Method for reconstituting liposomal anamycin

Abstract

Methods for reconstituting lyophilized anamycin and methods for treating patients are described.

Description

Technical Field

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 868,190, filed 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 has been formulated as liposomes (see U.S. Patent No. 7,238,366). Clinical trials regarding liposomal Annamycin have been described in 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 in the treatment of 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 the accurate administration of the dosage, 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 before administration and lose its effect, resulting in waste of expensive active ingredients. Regulatory standards also require that the purity and amount of the drug be provided at a certain level by following the procedures specified on the label.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The formulation of anamycin is a particularly challenging task due to its inherent physical properties, as well as the fact that both the parent drug and the liposomal formulation are inherently unstable under certain conditions. Therefore, finding a stable method for formulating a liposomal anamycin dosage form that ensures purity and avoids degradation would be advantageous. [Means for solving the problem]

[0005] This invention provides a method for forming a liposomal anamycin suspension by reconstituting a lyophilized preliposomal anamycin precursor.

[0006] A method for reconstituting anamycin, and this type of method is By adding pre-warmed physiological saline to one or more containers of lyophilized liposomal anamycin precursor, a suspended liposomal anamycin with a concentration of approximately 0.5 to 1.5 mg / mL is produced; Maintaining one or more of the aforementioned containers at 24-45°C; The aforementioned suspended liposomal anamycin is diluted with pre-warmed physiological saline to obtain liposomal anamycin with a concentration of approximately 0.5 to 1.5 mg / mL in physiological saline; Maintain the one or more containers of the suspended liposomal anamycin and the diluted liposomal anamycin at 24-45°C during and after dilution. We also provide methods that include this.

[0007] A method for preparing a liposomal formulation of reformulated anamycin in an effective dose, wherein this type of method is Syringes and non-PVC IV bags containing (approximately) 0.9% USP sodium chloride (aqueous injection USP sodium chloride), which is physiological saline, are heated in an oven or bag warmer at 37°C ± 4°C for at least approximately 3 hours. The volume of saline solution in the syringe (in mL) is equal to the weight of the liposomal anamycin precursor lyophilized material to be reconstituted (in mg). The volume of saline solution in the aforementioned IV bag corresponds to the total volume of saline solution in all syringes. The volume of the IV bag is sufficient to accommodate the total volume of saline and liposomal anamycin for diluting the total dose of liposomal anamycin; Remove vials containing a sufficient number of liposomal anamycin precursor lyophilized materials to provide the desired total dose of anamycin from the freezer, and allow the vials to stand at room temperature for 30 minutes to 2 hours; Each vial of the liposomal anamycin precursor lyophilized product is to be shaken vigorously to break up any dry solid; Inject air into each vial from an air-filled syringe until the pressure in each vial is equal; The process involves adding a certain volume of 37°C±4°C physiological saline from a syringe maintained at 37°C±4°C in an oven to a vial containing a corresponding weight of liposomal anamycin precursor lyophilized product, while maintaining negative pressure throughout the addition of the physiological saline, until the final anamycin concentration reaches approximately 1 mg / mL; Immediately shake the vial vigorously by hand for 15 seconds, then place it in a resealable polyethylene storage bag; Repeat the process of adding saline solution, shaking the vial, and loading the vial into the storage bag until all vials required for the total dose of anamycin are prepared in the same manner; Shake all vials a second time, 2 to 4 vials at a time, for 1 to 2 minutes; Store all reconstituted liposomal anamycin vials at 37°C ± 4°C for 30 minutes, up to a maximum of 1 hour, until ready for dilution; Remove no more than two vials of reconstituted liposomal anamycin at a time from a storage location at 37°C ± 4°C, gently invert each vial once, and while maintaining negative pressure throughout the withdrawal of the suspension, remove the bubble-free suspension from the vial; Slowly introduce the extracted, reconstituted, bubble-free suspension of liposomal anamycin into a non-PVC IV bag of pre-warmed saline prepared as described above, and repeat this procedure for the second vial; The steps of removing the vial, withdrawing the suspension, and introducing this suspension into a pre-warmed IV bag are repeated until all vials of reconstituted liposomal anamycin, sufficient to reach the total dose of anamycin, are introduced into a non-PVC IV bag, thereby obtaining an IV bag containing the final reconstituted liposomal anamycin at a concentration of approximately 0.5 mg / mL; By gently inverting the container several times without creating foam, the physiological saline and reconstituted liposomal anamycin are mixed within the IV bag. We also provide methods that include this.

[0008] A method of treating cancer, By adding pre-warmed physiological saline to one or more containers of lyophilized liposomal anamycin precursor, a liposomal anamycin suspension with a concentration of approximately 0.5 to 1.5 mg / mL is produced; Maintaining one or more of the aforementioned containers at 24-45°C; The aforementioned suspended liposomal anamycin is diluted with pre-warmed physiological saline to obtain liposomal anamycin with a concentration of approximately 0.3 to 0.7 mg / mL in physiological saline; Maintain the one or more containers of the suspended liposomal anamycin and the diluted liposomal anamycin at 24-45°C during and after dilution. Also provided is a method for treating cancer, which comprises administering to a patient in need thereof a diluted liposomal amikacin prepared by

[0009] Also provided is the use of a diluted reconstituted liposomal amikacin prepared according to the above method in the manufacture of a medicament for treating cancer.

[0010] Also provided is a diluted reconstituted liposomal amikacin prepared according to the above method for use in the treatment of cancer.

Brief Description of the Drawings

[0011] [Figure 1] It is a schematic diagram illustrating a method for reconstituting liposomal amikacin from a freeze-dried product of a liposomal amikacin precursor. [Figure 2] It is a superimposed histogram result of samples at 20 to 25°C. [Figure 3] It is a superimposed histogram result of samples at 28 to 42°C. [Figure 4] It is a superimposed histogram result of samples at 20 to _{42}°C.

Mode for Carrying Out the Invention

[0012] A method for preparing a formulation of liposomal amikacin having improved stability and high purity is provided.

[0013] Definition 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" has the following structure:

Chem.

[0016] The terms "liposome", "liposomal", and similar terms shall refer to a substantially spherical structure including lipids, fatty acids, lipid bilayer-type structures, unilamellar vesicles, and amorphous lipid vesicles. Classically, a liposome is a completely closed lipid bilayer membrane that encapsulates a certain volume of aqueous substance. Liposomes include non-classical forms where anamycin 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 (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 (nonpolar) "tails" of the lipid monolayers face towards the center of the bilayer and the hydrophilic "heads" face towards the aqueous layer.

[0017] The terms “liposome precursor-lyophilized product” and “liposomal precursor-lyophilized product” refer to a non-aqueous material that will form liposomes upon the addition of an aqueous solution. In some embodiments, the non-aqueous material is a dry (non-liquid, non-gel, etc.) material. The term "lyophilized product" is used in a broad sense to include drying residues obtained by sublimating a frozen liquid from a non-volatile material, residues obtained by rotary evaporation and similar procedures, and dry compositions that will yield liposomes upon the addition of an aqueous layer (with or without stirring). In particular, “liposome precursor-lyophilized product” should be understood as a product that is not in a liposomal form after lyophilization.

[0018] The term “liposomal anamycin precursor lyophilized product” refers to a type of liposomal precursor lyophilized product in which the drug is anamycin. In some embodiments, the lyophilized product is prepared according to the procedure described in U.S. Patent Application Publication No. 2005 / 0238707, which is incorporated herein by reference for all purposes.

[0019] The phrase "preheated" means that the article, solution, or suspension has been heated to a temperature of approximately 24-45°C, for example, 30-45°C, for example, 37°C ± 4°C, or 37°C ± 2°C, or 37°C ± 1°C.

[0020] The term "high-purity anamycin liposome-encapsulated precursor lyophilized product" refers to a material whose purity is analyzed by HPLC using validated standard samples to be 95% or higher in anamycin content. In some embodiments, the purity of anamycin is at least 96%, or at least 97%, or at least 98%, or at least 99%.

[0021] The above section used many abbreviations and acronyms. Their full spellings 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 PLA liposomal anamycin precursor lyophilized PVC (Polyvinyl Chloride) WFI Water for Injection (compliant with USP standards)

[0022] Throughout this specification and the subsequent claims, unless otherwise required by context, the word “comprise” or its variations “comprises” or “comprising” should be understood to mean that it includes the described integer or process or group of integers or processes, but not to exclude any other integer or process or group of integers or processes. Where such a specified range is described herein, the described range is intended to include all specific integer quantities between the described range. For example, the range of approximately 24–45°C is intended to include 24, 30, 33, 34, 35, 36, 37, 38, 39, 40, and 45°C.

[0023] The entire disclosures of each U.S. patent and international patent application described herein are incorporated herein by reference in their entirety for any purpose.

[0024] We also provide a method for producing liposomal anamycin with excellent uniformity and stability. We also provide a novel method for producing liposomal anamycin with excellent uniformity and stability. In particular, a method for reconstituting anamycin, of which this type of method, By adding pre-warmed physiological saline to one or more containers of lyophilized liposomal anamycin precursor, a suspended liposomal anamycin with a concentration of approximately 0.5 to 1.5 mg / mL is produced; Maintaining one or more of the aforementioned containers at 24-45°C; The aforementioned suspended liposomal anamycin is diluted with pre-warmed physiological saline to obtain liposomal anamycin with a concentration of approximately 0.5 to 1.5 mg / mL in physiological saline; Maintain the one or more containers of the suspended liposomal anamycin and the diluted liposomal anamycin at 24-45°C during and after dilution. This provides a method that includes this.

[0025] In some embodiments, the concentration of the liposomal anamycin suspension is 0.5 to 1.5 mg / mL. In some embodiments, the concentration of the liposomal anamycin suspension is 0.8 to 1.2 mg / mL. In some embodiments, the concentration of the liposomal anamycin suspension is approximately 1 mg / mL.

[0026] In some embodiments, the total weight of anamycin in the diluted suspension, added to preheated saline contained in an IV bag, is equal to the required effective dose.

[0027] In some embodiments, the suspended liposomal anamycin is diluted by adding it to an IV bag containing pre-warmed saline. In some embodiments, the temperature of the IV bag containing saline is 30-45°C. In some embodiments, the temperature of the IV bag containing saline is 37°C ± 4°C.

[0028] In some embodiments, reconstituted liposomal anamycin is generated at temperatures above 24°C. In some embodiments, the reconstituted liposomal anamycin is maintained at temperatures above 24°C before and during dilution, and before and during injection. Surprisingly, liposomal anamycin was found to be unstable below 24°C.

[0029] In some embodiments, the reconstituted liposomal anamycin is maintained at a temperature of 30–45°C. In some embodiments, the reconstituted liposomal anamycin is maintained at a temperature of 30–42°C. In some embodiments, the reconstituted liposomal anamycin is maintained at a temperature of 37°C ± 4°C. In some embodiments, the liposomal anamycin suspension is maintained at or near 37°C, and the diluted liposomal anamycin is maintained at or near 37°C.

[0030] In some embodiments, the concentration of the diluted liposomal anamycin is approximately 0.5 mg / mL.

[0031] In some embodiments, a protective sleeve is used on the IV infusion line to ensure that diluted liposomal anamycin is delivered at a temperature of 37°C ± 4°C or higher. In some embodiments, the sleeve is heated. In some embodiments, the sleeve is insulated.

[0032] In some embodiments, a method for reconstituting anamycin is, By adding physiological saline to one or more containers of lyophilized liposomal anamycin precursor at 37°C ± 4°C, a suspended liposomal anamycin with a concentration of approximately 1 mg / mL is produced; Maintaining one or more of the aforementioned containers at 37°C ± 4°C; By diluting the aforementioned suspended liposomal anamycin with physiological saline at 37°C ± 4°C, liposomal anamycin with a concentration of approximately 0.5 mg / mL in physiological saline is obtained; Maintain the one or more containers of the suspended liposomal anamycin and the diluted liposomal anamycin at 37°C ± 4°C during and after dilution. Includes.

[0033] In some embodiments, the diluted liposomal anamycin is maintained at 37°C ± 4°C before and during administration.

[0034] In some embodiments, the container for lyophilized anamycin is a vial having at least 4 mL of headspace after adding pre-warmed saline to allow for sufficient mixing to form liposomes. In some embodiments, the vial size is 50 mL.

[0035] In some embodiments, a method for preparing a liposomal formulation of reformulated anamycin in an effective dose is: Syringes and non-PVC IV bags containing (approximately) 0.9% USP sodium chloride water for injection, respectively, are heated in an oven or bag warmer at 37°C ± 4°C for at least approximately 3 hours. The volume of saline solution in the syringe (in mL) is equal to the weight of the liposomal anamycin precursor lyophilized material to be reconstituted (in mg). The volume of saline solution in the aforementioned IV bag corresponds to the total volume of saline solution in all syringes. The volume of the IV bag is sufficient to accommodate the total volume of saline and liposomal anamycin for diluting the total dose of liposomal anamycin; Remove vials containing a sufficient number of liposomal anamycin precursor lyophilized materials to provide the desired total dose of anamycin from the freezer, and allow the vials to stand at room temperature for 30 minutes to 2 hours; Each vial of the liposomal anamycin precursor lyophilized product is to be shaken vigorously to break up any dry solid; Inject air into each vial from an air-filled syringe until the pressure in each vial is equal; From a syringe maintained at 37°C±4°C in an oven, a certain volume of physiological saline at 37°C±4°C is added to a vial containing a corresponding weight of liposomal anamycin precursor lyophilized material, while maintaining negative pressure throughout the addition of the physiological saline, until the final anamycin concentration is approximately 1 mg / mL; Immediately shake the vial vigorously by hand for 15 seconds, then place it in a resealable polyethylene storage bag; The steps of adding saline solution, shaking the vial, and loading the vial into the storage bag are repeated until all vials required for the total dose of anamycin are prepared in the same manner; Shake all vials a second time, 2 to 4 vials at a time, for 1 to 2 minutes; Store all reconstituted liposomal anamycin vials at 37°C ± 4°C for 30 minutes, up to a maximum of 1 hour, until ready for dilution; Remove no more than two vials of reconstituted liposomal anamycin at a time from a storage location at 37°C ± 4°C, gently invert each vial once, and while maintaining negative pressure throughout the withdrawal of the suspension, remove the bubble-free suspension from the vial; Slowly introduce the extracted, reconstituted, bubble-free suspension of liposomal anamycin into a non-PVC IV bag of pre-warmed saline prepared as described above, and repeat this procedure for the second vial; The steps of removing the vial, withdrawing the suspension, and introducing this suspension into a pre-warmed IV bag are repeated until all vials of reconstituted liposomal anamycin, sufficient to reach the total dose of anamycin, are introduced into a non-PVC IV bag, thereby obtaining an IV bag containing the final reconstituted liposomal anamycin at a concentration of approximately 0.5 mg / mL; By gently inverting the container several times without creating foam, the physiological saline and reconstituted liposomal anamycin are mixed within the IV bag. Includes.

[0036] In some embodiments, the liposomal anamycin precursor lyophilized product comprises anamycin, one or more lipids, and one or more nonionic surfactants. In some embodiments, the lipids include DMPC and DMPG, and the nonionic surfactants include polysorbate-type surfactants produced by ethoxylation of sorbitan followed by the addition of a carboxylic acid. In some embodiments, the nonionic surfactants include polyoxyethylene sorbitan monolaurate. In some embodiments, the pre-liposomal anamycin lyophilized product comprises 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. In some embodiments, DMPC is 65.3-67.3 wt% and DMPG is 27.1-29.9 wt%.

[0037] The present invention also provides a method of treatment by injecting a patient in need of liposomal anamycin in an effective dose, prepared by the method described above, from a lyophilized liposomal anamycin precursor, wherein the liposomal formulation is maintained at a temperature of 37°C ± 4°C throughout the reconstitution and administration process and is used within 24 hours.

[0038] The invention also provides a use of the reconstituted liposomal anamycin described above for the treatment of cancer, comprising administering an effective dose of diluted liposomal anamycin, prepared as described above, to a patient in need thereof. In some embodiments, the cancer is selected from breast cancer, leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, gastric cancer, Hodgkin lymphoma and non-Hodgkin lymphoma, ovarian cancer, thyroid cancer, colon cancer, traditional cell bladder cancer, and Wilms' tumor.

[0039] Reconstituted liposomal anamycin, prepared as described herein, is useful for the treatment of certain human cancers, including breast cancer, leukemias including acute myeloid leukemia (AML) and acute lymphoblastic leukemia, gastric cancer, Hodgkin lymphoma and non-Hodgkin lymphoma, ovarian cancer, thyroid cancer, colon cancer, traditional cell bladder cancer, and Wilms' tumor.

[0040] Dosage and route of administration Generally, reconstituted liposomal formulations are used within 24 hours of reconstitution to treat patients in conditions where the treatment would be beneficial. Intravenous administration of the drug is achieved by standard means and is completed within 110-130 minutes, depending on the dose.

[0041] Liposomal anamycin is used in methods for treating cancer and for inhibiting tumor growth in mammals, particularly humans. Cancers that can be treated include leukemia and lymphoma. In particular, anamycin can be used to treat leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). Liposomal anamycin can also be used to treat lymphomas such as Hodgkin lymphoma and non-Hodgkin lymphoma. This method involves administering an effective amount of a drug composition to a mammal.

[0042] The administration step can preferably be parenteral, by intravenous, intra-arterial, intramuscular, intralymphatic, intraperitoneal, subcutaneous, intrapleural, intrathecal injection, or by topical application. In some embodiments, anamycin is administered intravenously. In some embodiments, this type of administration is a repeated-dose regimen until tumor reduction or disappearance is achieved and can be used in combination with forms of tumor treatment such as surgical procedures or chemotherapy using different drugs. In some embodiments, the dose of the composition to be administered is about 120-250 mg / m² to the target mammal. 2 It is located between them.

[0043] The present invention will be further illustrated by the following embodiments, but this should not be construed as limiting the scope of the invention in any way. On the contrary, it should be clearly understood that, after reading this specification, a person skilled in the art may conceive of various other embodiments, modifications, and equivalents that do not depart from the spirit of the invention and / or the appended claims. [Examples]

[0044] Example 1 General method A. Material A lyophilized liposomal anamycin precursor was prepared as a powder containing DMPC, DMPG, polysorbate 20, and anamycin.

[0045] Dimyristoylphosphatidylcholine (DMPC) and 1,2-dimiristoyl-sn-glycero-3-[phosphor-rac-(1-glycerol)]sodium salt (DMPG) were obtained as dry powders from Nippon Fine Chemicals, Inc. in Osaka Prefecture, Japan.

[0046] Anamycin (purity >95%) (MW = 640.39) as a THF solvate was synthesized with slight modifications as previously described (Horton, D., Priebe, W., 4-demethoxy-3'-desamino-2'-halo-anthracyclines and pharmaceutical compositions containing the same, U.S. Patent No. 4,537,882, 1985).

[0047] Physiological saline solution was obtained from Abbott Laboratories, North Chicago, Ill.

[0048] In the following examples, the liposomal anamycin precursor was reconstituted according to the method described below.

[0049] Step 1: Prepare the saline syringe and IV bag. All procedures were performed using aseptic techniques in a laminar flow hood or cleanroom.

[0050] One 45 mL syringe of physiological saline (0.9% sodium chloride, for injection, USP) was prepared for each 45 mg vial of liposomal anamycin. These saline syringes were used to reconstitute the liposomal anamycin precursor. For example, eight syringes were prepared for a total dose of 360 mg of liposomal anamycin.

[0051] The syringe was kept in a 37°C oven or IV bag warmer for at least 2-3 hours.

[0052] A non-PVC IV bag was prepared containing an additional volume of saline equal to the total volume of saline in the syringe used for reconstituting the liposomal anamycin precursor. The IV bag was sized to accommodate twice the volume of saline in the syringe. For example, if the total dose of the test drug was 360 mg, eight 45 mg vials of liposomal anamycin precursor were required, each containing 45 mL of saline, for a total of 360 mL of saline. Accordingly, a non-PVC IV bag containing 360 mL of saline was also prepared. This was done by removing 640 mL of saline from a standard 1,000 mL non-PVC IV bag of saline. The 1,000 mL IV bag was large enough to accommodate twice the volume of saline remaining in the bag, i.e., 2 × 360 mL = 720 mL.

[0053] The prepared saline IV bag was warmed in a commercially available IV bag warmer preheated to approximately 37°C or in an oven preheated to 37°C, according to the manufacturer's instructions. Before using the IV bag in the next step, it was kept in the bag warmer or oven for approximately 2-3 hours to allow the IV bag temperature to reach approximately 37°C.

[0054] Step 2: Reconstitution of preliposome-encapsulated anamycin lyophilized product Vials of liposomal anamycin precursor lyophilized product (each containing 45 mg of anamycin, excluding the weight of solvated THF) with a total weight sufficient to provide the required total dose of the drug were removed from the freezer and allowed to stand at room temperature for approximately 30 minutes to 2 hours. The vials were vigorously shaken to break up any dry solids that may have formed in the liposomal anamycin precursor lyophilized product.

[0055] The vials were moved to a laminar flow hood, and air was injected into each using an air-filled syringe until the air pressure was equal. One 45 mL syringe of saline was taken at a time from a 37°C oven or IV bag warmer preheated to 37°C and immediately used for reconstitution. To minimize the possibility of cooling, the bag warmer or oven was placed inside or next to the hood in which the lyophilized preliposomal anamycin was being reconstituted. The saline used for reconstitution was kept at 37°C (±4°C) when mixed with the lyophilized formulation in the vials.

[0056] One syringe at a time was removed from the bag warmer or oven and immediately mixed with the preliposomal anamycin lyophilized product. For example, a 45 mL syringe of saline was removed from the 37°C IV bag warmer or oven, and 45 mL of 37°C saline was added to the first 45 mg vial of preliposomal anamycin lyophilized product to achieve a final anamycin concentration of approximately 1 mg / mL. After addition, the vial was brought to atmospheric pressure by simply puncturing the septum with a needle. The needle was removed, the vial was immediately shaken vigorously by hand for 2 minutes, placed in a zip-locked plastic bag, and returned to the 37°C oven or bag warmer.

[0057] Next, in the same manner, additional syringes of 45 mL of physiological saline were taken one at a time from the 37°C IV bag warmer or oven, and 45 mL of 37°C physiological saline was added to each of the additional 45 mg vials of lyophilized liposomal anamycin precursor. The vials were shaken and placed into sealed plastic bags. Each vial was placed into a vial bag and kept in the oven or bag warmer until all the vials required for the total dose of liposomal anamycin precursor had been reconstituted.

[0058] After reconstituting all vials and placing them in an oven or bag warmer, remove 2-4 vials, shake them for another 1-2 minutes, and then return them to the oven or bag warmer. Repeat this process until all vials have been shaken a second time. Allow the vials to stand at 37°C for 30 minutes to 1 hour, then begin the final dilution in the IV bag as described in step 2.

[0059] Particle size analysis of the reconstituted anamycin samples revealed that the average particle size was consistently around 8 μm, with 50% distributed in the range of approximately 5–10 μm and 90% distributed in the range of approximately 2–20 μm.

[0060] Step 3: Dilution of liposomal anamycin in an IV bag Dilution of liposomal anamycin was completed within approximately one hour of reconstitution. The reconstituted liposomal anamycin vials, prepared as described in Step 2, were maintained at approximately 37°C before being transferred to IV bags for dilution. These bags were not removed from the 37°C oven until immediately before dilution.

[0061] The reconstituted liposomal anamycin vials (45 mg in 45 mL of saline, one vial per dose) containing the measured volume of approximately 37°C saline, prepared as described in Step 2 above, were removed from the 37°C oven for dilution. The vials were gently inverted, and the liposomal suspension was withdrawn with a syringe. If it was necessary to equalize the pressure, the septum was punctured for 1 second, the needle was withdrawn, and the drug withdrawal was continued. The suspension was immediately transferred slowly (avoiding foaming) to a preheated non-PVC IV bag prepared as described in Step 1. Care was taken to avoid transferring any foam.

[0062] Similarly, a sufficient number of additional vials to provide the required total dose of pre-liposomal anamycin were removed from the oven one at a time, gently inverted, and the liposomal anamycin suspension was withdrawn with a syringe and immediately transferred gently into the contents of a pre-warmed non-PVC IV bag. This process was repeated until all vials had been transferred to a non-PVC IV bag containing diluent saline. The saline and liposomal anamycin in the IV bag were mixed by gently inverting the container several times, taking care not to create bubbles. The time was recorded in the IV bag. The final liposomal anamycin concentration was approximately 0.5 mg / mL.

[0063] Throughout this step, the IV bags containing the reconstituted liposomal anamycin were kept at approximately 37°C by immediately transferring them to a preheated IV bag warmer.

[0064] The IV bag warmer was unplugged when it was ready to be moved to the patient's infusion room, moved within 30 to 60 minutes, and immediately plugged back in next to the patient receiving the infusion. The diluted liposomal anamycin suspension was maintained at approximately 37°C until and during the infusion to the patient.

[0065] Reconstituted liposomal anamycin diluted with physiological saline was stable for 24 hours at approximately 37°C; however, after that time, the drug became unacceptable for administration to the subjects. Only non-PVC and non-DEHP tubing was used for drug administration.

[0066] Care was taken to continue monitoring the time from the start of reconfiguration until the completion of the 2-hour infusion. No more than 24 hours should have elapsed between the start of reconfiguration and the completion of the infusion.

[0067] Example 2 Temperature-related tests Visual inspections were performed on each sample. Furthermore, the appearance of the samples was observed immediately after reconstitution at each temperature. Any changes in appearance during particle size testing were also recorded.

[0068] The results regarding the appearance of the sample material before and immediately after reconstruction, along with the appearance after particle size testing, are shown below. All pre-reconstruction appearance results were consistent with the target limit. However, the appearance after reconstruction differed depending on the temperature. As the sample temperature increased, the likelihood of undispersed particles decreased. Clumps of undispersed particles were observed at temperatures of 20–25°C, while no evidence of such clumps was shown at temperatures of 28–42°C. Furthermore, this table shows that changes in appearance from reconstruction to the end of particle size testing were limited to lower temperatures. In these lower-temperature samples, clumps of particles present immediately after reconstruction tended to disperse, but not necessarily completely. It is not surprising that the appearance of higher-temperature samples remained unchanged, as no undispersed clumps were present immediately after reconstruction.

[0069] [Table 1]

[0070] Particle size testing was performed using a laser diffraction particle size analyzer. A single sample was prepared and analyzed at temperatures of 37°C (nominal), 20°C, 23°C, 25°C, 28°C, 30°C, and 42°C. Each step of sample preparation (i.e., incubation temperature, etc.) and analysis (i.e., temperature of the instrument's dispersion medium, etc.) was performed at the specified temperature.

[0071] The results obtained from temperature tests in particle size analysis are shown below. Histograms of the results are shown overlaid in Figures 2-4. Overall, the particle size results for samples at 20-25°C differed significantly from the target limit, while the results for samples at 28-42°C were similar. The very large particle size of the 20°C sample suggests that the sample material did not form liposomes during reconstitution. The results for the 23-25°C sample were significantly lower than the target limit, indicating that liposome formation was in progress. The results for the 28-42°C sample were much more stable and closer to the target limit. This indicates that liposomes were fully formed and were quite stable within this temperature range. However, it should be noted that the 28°C sample did not fall within the target limit range. From this, it appears that although the particle size changed in the 25-28°C temperature range, liposome formation was not fully completed until the temperature reached approximately 30°C. Finally, looking at the results for the 28-42°C samples, it should be noted that the Dv90 values ​​increase with increasing temperature.

[0072] [Table 2]

[0073] Both the appearance and particle size results indicate that temperature affects the particle size distribution of liposomes in the reconstituted formulation. Reconstitution at temperatures of 20–25°C results in clumps of undispersed particles, and the particle size results at these temperatures differ significantly. The appearance and particle size results at temperatures of 28–42°C are far more stable, suggesting that the transition temperature range is approximately 25–28°C. All of this data suggests that the formulation should be reconstituted and maintained at temperatures between approximately 30–42°C.

[0074] Although the present 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 present invention without departing from its spirit and scope.

[0075] All references cited herein, including patents, patent applications, and publications, are incorporated herein by reference, whether or not they were specifically incorporated previously.

[0076] The various features and embodiments of the present invention mentioned in the individual sections above may be applied to other sections with the necessary modifications, where appropriate. Thus, features specified in one section may be combined with features specified in other sections, where appropriate.

[0077] The above descriptions relating to certain specific embodiments provide sufficient information so that others, by applying their current knowledge, can readily modify or adapt such specific embodiments for various uses without departing from the broader concept, and such adaptations and modifications should be understood, and are intended, to fall within the meaning and equivalents of the disclosed embodiments. It should be understood that the expressions or technical terms used herein are for illustrative purposes only and not for limitation. While exemplary embodiments and specific terms are disclosed and used in the drawings and text of the specification, these are used in a general and descriptive sense only, unless otherwise specified, and not for the purpose of limiting the claims. Furthermore, those skilled in the art will understand that certain steps of the methods discussed herein can be arranged in an alternative order or combined. Therefore, 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 verify many equivalents of the embodiments of the present invention described herein without exceeding the scope of routine experimentation. Such equivalents are encompassed in the claims set forth below.

Claims

1. A method for reconstituting anamycin, and this method is By adding physiological saline solution preheated to 30-42°C to one or more containers of lyophilized liposomal anamycin precursor, a suspended liposomal anamycin with a concentration of 0.5-1.5 mg / mL is produced; Maintaining one or more of the above containers at 30-42°C; The aforementioned suspended liposomal anamycin is diluted with physiological saline preheated to 30-42°C to obtain diluted liposomal anamycin with a concentration of 0.3-0.7 mg / mL in physiological saline; Maintain the one or more containers of the suspended liposomal anamycin and the diluted liposomal anamycin at 30-42°C during and after dilution. Methods that include...

2. The method according to claim 1, wherein the concentration of the suspended liposomal anamycin is approximately 1 mg / mL.

3. The method according to claim 1 or 2, wherein the total weight of the diluted liposomal anamycin is equal to the required effective dose.

4. The method according to any one of claims 1 to 3, wherein the suspended liposomal anamycin is diluted by adding it to an IV bag containing physiological saline that has been preheated to 30 to 42°C.

5. The method according to any one of claims 1 to 4, wherein the suspended liposomal anamycin is maintained at a temperature of 37°C ± 4°C before, during, and after dilution.

6. The method according to any one of claims 1 to 5, wherein the concentration of the diluted liposomal anamycin is about 0.5 mg / mL.

7. The method according to any one of claims 1 to 6, wherein the suspension of liposomal anamycin is maintained at 37°C or near 37°C, and the diluted liposomal anamycin is maintained at 37°C or near 37°C.

8. A method for preparing a liposomal formulation of reformulated anamycin in an effective dose, the method comprising: Syringes and non-PVC IV bags containing (approximately) 0.9% USP sodium chloride water for injection, respectively, are heated in an oven or bag warmer at 37°C ± 4°C for at least approximately 3 hours. The volume of saline solution in the syringe (in milliliters) is equal to the weight of the liposomal anamycin precursor lyophilized material to be reconstituted (in milligrams). The volume of saline solution in the aforementioned IV bag corresponds to the total volume of saline solution in all syringes. The volume of the IV bag is sufficient to accommodate the total volume of saline solution and liposomal anamycin for diluting the total dose of liposomal anamycin; Remove vials containing a sufficient number of liposomal anamycin precursor lyophilized materials to provide the desired total dose of anamycin from the freezer, and allow the vials to stand at room temperature for 30 minutes to 2 hours; Each vial of the liposomal anamycin precursor lyophilized product is to be shaken vigorously to break up any dry solid; Inject air into each vial from an air-filled syringe until the pressure in each vial is equal; Adding a certain volume of 37°C ± 4°C physiological saline from a syringe maintained at 37°C ± 4°C in an oven to a vial containing a corresponding weight of liposomal anamycin precursor lyophilized product, while maintaining negative pressure throughout the addition of the physiological saline, until the final anamycin concentration is approximately 1 mg / mL; Immediately shake the vial vigorously by hand for 15 seconds, then place it in a resealable polyethylene storage bag; The steps of adding saline solution, shaking the vial, and loading the vial into the storage bag are repeated until all the vials required for the total dose of anamycin are prepared in the same manner; Shake all vials a second time, 2 to 4 vials at a time, for 1 to 2 minutes; Store all reconstituted liposomal anamycin vials at 37°C ± 4°C for 30 minutes, up to a maximum of 1 hour, until ready for dilution; The reconstituted liposomal anamycin vials are removed from a storage location at 37°C ± 4°C, no more than two vials at a time; each vial is gently inverted once; and while maintaining negative pressure throughout the withdrawal of the suspension, the bubble-free suspension is withdrawn from the vials; The extracted, reconstituted liposomal anamycin suspension, free of bubbles, is slowly introduced into a non-PVC IV bag of pre-warmed saline prepared as described above, and this step is repeated in the second vial; The steps of removing the vial, withdrawing the suspension, and introducing the suspension into the preheated IV bag are repeated until all vials of reconstituted liposomal anamycin, sufficient to reach the total dose of anamycin, are introduced into the non-PVC IV bag, thereby obtaining an IV bag containing a final reconstituted liposomal anamycin with a concentration of approximately 0.5 mg / mL; The physiological saline and reconstituted liposomal anamycin are mixed in the IV bag by gently inverting the container several times without creating foam. Methods that include...

9. The method according to claim 8, wherein the syringe and the non-PVC IV bag are heated in the oven or the bag warmer for about 2 to 3 hours.

10. The method according to claim 8 or 9, wherein the IV bag includes an IV infusion line, and the IV infusion line includes a heated protective sleeve.

11. The method according to claim 10, wherein the heated protective sleeve is insulated.

12. The method according to claim 8, wherein the IV bag warmer is unplugged while the patient is being moved to the infusion room, and plugged in immediately after the patient is moved to the infusion room.

13. A method for producing a pharmaceutical composition for use in the treatment of cancer, comprising an effective dose of diluted liposomal anamycin, the method comprising preparing the liposomal anamycin by the method of any one of claims 1 to 12.

14. The method according to claim 13, comprising preparing the liposomal anamycin by the method described in claim 5 or 7.

15. The method according to claim 13 or 14, wherein the cancer is a cancer selected from leukemia and lymphoma in humans.

16. The method according to claim 15, wherein the cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, and non-Hodgkin lymphoma.

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