Preparation of freeze-dried precursor of annamycin liposome

Abstract

Provided is a method for preparing a freeze-dried anamycin liposome precursor, a composition prepared by this method, and the use of the composition thus prepared in the treatment of cancer. More particularly, the method for preparing freeze-dried anamycin comprises the steps of: preparing a solution of anamycin, one or more lipids, one or more non-ionic surfactants, and one or more solvents, aseptically filtering the anamycin solution containing lipids; and freeze-drying the anamycin solution containing lipids to provide a freeze-dried anamycin liposome precursor.

Description

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 868,184, filed on Jun. 28, 2019, the disclosure of which is hereby incorporated by reference as if set forth in full herein.

[0002] Annamycin is a cancer chemotherapeutic agent of the anthracycline structural family. Due to its physical and pharmacological properties, dosage forms of annamycin compositions have been described in which the drug is formulated into liposomes (see U.S. 7,238,366). Clinical trials of liposomal annamycin have been described for adult patients with relapsed refractory acute lymphoblastic leukemia (M. Wetzler et al., Clinical Lymphoma, Myeloma and Leukemia, 13(4), 430 - 434, August 2013) and in the treatment of doxorubicin-resistant breast cancer (D. J. Booser et al., Cancer Chemother. Pharmacol. 50; 6 - 8, 2002).

[0003] In drug delivery systems, it is desirable to be able to produce drug formulations with consistent purity and quality to ensure the correct dose is administered, thereby providing the most effective treatment while avoiding undesirable side effects caused by impurities. In addition, poorly maintained or improperly formulated drug materials may be decomposed before administration, rendering them ineffective and thus wasting expensive active pharmaceutical ingredients. Regulatory standards must also provide a consistent level of drug purity and drug quantity according to the procedures specified on the label.

[0004] Annamycin presents particularly difficult challenges for formulation due to its inherent physical properties and the inherent instability of both the parent drug and liposomal formulations under certain conditions. Liposomal formulations of annamycin typically degrade and form annamycin crystals, and / or form large liposomes or phase separation, rendering the formulation unsuitable for administration. Thus, it would be advantageous to find a consistent method for preparing a liposomal precursor dosage form of annamycin that ensures purity and avoids decomposition of annamycin. SUMMARY OF THE INVENTION

[0005] A method for preparing freeze-dried annamycin is provided, the method comprising the steps of:

[0006] preparing a solution comprising one or more lipids, one or more nonionic surfactants, and one or more solvents and having a pH of 4.8 - 5.9;

[0007] adding an annamycin solution to the lipid solution, the annamycin solution comprising about 8 - 12 wt.% annamycin in DMSO, to provide a lipid-containing annamycin solution;

[0008] Sterile filter the lipid-containing annamycin solution; and

[0009] Lyophilize the lipid-containing annamycin solution to provide a lyophilized annamycin liposome precursor.

[0010] A method for producing lyophilized annamycin is provided, the method comprising the steps of:

[0011] Prepare a first solution comprising water and tert-butanol, wherein the ratio of water to tert-butanol is from about 8:2 to about 9:1;

[0012] Add one or more lipids and one or more nonionic surfactants to the first solution to form a second solution;

[0013] Adjust the pH of the second solution such that the pH is 4.8 - 5.9;

[0014] Add a third solution to the second solution, the third solution comprising about 8 - 12 wt.% annamycin in DMSO, to provide a lipid-containing annamycin solution;

[0015] Sterile filter the lipid-containing annamycin solution; and

[0016] Lyophilize the lipid-containing annamycin solution to provide a lyophilized annamycin liposome precursor.

[0017] A method for producing a lyophilized annamycin liposome precursor is also provided, the method comprising the steps of:

[0018] Add DMPC, DMPG and polysorbate 20 to a pre-warmed mixture of water and tert-butanol to prepare a first solution, wherein the pre-warming temperature is from about 35°C to about 42°C, and wherein the ratio of water to tert-butanol is from about 8:2 to about 9:1;

[0019] Adjust the pH of the first solution with a pharmaceutically acceptable acid to keep the pH of the solution constant for at least 15 minutes;

[0020] Add a second solution to the first solution, the second solution comprising about 8 - 12 wt% annamycin in DMSO, to provide a lipid-containing annamycin solution;

[0021] Sterile filter the lipid-containing annamycin solution; and

[0022] Lyophilize the lipid-containing annamycin solution in separate aliquots to provide a lyophilized liposome precursor.

[0023] A lyophilized annamycin liposome precursor prepared by any of the methods described herein is also provided.

[0024] Also provided is a method for treating cancer, the method comprising administering to a patient in need an effective dose of liposomal annamycin, which is prepared using a lyophilized annamycin liposome precursor prepared by any of the methods described herein.

[0025] Also provided is the use of a lyophilized annamycin liposome precursor prepared by any of the methods described herein in the manufacture of a medicament for treating cancer.

[0026] Also provided is a lyophilized annamycin liposome precursor prepared by any of the methods described herein for use in treating cancer. Detailed Description

[0027] Methods for preparing lyophilized annamycin liposome precursors with improved stability and high purity are provided. As described in US 7,238,366 (which is incorporated herein by reference in its entirety), the lyophilized annamycin liposome precursor composition can be reconstituted into an aqueous liposome composition by hydration for all purposes, such as for subsequent use in treating cancer.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0029] As used herein, the following terms have the meanings defined below.

[0030] The term "annamycin" means the compound (7S,9S)-7-(((2R,3R,4R,5R,6S)-4,5-dihydroxy-3-iodo-6-methyltetrahydro-2H-pyran-2-yl)oxy)-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-7,8,9,10-tetrahydrotetracene-5,12-dione, which has the following structure:

[0031]

[0032] The term "high purity lyophilized annamycin liposome precursor" means a material with a purity of annamycin of not less than 95% as analyzed by HPLC using a validated standard sample. In some embodiments, the annamycin is at least 96% pure, or at least 97% pure, or at least 98% pure, or at least 99% pure.

[0033] The term "liposomes, liposomal", etc. means a generally spherical structure that includes lipids, fatty acids, lipid bilayer-type structures, monolayer vesicles, and amorphous lipid vesicles. Classically, a liposome is a completely enclosed lipid bilayer membrane that contains an entrapped volume of water. Liposomes include non-classical forms in which annamycin can be within the bilayer, part of the bilayer, and adsorbed to the bilayer. Liposomes can be monolayer vesicles (having a single bilayer membrane) or multilamellar vesicles (characterized by an onion-like structure of multiple membrane bilayers, each bilayer separated from the next by a layer of water). The bilayer is composed of two lipid monolayers having hydrophobic "tail" regions and hydrophilic "head" regions. The structure of the membrane bilayer is such that the hydrophobic (non-polar) "tails" of the lipid monolayers face the center of the bilayer, while the hydrophilic "heads" face the aqueous phase.

[0034] The term "preliposome-lyophilizate and preliposomallyophilizate" means a non-aqueous material that will form liposomes upon addition of an aqueous solution. In some embodiments, the non-aqueous material is a dry (e.g., in a non-liquid, non-gel) material. Lyophilizates are widely used and include dry residues from the sublimation of frozen liquids from non-volatile materials, residues from rotary evaporation and similar procedures, and dry compositions that will produce liposomes upon addition of an aqueous phase (with or without agitation). It is to be specifically understood that "preliposome-lyophilizate" is not the lyophilized form of liposomes.

[0035] The term "lipid" refers to any pharmaceutically acceptable organic compound that is a fatty acid or its derivative. In some embodiments, the lipid is a phospholipid, such as phosphatidylcholine including DMPC and DPMG, but may also include other lipids such as phosphatidylethanolamine.

[0036] The term "nonionic surfactant" refers to a pharmaceutically acceptable surfactant having covalently bonded oxygen-containing hydrophilic groups that are bonded to a hydrophobic parent structure. Suitable nonionic surfactants include ethoxylates, fatty alcohol ethoxylates, alkylphenol ethoxylates, fatty acid ethoxylates, ethoxylated fatty esters and oils, ethoxylated amines, fatty acid amides, end-capped ethoxylates, poloxamers, fatty acid esters of polyhydroxy compounds, fatty acid esters of glycerol, and fatty acid esters of sorbitol. In some embodiments, the nonionic surfactant is a polysorbate-type surfactant formed by ethoxylating sorbitan and then adding carboxylic acid. In some embodiments, the nonionic surfactant comprises polyoxyethylene sorbitan monolaurate (polysorbate 20) and polyethoxylated sorbitan monooleate (polysorbate 80).

[0037] "Polysorbate 20" refers to a commercially available nonionic surfactant (ICI Americas Inc.), which consists of a mixture of polyoxyethylene chains of different lengths attached to sugars that are linked to sorbitan. These polyoxyethylene sugars are also attached to fatty acids. The trade name of this material is Tween TM 20; the composition is polyoxyethylene sorbitan monolaurate (MW about 1300). Polysorbate 20 is shown below, where w + x + y + z = 20.

[0038]

[0039] The term "pharmaceutically acceptable acid" refers to any organic and inorganic acids known in the art to have good tolerance and be suitable for administration to human patients. Such salts include 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid (L), aspartic acid (L), benzenesulfonic acid, benzoic acid, camphoric acid (+), camphor-10-sulfonic acid (+), decanoic acid (capric acid), caproic acid, caprylic acid (caprylic acid or octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid (D), gluconic acid (D), glucuronic acid (D), glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid (DL), lactobionic acid, lauric acid, maleic acid, malic acid (-L), malonic acid, mandelic acid (DL), methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyroglutamic acid (-L), salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid (+L), thiocyanic acid, toluenesulfonic acid (p), undecylenic acid. Pharmaceutically acceptable acids include hydrochloric acid and sulfuric acid.

[0040] In the previous sections, many abbreviations and acronyms were used, and their complete descriptions are provided below:

[0041] DMPC dipalmitoylphosphatidylcholine

[0042] DMPG 1,2-dimyristoyl-sn-glycero-3-phosphate-(1'-rac-glycerol) (sodium salt)

[0043] DMSO dimethyl sulfoxide

[0044] IV intravenous

[0045] DEHP bis(2-ethylhexyl) phthalate

[0046] PVC Polyvinyl Chloride

[0047] WPI Water for Injection, USP

[0048] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprising", or variations such as "comprises" or "comprising", shall be understood to imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps. It is intended by the recitation of such a specified range that all those specific integer amounts between the recited ranges are also included. For example, a range of about 35°C - 42°C is also intended to cover 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, and 42°C.

[0049] The entire disclosure of each U.S. patent and international patent application mentioned in this patent specification is hereby incorporated by reference in its entirety for the purposes for which it is used.

[0050] A method for preparing a freeze-dried anamycin liposome precursor with superior purity and stability is provided. In particular, a method for preparing freeze-dried anamycin is provided, which method comprises the following steps:

[0051] Preparing a first solution comprising water and tert-butanol, wherein the ratio of water to tert-butanol is from about 8:2 to about 9:1;

[0052] Adding one or more lipids and one or more nonionic surfactants to the first solution to form a second solution;

[0053] Adjusting the pH of the second solution such that the pH is 4.8 - 5.9;

[0054] Adding a third solution to the second solution, the third solution comprising about 8 - 12 wt.% anamycin in DMSO, to provide a lipid-containing anamycin solution;

[0055] Sterile filtering the lipid-containing anamycin solution; and

[0056] Freeze-drying the lipid-containing anamycin solution to provide a freeze-dried anamycin liposome precursor.

[0057] In some embodiments, the second solution comprises DMPC, DMPG, and a polyoxyethylene sorbitan surfactant. In some embodiments, the second solution comprises DMPC, DMPG, and polyoxyethylene sorbitan monolaurate.

[0058] In some embodiments, each lipid and each nonionic surfactant are added separately.

[0059] In some embodiments, DMPC, DMPG, and polysorbate 20 are sequentially added to the first solution.

[0060] In some embodiments, the method comprises adjusting the pH of the second solution to 5.3 ± 0.2 with a pharmaceutically acceptable acid selected from the group consisting of HCl and H 2 SO 4 .

[0061] In some embodiments, the method comprises adding a third solution to the second solution, the third solution comprising about 10 wt.% annamycin in DMSO, to provide a lipid-containing annamycin solution.

[0062] In some embodiments, the annamycin liposome precursor lyophilizate comprises: 1.8 - 2.2 wt% annamycin, 3.0 - 3.4 wt.% polysorbate 20, and 94.4 - 95.2 wt.% lipids selected from DMPC and DMPG. In some embodiments, DMPC is 65.3 - 67.3 wt.% and DMPG is 27.1 - 29.9 wt.%.

[0063] In some embodiments, the annamycin in the resulting lyophilizate is at least 98% pure. In some embodiments, the annamycin in the resulting lyophilizate is at least 99% pure.

[0064] In some embodiments, a method of preparing lyophilized annamycin comprises the steps of:

[0065] Preparing a first solution comprising water and tert-butanol, wherein the ratio of water to tert-butanol is from about 8:2 to about 9:1;

[0066] Adding one or more lipids and one or more nonionic surfactants to the first solution to form a second solution comprising DMPC, DMPG, and polysorbate 20;

[0067] Adjusting the pH of the second solution such that the pH is 5.3 ± 0.2;

[0068] Equilibrating the solution for at least 15 minutes;

[0069] Measuring the pH and adjusting the pH with an acid when the pH is above 5.3;

[0070] Equilibrating the solution for at least 15 minutes;

[0071] Repeating the steps of measuring the pH, adjusting the pH, and equilibrating the solution until the pH of the solution remains at pH 5.3 ± 0.2;

[0072] Add a third solution to the second solution, the third solution comprising about 8 - 12 wt.% annamycin in DMSO, to provide a lipid-containing annamycin solution;

[0073] Sterile filter the lipid-containing annamycin solution; and

[0074] Lyophilize the lipid-containing annamycin solution to provide a lipid-containing annamycin lyophilizate.

[0075] In some embodiments, the acid used to adjust the pH is selected from the group consisting of: HCl and H 2 SO 4 . In some embodiments, the acid is HCl, such as 1M HCl.

[0076] In some embodiments, the method further comprises measuring the pH after adding the annamycin-containing solution and adjusting the pH to 5.3 ± 0.2 if necessary.

[0077] In some embodiments, after each addition of acid, the lipid-containing solution is equilibrated for at least 30 minutes. In some embodiments, after each addition of acid, the lipid-containing solution is equilibrated for about 45 minutes.

[0078] In some embodiments, each solution is maintained at a temperature from about 38°C to about 42°C until lyophilization begins.

[0079] In some embodiments, producing a lipidosome precursor annamycin lyophilizate comprises the steps of:

[0080] Add DMPC, DMPG, and polysorbate 20 to a pre-warmed mixture of water and tert-butanol to prepare a first solution, wherein the pre-warmed temperature is from about 35°C to about 42°C, and wherein the ratio of water to tert-butanol is from about 8:2 to about 9:1;

[0081] Adjust the pH of the first solution with one or more pharmaceutically acceptable acids to keep the pH of the solution constant for about 15 - 90 minutes;

[0082] Add a second solution to the first solution, the second solution comprising about 8 - 12 wt.% annamycin in DMSO, to provide a lipid-containing solution;

[0083] Sterile filter the lipid-containing solution; and

[0084] Lyophilize the lipid-containing annamycin solution in separate aliquots to provide a lipidosome precursor lyophilizate.

[0085] In some embodiments, the method comprises using an acid selected from HCl and H 2 SO 4The pH of the first solution is adjusted to 5.3 ± 0.2 with a pharmaceutically acceptable acid of the composition of the group.

[0086] In some embodiments, the method includes adding a third solution to a second solution, the third solution comprising about 10 wt.% annamycin in DMSO, to provide a lipid-containing annamycin solution.

[0087] In some embodiments, the annamycin liposome precursor lyophilizate comprises: 1.8 - 2.2 wt.% annamycin, 3.0 - 3.4 wt.% polysorbate 20, and 94.4 - 95.2 wt.% lipids selected from DMPC and DMPG. In some embodiments, DMPC is 65.3 - 67.3 wt.% and DMPG is 27.1 - 29.9 wt.%.

[0088] In some embodiments, the annamycin in the resulting lyophilizate is at least 98% pure. In some embodiments, the annamycin in the resulting lyophilizate is at least 99% pure.

[0089] Also provided is a composition of annamycin liposome precursor, the composition being prepared by any of the methods described herein and comprising 1.8 - 2.2 wt.% annamycin, 3.0 - 3.4 wt.% polysorbate 20, and 94.4 - 95.2 wt.% lipids selected from DMPC and DMPG. In some embodiments, DMPC is 65.3 - 67.3 wt.% and DMPG is 27.1 - 29.9 wt.%.

[0090] In some embodiments, the composition of the annamycin liposome precursor lyophilizate is at least 98% pure.

[0091] Also provided is a method for preparing a lipid-containing annamycin solution by adding a DMSO solution of annamycin to a pH-adjusted solution (the solution comprising DMPG, DMPC, and polysorbate 20 in water / t-butanol), wherein the pH is maintained at pH 4.8 - 5.9 for 15 - 75 minutes. In another embodiment, the pH is maintained at 5.3 + / - 0.2. In another embodiment, the pH is maintained for about 45 minutes.

[0092] Also provided is a method for preparing a separately viable amount of a high-purity annamycin liposome lipid precursor lyophilizate, the method comprising lyophilizing a predetermined separate aliquot of the lipid-containing solution for about 48 hours.

[0093] Particular embodiments include preparing a high-purity annamycin liposome precursor lyophilizate, wherein a separately viable amount is prepared in a 50 mL vial containing 45 mg annamycin.

[0094] Also provided is a method of treating cancer comprising administering to a patient in need thereof an effective dose of liposomal anamycin prepared using an anamycin liposome prolyophilisate prepared by any of the methods described herein.

[0095] In some embodiments, there is provided a use of an anamycin liposome prolyophilisate prepared according to any of the methods described herein in the manufacture of a medicament for treating cancer.

[0096] In some embodiments, an anamycin liposome pro-lyophilizate prepared according to any of the methods described herein is provided for use in treating cancer.

[0097] General experimental methods

[0098] Dimyristoylphosphatidylcholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1 glycerol)] sodium salt (DMPG) were each obtained as dry powders from Nippon Fine Chemicals, Inc., Osaka, Japan.

[0099] Anamycin (purity>95%) (MW=640.39) was synthesized as described previously with minor modifications (Horton, D., Priebe, W. 4-demethoxy-3'-desamino-2'-halo-anthracyclines and pharmaceutical compositions containing same. U.S. Pat. No. 4,537,882, 1985).

[0100] Polysorbate 20, DMSO, chloroform, and tert-butyl alcohol were obtained from Aldrich Chemical Company, Inc., Milwaukee, WI. Physiological saline was obtained from Abbott Laboratories, North Chicago, IL.

[0101] Dosage and route of administration.

[0102] The lyophilized powder of the liposomal precursor of annamycin is usually used on the day of administration to prepare an effective dose of liposomal annamycin, for example, liposomal annamycin suspended in saline in a non-PVC IV bag. The stability of the liposomal solution requires storing the drug as a lyophilized liposomal precursor at low temperature (stored frozen at -80°C to 0°C) until immediately before administration, and once the liposomal solution is formed, it should be used or discarded within 24 hours. Liposomal annamycin is used in methods for treating cancer in mammals (especially humans) and in methods for inhibiting tumor growth in mammals (especially humans). Cancers that can be treated include leukemia and lymphoma. In particular, annamycin can be used to treat leukemia, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). Liposomal annamycin can be used to treat lymphoma, including Hodgkin lymphoma and non-Hodgkin lymphoma.

[0103] The method includes administering to a mammal an effective amount of the pharmaceutical composition. The administration step can suitably be parenteral and can be by intravenous injection, intraarterial injection, intramuscular injection, intralymphatic injection, intraperitoneal injection, subcutaneous injection, intrapleural injection, intrathecal injection, or by topical application of a dose. In some embodiments, such administration is a repeated regimen until tumor regression or disappearance is achieved and can be used in combination with forms of tumor therapy such as surgery or chemotherapy with different agents. In some embodiments, for a mammalian subject to whom the composition is administered, the administered dose of the composition is between about 125 and 280 mg / m2.

[0104] Examples

[0105] The embodiments will now be described by way of example only with respect to the following examples.

[0106] General procedure.

[0107] Example 1. Preparation of the precursor of the freeze-dried annamycin liposome

[0108] Before use, warm tert-butanol and WFI (water for injection) in a water bath at 40°C ± 5°C.

[0109] Add tert-butanol (9181.2 g ± 1%) to a 15 L beaker with stirring and add water for injection (WFi, 1465.8 g ± 1%). During the addition, maintain the beaker at about 40°C. Remove approximately 2 L of the mixed solution and pour it into a sterile beaker for use as a "rinse" wash in subsequent steps. Continue stirring the bulk alcohol / aqueous solution.

[0110] Add the DMPC (743.554 g ± 1%) contained in the beaker to the bulk alcohol / water solution. After adding all of the DMPC, use a portion (about 1 / 4) of the "rinse" liquid to wash out any remaining residue in the beaker and add the washings to the bulk mixture. Stir the mixture until all of the materials are dissolved.

[0111] Add the DMPG sodium salt (318.654 g ± 1%) contained in the beaker to the bulk alcohol / water solution. After adding all of the DMPG, use a portion (about 1 / 4) of the "rinse" liquid to wash out any remaining residue in the beaker and add the washings to the bulk mixture. Stir the entire mixture until all of the materials are dissolved.

[0112] Carefully dispense the polysorbate 20 (36.12 g ± 1%) contained in a 100 mL beaker into the bulk solution. After adding all of the polysorbate 20, use a portion (about 1 / 4) of the "rinse" liquid to wash out any remaining residue in the beaker and add the washings to the bulk mixture. Stir the entire mixture until all of the materials are dissolved and maintain at 40 °C + / - 5 °C.

[0113] Measure the pH of the bulk solution and adjust it to 5.3 ± 0.2 with 1.0 M HCl. Measure the pH at 2 - 3 minute intervals and readjust the pH to 5.3 ± 0.2 with 1.0 M HCl again. Repeat this step until the pH of the mixture remains stable at pH approximately 5.3 ± 0.2 for 2 - 3 minutes. Then stir the solution for 45 minutes, check its pH again, and adjust the pH to pH 5.3 ± 0.2 with 1.0 M HCl if necessary. After stirring the mixture for another 45 minutes, measure the pH. Continue the pH adjustment process if necessary until the pH reading is pH 5.3 ± 0.2 after stirring for 2 - 3 minutes. If the pH has not changed compared to the initial reading, the bulk solution is ready for further processing. Otherwise, repeat this step until the pH remains at pH 5.3 ± 0.2 for approximately 45 minutes.

[0114] Prepare annamycin (as a THF complex) in purified form (by HPLC, >98%) according to the procedure in US,977,327 (Example VIII, columns 7 - 8).

[0115] Add DMSO (234.08 g ± 1%) to the annamycin (THF complex, API, 23.16 g ± 1%) in a sterile beaker and stir the materials until dissolved. Add the DMSO / API solution to the bulk solution with stirring and add any remaining "rinse" liquid directly to the bulk solution and continue stirring.

[0116] Pass the solution twice through a sterile 0.2 micron filter. Store the filtered product solution overnight at ambient temperature. The total volume of the product solution is approximately 13.83 L.

[0117] Load the filtered product solution (25 mL by weight, as determined by density testing as part of process testing) into a 50 mL clear glass lyophilization vial. Lyophilize for 48 hours to remove all DMSO, tert-butanol, and water, thereby providing the liposome precursor powder.

[0118] Measure the purity of annamycin in the lyophilized powder by HPLC using a validated reference standard sample.

[0119] Although the invention has been described with reference to specific embodiments and examples, those skilled in the art will recognize that various modifications can be made to the invention without departing from its spirit and scope.

[0120] All references cited herein, including patents, patent applications, and publications, are hereby incorporated by reference in their entirety. Whether previously specifically incorporated or not.

[0121] The various features and embodiments of the invention mentioned in the above sections are appropriately applicable to other sections with necessary modifications. Thus, the features specified in one section can be appropriately combined with the features specified in other sections.

[0122] The above description of certain specific embodiments provides sufficient information such that others can, without departing from the general concept, readily modify and / or adapt such embodiments for various applications by applying existing knowledge. Therefore, such adaptations and modifications should and are intended to be covered within the meaning and scope of the equivalents of the disclosed embodiments. It should be understood that the language or terminology employed herein is for the purpose of description and not of limitation. In the drawings and the specification, exemplary embodiments have been disclosed, and although specific terms may be employed, these terms are used only in a general and descriptive sense and not for the purpose of limiting the scope of the claims. Additionally, those skilled in the art will understand that certain steps of the methods discussed herein can be sequenced in alternative orders or the steps can be combined. Accordingly, it is intended that the appended claims not be limited to the specific embodiments disclosed herein. Many equivalents of the embodiments of the invention described herein will be recognized or can be determined by those of ordinary skill in the art using only routine experimentation. Such equivalents are covered by the following claims.

Claims

1. A method for preparing freeze-dried annamycin, the method comprises the following steps: Preparing a solution comprising one or more lipids, a non-ionic surfactant and one or more solvents and having a pH of 4.8 - 5.9; Adding an annamycin solution to the lipid solution, the annamycin solution comprising 8 - 12 wt.% annamycin in DMSO, to provide a lipid-containing annamycin solution; Sterile filtering the lipid-containing annamycin solution; and Freeze-drying the lipid-containing annamycin solution to provide a freeze-dried annamycin liposome precursor; wherein the one or more lipids are selected from DMPC and DMPG, and the non-ionic surfactant is polysorbate 20.

2. A method for preparing freeze-dried annamycin, the method comprises the following steps: Preparing a first solution comprising water and tert-butanol, wherein the ratio of water to tert-butanol is from 8:2 to 9:1; Adding one or more lipids and a non-ionic surfactant to the first solution to form a second solution; Adjusting the pH of the second solution such that the pH is 4.8 - 5.9; Adding a third solution to the second solution, the third solution comprising 8 - 12 wt.% annamycin in DMSO, to provide a lipid-containing annamycin solution; Sterile filtering the lipid-containing annamycin solution; and Freeze-drying the lipid-containing annamycin solution to provide a freeze-dried annamycin liposome precursor; wherein the one or more lipids are selected from DMPC and DMPG, and the non-ionic surfactant is polysorbate 20.

3. The method according to claim 2, wherein the pH of the second solution is adjusted to 5.3 ± 0.2 with a pharmaceutically acceptable acid selected from the group consisting of HCl and H 2 SO 4 .

4. The method according to any one of claims 2 - 3, wherein the third solution comprises 10 wt.% annamycin in DMSO.

5. The method according to any one of claims 2 - 3, wherein each lipid and surfactant is added separately.

6. The method according to any one of claims 2 - 3, wherein DMPC, DMPG and polysorbate 20 are added sequentially to the first solution.

7. The method according to any one of claims 2 - 3, wherein the freeze-dried annamycin liposome precursor comprises: 1.8 - 2.2 wt.% annamycin, 3.0 - 3.4 wt.% polysorbate 20, and 94.4 - 95.2 wt.% lipids selected from DMPC and DMPG.

8. The method according to claim 7, wherein the annamycin in the resulting freeze-dried product is at least 98% pure.

9. The method according to claim 8, wherein the annamycin in the resulting freeze-dried product is at least 99% pure.

10. The method according to any one of claims 2 - 3, the method further comprises: After initially adjusting the pH of the second solution to pH 5.3 ± 0.2, equilibrating the solution for at least 15 minutes; Measuring the pH, and when the pH is higher than 5.3, adjusting the pH with an acid; Equilibrating the solution for at least 15 minutes; and Repeating the following steps: measuring the pH, adjusting the pH, and equilibrating the solution until the pH of the solution remains at pH 5.3 ± 0.

2.

11. The method according to claim 10, wherein the acid used to adjust the pH is selected from the group consisting of: HCl and H 2 SO 4 .

12. The method according to claim 10, wherein after each addition of acid, the second solution is equilibrated for at least 30 minutes.

13. The method according to any one of claims 2 - 3, wherein each solution is maintained at a temperature from 38°C to 42°C until lyophilization is initiated.

14. A method for producing a freeze - dried anamycin liposome precursor, the method comprising the steps of: adding DMPC, DMPG, and polysorbate 20 to a pre - warmed mixture of water and tert - butanol to prepare a first solution, wherein the pre - warming temperature is from 35°C to 42°C, and wherein the ratio of water to tert - butanol is from 8:2 to 9:1; adjusting the pH of the first solution with one or more pharmaceutically acceptable acids to keep the pH of the solution constant for 15 - 90 minutes; adding a second solution to the first solution, the second solution comprising 8 - 12 wt.% anamycin in DMSO, to provide a lipid - containing anamycin solution; sterile - filtering the lipid - containing anamycin solution; and lyophilizing the lipid - containing solution in separate aliquots to provide a freeze - dried anamycin liposome precursor; wherein the second solution has a pH of 5.3 ± 0.

2.

15. The method according to claim 14, wherein the second solution comprises 10 wt.% anamycin in DMSO.

16. A composition of a freeze - dried anamycin liposome precursor prepared by the method according to claim 1, the composition comprising: anamycin, one or more lipids, and a non - ionic surfactant.

17. The composition according to claim 16, the composition comprising: 1.8 - 2.2 wt% anamycin, 3.0 - 3.4 wt.% polysorbate 20, and 94.4 - 95.2 wt.% lipids selected from DMPC and DMPG.

18. The composition according to claim 16, wherein the DMPC is 65.3 - 67.3 wt.% and the DMPG is 27.1 - 29.9 wt.%.

19. The composition according to any one of claims 16 - 18, wherein the anamycin in the produced freeze - dried product is at least 98% pure.

20. Use of liposomal anamycin in the preparation of a medicament for treating cancer in a patient in need thereof, the liposomal anamycin being prepared by reconstituting a freeze - dried anamycin liposome precursor prepared according to any one of claims 1 - 15.

21. The use according to claim 20, wherein the patient is a human, and the cancer is selected from the group consisting of leukemia and lymphoma.

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