Preparation of Freeze-Dried Liposomal Anamycin Precursor

Abstract

The present invention provides a method for producing a lyophilized liposomal anamycin precursor, a composition produced by the method, and the use of the composition produced by the method in the treatment of cancer. More specifically, the method for producing lyophilized anamycin includes the steps of preparing a solution containing anamycin, one or more lipids, one or more nonionic surfactants, and one or more solvents, sterile filtering the lipid-containing solution of anamycin, and lyophilizing the lipid-containing solution of anamycin to obtain a lyophilized liposomal anamycin precursor.

Description

Technical Field

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

Background Art

[0002] Annamycin is a cancer chemotherapeutic agent and belongs to a group having an anthracene structure. Compositions for formulating Annamycin have been described according to its physical and pharmacological properties, and this drug is formulated as liposomes (see U.S. Patent No. 7,238,366). Clinical trials regarding liposomal Annamycin have been described in the treatment of adult patients with relapsed and refractory acute lymphoblastic leukemia (M. Wetzler, et al., Clinical Lymphoma, Myeloma and Leukemia, 13(4), 430-434 August 2013) and doxorubicin-resistant breast cancer (D. J. Booser et al., Cancer Chemother. Pharmacol. 50; 6-8, 2002).

[0003] In the drug delivery system, it is desirable to be able to manufacture a formulation with constant purity and quality for the purpose of ensuring accurate dosing and thus providing the most effective treatment while avoiding unwanted side effects caused by impurities. Furthermore, a drug material with poor maintenance or inappropriate formulation may decompose and lose its effect before administration, resulting in waste of expensive active pharmaceutical ingredients. Regulatory standards also require that the purity and amount of the drug be provided at a constant level by following the procedures specified on the label.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The formulation of annamycin is a particularly difficult problem due to its inherent physical properties and because both the parent drug and the liposomal formulation are inherently unstable under certain conditions. Liposomal formulations of annamycin often become unsuitable for administration because they decompose to form annamycin crystals and / or form large liposomes or phase separate. Therefore, it would be advantageous to find a stable method for formulating a dosage form of a liposomal annamycin precursor that ensures purity and avoids degradation. **Means for Solving the Problems**

[0005] A method for producing freeze-dried annamycin, comprising the step of preparing a solution having a pH of 4.8 to 5.9, the solution containing one or more lipids, one or more nonionic surfactants, and one or more solvents; obtaining a lipid-containing solution of Annamycin by adding an annamycin solution containing about 8 to 12 wt% annamycin in DMSO to this lipid solution; sterile filtering this lipid-containing solution of annamycin; and obtaining a freeze-dried product of a liposomal annamycin precursor by freeze-drying this lipid-containing solution of annamycin A method is provided that includes these steps.

[0006] A method for manufacturing freeze-dried annamycin, comprising the step of preparing a first solution containing water and t-butanol, wherein the ratio of water to t-butanol is about 8:2 to about 9:1; generating a second solution by adding one or more lipids and one or more nonionic surfactants to the first solution; adjusting the pH of the second solution to a pH of 4.8 to 5.9; Adding a third solution containing about 8-12 wt% of amikacin in DMSO to the second solution to obtain a lipid-containing solution of amikacin; Sterile filtering the lipid-containing solution of amikacin; Freeze-drying the lipid-containing solution of amikacin to obtain a freeze-dried product of liposomal amikacin precursor A method comprising is provided.

[0007] A method for producing a freeze-dried product of liposomal amikacin precursor, comprising: Preparing a first solution by adding DMPC, DMPG, and polysorbate 20 to a mixture of pre-warmed water and t-butanol, wherein the pre-warming temperature is about 35°C to about 42°C and the water to t-butanol ratio is about 8:2 to about 9:1; Adjusting the pH of the first solution using a pharmaceutically acceptable acid so that the pH of the solution remains constant for at least 15 minutes; Adding a second solution containing about 8-12 wt% of amikacin in DMSO to the first solution to obtain a lipid-containing solution of amikacin; Sterile filtering the lipid-containing solution of amikacin; Freeze-drying the lipid-containing solution of amikacin by aliquoting and freeze-drying to obtain a freeze-dried product of liposomal precursor A method comprising is also provided.

[0008] A freeze-dried product of liposomal amikacin precursor prepared by any of the methods described herein is also provided.

[0009] A method for treating cancer, comprising administering an effective dose of liposomal amikacin prepared using a freeze-dried product of liposomal amikacin precursor prepared by any of the methods described herein to a patient in need thereof, is also provided.

[0010] Also provided is the use of a freeze-dried liposomal amikacin precursor prepared according to any of the methods described herein in the manufacture of a medicament for treating cancer.

[0011] Also provided is a freeze-dried liposomal amikacin precursor prepared according to any of the methods described herein for use in the treatment of cancer.

DETAILED DESCRIPTION OF THE INVENTION

[0012] Provided is a method for creating a freeze-dried liposomal amikacin precursor having improved stability and high purity. This freeze-dried liposomal amikacin precursor composition can be reconstituted into an aqueous liposomal composition by hydrating, for example, as described in U.S. Patent No. 7,238,366, which is hereby incorporated by reference in its entirety for all purposes, and then used in the treatment of cancer.

[0013] 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.

[0014] The following terms used herein have the meanings defined below.

[0015] The term "amikacin" shall mean the compound having the following structure:

CHEMICAL STRUCTURE

[0016] The term "lyophilized high-purity liposomal amikacin precursor" shall mean the purity of a material analyzed by HPLC using a verified reference sample and found to contain 95% or more amikacin. In some embodiments, the purity of amikacin is at least 96%, or at least 97%, or at least 98%, or at least 99%.

[0017] The terms "liposome", "liposomal", and the like shall mean a substantially spherical structure including lipids, fatty acids, lipid bilayer structures, unilamellar vesicles, and amorphous lipid vesicles. Classically, a liposome is a completely enclosed lipid bilayer membrane that encapsulates a volume of aqueous material. Liposomes include non-classical forms in which amikacin may be inside the bilayer, part of the bilayer, or absorbed into the bilayer. A liposome may be a unilamellar vesicle (having a single bilayer membrane) or a multilamellar vesicle (an onion-like structure characterized by multiple bilayer membranes separated from adjacent layers by aqueous layers). The bilayer is composed of two lipid monolayers having hydrophobic "tail" regions and hydrophilic "head" regions. The bilayer membrane has a structure such that the hydrophobic (non-polar) "tails" of the lipid monolayers face towards the center of the bilayer and the hydrophilic "heads" face towards the aqueous layer.

[0018] The terms "preliposome - lyophilizate" and "preliposomal lyophilizate" shall mean non - aqueous materials that will form liposomes upon addition of an aqueous solution. In some embodiments, the non - aqueous material is a dried (non - liquid, non - gel - like) material. The lyophilizate is used in a broad sense to include the dry residue obtained by sublimating frozen liquid from non - volatile materials, the residue by rotary evaporation and similar procedures, and dry compositions that will yield liposomes (with or without stirring) upon addition of an aqueous layer. In particular, "preliposome - lyophilizate" should be understood as not being in a liposomal form after lyophilization.

[0019] The term "lipid" refers to any of the classifications of pharmaceutically acceptable organic compounds that are fatty acids or their derivatives. In some embodiments, the lipid is a phospholipid such as phosphatidylcholine including DMPC and DPMG, but other lipids such as phosphatidylethanolamine from eggs can also be included.

[0020] The term "non - ionic surfactant" refers to a pharmaceutically acceptable surfactant having a hydrophilic group containing a covalently - bonded oxygen, which is bonded to a hydrophobic parent structure. Suitable non - ionic 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 non - ionic surfactant is a polysorbate - type surfactant produced by ethoxylating sorbitan and then adding a carboxylic acid. In some embodiments, the non - ionic surfactant includes polyoxyethylene sorbitan monolaurate (polysorbate 20) and polyethoxylated sorbitan monooleate (polysorbate 80).

[0021] "Polysorbate 20" refers to a commercially available nonionic surfactant (ICI Americas Inc.) composed of common sorbitan sugars to which mixtures of polyoxyethylene of different chain lengths are attached. This polyoxyethylene sugar is also attached to fatty acids. The trade name of this material is Tween™ 20; the composition is polyoxyethylene sorbitan monolaurate (MW approximately 1300). As polysorbate 20, the following is shown. w + x + y + z = 20. [Chemical formula]

[0022] The term "pharmaceutically acceptable acid" refers to any organic and inorganic acid that is known in the art to be highly tolerated and suitable for administration to human patients. Examples of salts of this type 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 (+), capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid (D), gluconic acid (D), glucuronic acid (D), glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid (DL), lactobionic acid, lauric acid, maleic acid, malic acid (-L), malonic acid, mandelic acid (DL), methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, 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.

[0023] In the above section, many abbreviations and acronyms were used. Their full notations are shown below. DMPC dimyristoylphosphatidylcholine DMPG 1,2-dimyristoyl-sn-glycero-3-[phospho-(1'-rac-glycerol)(sodium salt) DMSO dimethyl sulfoxide IV intravenous DEHP bis(2-ethylhexyl) phthalate PVC polyvinyl chloride WPI water for injection (USP)

[0024] Throughout this specification and the appended claims, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will 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. When a specified range is described herein, it is intended that the described range includes all specific integral amounts falling within the described range. For example, the range of about 35 to 42 °C is intended to include 35, 36, 37, 38, 39, 40, 41, and 42 °C.

[0025] The entire disclosure content of each U.S. patent and international patent application described in this patent specification is hereby incorporated by reference herein in its entirety for all purposes.

[0026] Provided is a method for producing a freeze-dried liposomal amikacin precursor with very high purity and stability. In particular, it is a method for producing freeze-dried amikacin, comprising: preparing a first solution containing water and t-butanol, wherein the ratio of water to t-butanol is about 8:2 to about 9:1; generating a second solution by adding one or more lipids and one or more nonionic surfactants to the first solution; adjusting the pH of the second solution to a pH of 4.8 to 5.9; obtaining a lipid-containing solution of amikacin by adding a third solution containing about 8 to 12 wt% of amikacin in DMSO to the second solution; sterile filtering the lipid-containing solution of amikacin; obtaining a freeze-dried liposomal amikacin precursor by freeze-drying the lipid-containing solution of amikacin A method comprising the above is provided.

[0027] 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.

[0028] In some embodiments, each lipid and each nonionic surfactant are added separately.

[0029] In some embodiments, DMPC, DMPG, and polyoxyethylene sorbitan monolaurate are sequentially added to the first solution.

[0030] In some embodiments, the method includes adjusting the pH of the second solution to 5.3 ± 0.2 using a pharmaceutically acceptable acid selected from the group consisting of HCl and H 2 SO 4 2SO4.

[0031] In some embodiments, the method includes obtaining a lipid-containing solution of amikacin by adding a third solution containing about 10 wt% amikacin in DMSO to the second solution.

[0032] In some embodiments, the lyophilized liposomal amikacin precursor comprises 1.8 - 2.2 wt% amikacin; 3.0 - 3.4 wt% polysorbate 20; and 94.4 - 95.2 wt% of a lipid selected from DMPC and DMPG. In some embodiments, DMPC is 65.3 - 67.3 wt% and DMPG is 27.1 - 29.9 wt%.

[0033] In some embodiments, the purity of amikacin in the resulting lyophilized product is at least 98%. In some embodiments, the purity of amikacin in the resulting lyophilized product is at least 99%.

[0034] In some embodiments, a method for preparing lyophilized amikacin, Preparing a first solution comprising water and t-butanol, wherein the ratio of water to t-butanol is from about 8:2 to about 9:1; Adding one or more lipids and one or more nonionic surfactants to the first solution to produce a second solution comprising DMPC, DMPG, and polysorbate 20; Adjusting the pH of the second solution to a pH of 5.3 ± 0.2; Equilibrating this solution for at least 15 minutes; Measuring the pH and adjusting the pH with an acid if the pH exceeds 5.3; Equilibrating this solution for at least 15 minutes; 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; Adding a third solution comprising about 8 - 12 wt% of anamycin in DMSO to the second solution to obtain a lipid-containing solution of anamycin; Sterile filtering the lipid-containing solution of anamycin; Obtaining a lipid-containing anamycin lyophilizate by lyophilizing the lipid-containing solution of anamycin comprising.

[0035] In some embodiments, the acid used for pH adjustment is selected from the group consisting of HCl and H 2 SO 4 In some embodiments, the acid is HCl such as 1M HCl.

[0036] In some embodiments, the method further comprises measuring the pH after adding the anamycin-containing solution and adjusting the pH to 5.3 ± 0.2 if necessary.

[0037] In some embodiments, the lipid-containing solution is equilibrated for at least 30 minutes each time an acid is added. In some embodiments, the lipid-containing solution is equilibrated for about 45 minutes each time an acid is added.

[0038] In some embodiments, each solution is maintained at a temperature of about 38°C to about 42°C until lyophilization is initiated.

[0039] In some embodiments, the liposomal amikacin precursor lyophilizate Preparing a first solution by adding DMPC, DMPG, and polysorbate 20 to a pre-warmed mixture of water and t-butanol, wherein the pre-warming temperature is about 35°C to about 42°C and the water to t-butanol ratio is about 8:2 to about 9:1; Adjusting the pH of the first solution using one or more pharmaceutically acceptable acids such that the pH of the solution remains constant for about 15 - 90 minutes; Adding a second solution containing about 8 - 12 wt% amikacin in DMSO to the first solution to obtain a lipid-containing solution; Sterile filtering the lipid-containing solution; Obtaining a liposomal precursor lyophilizate by aliquoting and lyophilizing the lipid-containing solution of amikacin is produced by.

[0040] In some embodiments, the method includes adjusting the pH of the first solution to 5.3 ± 0.2 using a pharmaceutically acceptable acid selected from the group consisting of HCl and H 2 SO 4 In some embodiments, the method includes obtaining a lipid-containing solution of amikacin by adding a second solution containing about 10 wt% amikacin in DMSO to the second solution.

[0041]

[0042] ​In some embodiments, the lyophilized liposomal amikacin precursor contains 1.8 - 2.2 wt% amikacin, 3.0 - 3.4 wt% polysorbate 20, and 94.4 - 95.2 wt% lipid selected from DMPC and DMPG. In some embodiments, DMPC is 65.3 - 67.3 wt% and DMPG is 27.1 - 29.9 wt%.

[0043] In some embodiments, the purity of amikacin in the resulting lyophilized product is at least 98%. In some embodiments, the purity of amikacin in the resulting lyophilized product is at least 99%.

[0044] Also provided is a composition of a liposomal amikacin precursor prepared by any of the methods described herein and containing 1.8 - 2.2 wt% amikacin, 3.0 - 3.4 wt% polysorbate 20, and 94.4 - 95.2 wt% lipid selected from DMPC and DMPG. In some embodiments, DMPC is 65.3 - 67.3 wt% and DMPG is 27.1 - 29.9 wt%.

[0045] In some embodiments, the purity of the composition of the lyophilized liposomal amikacin precursor is at least 98%.

[0046] Also provided is a process for preparing a lipid-containing solution of amikacin by adding a DMSO solution of amikacin to a pH-adjusted solution containing 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 other embodiments, the pH is maintained at 5.3 ± 0.2. In other embodiments, this pH is maintained for about 45 minutes.

[0047] A process for preparing a freeze-dried product of high-purity liposomal amikacin precursor in individual practicable quantities, which includes lyophilizing a lipid-containing solution of individually measured aliquots for about 48 hours, is also provided.

[0048] Certain embodiments include the preparation of a freeze-dried product of high-purity liposomal amikacin precursor, wherein the individual practicable quantity is prepared in a 50 mL vial containing 45 mg of amikacin.

[0049] A method for treating cancer, which includes administering an effective dose of liposomal amikacin prepared using a freeze-dried product of liposomal amikacin precursor prepared by any of the methods described herein to a patient in need thereof, is also provided.

[0050] In some embodiments, provided is the use of a freeze-dried product of liposomal amikacin precursor prepared according to any of the methods described herein in the manufacture of a medicament for treating cancer.

[0051] In some embodiments, provided is a freeze-dried product of liposomal amikacin precursor prepared according to any of the methods described herein for use in the treatment of cancer.

[0052] General experimental methods Dimyristoylphosphatidylcholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-[phosphor-rac-(1-glycerol)] sodium salt (DMPG) were obtained as dry powders from Nippon Fine Chemicals, Inc., Osaka, Japan.

[0053] Anamycin (purity > 95%) (MW = 640.39) was synthesized as described previously (with slight modification) (Horton, D., Priebe, W, 4-demethoxy-3'-desamino-2'-halo-anthracyclines and pharmaceutical compositions containing same, U.S. Patent No. 4,537,882, 1985).

[0054] Polysorbate 20, DMSO, chloroform, and t-butyl alcohol were obtained from Aldrich Chemical Company, Inc., Milwaukee, Wis. Physiological saline was obtained from Abbott Laboratories, North Chicago, Ill.

[0055] Dosage and Route of Administration The lyophilized powder of the liposomal precursor of anamycin is usually used on the day of administration to prepare an effective dose of liposomal anamycin, for example, suspended in physiological saline in a non-PVC IV bag. For the stability of the liposomal solution, it is necessary to store the drug as a lyophilized powder of the liposomal precursor at a low temperature (frozen and stored at -80°C to 0°C) until immediately before administration, use it within 24 hours once formed, and discard it if not used. Liposomal anamycin is used in methods for treating cancer and methods for suppressing tumor growth in mammals, particularly humans. Cancers that can be treated include leukemia and lymphoma. In particular, anamycin can be used for the treatment of leukemia such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). Liposomal anamycin can be used for the treatment of lymphoma such as Hodgkin lymphoma and non-Hodgkin lymphoma.

[0056] This method involves administering an effective amount of a drug composition to a mammal. The administration step can preferably be carried out parenterally, by intravenous, intraarterial, intramuscular, intralymphatic, intraperitoneal, subcutaneous, intrathoracic, intrathecal injection or by topical application. In some embodiments, this type of administration is a repeated dosing regimen until tumor shrinkage or disappearance is achieved and can be used in combination with forms of tumor treatment such as surgical procedures or chemotherapy using different agents. In some embodiments, the dosage of the composition administered is between about 125 - 280 mg / m 2 in the subject mammal being administered.

Examples

[0057] Here, the embodiments will be described by taking the following examples as just one example.

[0058] General procedure Example 1 Preparation of an anamycin liposome lyophilized precursor t-butanol and WFI (Water for Injection) were heated to 40°C ± 5°C in a water bath before use.

[0059] t-butanol (9181.2 g ± 1%) was added to a 15 L beaker, and while stirring, water for injection (WFi, 1465.8 g ± 1%) was added. During this addition, the beaker was maintained at about 40°C. Approximately 2 L of the mixed solution was withdrawn and placed in a sterilized beaker for use as a "rinse" wash solution in subsequent steps. Stirring of the main alcohol / aqueous solution was continued.

[0060] DMPC (743.554 g ± 1%) placed in the beaker was added to the main alcohol / aqueous solution. After all of the DMPC was added, a portion (about 1 / 4) of the "rinse" solution was used to wash the residue remaining in the beaker and added to the main mixture. The mixture was stirred until all materials were dissolved.

[0061] The sodium salt of DMPG (318.654 g ± 1%) placed in a beaker was added to the main alcohol / aqueous solution. After all of the DMPG had been added, a portion (about 1 / 4) of the "rinse" solution was used to wash away the residue remaining in the beaker and added to the main mixture. The entire mixture was stirred until all materials were dissolved.

[0062] Polysorbate 20 (36.12 g ± 1%) placed in a 100 mL beaker was carefully added in portions to the main solution. After all of the polysorbate 20 had been added, a portion (about 1 / 4) of the "rinse" solution was used to wash away the residue remaining in the beaker and added to the main mixture. The entire mixture was stirred until all materials were dissolved and maintained at 40 ± 5 °C.

[0063] The pH of the main solution was measured and adjusted to 5.3 ± 0.2 with 1.0 M HCl. The pH was measured at 2 - to 3-minute intervals and readjusted to 5.3 ± 0.2 with 1.0 M HCl again. This step was repeated until the pH of the mixture remained at about 5.3 ± 0.2, a constant pH, for about 2 - 3 minutes. The solution was then stirred for 45 minutes, the pH was examined again, and adjusted to pH 5.3 ± 0.2 with 1.0 M HCl if necessary. After the mixture was stirred for an additional 45 minutes, the pH was measured. If necessary, the procedure for adjusting the pH was continued until the pH reading after stirring for 2 - 3 minutes was 5.3 ± 0.2. When the pH no longer changed from the initial reading, the main solution was in a state ready for further processing. If not, this step was repeated until the pH remained at pH 5.3 ± 0.2 for about 45 minutes.

[0064] Anamycin (as a THF complex) was prepared in a purified form (>98% by HPLC) according to the procedure of U.S. Patent No. 977,327 (Example VIII, columns 7 - 8).

[0065] In a sterilized beaker, DMSO (234.08 g ± 1%) was added to anamycin (THF complex, API 23.16 g ± 1%), and the mixture was stirred until the material dissolved. The DMSO / API solution was added to the main solution while stirring, and all of the remaining "rinse" liquid was added directly to the main solution, and stirring was continued.

[0066] This solution was passed through a 0.2 micron sterilizing filter twice. The filtered product solution was stored overnight at ambient temperature. The bulk volume of the resulting solution was approximately 13.83 L.

[0067] The filtered product solution (25 mL, by weight determined from a density test conducted as part of the tests performed during processing) was filled into 50 mL clear glass lyophilization vials. By performing lyophilization for 48 hours, all of the DMSO, t-butyl alcohol, and water were removed to obtain a liposome precursor powder.

[0068] The purity of the anamycin in the lyophilized powder was measured by HPLC using a validated standard sample.

[0069] Although the present invention has been described with reference to specific embodiments and examples, those skilled in the art recognize that various modifications can be made to the invention without departing from its spirit and scope.

[0070] The entire contents of all references, including patents, patent applications, and publications, cited herein are hereby incorporated by reference into this specification, whether or not they have been specifically incorporated already.

[0071] The various features and embodiments of the invention mentioned in the above individual paragraphs are applicable to other paragraphs with the necessary modifications, if appropriate. Accordingly, the features specified in one paragraph can be combined with the features specified in other paragraphs, if appropriate.

[0072] The foregoing description of certain embodiments is sufficient to enable others skilled in the art, applying current knowledge, to modify or adapt such specific embodiments for various uses without departing from the general concept, and, accordingly, such adaptations and modifications are intended to be within the meaning and equivalent scope of the disclosed embodiments. It should be understood that the terminology and phraseology used herein is for the purpose of description only and not of limitation. Although exemplary embodiments have been disclosed in the drawings and the detailed description, and specific terms have been used, these terms are used only in a general and descriptive sense and not for the purpose of limiting the scope of the claims. Further, those skilled in the art will appreciate that the specific steps of the methods discussed herein can be arranged in alternative orders or combined. Accordingly, the appended claims are not intended to be limited to the specific embodiments disclosed herein. Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method for producing freeze-dried amikacin, comprising: preparing a solution containing one or more lipids, one or more nonionic surfactants, and one or more solvents, with a pH of 4.8 to 5.9; obtaining a lipid-containing solution of amikacin by adding an amikacin solution containing about 8 to 12 wt% of amikacin in DMSO to the lipid solution; sterile filtering the lipid-containing solution of amikacin; obtaining a freeze-dried liposomal amikacin precursor by freeze-drying the lipid-containing solution of amikacin and a method comprising the steps.

2. A method for producing freeze-dried amikacin, comprising: preparing a first solution containing water and t-butanol, wherein the ratio of water to t-butanol is about 8:2 to about 9:1; generating a second solution by adding one or more lipids and one or more nonionic surfactants to the first solution; adjusting the pH of the second solution to a pH of 4.8 to 5.9; obtaining a lipid-containing solution of amikacin by adding a third solution containing about 8 to 12 wt% of amikacin in DMSO to the second solution; sterile filtering the lipid-containing solution of amikacin; obtaining a freeze-dried liposomal amikacin precursor by freeze-drying the lipid-containing solution of amikacin and a method comprising the steps.

3. The method according to claim 2, wherein the second solution contains DPPC, DPPG, and a polyoxyethylene sorbitan surfactant.

4. The method according to claim 2 or 3, wherein the second solution contains DPPC, DPPG, and polyoxyethylene sorbitan monolaurate.

5. The pH of the second solution is adjusted to 5.3 ± 0.2 using a pharmaceutically acceptable acid selected from the group consisting of HCl and H ２ SO ４ The method according to any one of claims 2 to 4.

6. The method according to any one of claims 2 to 5, wherein the third solution contains about 10 wt% of amikacin in DMSO.

7. The method according to any one of claims 2 to 6, wherein each of the lipids and each of the surfactants are added separately.

8. The method according to any one of claims 2 to 7, wherein DPPC, DPPG, and polyoxyethylene sorbitan monolaurate are sequentially added to the first solution.

9. The lyophilized liposomal amikacin precursor according to any one of claims 2 to 8 contains 1.8 to 2.2 wt% of amikacin; 3.0 to 3.4 wt% of polysorbate 20; and 94.4 to 95.2 wt% of a lipid selected from DMPC and DMPG.

10. The method according to claim 9, wherein the purity of the amikacin in the produced lyophilized product is at least 98%.

11. The method according to claim 10, wherein the purity of the amikacin in the produced lyophilized product is at least 99%.

12. First, adjust the pH of the second solution to pH 5.3 ± 0.2, and then equilibrate the solution for at least 15 minutes; Measure the pH, and if the pH exceeds 5.3, adjust the pH with an acid; Equilibrate the solution for at least 15 minutes; Repeat 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. The method according to any one of claims 4 to 11, further comprising the above steps.

13. The acid used for adjusting the pH is selected from HCl and H ２ SO ４ The method according to claim 12

14. The method according to claim 12 or 13, wherein the second solution is equilibrated for at least 30 minutes each time an acid is added.

15. The method according to any one of claims 2 to 14, wherein each solution is maintained at a temperature of about 38°C to about 42°C until lyophilization is started.

16. A method for producing a lyophilized liposomal amikacin precursor, comprising: Preparing a first solution by adding DMPC, DMPG, and polysorbate 20 to a mixture of pre-warmed water and t-butanol, wherein the pre-warming temperature is about 35°C to about 42°C, and the water-to-t-butanol ratio is about 8:2 to about 9:1; Adjusting the pH of the first solution using one or more pharmaceutically acceptable acids so that the pH of the solution remains constant for about 15 to 90 minutes; Adding a second solution containing about 8 to 12 wt% of amikacin in DMSO to the first solution to obtain a lipid-containing solution of amikacin; Sterile filtering the lipid-containing solution of amikacin; Obtaining a lyophilized liposomal amikacin precursor by separately aliquoting and lyophilizing the lipid-containing solution. The method comprising the above steps.

17. The method according to claim 16, wherein the second solution contains about 10 wt% of amikacin in DMSO and the pH is 5.3 ± 0.

2.

18. A composition of a freeze-dried liposomal amikacin precursor prepared by the method according to claim 1, comprising amikacin, one or more lipids, and one or more nonionic surfactants.

19. The composition according to claim 18, comprising 1.8 to 2.2 wt% of amikacin, 3.0 to 3.4 wt% of polysorbate 20, and 94.4 to 95.2 wt% of a lipid selected from DMPC and DMPG.

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

21. The composition according to any one of claims 18 to 20, wherein the purity of the amikacin in the produced freeze-dried product is at least 98%.

22. A medicament for the treatment of cancer, comprising the freeze-dried liposomal amikacin precursor produced according to any one of claims 1 to 17, and administering an effective amount of liposomal amikacin prepared by reconstituting the freeze-dried liposomal amikacin precursor to a patient in need thereof, which is used in a method for treating cancer.

23. The medicament according to claim 22, which is a medicament for the treatment of cancer in a human patient, wherein the cancer is selected from the group consisting of leukemia and lymphoma.

24. 1.8 to 2.2 wt% of amikacin; 65.3 to 67.3 wt% of DMPC (dimyristoylphosphatidylcholine); 27.1 to 29.9 wt% of DMPG (1,2-dimyristoyl-sn-glycero-3-[phospho-(1'-rac-glycerol)] sodium salt); and 3.0 to 3.4 wt% of polysorbate 20, A freeze-dried liposomal amikacin precursor composition produced by the method according to claim 1, 2 or 16.

25. The composition according to claim 24, having a purity of at least 98%.

26. The composition according to claim 25, wherein the purity is measured by HPLC using a verified standard sample.

27. The composition according to claim 24, having a purity of at least 99%.

28. The composition according to claim 27, wherein the purity is measured by HPLC using a verified reference sample.

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