85:15, about 80:20, about 75:25, about 70:30, about 65:35, about 60:40, about 55:45, about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, or about 10:90, including all ranges and subranges therebetween. In further embodiments, the solid dosage form comprises and 44/14 (as described herein). In embodiments, the ratio to 44/14 ranges from about 90:10 to about 10:90, including about 90:10, about 85:15, about 80:20, about 75:25, about 70:30, about 65:35, about 60:40, about 55:45, about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, or about 10:90, including all ranges and subranges therebetween.

Amphotericin B formulations disclosed herein optionally comprise a stabilizer. In some embodiments, the stabilizer is a thermal stabilizer, such as tocopherol polyethylene glycol succinate (e.g., TPGS or vitamin E TPGS). In some embodiments, the stabilizer is an antioxidant, such as Butylated Hydroxyanisole (BHA) or Butylated Hydroxytoluene (BHT). Such thermal stabilizers and/or antioxidants enhance the thermal stability of the formulation, which in turn can increase the shelf life of the formulation, which is particularly important in tropical regions of the world where long term exposure to high temperatures is common and refrigerated medicinal storage is difficult.

Structurally, tocopheryl polyethylene glycol succinate has polyethylene glycol (PEG) covalently coupled to a tocopherol (e.g., alpha-tocopherol or vitamin E) through a succinate linker. Because PEG is a polymer, various polymer molecular weights can be used to prepare TPGS. In one embodiment, TPGS is tocopherol polyethylene glycol succinate 1000, wherein the average molecular weight of the PEG is 1000. One suitable tocopherol polyethylene glycol succinate is vitamin E TPGS commercially available from Eastman corporation (Eastman).

In some embodiments, the dose of amphotericin B in a solid dosage form of the present disclosure ranges from about 1mg to about 500mg, including about 1mg, about 5mg, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 55mg, about 60mg, about 65mg, about 70mg, about 75mg, about 80mg, about 85mg, about 90mg, about 95mg, about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 420mg, about 370mg, about 380mg, about 390mg, about 380mg, about 390mg, about 380mg, about 180mg, about, About 450mg, about 460mg, about 470mg, about 480mg, about 490mg, or about 500mg, including all ranges and subranges therebetween.

In some embodiments, the% w/w of amphotericin B in the solid dosage form is at least about 1%, or at least about 5%, or at least about 10%, or at least about 15%, or about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%. In some embodiments, the% w/w of amphotericin B in the solid dosage form ranges from about 1% to about 70%, or from about 5% to about 60%, or from about 5% to about 50%, or from about 5% to about 40%, or from about 10% to about 40%, or from about 15% to about 40%, or from about 20% to about 35%, or from about 20% to about 30%.

The solid dosage forms of the present disclosure can be prepared by any suitable method, including granulating a therapeutic agent (e.g., amphotericin B) with an excipient (e.g., fillers, glidants, lubricants, etc. known in the art and described herein), extruding the therapeutic agent with an excipient, directly compressing the therapeutic agent with an excipient to form a tablet, and the like.

In particular embodiments, the solid dosage forms of the present disclosure can be prepared by coating the active agent, for example, amphotericin B on a solid carrier. The solid carrier can be any material that can be coated with the pharmaceutical-containing composition and is suitable for human consumption. Any conventional coating process may be used. For example, the therapeutic agent (e.g., amphotericin B) can be dissolved or suspended in a suitable solvent (e.g., ethanol) with an optional binder or alternatively with one or more of the lipophilic components described herein, and can be deposited on the solid carrier by methods known in the art (e.g., fluidized bed coating methods or pan coating methods). The solvent may be removed, or removed in situ, for example by drying during the coating process (e.g., during fluidized bed coating) and/or in a subsequent drying step.

In some embodiments, the solid carrier may be inert beads or inert particles. In other embodiments, the solid carrier is a core-sheath (non-pareil seed), an acidic buffer crystal, a basic buffer crystal, or an encapsulated buffer crystal.

In some embodiments, the solid carrier can be a sugar sphere, a fiber sphere, a lactose-Microcrystalline (MCC) sphere, a mannitol-MCC sphere, or a silicon dioxide sphere.

In other embodiments, the solid carrier may be a saccharide, a sugar alcohol, or a combination thereof. Suitable sugars include lactose, sucrose, maltose and combinations thereof. Suitable sugar alcohols include mannitol, sorbitol, xylitol, maltitol, arabitol, ribitol, dulcitol, iditol, isomaltitol, lactitol, erythritol, and combinations thereof.

In embodiments, the solid carrier may be formed by combining any of the above materials with a filler. Examples of suitable fillers that can be used to form the solid carrier include lactose, microcrystalline cellulose, silicified microcrystalline cellulose, mannitol-microcrystalline cellulose, and silicon dioxide.

In other embodiments, the dosage forms disclosed herein do not comprise a solid carrier.

In embodiments, the solid dosage forms disclosed herein may comprise one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients include fillers, diluents, glidants, disintegrants, binders and lubricants. Other pharmaceutically acceptable excipients include acidulants, alkalinizing agents, preservatives, antioxidants, buffering agents, chelating agents, coloring agents, complexing agents, emulsifying and/or solubilizing agents, flavoring agents, humectants, sweeteners, and wetting agents.

Examples of suitable fillers and/or binders include lactose (e.g., spray-dried lactose, alpha-lactose, beta-lactose, various grades of or), microcrystalline cellulose (various grades of or), hydroxypropyl cellulose, L-hydroxypropyl cellulose (low substituted), low molecular weight hydroxypropyl methylcellulose (HPMC) (e.g., Methocel E, F, and K from Dow Chemical, Inc. (Shin-Etsu, Ltd.), hydroxyethyl cellulose, sodium carboxymethyl cellulose, carboxymethyl hydroxyethyl cellulose, and other cellulose derivatives, sucrose, agarose, sorbitol, mannitol, xylitol, dextrin, maltodextrin, starch or modified starches (including potato, corn, and rice starches), calcium phosphate (e.g., basic calcium phosphate, and the like, Calcium hydrogen phosphate, dicalcium phosphate hydrate), calcium sulfate, calcium carbonate, sodium alginate, polyvinylpyrrolidone, and polyethylene glycol.

Examples of pharmaceutically acceptable diluents include calcium carbonate, calcium hydrogen phosphate, tricalcium phosphate, calcium sulfate, microcrystalline cellulose, powdered cellulose, dextran, dextrin, glucose, fructose, kaolin, lactose, mannitol, sorbitol, starch, pregelatinized starch, sucrose, and sugar.

Pharmaceutically acceptable disintegrants include alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium (e.g., and), colloidal silicon dioxide, croscarmellose sodium, crospovidone (e.g., and), guar gum, magnesium aluminum silicate, methylcellulose, microcrystalline cellulose, polacrilin potassium, powdered cellulose, pregelatinized starch, sodium alginate, sodium starch glycolate (e.g., Explotab), potato starch, and starch.

Examples of pharmaceutically acceptable glidants include colloidal silicon dioxide, magnesium trisilicate, powdered cellulose, starch, talc and tricalcium phosphate.

Pharmaceutically acceptable lubricants include stearic acid, magnesium stearate, calcium stearate or other metal stearates (e.g., zinc stearate), glyceryl monostearate, glyceryl palmitostearate, waxes and glycerides, hydrogenated castor oil, hydrogenated vegetable oil, light mineral oil, polyethylene glycol, glyceryl behenate, colloidal silica, hydrogenated vegetable oil, corn starch, sodium lauryl sulfate, sodium stearyl fumarate, polyethylene glycol, alkyl sulfates, sodium benzoate, talc and sodium acetate.

Flavoring agents and flavor enhancers make the dosage form more palatable to the patient. Common flavoring agents and flavor enhancers for pharmaceutical products that may be included in the compositions and/or combinations of the present invention include maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol, and tartaric acid.

The solid composition may also be dyed using any pharmaceutically acceptable colorant to improve its appearance and/or to facilitate patient identification of the product and unit dosage level.

The compositions disclosed herein may be formulated into solid dosage forms. Suitable solid dosage forms include tablets and capsules, such as gelatin capsules or suitable synthetic capsules known in the art, such as HPMC (hydroxypropylmethylcellulose) capsules.

In an embodiment, the solid dosage forms described herein can be prepared by:

(a) Dissolving at least one lipid and a therapeutic agent in a solvent, thereby forming a liquid mixture comprising the therapeutic agent;

(b) Coating a mixture comprising a therapeutic agent on a solid carrier; and

(c) Removing the solvent;

Thereby forming drug-coated particles.

Any solvent in which the lipophilic component and therapeutic agent can be dissolved can be used to prepare the solid dosage forms described herein. Examples of suitable solvents include lipophilic solvents, such as lipophilic organic solvents. Non-limiting examples of solvents include alcohols (e.g., ethanol, propanol, isopropanol, etc.), ketones (e.g., acetone, etc.), dimethyl sulfoxide, methylene chloride, and the like.

The drug-coated particles can be milled as desired and passed through one or more mesh screens to produce particles having the desired size range. In various embodiments, the average particle size of the drug-coated particles may range from 10 to 2000 μm, such as 100-1000 μm or 500-1000 μm.

In embodiments, the drug-coated particles may be filled into capsules or compressed into tablets, optionally in combination with various excipients as described herein.

In other embodiments, a therapeutic agent (e.g., amphotericin B) and a melt of a suitable amount of room temperature solid lipophilic component (as described herein) can be mixed together (e.g., using methods other than the compositions disclosed in u.s.8,592,382 and u.s.8,673, 866), optionally with a suitable amount of solvent (e.g., ethanol), until a homogeneous mixture or solution is formed. The resulting mixture or solution is then allowed to cool, thereby forming a semi-solid composition. The semi-solid composition can then be filled into a gelatin capsule, thereby providing a solid dosage form.

In embodiments, the amphotericin B dosage forms disclosed herein are bioequivalent to conventional liquid formulations. That is, the solid dosage form has a mean maximum plasma concentration (Cmax), a mean AUC, and a mean Tmax that are within about 80% to about 125% of each of the mean Cmax, mean AUC, and mean Tmax of a conventional liquid composition when administered to a human or animal (e.g., a rat model or a beagle model). As used herein, Cmax, AUC, and Tmax refer to the average of such values measured for a population of subjects.

Conventional liquid dosage forms provided a Cmax of amphotericin B of 71 + -10 ng/mL when orally administered to male Sprague-Duller (Sprague Dawley) rats at a dose of 4.5mg/kg, or 96 + -15 ng/mL when orally administered to male Sprague-Duller rats at a dose of 10 mg/kg.

In some embodiments, a solid dosage form described herein provides a Cmax of amphotericin B in the range of about 80% to about 125% (i.e., about 71 ± 10ng/mL) of 61ng/mL to 81ng/mL, e.g., about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL, about 105ng/mL, including all values and subranges therebetween, when orally administered to male sprag-duller rats at a dose of 4.5 mg/kg.

In other embodiments, when orally administered to male Sprague-Duler rats at a dose of 10mg/kg, the solid dosage forms described herein provide a Cmax for amphotericin B in the range of about 80% to about 125% of 81ng/mL to 111ng/mL (i.e., 96 + -15 ng/mL), for example, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL, about 105ng/mL, about 110ng/mL, about 115ng/mL, about 120ng/mL, about 125ng/mL, about 130ng/mL, about 135ng/mL, about 140ng/mL, about 145ng/mL, including all values and subranges therebetween.

Conventional liquid dosage forms provide an AUC (0-24) of amphotericin B of 991 ± 170h · ng/mL when orally administered to male sprague-doller rats at a dose of 4.5mg/kg, or 1534 ± 229h · ng/mL when orally administered to male sprague-doller rats at a dose of 10 mg/kg.

In some embodiments, a solid dosage form described herein provides an AUC (0-24) of amphotericin B in the range of about 80% to about 125% of about 821 h-ng/mL to about 1161 h-ng/mL (i.e., 991 ± 170 h-ng/mL), e.g., about 600 h-ng/mL, about 650 h-ng/mL, about 700 h-ng/mL, about 750 h-ng/mL, about 800 h-ng/mL, about 850 h-ng/mL, about 900 h-ng/mL, about 950 h-ng/mL, about 1000 h-ng/mL, about 1050 h-ng/mL, about 1100 h-ng/mL, about 1150 h-ng/mL, about 1200 h-ng/mL, about 1250 h-ng/mL, about 1300 h-ng/mL, when orally administered to male sprag-duller rats at a dose of 4.5mg/kg, About 1350 h-ng/mL, about 1400 h-ng/mL, about 1450 h-ng/mL, about 1500 h-ng/mL, or about 1550 h-ng/mL, including all values and subranges therebetween.

In other embodiments, a solid dosage form described herein provides an AUC (0-24) of amphotericin B in the range of about 80% to about 125% of about 1305 h-ng/mL to about 1763 h-ng/mL (i.e., 1534 ± 229 h-ng/mL) when administered orally to male sprague-duller rats at a dose of 10mg/kg, e.g., about 1000 h-ng/mL, about 1050 h-ng/mL, about 1100 h-ng/mL, about 1150 h-ng/mL, about 1200 h-ng/mL, about 1250 h-ng/mL, about 1300 h-ng/mL, about 1350 h-ng/mL, about 1400 h-ng/mL, about 1450 h-ng/mL, about 1500 h-ng/mL, about 1550 h-ng/mL, about 1600 h-ng/mL, about 1650 h-ng/mL, about 1700 h-ng/mL, about 1600 h-ng/mL, or, About 1750 h-ng/mL, about 1800 h-ng/mL, about 1850 h-ng/mL, about 1900 h-ng/mL, about 1950 h-ng/mL, about 2000 h-ng/mL, about 2050 h-ng/mL, about 2100 h-ng/mL, about 2150 h-ng/mL, about 2200 h-ng/mL, or about 2250 h-ng/mL, including all values and subranges therebetween.

Conventional liquid dosage forms provide an AUC (0-48) of amphotericin B of 2695 ± 433h · ng/mL when orally administered to male sprague-doller rats at a dose of 10 mg/kg.

In embodiments, a solid dosage form of amphotericin B disclosed herein provides an AUC (0-48) of amphotericin B in the range of about 80% to about 125% of about 2262h ng/mL to about 3128h ng/mL, e.g., about 1750h ng/mL, about 1800h ng/mL, about 1850h ng/mL, about 1900h ng/mL, about 1950h ng/mL, about 2000h ng/mL, about 2050h ng/mL, about 2100h ng/mL, about 2150h ng/mL, about 2200h ng/mL, about 2250h ng/mL, about 2300h ng/mL, about 2350h ng/mL, about 2400h ng/mL, about 2500h ng/mL when administered orally to male Spragger-Duller rats at a dose of 10mg/kg, About 2550h ng/mL, about 2600h ng/mL, about 2650h ng/mL, about 2700h ng/mL, about 2750h ng/mL, about 2800h ng/mL, about 2850h ng/mL, about 2900h ng/mL, about 2950h ng/mL, about 3000h ng/mL, about 3050h ng/mL, about 3100h ng/mL, about 3150h ng/mL, about 3200h ng/mL, about 3250h ng/mL, about 3300h ng/mL, about 3350h ng/mL, about 3400h ng/mL, about 3450h ng/mL, about 3500h ng/mL, about 3550h ng/mL, about 3600h ng/mL, about 3650h ng/mL, about 0h ng/mL, about 3750h ng/mL, about 3700h ng/mL, about 3750h ng/mL, about 3800h ng/mL, About 3850 h-ng/mL, about 3900 h-ng/mL, about 4000 h-ng/mL, including all values and subranges therebetween.

Conventional liquid dosage forms provide a Tmax for amphotericin B of 6.3 + 0.9h when orally administered to male sprague-doller rats at a dose of 4.5mg/kg, or 12.5 + 2.7h when orally administered to male sprague-doller rats at a dose of 10 mg/kg.

In embodiments, the solid dosage form of amphotericin B disclosed herein provides a Tmax for amphotericin B in the range of about 80% to about 125% of about 5.4h to about 7.2h, e.g., about 4.1h, about 4.2h, about 4.3h, about 4.4h, about 4.5h, about 4.6h, about 4.7h, about 4.8h, about 4.9h, about 5.0h, about 5.1h, about 5.2h, about 5.3h, about 5.4h, about 5.5h, about 5.6h, about 5.7h, about 5.8h, about 5.9h, about 6.0h, about 6.1h, about 6.2h, about 6.3h, about 6.4h, about 6.6h, about 6.7h, about 6.8h, about 7.9h, about 6.0h, about 6.1h, about 6.2h, about 6.3h, about 6.4h, about 6.6h, about 6.7h, about 6.8h, about 7.8h, about 7.0h, about 7h, about 7.2h, about 7.8h, about 7h, about 7.0h, about 7h, about 7.2h, about 7.3h, about 6.3h, about 7, About 8.4h, about 8.5h, about 8.6h, about 8.7h, about 8.8h, about 8.9h, about 9h, about 9.1h, about 9.2h, about 9.3h, about 9.4h, or about 9.5h, including all values and subranges therebetween.

In embodiments, the solid dosage form of amphotericin B disclosed herein provides a Tmax for amphotericin B in the range of about 80% to about 125% of about 9.8h to about 15.2h, e.g., about 7.0h, about 7.1h, about 7.2h, about 7.3h, about 7.4h, about 7.5h, about 7.6h, about 7.7h, about 7.8h, about 7.9h, about 8.0h, about 8.1h, about 8.2h, about 8.3h, about 8.4h, about 8.5h, about 8.6h, about 8.7h, about 8.8h, about 8.9h, about 9h, about 9.1h, about 9.2h, about 9.3h, about 9.4h, about 9.5h, about 9.7h, about 10.8h, about 10.9h, about 10h, about 10.1h, about 9.2h, about 9.3h, about 9.4h, about 9.5h, about 9.9h, about 10.10 h, about 10.1h, about 10.2h, about 10.3h, about 10.9h, about 10h, about 10.9h, about 11.3h, about 11.4h, about 11.5h, about 11.6h, about 11.7h, about 11.8h, about 11.9h, about 12.0h, about 12.1h, about 12.2h, about 12.3h, about 12.4h, about 12.5h, about 12.6h, about 12.7h, about 12.8h, about 12.9h, about 13.0h, about 13.1h, about 13.2h, about 13.3h, about 13.4h, about 13.5h, about 13.6h, about 13.7h, about 13.8h, about 13.9h, about 14.0h, about 14.1h, about 14.2h, about 14.3h, about 14.4h, about 14.5h, about 14.6h, about 14.7h, about 14.8h, about 14.9h, about 15.1h, about 15.2h, about 16.1h, about 16.2h, about 16.6h, about 17.6h, about 16.7h, about 15.0h, about 15.1h, about 15.2h, about 16.1h, about 16.2h, about 16, about 16.1h, about 16.6h, about 16.2h, about 17, about 16.6h, about 16.1h, about 16, about 16.2h, about 17, about 16.6h, about 16.2h, about 17.6h, about 16.6h, about 17, about 16.2h, about 17.6h, about 17, about 17.8h, about 17.9h, about 18.0h, about 18.1h, about 18.2h, about 18.3h, about 18.4h, about 18.5h, about 18.6h, about 18.7h, about 18.8h, about 18.9h, about 19.0h, about 19.1h, about 19.2h, about 19.3h, about 19.4h, about 19.5h, including all values and subranges therebetween.

The solid dosage forms described herein have been administered to beagle dogs and the mean plasma concentrations were measured after administration. In embodiments, between 1 hour and 24 hours after oral administration of a dose of 100mg to a beagle dog, the solid dosage forms described herein provide a plasma concentration of amphotericin B that is in the range of about 80% to about 125% of about 7.61ng/mL to about 52.21ng/mL, e.g., about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, including all values and subranges therebetween.

In some embodiments, a solid dosage form described herein provides a Cmax (in beagle dogs) of amphotericin B after oral administration of a 100mg dose to a beagle dog ranging from about 80% to about 125% of about 39.3ng/mL to about 53.5ng/mL (i.e., 46.4 ± 53.5ng/mL), e.g., about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, including all values and subranges therebetween. In other embodiments, the solid dosage forms described herein provide a Cmax (in beagle dogs) of amphotericin B after oral administration of a 100mg dose to a beagle dog ranging from about 80% to about 125% of about 45.3ng/mL to about 59.7ng/mL (i.e., 52.5 ± 7.2ng/mL), e.g., about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, including all values and subranges therebetween.

In other embodiments, upon oral administration of a 100mg dose to a beagle dog, the solid dosage forms described herein provide a Tmax for amphotericin B (in beagle dogs) ranging from about 80% to about 125% of about 9.5h to about 18.5h (i.e., 14.0 ± 4.5h), e.g., about 7.5h, about 8.0h, about 9.0h, about 9.5h, about 10h, about 10.5h, about 11h, about 11.5h, about 12h, about 12.5h, about 13h, about 13.5h, about 14h, about 14.5h, about 15h, about 15.5h, about 16h, about 16.5h, about 17h, about 17.5h, about 18h, about 18.5h, about 19h, about 19.5h, about 20h, about 20.5h, about 21h, about 21.5h, about 22h, about 22.5h, about 23.5h, about 24h, about 24.5h, about 24h, about 25.5h, and all ranges therebetween.

In other embodiments, the solid dosage forms described herein provide a Tmax for amphotericin B (in beagle dogs) ranging from about 80% to about 125% of about 4.7h to about 11.3h (i.e., 8.0 ± 3.3h), e.g., about 3.5h, about 3.6h, about 3.7h, about 3.8h, about 3.9h, about 4.0h, about 4.1h, about 4.2h, about 4.3h, about 4.4h, about 4.5h, about 4.6h, about 4.8h, about 4.9h, about 5.0h, about 5.1h, about 5.2h, about 5.3h, about 5.4h, about 5.5h, about 5.6h, about 5.7h, about 5.8h, about 5.9h, about 6.9h, about 6h, about 6.0h, about 6.2h, about 7.3h, about 5.4h, about 5.5h, about 6.6h, about 6.7h, about 6h, about 7.8h, about 6h, about 6.0h, about 7.7h, about 6h, about 6.7.7 h, about 6h, about 6.7h, about 6.7.7 h, about 6h, about 6.7.7.7 h, about 6h, about 6.7.7 h, about 6, About 8.0h, about 8.1h, about 8.2h, about 8.3h, about 8.4h, about 8.5h, about 8.6h, about 8.7h, about 8.8h, about 8.9h, about 9h, about 9.1h, about 9.2h, about 9.3h, about 9.4h, about 9.5h, about 9.6h, about 9.7h, about 9.8h, about 9.9h, about 10.0h, about 10.1h, about 10.2h, about 10.3h, about 10.4h, about 10.5h, about 10.6h, about 10.7h, about 10.8h, about 10.9h, about 11.0h, about 11.1h, about 11.2h, about 11.3h, about 11.4h, about 11.5h, about 11.6h, about 11.8h, about 12.12 h, about 13.1h, about 13.12 h, about 13.2h, about 13.1h, about 13.2h, about 13.3h, about 13.4h, about 11.4h, about 11.5h, about 11.7h, about 12.12 h, about 13.12 h, about 13.1h, about 13.12 h, about 13.2h, about 13h, about 13.2h, about 13.4h, about 13.2h, About 14.5h, including all values and subranges therebetween.

In some embodiments, a solid dosage form described herein provides an AUC0-Tlast (in beagle dogs) of amphotericin B ranging from about 1409ng hr/mL to about 1991ng hr/mL (i.e., 1700 ± 291ng hr/mL), such as about 1100ng hr/mL, about 1200ng hr/mL, about 1300ng hr/mL, about 1400ng hr/mL, about 1500ng hr/mL, about 1600ng hr/mL, about 1800ng hr/mL, about 1700ng hr/mL, about 1300ng hr/mL, about 1900ng hr/mL, about 2000ng hr/mL, about 2100ng hr/mL, about 2200ng hr/mL, about 2300ng hr/mL, about 2500ng hr/mL, including all values and subranges therebetween. In other embodiments, following oral administration of a dose of 100mg to a beagle dog, the solid dosage forms described herein provide amphotericin B having an AUC0-Tlast (ng × hr/mL) ranging from about 1777ng × hr/mL to about 125% of (i.e., 2146 ± 369ng × hr/mL), e.g., about 1400ng × hr/mL, about 1500ng × hr/mL, about 1600ng × hr/mL, about 1700ng × hr/mL, about 1800ng × hr/mL, about 1900ng × hr/mL, about 2000ng × hr/mL, about 2100ng × hr/mL, about 2200ng × hr/mL, about 2300ng × hr/mL, about 2400ng × hr/mL, about 2500ng × hr/mL, about 2700ng × hr/mL, about 2600ng × hr/mL, about 2500ng × hr/mL, about 2400ng × hr/mL, about 2515ng × hr/mL, about 10ng × mg × hr/mL, about 10mg × mg/mL, about 2900ng hr/mL, about 3000ng hr/mL, about 3100ng hr/mL, about 3200ng hr/mL, including all values and subranges therebetween.

In some embodiments, after oral administration of a 100mg dose to a beagle dog, the solid dosage forms described herein provide an MRTLast of amphotericin B (in beagle dogs) in the range of about 26.1hr to about 80% to about 125% of 27.3hr (i.e., 26.7 ± 0.6hr), e.g., about 20hr, about 20.5hr, about 21hr, about 21.5hr, about 22hr, about 22.5hr, about 23hr, about 23.5hr, about 24hr, about 24.5hr, about 25hr, about 25.5hr, about 26hr, about 26.5hr, about 27hr, about 27.5hr, about 28hr, about 28.5hr, about 29hr, about 29.5hr, about 30hr, about 31.5hr, about 32hr, about 32.5hr, about 33hr, about 33.5hr, about 34hr, about 34.5hr, and all ranges subsumed therein. In some embodiments, after oral administration of a 100mg dose to a beagle dog, the solid dosage forms described herein provide an MRTLast of amphotericin B (in beagle dogs) in the range of about 25.5hr to about 29.1hr (i.e., 27.3 ± 1.8hr) of about 80% to about 125%, e.g., about 20hr, about 20.5hr, about 21hr, about 21.5hr, about 22hr, about 22.5hr, about 23hr, about 23.5hr, about 24hr, about 24.5hr, about 25hr, about 25.5hr, about 26hr, about 26.5hr, about 27hr, about 27.5hr, about 28hr, about 28.5hr, about 29hr, about 29.5hr, about 30hr, about 31.5hr, about 32hr, about 32.5hr, about 33hr, about 33.5hr, about 34hr, about 34.5hr, about 35.5hr, about 30hr, about 31.5hr, about 32.5hr, about 36hr, and all ranges subsumed therein.

In some embodiments, the AUC, Cmax, Tmax and/or MRTLast does not vary by more than 20% between fed and fasted states. That is, in some embodiments, the percent difference in AUC0-Tlast (ng hr/mL) in the fed and fasted states is less than or equal to 20%, such as less than or equal to about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, and about 0.1%, including all values therebetween. In some embodiments, the percent difference in Cmax (ng hr/mL) in fed and fasted states is less than or equal to 20%, such as less than or equal to about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, and about 0.1%, including all values therebetween.

In some embodiments, a solid dosage form described herein provides a Cmax (in a beagle dog) ranging from about 80% to about 125% of about 40.63ng/mL to about 82.57ng/mL (i.e., 61.6 ± 20.97ng/mL), e.g., about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL, and about 105ng/mL, including all values and subranges therebetween, upon oral administration of a 500mg dose to a beagle dog in a fasted state.

In some embodiments, upon oral administration of a 500mg dose to a fed beagle dog, a solid dosage form described herein provides a Cmax (in the beagle dog) ranging from about 80% to about 125% of about 44ng/mL to about 88.75ng/mL (i.e., 66.5 ± 22.5ng/mL), e.g., about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, about 100ng/mL, and about 105ng/mL, including all values and subranges therebetween.

In some embodiments, upon oral administration of a dose of 500mg to a dog in a fasted state, the solid dosage forms described herein provide AUC0-Tlast (in the dog) ranging from about 568ng hr/mL to about 125% of, e.g., about 400ng hr/mL, about 500ng hr/mL, about 600ng hr/mL, about 700ng hr/mL, about 800ng hr/mL, about 900ng hr/mL, about 1000ng hr/mL, about 1100ng hr/mL, about 1200ng hr/mL, about 1300ng hr/mL, about 1400ng hr/mL, about 1600ng hr/mL, about 1700ng hr/mL, about 700ng hr/mL, about 800ng hr/mL, about 900ng hr/mL, about 1600ng hr/mL, about 1800ng hr/mL, about 1700ng hr/mL, about 700ng hr/mL, about 1400ng hr/mL, about 1600 ng/mL, about 1700 ng/mL, or the composition of the dog, About 1900ng x hr/mL, about 2000ng x hr/mL, about 2100ng x hr/mL, about 2200ng x hr/mL, about 2300ng x hr/mL, about 2400ng x hr/mL, about 2500ng x hr/mL, including all values and subranges therebetween.

In some embodiments, upon oral administration of a dose of 500mg to a fed dog, the solid dosage forms described herein provide AUC0-Tlast (in the dog) ranging from about 657ng hr/mL to about 1791ng hr/mL (i.e., 1224 ± 567ng/mL), e.g., about 500ng hr/mL, about 600ng hr/mL, about 700ng hr/mL, about 800ng hr/mL, about 900ng hr/mL, about 1000ng hr/mL, about 1100ng hr/mL, about 1200ng hr/mL, about 1300ng hr/mL, about 1400ng hr/mL, about 1500ng hr/mL, about 1600ng hr/mL, about 1700ng hr/mL, about 1900ng mL, about 1400 ng/mL, about 1500ng hr/mL, about 1600ng hr/mL, about 1800 ng/mL, about 1900 ng/mL, about, About 2000ng x hr/mL, about 2100ng x hr/mL, about 2200ng x hr/mL, about 2300ng x hr/mL, about 2400ng x hr/mL, about 2500ng x hr/mL, including all values and subranges therebetween.

In some embodiments, the solid dosage forms described herein provide a Tmax for amphotericin B (in beagle dogs) ranging from about 80% to about 125% of about 5h to about 13h (i.e., 9 ± 4h) after oral administration of a 500mg dose to a beagle dog in the fed or fasted state, e.g., about 3.9h, about 4.0h, about 4.1h, about 4.2h, about 4.3h, about 4.4h, about 4.5h, about 4.6h, about 4.8h, about 4.9h, about 5.0h, about 5.1h, about 5.2h, about 5.3h, about 5.4h, about 5.5h, about 5.6h, about 5.7h, about 5.8h, about 5.9h, about 6.0h, about 6.1h, about 6.2h, about 6.3h, about 6.6h, about 6.7h, about 7.8h, about 7.0h, about 6.1h, about 6.2h, about 6.3h, about 6.6h, about 7h, about 7.7h, about 7.8h, about 7h, about 7.8h, about 7.0h, about 7h, about 7.0h, about 6.0h, about 8.2h, about 8.3h, about 8.4h, about 8.5h, about 8.6h, about 8.7h, about 8.8h, about 8.9h, about 9h, about 9.1h, about 9.2h, about 9.3h, about 9.4h, about 9.5h, about 9.6h, about 9.7h, about 9.8h, about 9.9h, about 10.0h, about 10.1h, about 10.2h, about 10.3h, about 10.4h, about 10.5h, about 10.6h, about 10.7h, about 10.8h, about 10.9h, about 11.0h, about 11.1h, about 11.2h, about 11.3h, about 11.4h, about 11.5h, about 11.6h, about 11.7h, about 11.8h, about 11.0h, about 12.1h, about 13.2h, about 13.3h, about 13.4h, about 12.4h, about 13.5h, about 13.6h, about 13.7h, about 13.12 h, about 13.1h, about 13.2h, about 13.14 h, about 13.2h, about 13.14 h, about 13.1h, about 13.2h, about 13.14 h, about 13.2h, about 13h, about 13.2h, about 13.14 h, about 13.2h, about 13.1h, about 13.14 h, about 13.4h, about 13.2h, About 14.7h, about 14.8h, about 14.9h, about 15.0h, about 15.1h, about 15.2h, about 15.3h, about 15.4h, about 15.5h, about 15.6h, about 15.7h, about 15.8h, about 15.9h, about 16.0h, about 16.1h, about 16.2h, and about 16.3h, including all values and subranges therebetween.

In some embodiments, the solid dosage forms described herein provide an MRTLast (in beagle dogs) of amphotericin B ranging from about 10.29hr to about 14.69hr (i.e., 12.49 ± 2.2hr) of about 80% to about 125%, e.g., about 8.0h, about 8.1h, about 8.2h, about 8.3h, about 8.4h, about 8.5h, about 8.6h, about 8.7h, about 8.8h, about 8.9h, about 9h, about 9.1h, about 9.2h, about 9.3h, about 9.4h, about 9.5h, about 9.6h, about 9.7h, about 9.8h, about 9.9h, about 10.0h, about 10.1h, about 10.2h, about 10.3h, about 10.5h, about 10.6h, about 9.7h, about 9.8h, about 9.9h, about 10.0h, about 10.1h, about 10.2h, about 10.5h, about 10.1h, about 10.11 h, about 11.11 h, about 10.11 h, about 10.1h, about 10.11 h, about 10.6h, about 11h, about 11.11 h, about 9.11 h, about 9.6h, about 11h, about 11.6h, about 9.7h, about 9.8h, about 10.9h, about 10h, About 12.3h, about 12.4h, about 12.5h, about 12.6h, about 12.7h, about 12.8h, about 12.9h, about 13.0h, about 13.1h, about 13.2h, about 13.3h, about 13.4h, about 13.5h, about 13.6h, about 13.7h, about 13.8h, about 13.9h, about 14.0h, about 14.1h, about 14.2h, about 14.3h, about 14.4h, about 14.5h, about 14.6h, about 14.7h, about 14.8h, about 14.9h, about 15.0h, about 15.1h, about 15.2h, about 15.3h, about 15.4h, about 15.5h, about 15.6h, about 15.7h, about 15.8h, about 15.9h, about 16.0h, about 16.1h, about 16.2h, about 16.3h, about 16.4h, about 16.5h, about 16.6h, about 16.7h, about 16.8h, about 16.9h, about 17h, about 17.1h, about 17.2h, about 17.3h, about 17.4h, about 17.5h, about 17.6h, about 17.7h, about 17.8h, about 17.9h, about 18.0h, about 18.1h, about 18.2h, about 18.3h, about 18.4h, and about 18.5h, including all values and subranges therebetween.

In some embodiments, the solid dosage forms described herein provide an MRTLast (in beagle dogs) of amphotericin B ranging from about 10.29hr to about 14.69hr (i.e., 12.06 ± 1.4hr) of about 80% to about 125%, e.g., about 8.0h, about 8.1h, about 8.2h, about 8.3h, about 8.4h, about 8.5h, about 8.6h, about 8.7h, about 8.8h, about 8.9h, about 9h, about 9.1h, about 9.2h, about 9.3h, about 9.4h, about 9.5h, about 9.6h, about 9.7h, about 9.8h, about 9.9h, about 10.0h, about 10.1h, about 10.2h, about 10.5h, about 10.6h, about 9.7h, about 9.8h, about 9.9h, about 10.0h, about 10.1h, about 10.2h, about 10.5h, about 10.6h, about 11.11 h, about 10.1h, about 10.11 h, about 10.6h, about 10.11 h, about 11h, about 11.7h, about 10.7h, about 10.8h, about 10.9h, about, About 12.3h, about 12.4h, about 12.5h, about 12.6h, about 12.7h, about 12.8h, about 12.9h, about 13.0h, about 13.1h, about 13.2h, about 13.3h, about 13.4h, about 13.5h, about 13.6h, about 13.7h, about 13.8h, about 13.9h, about 14.0h, about 14.1h, about 14.2h, about 14.3h, about 14.4h, about 14.5h, about 14.6h, about 14.7h, about 14.8h, about 14.9h, about 15.0h, about 15.1h, about 15.2h, about 15.3h, about 15.4h, about 15.5h, about 15.6h, about 15.7h, about 15.8h, about 15.9h, about 16.0h, about 16.1h, about 16.2h, about 16.3h, about 16.4h, about 16.5h, about 16.6h, about 16.7h, about 16.8h, about 16.9h, about 17h, about 17.1h, about 17.2h, about 17.3h, about 17.4h, about 17.5h, about 17.6h, about 17.7h, about 17.8h, about 17.9h, about 18.0h, about 18.1h, about 18.2h, about 18.3h, about 18.4h, and about 18.5h, including all values and subranges therebetween.

The amphotericin B dosage forms described may be administered according to any suitable dosing regimen sufficient to treat a condition in a subject in need thereof. In particular embodiments, an amphotericin B formulation as described herein is administered to a subject one or more times, two or more times, three or more times, four or more times, five or more times, or six or more times, wherein there is a duration of time between each provision. In some embodiments, it may be desirable to administer multiple dosage forms simultaneously to provide the desired dosage. In particular embodiments, the amphotericin B formulation is provided to a subject (e.g., human) once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times, wherein there is a duration of time between each provision. In particular embodiments, the amphotericin B formulation is provided to the subject about once per day for about four days, about once per day for about five days, about once per day for about six days, or about once per day for about one week. In particular embodiments, the amphotericin B formulation is provided to the subject once a day, twice a day, three times a day, or four times a day, e.g., for any of the durations described herein. In particular embodiments, the amphotericin B formulation is provided to the subject about once a day, twice a day, three times a day, four times a day, or once every two days for about three days, four days, five days, six days, one week, two weeks, three weeks, one month, or two months or more. In particular embodiments, the number of days and/or weeks is continuous. In some embodiments, amphotericin B dosage forms described herein are formulated for once daily administration.

In some embodiments, the total daily dose of amphotericin B ranges from about 50 mg/day to about 1500 mg/day, e.g., about 100 mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 300 mg/day, 350 mg/day, 400 mg/day, 450 mg/day, 500 mg/day, 550 mg/day, 600 mg/day, 650 mg/day, 700 mg/day, 750 mg/day, 800 mg/day, 850 mg/day, 900 mg/day, 950 mg/day, 1000 mg/day, 1050 mg/day, 1100 mg/day, 1150 mg/day, 1200 mg/day, 1250 mg/day, 1300 mg/day, 1350 mg/day, 1400 mg/day, or, 1450 mg/day, or about 1500 mg/day, including all values and subranges therein.

In some embodiments, the amphotericin B formulations disclosed herein are provided to the subject multiple times per day. In some such embodiments, amphotericin B is present in a single dose in an amount ranging from about 50 mg/day to about 1500 mg/day, e.g., about 100 mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 300 mg/day, 350 mg/day, 400 mg/day, 450 mg/day, 500 mg/day, 550 mg/day, 600 mg/day, 650 mg/day, 700 mg/day, 750 mg/day, 800 mg/day, 850 mg/day, 900 mg/day, 950 mg/day, 1000 mg/day, 1050 mg/day, 1100 mg/day, 1150 mg/day, 1200 mg/day, 1250 mg/day, 1300 mg/day, 1350 mg/day, 1400 mg/day, B, or a combination thereof, 1450 mg/day, or about 1500 mg/day, including all values and subranges therein.

In some embodiments, a single dose of amphotericin B formulation disclosed herein comprises multiple dosage forms (e.g., multiple capsules). For example, in some embodiments, a single dose of an amphotericin B formulation may comprise at least about 1 dosage form, at least about 2 dosage forms, at least about 3 dosage forms, at least about 4 dosage forms, at least about 5 dosage forms, at least about 6 dosage forms, at least about 7 dosage forms, at least about 8 dosage forms, at least about 9 dosage forms, or at least about 10 dosage forms, and the like. In other embodiments, a single dose of an amphotericin B formulation comprises from about 1 dosage form to about 10 dosage forms, such as about 2 dosage forms, about 3 dosage forms, about 4 dosage forms, about 5 dosage forms, about 6 dosage forms, about 7 dosage forms, about 8 dosage forms, or about 9 dosage forms, including all values and subranges therein.

Amphotericin B dosage forms can be administered to treat any infection that responds to amphotericin B. In some embodiments, the amphotericin dosage forms described herein can be used to treat infectious diseases such as fungal infections, Human Immunodeficiency Virus (HIV), and parasitic infections. Infectious diseases that can be treated by the methods and formulations disclosed herein include fungal infections (aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, cryptococcosis, histoplasmosis, mucormycosis, paracoccidioidomycosis, and sporotrichosis), visceral leishmaniasis, cutaneous leishmaniasis, Chagas disease, and febrile neutropenia. Amphotericin B has been shown to bind to amyloid and prevent fibril formation. Accordingly, amphotericin B disclosed herein may be used to treat diseases associated with fibrillogenesis, such as alzheimer's disease.

in some embodiments, the present disclosure provides methods for treating visceral leishmaniasis, comprising orally administering a solid dosage form described herein comprising an effective amount of amphotericin B to a subject in need thereof. In another embodiment, the present disclosure provides a method of treating a fungal infection, the method comprising orally administering a solid dosage form described herein comprising an effective amount of amphotericin B described herein to a subject in need thereof. In particular embodiments, a therapeutically effective amount of amphotericin B is sufficient to achieve plasma levels of 0.01. mu.M to 10mM, 0.01. mu.M to 1mM, 0.01. mu.M to 100nM, or 0.01. mu.M to 10 mM. The therapeutically effective amount of amphotericin B administered may vary depending on the subject and the severity of the condition. In one embodiment, a therapeutically effective amount may range from about 0.01mg/kg to about 1000mg/kg, from about 0.1mg/kg to about 100mg/kg, from about 0.5mg/kg to about 50mg/kg, from about 1mg/kg to about 20mg/kg of the subject's body weight, or from about 5mg/kg to about 10mg/kg, such as about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, or about 10 mg/kg.

Examples of the invention

Example 1: materials and methods

Table 1 provides a description of the materials used for the analytical studies described herein. Amphotericin (AmpB) was stored at 2-8 ℃ protected from light and moisture. Other materials were stored at Room Temperature (RT).

Table 1: material

Sample preparation 1

The contents of the 2 capsules were emptied into a 200ml low light flask.

NMP (about 80% by volume) was added and sonicated for 15 minutes while an ice pack was added to the bath.

Shake for 15 minutes.

The solution was mixed and allowed to equilibrate to room temperature.

Diluted to volume with NMP.

5mL of the above solution was diluted to 50mL with diluent A (25% ammonium acetate solution/25% NMP/50% methanol).

The filter was made with a 0.45 μm nylon filter and the first 3ml was discarded.

Sample preparation 2

The contents of the 2 capsules were emptied into a 200ml low light flask.

Add 50ml of NMP and sonicate for 15 minutes while adding an ice bag to the bath.

Shake for 15 minutes.

About 90% by volume of diluent B (ammonium acetate solution/methanol 1:2) was added.

The solution was mixed well and allowed to equilibrate to room temperature.

Diluted to volume with diluent B.

5mL of the above solution was diluted to 50mL with diluent A (25% ammonium acetate solution/25% NMP/50% methanol).

The filter was made with a 0.45 μm nylon filter and the first 3ml was discarded.

Sample preparation 3

Transfer the contents of 4 capsules and empty gelatin capsules to a 500ml low light flask.

Add 125ml of NMP and sonicate for 30-45 minutes (add ice packs to minimize heating in the bath) until the sample is completely dispersed. During sonication, the device is shaken vigorously at regular intervals. Note that: the capsule shell remains intact.

About 90% by volume of diluent B (ammonium acetate solution/methanol 1:2) was added.

The solution was mixed well and allowed to equilibrate to room temperature (placed in a refrigerator for rapid cooling).

Diluted to volume with diluent B.

3mL of the above solution was diluted to 25mL with diluent A (25% ammonium acetate solution/25% NMP/50% methanol).

The filter was made with a 0.45 μm nylon filter and the first 3ml was discarded.

Sample preparation 4

Transfer the contents of the 3 capsules to a 500ml low light flask.

Fill volume with 0.5% SDS in water.

Add stir bar and stir for a minimum of 90 minutes.

8mL of the above solution was diluted to 50mL with 0.5% SDS in water.

The filter was made with a 0.45 μm nylon filter and the first 3ml was discarded.

Example 2: solid formulations

Amphotericin B with Gelucire/Peceol/TPGS containing formulations was prepared as shown in tables 2-4 based on reference iCo/Wasan liquid formulation in table 5.

The first Gelucire and TPGS were melted and weighed, both in the same vessel. The Peceol was weighed and added to the Gelucire-TPGS mixture. Ethanol was weighed and added to the Gelucire-TPGS-Peceol mixture and mixed using a stirring hotplate at a temperature of about 40 ℃ until all ingredients were dissolved (# 1 in fig. 1). The solution was added to the API (# 2 in fig. 1) and mixed using a pestle for about 5 minutes. This mixture was 'creamy' (# 3 in fig. 1) at 25 ℃. The internal phase powder excipients were separately mixed using a V blender at 25rpm for 2 minutes. The two mixtures were mixed using a pestle/mortar for about 5 minutes. The resulting mixture (# 4 in fig. 1) was placed in an oven at 40 ℃ for 1-2 hours to evaporate ethanol, and then removed from the oven and held at 22-25 ℃ for about 2 hours. The particles were obtained by grinding through a 20 mesh (850 μm) sieve. A lubricant (e.g., magnesium stearate) is mixed with the granules using a V blender for 2 minutes. The final blend (# 5 in fig. 1) was encapsulated into hard shell gelatin capsules of size "0" (435 mg/capsule). The capsules were filled by volume using a tapping/tamping technique.

Table 2: formulation 1

Items a-h are internal phase components and item i is an external phase component.

Table 3: formulation 2

Items a-g are internal phase components and item h is an external phase component.

Table 4: formulation 3

Items a-g are internal phase components and item h is an external phase component.

Table 5: comparison of iCo/Wasan formulation with formulations 1-3

EXAMPLE 2 Scale-Up test of solid formulations from example 1

Formulation 1 and formulation 2 were scaled up from 20g to 100g (tables 6-7; formulation 1A and formulation 2A, respectively). Granulation was performed using a GMX top drive high shear granulation/mixing system, where Gelucire, Peceol and TPGS were dissolved in ethanol and this solution was added at 26 g/min and mixed to the API at 60rpm for 3 minutes. The powdered ingredients were separately mixed for 2 minutes using a V blender. This powder blend was added to the Gelucire/Peceol/TPGS/ethanol/drug mixture and mixed for 6 minutes at impeller/chopper speed of 850/1800 rpm. The ethanol was then removed using a fluid bed dryer at 40 ℃. Fluidization is maintained until the volatile compound content is less than or equal to 3% (about 20 minutes). The volatile compound content was determined by Loss On Drying (LOD) technique. The dried granules were sized by screening through an 18 mesh screen and then finally lubricated.

table 6: formulation 1A

Items a-h are internal phase components and item i is an external phase component.

Table 7: formulation 2A

Items a-g are internal phase components and item h is an external phase component.

EXAMPLE 3 semi-solid formulation

Semi-solid lipid-based formulations (formulation 4 and formulation 5, table 8A) filled into capsules for oral administration were prepared according to iCo formulation composition (table 8). However, in contrast to the iCo/Wasan liquid process, a melt process is employed. In practice, the semisolid excipient (Gelucire/TPGS) is melted at 35-40 ℃ using a hot plate magnetic stirrer, weighed and mixed with Peceol (liquid excipient) until a clear solution is obtained. The heating was stopped and AmpB was added and mixed for 5 minutes. The final liquid blend was kept under stirring and hot-filled into hard gelatin capsules of size 00 (fill weight: 804mg) containing 4mg AmpB. An additional batch (formulation 5) containing the same amount of lipid excipient was made, but 'spiked' with more AmpB to produce a 100mg dose of capsules (fill weight: 900 mg).

Table 8A: iCo/Wasan liquid formulation, formulation 4, and formulation 5

*＝0.95mL

EXAMPLE 4 Scale-Up test of the semi-solid formulation from example 3

The batch size of lipid-based formulation 5 was scaled up from 23g to 360g (table 8B). Each excipient was melted in its original container and then stirred to ensure homogeneity prior to sampling. Weighed molten samples were mixed together and AmpB was added with stirring. The mixture was kept at 40 ℃ and under constant stirring for at least 30 minutes to ensure complete dispersion/dissolution. The final mixture was filled into hard gelatin capsules. Once the contents of the capsule have cooled to room temperature, the capsule is sealed using a mixture of purified water and ethanol (50:50 v/v). A few drops of the solution were gently applied around the junction of the body and the lid of the closed capsule. Excess solution was immediately wiped off using a clean and dry cloth. The capsules were individually dried by resting vertically on a copper plate. The sealed capsules were stored at 4 ℃ until stability studies were initiated.

Table 8B: 100mg amphotericin B lipid formulation 5A

Composition (I)	mg/dose	％w/w	g/batch
				Amphotericin B	100	11.1	40.0
TPGS	40	4.4	16.0
				Peceol	380	42.2	152.0
Gelucire 44/14	380	42.2	152.0
				In total:	900	100	360

Example 5 physical and chemical Properties

The final mixture was evaluated by bulk/tap density and powder flow properties as well as residual solvent.

Bulk density/tap density-powder flow properties-USP <616>

Bulk and tap densities were determined by Vanderkamp tap density tester model 10700(VanKel Industries, Inc.) and Mettler Toledo equilibrium model AT200 using the USP <616> method. Each parameter was determined in duplicate using a 50mL graduated glass cylinder. Bulk density was determined by measuring the volume of a powder sample of known mass in a graduated cylinder, while tap density was measured by mechanically tapping the cylinder until no further volume change was observed. Powder flow properties were evaluated using the Carr's Compressibility Index (Carr's compression Index) and the Hausner ratio (Hausner ratio) as described in the following paragraphs.

Karnst Compressibility Index (CI): this flow property was calculated using bulk density data and tap density data when fitted to the following equation:

Compressibility index ═ tap density-bulk density)/tap density × 100%

Hausner Ratio (HR): this flow property was calculated as the ratio of tap density to bulk density.

Table 9 presents Compressibility Index (CI) and Hausner Ratio (HR) values interpretations and descriptive qualitative examples according to USP <1174 >.

Table 9: fluidity scale

Compressibility index (%)	Flow characteristics	Haosner ratio	Examples of the invention
				≤10	Superior food	1.00-1.11	Free flowing particles
11-15	Good wine	1.12-1.18	Powdery particles
				16-20	In general	1.19-1.25	Coarse powder
21-25	Go and can	1.26-1.34	Fine powder of
				26-31	Difference (D)	1.35-1.45	Fluidizable powder
32-37	is very poor	1.46-1.59	Adhesive powder
				>38	Is very poor	>1.60	Very adherent powders

Table 10 shows the density and powder flow properties. The formulation exhibits sufficient flowability. To fill 435mg of the final blend from formulation 1 into a size 0 capsule, it was necessary to tap and tamp. Using higher density excipients (e.g., microcrystalline cellulose type 200 or 302 or high functionality and multifunctional excipients such as silicified microcrystalline cellulose (a combination of microcrystalline cellulose and colloidal silicon dioxide)), bulk density can be increased by high shear granulation. Silicified microcrystalline cellulose (Prosolv HD 90) has a bulk density of 0.38-0.50g/cm3 and is used in formulation 2, resulting in an increase in bulk density.

Table 10: density and flow Properties (n ═ 2)

Uniformity of capsule weight

As an example to show sufficient flow properties, 12 capsules of formulation 1 were statistically tested (table 11). RSD for weight uniformity was confirmed to be < 6.0% and no units were outside the range of 85-115% of the labeled amount.

Table 11: encapsulation statistics for 100mg amphotericin B in 0-size capsules (n-12) of formulation 1

Statistical data	Value (mg, total weight)
		Mean value of	527.8
Standard deviation of	1.8
		RSD(％)	0.3
Minimum value	525.0
		Maximum value	529.9

Statistics for the semi-solid formulations are shown in table 12. Formulation 4 was filled into approximately 90% of the capsule volume by weight to give 4mg amphotericin B/capsule. Formulation 5 and formulation 5A with 100mg amphotericin B/capsule were filled to 100% of the capsule volume.

Table 9: statistical encapsulation of amphotericin B semisolid formulation in size 00 capsules (n ═ 6) for formulation 4, formulation 5, and formulation 5A

Residual solvent

The residual solvent was determined by thermogravimetric analysis (TGA) using a TA instruments Q50 thermogravimetric analyzer at a scan rate of 10 ℃ min-1 over a temperature range of 25 ℃ to 200 ℃. Samples (11-13mg) were heated in a platinum open pan under nitrogen (60mL min-1).

The TGA profile of the final mixture of amphotericin B (23%) solid oral dosage formulation is shown in figure 2. TGA shows that between 25 ℃ and 100 ℃ weight loss is 2.4-3.8%, which is usually associated with evaporation of volatile compounds (solvent and moisture). This weight loss is low considering that the moisture content of microcrystalline cellulose is between 3% and 5% (moisture data from CofA). Thus, for samples containing higher amounts of microcrystalline cellulose (e.g., formulation 2 and formulation 3), a slight increase in weight loss (3.8%) appeared to be normal compared to formulation 1 (2.4%).

example 6. analytical testing:

Formulation 1

The results of the amphotericin B formulation assay and related materials are shown in table 13. Replica 1 and replica 2 were prepared using sample preparation 1 and replica 3 was prepared using sample preparation 2 in an attempt to minimize NMP consumption in the diluent. Similar extraction efficiencies were obtained with both sample preparation procedures. The assay and related substance extraction procedure achieved about 95% recovery. The dissolution profile varied rather rapidly with 95% release at 45 minutes (figure 3). After increasing the paddle speed, 100% release was achieved.

Table 13: analysis results of 100mg amphotoxin B capsules for formulation 1

Formulation 2 and formulation 3

The amphotericin B assay and related material results using the modified extraction procedure are shown in table 14. Sample preparation 3 (see methods for details) was used to extract the contents from 4 capsules and the empty capsule shell of each sample replica. Some samples required longer sonication time to break up the agglomerates formed. Longer sonication times also increase the amount of degradation that occurs during sample preparation. The dissolution profile is shown in figure 3. Formulation 3 initially appeared somewhat slow, but quickly re-incorporated the properties of the other formulations.

Table 15 shows impurity profiles of AmpB for the formulations indicating that the methods used to create the dosage forms do not adversely affect AmpB.

Table 14: analysis results of 100mg amphotoxin B capsules for formulation 2 and formulation 3

Peak is more than or equal to 0.10 percent of the report

A sonicates one replica for a significantly longer time to break up agglomerates present in the sample, which results in a higher level of degradation.

Table 10: impurity profile of AMpB

Peak is more than or equal to 0.10 percent of the report

Formulation 1A and formulation 2A

The determination of amphotericin B in the capsules and related substances was performed at the initial time point (T ═ 0) using a modified extraction procedure (table 16). The contents from 3 capsules of each sample replica were extracted using sample preparation 4 (see methods for details). The dissolution profile of the scaled-up batch was comparable to the previous batch (fig. 4).

Table 11: analysis results of 100mg amphotoxin B capsules for formulation 1A and formulation 2A

Peak is more than or equal to 0.10 percent of the report

Formulation 4 and formulation 5

Semisolid lipid based formulations in capsules for oral administration were prepared according to iCo formulation composition and Corealis modification method. The dissolution profile of the capsules was analyzed in current 0.1N HCl + 0.5% SDS medium and simulated fed intestinal fluid (FeSSIF pH 5.8) (table 17, fig. 5).

Semi-solid formulation-formulation 4 (drug load of 0.5%) and formulation 5 (drug load of 11.1%) showed slightly slower dissolution profiles in 0.5% SDS in water, with dissolution times of up to 30 minutes, compared to 'solid' capsule formulation 1A (drug load of 23%). In FeSSIF medium at pH 5.8, where solubility of amphotericin B may be limited, the dissolution profile may reach up to about 35% dissolution for 'solid' capsule formulations and less than 15% dissolution for semi-solid formulations (fig. 6). In this in vitro model, the semi-solid formulation with increased lipid concentration showed no improvement in the dissolution profile. Both formulation 4 and formulation 5 showed similar end results compared to solid oral dosage formulation 1A.

Table 12: dissolution results for amphotericin B semi-solid capsules of formulation 1A, formulation 4 and formulation 5.

Sample L268-01021, injected once at 15 minutes, showed 130% dissolution, with approximately 90% dissolution observed at subsequent time points. The vial was re-filled and the peak area did not change. The atypical value is not reported

Formulation 5 and formulation 5A

Formulation 5 and formulation 5A were the same composition, but prepared on a different scale. Thus, the mixing time increases. Further, formulation 5A and formulation 5A-1 capsules were from the same final blend with only one difference, with formulation 5A capsule sealed and formulation 5A-1 subsequently filled and not sealed. The initial/T-0 data shown in figure 7 shows that the dissolution profiles for all three batches are different. However, after 60 minutes 90-100% of the AmpB was dissolved. Subsequently, it was also found that lower dissolution profiles were observed for formulation 5A stored at 40 ℃/75% RH for 1 month and for formulation 5 stored at 5 ℃ for about 5 months.

Without being bound by theory, the decline in dissolution profile over time may be due to the varying degrees of dissolving AmpB during processing of different batch batches.

Example 7 stability Studies

Formulation 1A and formulation 2A

Stability studies were initiated on formulation 1A and formulation 2A. The capsules were packaged in 30cc HDPE bottles with induction sealed PP caps and the bottles were stored in a humidity chamber of 25 ℃/60% RH under ICH stability conditions and 40 ℃/75% RH under accelerated conditions. After preparation, the capsules were stored directly at 4-8 ℃ before being placed in the stability chamber.

The stability test results for 100mg amphotericin B capsule formulation 1A and formulation 2A are summarized in tables 18 to 20. The dissolution profiles are compared in figure 8. Both formulations were stable at 25 ℃/60% RH and 40 ℃/75% RH for up to 6 months with no significant change in assay, related substances and dissolution profile compared to the initial (T ═ 0) results.

Table 13: stability test results for formulation 1A and formulation 2A

Note that: the assay was only quantitative for API. Chromatographic impurities are not considered.

Table 19: related substances of formulation 1A and formulation 2A

Relative to AmpB

Note that: peaks are greater than or equal to 0.10% of reported; the reported impurity characteristics (% area) are equivalent in the API solution and the sample solution.

As the peaks were more extensive, they were not integrated at previous time points, sharper peaks were now observed and reported.

table 20: stability test results (solubility) of formulation 1A and formulation 2A

Formulation 5A

Another stability study was initiated on formulation 5A. Capsules were packaged according to formulation 1A and formulation 2A and stored under the same conditions.

Amphotericin B/TPGS/Peceol/Gelucire44/14 semisolid lipid-based formulation in hard shell capsules (formulation 5A) was prepared and stored under ICH controlled stability conditions. After 3 months of storage, the formulations remained stable with no loss of efficacy (table 21) and no increase in related substances (table 22). However, the dissolution profile was reduced (table 23 and fig. 9). As previously mentioned, this behavior may be the result of AmpB recrystallization or aggregation.

Table 21: stability results for formulation 5A

A does not have enough sample units available for n-6

Table 22: material stability results for formulation 5A

Note that: peaks are greater than or equal to 0.10% of reported; the reported impurity characteristics (% area) are equivalent in the API solution and the sample solution.

The two peaks observed by B at RRT 0.73 and RRT 0.74 were previously observed as one peak at RRT 0.73.

The peak observed for E at RRT 0.74 consists of 2 co-eluting peaks observed alone when analyzed at only 2 minute time points.

Table 23: dissolution Curve stability results for formulation 5A

After 45 minutes at 50rpm, the capsule pieces and their contents remained in the sinkers.

D the capsules disintegrate slowly compared to the samples at 40 ℃/75% RH (3 months).

Example 8 pharmacokinetics of formulation 1A and formulation 5A

The pharmacokinetics of formulation 1A and formulation 5 were evaluated in beagle dogs after a single oral administration and compared to a liquid formulation (i.e., the iCo/Wasan liquid formulation described above). Tissue distribution of these amphotericin B capsule formulations was also evaluated 24 hours after three days of once daily repeated oral dosing in beagle dogs.

As outlined in table 24 below, amphotericin B in formulation 1A, formulation 5A, and the liquid formulation (i.e., iCo/Wasan formulation) was administered to male dogs:

Table 24: beagle research design

Blood was collected for TK evaluation on days 1,2 and 3 (up to 72 hours post-dose). The dogs receiving the capsule formulation then received a single oral administration for more than three days (4-6 days) and were euthanized 24 hours after the last administration and subsequent tissue samples were collected (approximately 1g, except for mesenteric lymph nodes): brain (brain, cerebellum, medulla), heart, kidney (cortex and medulla), liver, lung, spleen, testis, mesenteric lymph nodes and gastrointestinal tissue (duodenum, jejunum, ileum and colon). Samples of intestinal contents were also collected. Plasma samples and tissues of amphotericin B content were analyzed using a qualified LC/MS analysis method.

amphotericin B in all formulations administered orally at a dose of 100mg was well tolerated by dogs and no associated adverse clinical signs were observed.

Following oral administration of amphotericin B in three different formulations, mean plasma levels of amphotericin B initially rose rapidly and in a similar manner (up to 2 hours post-administration) and then rose at a slower rate to reach a plateau (6-24 hours post-administration), and then declined slowly thereafter. The pharmacokinetic parameters (± SE) for Wasan formulation, formulation 1A and formulation 5A determined in all dogs after a single administration of amphotericin B are summarized in table 25 below. The mean Cmax value, Tmax value, AUC0-Tlast value, and MRTLast value were not significantly different from each other for the three formulations.

TABLE 25 pharmacokinetics

Plasma concentrations of amphotericin B were measured after administration of formulation 1A, formulation 5A, and oral liquid dosage forms (i.e., the iCo/Wasan formulation described above). These results are reported in table 26.

TABLE 26 plasma concentration of amphotoxin B in beagle dogs

NPD ═ undetected peak

All concentrations shown are below LLOQ (100ng/mL)

The quantitation limit was defined as 100ng/mL and this quantitation limit was used to establish the bioanalytical method. No plasma levels above the limit of quantitation of 100ng/mL were observed for all dogs after a single dose and 24 hours after repeated dosing of the formulation.

Plasma concentrations of amphotericin B24 hours post-administration with formulation 1A and formulation 5A were 56.0 + -6.9 ng/mL and 52.3 + -4.6 ng/mL, respectively (see also FIG. 11).

To verify that there were no stability problems with the cryopreserved plasma samples, a single capsule (formulation 1A) was administered to one dog, and blood samples were taken at2 and 4 hours post-administration and amphotericin B was measured in both fresh and frozen samples. The levels of amphotericin B in the frozen and fresh samples were similar and in the same range as the plasma levels presented above.

As an additional part of the study, the literature reviews pharmacokinetic studies of amphotericin. Two reports were found. Amphotericin B was quantified by both HPLC and LC-MS using a lower quantitation limit set to 20 ng/mL. In the first publication (S. Kalbag et al, Cambridge (CAmB) -Focus-Tox-Poster, 4 months 2013), dogs (male and female) were orally administered formulated amphotericin B at 15mg/kg, 30mg/kg and 45 mg/kg; a plasma Cmax of 15mg/kg close to the dose in this study (about 10mg/kg), ranging from 51.9-67.3ng/mL (male-female) values close to the plasma Cmax observed in this study, and a similar plasma concentration curve relates to 24 hours. In a study conducted in rats following oral administration of amphotericin B in a novel lipid formulation (e.k. wasan, journal of antibacterial Chemotherapy 64:101 @, 108,2009), Cmax was 96ng/mL after administration of 10mg/kg, which was in the range of the plasma levels we observed for the original solution. Thus, based on the studies and plasma profiles obtained, the plasma concentrations measured between the detection and quantification limits represent the actual plasma concentrations obtained.

In a laboratory management protocol (GLP)/dose range exploration (DRF) study, the tissue concentration of amphotericin B in beagle dogs was measured after administration of formulation 1A and formulation 5. Table 27 shows the tissue levels of amphotericin B (see also fig. 10).

TABLE 27 tissue concentration of amphotericin B in canine models.

Values are expressed as mean ± SE of n-6 and include plasma concentrations above the detection limit but below the quantification limit and values above the upper quantification limit; samples in which no peak was detected were included as zero values in the mean.

the tissue partition coefficient of 1Kp was calculated by dividing the mean tissue level by the mean plasma level of amphotericin B observed after repeated dosing of prototype formulation 1A and formulation 5A and assuming that 1g of tissue represents a tissue volume of 1 mL.

After repeated dosing of amphotericin B in formulations 1A and 5, the distribution of amphotericin B in tissue and intestinal contents was similar. Gastrointestinal tissue and content contain the highest levels, with intestinal content levels ranging from 1938.8-3106.6ng/g w/w. samples, and tissue levels and tissue/plasma ratios ranging from 69.0-703.3ng/g w/w. tissue and 1.24-12.55, respectively. In non-gastrointestinal tissues, the renal cortex and medulla behind the liver and mesenteric lymph nodes had the highest levels, with tissue levels and tissue/plasma ratios ranging from 21.7-157.8ng/g w/w. and 0.42-3.03, respectively. The remaining tissue had very low levels of amphotericin B, with tissue levels and tissue/plasma ratios ranging from 0.0-7.6ng/g w.w. and 0.00-0.17ng/g w.w. respectively.

In summary, oral administration of formulation 1A, 100mg of amphotericin B contained in formulation 5, and the liquid formulation had good resistance to canine. Oral bioavailability of amphotericin B from iCo-010(iCo/Wasan liquid formulation) and formulation 1A and formulation 5 (capsule formulation) was similar, noting that there were no significant differences in Cmax, Tmax and AUC0-Tlast between the formulation groups. The tissue distribution of amphotericin B was similar after administration of formulation 1A and formulation 5, with the highest levels found in gastrointestinal tissue, followed by kidney, liver and mesenteric lymph nodes, with lower levels observed in lung, spleen and testis, and very low levels observed in brain and heart regions.

Example 9 pharmacokinetics of amphotericin B after oral administration to fed and fasted beagle dogs

This study involved the administration of test items by the oral route at two different stages and in a crossover design as outlined in table 28.

TABLE 28 summary of the study

Sequence of	Numbering of dogs	dose of amphotericin B (mg)	Stage 1	Rinsing and Observation phases	Stage 2
						I	2M	500	Eating food	7 days	Fasting
II	2M	500	fasting	7 days	Eating food

M ═ male

Administration of test items

Test item capsules were administered at each dose stage as follows.

Fasting administration:

Dogs were fasted overnight. The following morning dosing was started at approximately 8:30 to 9: 00. Five (5) capsules, each containing 100mg of formulated amphotericin B, were administered orally by placing one capsule on the back of the tongue at a time. Immediately after the last capsule was administered, about 20mL of tap water was slowly administered through a syringe in the mouth corner to ensure swallowing. The dog was again inspected for oral cavities to ensure that there were no traces of capsules in the oral cavity. Food was administered after a blood sampling time of 2 hours (Lab Diet certified dog feed # 5007). The water is freely drunk.

And (4) feeding dose:

Dogs were fasted overnight. The following morning dosing was started at approximately 8:30 to 9: 00. Five (5) capsules, each containing 100mg of formulated amphotericin B, were administered orally by placing one capsule on the back of the tongue at a time. Immediately after dosing, approximately 20mL of tap water was slowly administered through a syringe in the mouth corner to ensure swallowing. The dog's mouth was again checked to ensure no traces of capsules in the mouth and 300 (+ -5) grams of wet dog food (meat bread with real chicken) was supplied from the food bowl. All dogs consumed 300 + -5 grams of canned wet dog food within 4-5 minutes. The water is freely drunk.

Observation of Life time

Mortality rate: mortality checks were performed and recorded twice daily on dosing days and once daily on non-dosing days during the study. On the day of dosing, animals were closely monitored during the first 60 minutes after dosing.

Weight: body weights were recorded before each dose and at the end of the 7 day observation period.

Pharmacokinetic blood sampling: blood samples were collected from all animals after dosing as follows: blood sample collection was performed at 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, and 24 hours after administration on days 1 and 8 of administration.

For the purpose of collecting the above samples, blood was taken from the jugular vein of each animal. Each blood sample (approximately 2mL) was collected into a vacuum tube containing anticoagulant (K2 EDTA). The time of each sample was recorded (actual time, recorded along with the date and time of administration).

After collection, the blood was placed in a refrigerated centrifuge at 2000rpm for 20 minutes to separate the plasma. The recovered plasma was stored in duplicate vials and frozen (at-80 ± 10 ℃) for analysis. For each step in the preparation of plasma, the sample is protected as much as possible from ambient light.

After the last blood draw, each animal was returned to a canine flock of Nucro-Technics.

And (3) sample analysis: plasma sample analysis was performed at the Nucro-Technics bioanalytical laboratory using a qualified LC-MS/MS method to determine amphotericin B. Plasma samples will remain for three months after the final report is issued.

Pharmacokinetic analysis: plasma concentration-time data were analyzed by a non-compartmental method to obtain pharmacokinetic parameters using validated version 6.3 software (Pharsight Corp).

The main parameters were calculated (as listed below):

AUC 0-Tlast: the area under the plasma concentration-time curve from zero to the last quantifiable concentration time at time tlast was calculated using a linear trapezoidal method.

AUC0- ∞: extrapolated from zero to infinite area under the plasma concentration curve. AUC0- ∞ is calculated as AUC0-Tlast + (Clast/ke)

Cmax: maximum plasma concentration

Tmax: maximum concentration time determined from nominal time of blood sampling

And ke: the rate constant is eliminated. The elimination rate constant was estimated using linear regression at the end of the semilog concentration-time curve. A minimum of three data points will be used to calculate ke. No weighting is applied to the regression line.

t1/2 (e): terminal elimination half-life calculated according to ln (2)/ke

Additional parameters such as Mean Residence Time (MRT), clearance after oral administration (CL/F), and software generated distribution/volume after oral administration (Vz/F) can be reported by the study supervisor as appropriate.

Statistical analysis: AUC0-Tlast and Cmax were used as the main outcome variables to compare bioavailability between fed and fasted states, and Tmax was considered for absorption. Significant differences between the two formulation groups with respect to AUC0-Tlast, Cmax, and Tmax were evaluated using a student t-test and using a level of p <0.05 as an indicator of statistically significant differences between the two formulation groups. Significant differences between the variances of the grouped data were evaluated by applying the F-test to both groups and accepting a level of p <0.05 as an indicator of significant differences between variances.

Results

Clinical observations

Amphotericin B formulated as formulation 1A was well tolerated by dogs by oral administration at a dose of 500mg (contained in five capsules) and no associated adverse clinical signs were observed. Body weight was maintained throughout the treatment period (table 29).

TABLE 29 weight summary

Pharmacokinetics of fasted and fed dogs

The mean plasma concentrations of amphotericin B are presented in table 30, and individual plasma concentrations and mean plasma concentrations versus time are presented in figures 12 and 13. Pharmacokinetic parameters derived from plasma concentration versus time curves are presented in table 31.

Following oral administration of amphotericin B formulated into formulation 1A, mean plasma levels of amphotericin B initially rose rapidly and in a similar manner (up to 2 hours post-administration) and then rose at a slower rate to reach a plateau (6-24 hours post-administration) or peaked 4 hours post-administration, and then declined. In most cases, the elimination phase definition is ambiguous, resulting in an inability to determine pharmacokinetic parameters for the terminal elimination phase. A review of the pharmacokinetic parameters presented in table 31 shows that the mean Cmax value, Tmax value and AUC0-Tlast values did not differ significantly from each other for the fasted and fed states. One different dog in each of the fasted and fed groups had a lower AUC0-Tlast due to a significant drop in plasma concentration of amphotericin B4 hours after administration. The reported changes in pharmacokinetic parameters were not different between the fasted and fed groups.

The lack of significant differences between the pharmacokinetic parameters of the fasted and fed states and their variances indicates that the presence of food had little effect on the absorption of amphotericin B from formulations iCo-019.

TABLE 30 plasma concentration of amphotericin B after oral administration of formulation 1A to fasted and fed dogs

This data is expressed as the mean ± SE of n ═ 4; n.a. -not applicable

TABLE 31 PK parameters of amphotericin B after oral administration of 500mg amphotericin B formulated as formulation 1A to fasted and fed dogs

There were no significant differences in statistical parameters and their variances between the fasted and fed states.

Conclusion

In conclusion, oral capsule administration of 500mg of amphotericin B as formulation 1A was well tolerated by dogs. No significant difference in Cmax, Tmax, AUC0-Tlast and MRTlast was considered for fasted and fed dogs. Thus, this study showed that the presence of food did not affect oral absorption of amphotericin B from formulation 1A in beagle dogs.