Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
  • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1277—Preparation processes; Proliposomes
  • A61K9/1278—Post-loading, e.g. by ion or pH gradient
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

• Liposomes are well established nanoparticles that can enhance the efficacy of therapeutically active drugs by improving the plasma distribution and pharmacokinetics of the drugs over non-encapsulated forms (e.g., Weinstein, Liposomes: From Biophysics to Therapeutics, (Ostro, M. J., ed.), Marcel Dekker, Inc., N.Y., pp. 277-338, (1987).
• Vincristine Sulfate Liposome Injection is a liposome formulation of the anti-cancer therapeutic vincristine sulfate encapsulated in sphingomyelin-cholesterol liposomes which provides greater efficacy than standard vincristine sulfate injection USP (VSI).
• VSI standard vincristine sulfate injection USP
• Clinical trials have also shown that VSLI facilitates dose intensification by significantly extending vincristine's circulation half-life compared to non-encapsulated vincristine.
• the liposome provides the mechanism for delayed drug release and the liposome size allow the drug to accumulate in cancer tissues by extravasation (Webb et al., Cancer
• Encapsulation of vincristine sulfate into sphingomyelin-cholesterol liposomes is typically achieved by using an acidic intraliposomal pH (e.g., pH of 4) and an exterior medium at a neutral pH (e.g., pH of 7).
• pH of 4 an acidic intraliposomal pH
• pH of 7 an exterior medium at a neutral pH
• This pH gradient allows the weakly basic vincristine to diffuse into the liposome interior with high efficiency (Cullis et al., Trends in Biotech 9: 268-272, 1991; Boman et al, Bioch Biophys Acta, 1152:253-258, 1993).
• the liposome membrane In order for vincristine to accumulate in the liposome interior with the transmembrane pH gradient, the liposome membrane must become temporarily permeable to the steric bulk of vincristine.
• the temperature of the sphingomyelin-cholesterol liposome must be increased in order for the transmembrane H gradient to function with vincristine.
• the liposome bilayer which is an orientation of interlocking sphingomyelin and cholesterol molecules, requires a unique transient heat pattern to create thermotropic disorder transition states. These transition states essentially abate the weak intermolecular bonding between the membrane lipids, creating gaps in interlocking lipids and allow the liposome biolayer to become temporarily permeable.
• the encapsulation process takes advantage of the spontaneous self reassembly of the sphingomyelin-cholesterol lipids that occurs on cooling back to ambient temperature, which restores the membrane integrity.
• VLSI vincristine sulfate injection
• Vincristine Sulfate Liposome Injection is prepared from the individual components at a pharmacy according to the directions provided on the FDA-approved label (www.accessdata.fda.gov; Reference ID: 3172211, 2012). These directions include a heating procedure that requires the use of a water bath in order to achieve efficient encapsulation of vincristine in the sphingomyelin-cholesterol liposomes and maintain chemical purity of vincristine. The excellent heat transfer properties of water allow greater than 95% encapsulation of vincristine with no appreciable chemical degradation of the drug. Since VSLI is an injectable drug, the manufacture of the components and pharmacy preparation are strictly regulated to maintain sterility.
• VSLI open water bath during preparation of VSLI requires additional resources, planning, and equipment (e.g., floating ring), including an aseptic hood or "clean" room in order to maintain an aseptic environment.
• additional resources, planning, and equipment e.g., floating ring
• aseptic hood or "clean" room in order to maintain an aseptic environment.
• the constitution of VSLI cannot be done due to the restrictions on maintaining a sterile environment. Accordingly, there remains a need for improved methods of preparing VSLI that can be efficiently and reproducibly carried out without the additional resources and equipment currently required.
• the current invention is based, at least in part, on the development of a method for preparing VSLI that circumvents the need for the use of a heated water bath during the encapsulation process.
• the invention provides efficient, reproducible methods of preparing VSLI which may be widely used with unexpected ease and reduced risk of contamination.
• the invention features a method of preparing a pharmaceutically acceptable liquid composition comprising liposome-encapsulated vincristine which is free of substantial degradation products, the method comprising the steps of (a) constituting in a single vial (i) a first solution comprising vincristine sulfate at a concentration of about 1 mg/ml to about 5 mg/ml, wherein the first solution has a pH of about 3.5 to about 5.5; and (ii) a second solution comprising sphingomyelin/cholesterol liposomes at a low pH; (b) raising the pH of the constituting solution in the single vial to a pH of about 7.0 to 7.5; (c) heating the single vial comprising the constituting solution in a dry heat block equilibrated at about 75°C for at least about 13 to about 18 minutes, wherein said heat block comprises one or more bores about 1-5% larger than the average length or diameter of the single vial to produce a solution comprising constituted liposome en
• the constituted solution comprising liposome encapsulated vincristine comprises (i) less than about 2.5% free vincristine; and (ii) less than about 1.5% N-desformylvincristine.
• the first solution comprising vincristine sulfate has pH of about 4.5 to about 4.7. In one embodiment, the first solution further comprises mannitol at a concentration of about 100-200 mg/ml. In some embodiments, the pH of the second solution comprising the liposomes is about 4.0. In one embodiment, the second solution further comprises a citrate buffer.
• the pH of the constituting solution is raised by the addition of a third solution comprising a buffer at a pH of about 9.0.
• the third solution comprises sodium phosphate buffer.
• the constituting solution comprises a concentration ratio of about 0.1/1.0 to about 0.2/2.0 vincristine sulfate to lipid.
• the concentration of vincristine sulfate in the constituted solution is about 0.1 mg/mL to about 0.5 mg/mL. In some embodiments, the concentration of vincristine sulfate in the constituted solution is about at about 0.15 mg/mL to about 0.2 mg/mL. In one embodiment, the concentration of vincristine sulfate in the constituted solution is about 0.16 mg/mL.
• the first solution comprises vincristine sulfate USP (5 mg/5 mL), which is equivalent to 4.5 mg/5 mL vincristine free base, and 500 mg/5 mL mannitol
• the second solution comprises sphingomyelin/cholesterol liposomes consisting of 73.5 mg/mL sphingomyelin, 29.5 mg/mL cholesterol, 33.6 mg/mL citric acid, 35.4 mg/mL sodium citrate
• the third solution comprises 355 mg/25 mL dibasic sodium phosphate and 225 mg/25 mL sodium chloride.
• the ratio of sphingomyelin to cholesterol in the liposome is between about 75/25 mol %/mol sphingomyelin/cholesterol to 30/50 mol %/mol % sphingomyelin/cholesterol. In some embodiments, the liposomes comprise about 70/30 mol %/mol sphingomyelin/cholesterol to 40/45 mol %/mol % sphingomyelin/cholesterol. In one embodiment, the liposomes comprise approximately 55/45 mol %/mol %
• the liposomes comprising about 60/40 mol %/mol % sphingomyelin/cholesterol.
• the liposomes have a size range of about 0.05-0.5 microns. In some embodiments, the liposomes have a mean diameter of about 50-200 nm. In one embodiment, the liposomes have a mean diameter of about 90-125 nm. In some embodiments, the constituting solution has a volume of between about 20-50 mL. In some embodiments, the constituting solution has a volume of between 30-35 mL.
• the heat block is equilibrated to 75 ⁇ 2°C for about 15 minutes prior to insertion of the vial containing the constituting solution.
• the constituting solution is heated for about 13-15 minutes within the caliber bore of a dri -block equilibrated at 75 ⁇ 2°C.
• the constituting solution is heated for 14 minutes ⁇ 30 seconds within the caliber bore of a dri-block equilibrated at 75 ⁇ 2°C.
• the bores in the heat block are less than about 3% larger than the average length or diameter of the single vial containing the constituting solution.
• the single vial containing the constituting solution has a diameter between about 35.8 to about 37.3 mm
• the caliber bores in the heat block are cylindrical with a diameter between 37.2 to 37.8 mm in diameter.
• the constituted solution is equilibrated to room temperature for at least about 30 minutes.
• the volume of the constituted solution comprising a dose of liposome encapsulated vincristine for the patient of about 1.5 to about 2.4 mg/m is diluted with standard pharmaceutical diluents suitable for intravenous administration, to produce the pharmaceutically acceptable liquid composition.
• the volume of the patients calculated dose is removed from an infusion container and replaced with the calculated volume of the constituted VSLI solution.
• the pharmaceutically acceptable liquid composition comprising liposome-encapsulated vincristine is administered to the patient within no more than 24 hours after constitution.
• the VSLI produced according to the methods of the invention are typically administered to a patient having cancer.
• the cancer is lymphoma, leukemia, myeloma, brain cancer or neuroblastoma.
• the pharmaceutically acceptable liquid composition comprising liposome-encapsulated vincristine is administered by intravenous infusion over a period of about 30 to 60 minutes. In some embodiments, the pharmaceutically acceptable liquid composition comprising liposome-encapsulated vincristine is administered by intravenous infusion once every 7-28 days. In one embodiment, the pharmaceutically acceptable liquid composition comprising liposome-encapsulated vincristine is administered by intravenous infusion once every 7 days.
• Figure 1 is a graph depicting the internal heating profile of a constituted solution containing vincristine sulfate and sphingomyelin/cholesterol liposomes using a dry block and water bath.
• the present invention overcomes the deficiencies associated with the use of a water bath during the currently used methods of preparing VLSI. Surprisingly, it was discovered that the use of a heating block with a custom designed insert designed to conform to the container used for constituting the VSLI provides the heat profile needed to achieve uniform encapsulation of vincristine without significant degradation.
• the improved ability of the methods of preparing VSLI described herein to demonstrate parametric release is significant. Since the stability of VSLI warrants "just in time” preparation, the constitution process must be highly efficient and reproducible by the pharmacist. The pharmacist needs to know (i.e., parametrically) that the encapsulation has been achieved by the heat induced process because it provides "a system of release that gives the assurance that a product is of the intended quality based on the review of information collected during the constitution process and on the compliance with specific GMP requirements related to parametric release" (Annex 17 EU guidance).
• the heating block procedure of the present invention provides a convenient and compliant process to achieve constitution of VSLI with greater than 95% encapsulation efficiency. Since it involves fewer steps, the probability of operational errors is decreased. The process is overall more straightforward, takes less time, uses fewer resources, and is convenient for routine pharmacy operation. Additionally, the individual preparing the VSLI need not deal with potential microbial contamination from microbes growing in the water bath or water vapor from the heated water bath. Definitions
• a “sterile" composition or container, as used herein, is free of viable microbes as determined using the USP sterility test. (See, "The United States Pharmacopeial Convention: 2008) .
• Liposome vesicle and “liposome vesicle” will be understood to indicate structures having lipid-containing membranes enclosing an aqueous interior.
• the structures may have one or more lipid membranes unless otherwise indicated, although generally the liposomes will have only one membrane.
• Such single-layered liposomes are referred to herein as “unilamellar”.
• Multilayer liposomes are referred to herein as "multilamellar”.
• a “standard” therapeutic agent, or “free” therapeutic agent refers to a therapeutic agent that is not liposome-encapsulated. Usually, a drug is presumed to be “standard or “free” unless specified otherwise. A standard vinca alkaloid in free form may still be present in combination with other reagents, however, such as other chemotherapeutic compounds, a pharmaceutical carrier, or complexing agents, i.e. as used herein the term only specifically excludes lipid formulations of the vinca alkaloids.
• a drug product e.g., VSLI
• Systemic delivery refers to delivery that leads to a broad bio- distribution of a compound within an organism. Systemic delivery means that a useful, preferably therapeutic, amount of a compound is exposed to most parts of the body. To obtain broad bio-distribution generally requires a route of introduction such that the compound is not rapidly degraded or cleared (such as by first pass organs (liver, lung, etc.) or by rapid, nonspecific cell binding) before reaching a disease site. Systemic delivery of liposome- encapsulated vinca alkaloids is preferably obtained by intravenous delivery.
• terapéuticaally effective amount refers to an amount of drug (e.g., VSLI) effective to treat a disease or disorder (e.g., cancer) in a mammal, for example, resulting in stable disease, partial remission or complete remission of the cancerous state.
• a disease or disorder e.g., cancer
• a “stable disease,” as used herein, refers to a state wherein administration of the drug (e.g., VSLI) causes cessation of the growth or prevalence of a tumor or cancer as measured by standard clinical, radiological and/or biochemical means, although there is no regression or decrease in the size or prevalence of the cancer.
• VSLI stable disease
• a “Partial response” or “partial remission” refers to the amelioration of a cancerous state, as measured by standard clinical, radiological and/or biochemical means, in response to treatment.
• a “partial response” means that the size of a tumor or the level of a cancer-indicating blood marker has decreased from a baseline level (e.g., 20%, 30%, 40%> or 50%) in response to treatment.
• the responses are assessed based on the international working group criteria (International Working Group (IWS) criteria; BD Cheson et al, J Clin Oncol 15:4642-4649).
• a “complete response” or “complete remission” means that a cancerous state, as measured by, for example, tumor size and/or cancer marker levels, is undetectable following treatment.
• Neurological toxicity includes symptoms of neuropathy, such as hypoesthesia, hyperesthesia, paresthesia, hyporeflexia, areflexia, neuralgia, jaw pain, decreased vibratory sense, cranial neuropathy, ileus, burning sensation, arthralgia, myalgia, muscle spasm, weakness, and/or orthostatic hyptension both before and during treatment. Orthostatic hypotension may occur.
• a Neurological toxicity is assessed as being Grade 1 to Grade 3 based on the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.03 (http://ctep.cancer.gov/reporting/etc.html). Vincristine Sulfate
• Vincristine sulfate is a member of a vinca alkaloid family originally isolated from the periwinkle plant ⁇ Catharanthus roseus). Vincristine Sulfate has cell-cycle specific anticancer activity. The vinca alkaloid bind to tubulin, altering tubulin polymerization leading to metaphase arrest, inhibition of cell mitosis, and cell death. As a cell cycle specific agent its' therapeutic response is advanced by liposome encapsulation which maintains extended drug levels.
• Vincristine sulfate is commonly isolated as a 1 : 1 sulfate salt. It is a hygroscopic, white to slightly yellowish crystalline powder that is soluble in water. It has a molecular weight of 923.04 (salt form) / 824.98 (base form) and a molecular formula of C46H56N4O10 ⁇ H 2 SO 4 .
• the chemical name for vincristine sulfate is 22-oxovincaleukoblastine and it has the following chemical structure:
• Vincristine sulfate is prescribed as vincristine sulfate injection USP (e.g., as a 1 mg/mL solution) and is also known as leurocristine sulfate, Kyocristine, vincosid, vincrex, Oncovin, Vincasar PFS ® , is commercially available from any of a number of sources. Liposomes
• the liposome carrier component of the present invention are comprised of sphingomyelin and cholesterol liposomes injection (SCLI).
• SCLI cholesterol liposomes injection
• the ratio of sphingomyelin to cholesterol present in the liposome may vary, but generally is in the range of from 75/25 mol %/mol sphingomyelin/cholesterol to 30/50 mol % mol % sphingomyelin/cholesterol.
• the liposome composition comprise about 70/30 mol % mol
• the liposome compositions comprise approximately 55/45 mol % mol % sphingomyelin/cholesterol. In still another embodiment the liposome compositions comprise about 60/40 mol % mol % sphingomyelin/cholesterol.
• additional lipids may be present in the formulations, for example, to prevent lipid oxidation or to attach ligands onto the liposome surface.
• additional lipids will result in a decrease in the sphingomyelin/cholesterol ratio.
• the sphingomyelin/cholesterol liposomes used in the present invention can be multilamellar or unilamellar. Suitable methods for preparing the liposomes include, but are not limited to, sonication, extrusion, high pressure/homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small liposome vesicles, thin film
• the liposomes can be sized to achieve the desired particle size range using standard methods well-known in the art (e.g., see US 6,723,338).
• liposomes which can be used in the VSLI preparations described herein have a size range of about 0.05-0.5 microns (50-500 nm), 0.2-0.4 microns (200-400 nm), about 0.1-0.4 microns (100-400 nm), about 0.05-0.2 (50-200 nm) or about 0.5 (500 nm) to about 0.15 microns(150 nm).
• the liposomes have a particle size having a mean particle diameter of about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, 170 nm, about 180 nm, about 190 nm, or about 200 nm.
• the average particle size is between 90 and 125nm with the preferred average particle size about 107.5 nm, where 25% of the particle size distribution is not less than 70nm and where 90% of the distribution has a particle size of not more than 170 nm.
• the sphingomyelin/cholesterol liposomes function as the liposome component used in the VSLI preparations described herein and are manufactured so that the liposome interior has a low pH.
• VSI having a low pH
• SPLI having a low pH
• a buffer of a higher pH whereby the final pH of the external VSLI solution is about physiologically neutral.
• the result is the creation of a pH gradient across the lipid membrane in which the pH is lower in the interior core of the liposomes than the exterior surrounding solution.
• Such gradients are achieved according to known methods (e.g., US 6,723,338).
• gradients can be achieved by formulating the liposomes in the presence of a buffer with a pH between about 2 and about 6, a pH between about 3 and about 5, and then subsequently transferring the liposomes to a higher pH, for example, of about 7.0 to about 7.5.
• the liposomes have an interior pH of about 4.0.
• Any number of dilution buffers can be used, such as sodium phosphate.
• the buffer has a pH of 8-10, preferably 9.0, so that the final diluted external liposome solution when mixed with VSI and SPLI will have a physiologic neutral pH.
• the SPLI liposomes Prior to use in the preparation of VSLI according to the methods described herein, the SPLI liposomes can be stored at refrigerated conditions for substantial periods of time prior to drug encapsulation and constitution of VSLI for administration to a patient. Alternatively, the liposomes can be dehydrated, stored and then rehydrated prior to use in accordance with well-known methods (See, e.g., US Patents 5,077,056 or 5,736,155).
• VSLI is prepared with strict aseptic techniques, for example, in a biological safety cabinet or by established pharmacy safety procedures for the preparation of sterile injectable formulations and hazardous drugs. Procedures for handling and disposal of anticancer drugs must be strictly followed (NIOSH Alert: Preventing occupational exposure to antineoplastic and other hazardous drugs in healthcare settings. 2004. U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, DHHS (NIOSH) Publication No. 2004- 16; OSHA Technical Manual, TED 1-0.15 A, Section VI: Chapter 2. Controlling Occupational Exposure to Hazardous Drugs. OSHA, 1999; American Society of Health- System Pharmacists. ASHP guidelines on handling hazardous drugs. Am J Health-Syst Pharm. (2006) 63: 1172-1193; Polovich M, White JM, Kelleher LO (eds.) 2005.
• the process for preparing constituted VSLI includes the following general steps:
• a constituting solution is prepared by mixing in a sterile container a first solution of vincristine sulfate containing between about 1 mg/mL, about 2 mg/mL or about 5 mg/mL in a buffer containing about 100 to about 200 mg/mL mannitol (other pharmaceutically acceptable excipients in which vincristine sulfate remains stable can also be used) at a pH of about 3.5 to about 5.5, or about 4.5 to about 4.7, with a second solution of liposomes suspended in a buffer at low pH (e.g., about 4.0) at an appropriate concentration ratio, for example, 0.1/1.0 to 0.2/1.0 (weight vincristine sulfate to weight lipid).
• a first solution of vincristine sulfate containing between about 1 mg/mL, about 2 mg/mL or about 5 mg/mL in a buffer containing about 100 to about 200 mg/mL mannitol (other pharmaceutically acceptable excipients in which vin
• the pH of the constituting solution containing the vincristine sulfate and liposomes is then raised to about 7.0 to about 7.5 to create a pH gradient. This can be
• a buffer e.g., sodium phosphate
• a higher pH e.g., about 9.0
• the constituting solution is then heated for at least about 13 to about 18 minutes in a dry heat block equilibrated to about 75°C, which contains caliber wells less than about 5%, larger than the average length or diameter of the container containing the constituting solution to produce the constituted product, VSLI.
• the heated, constituting solution comprising the constituted product is then allowed to equilibrate for at least about 30 minutes, at least about 45 minutes or at least about 60 minutes to room temperature (15°C to 30°C,).
• a volume of the constituting solution corresponding to the dose of the constituted VSLI to be administered to the patient is then admixed with a solution suitable for intravenous administration to a final volume of about 100 mL.
• solutions of vincristine sulfate, liposomes and high pH buffer are provided in three separate containers.
• the three solutions are constituted into one sterile container with a capacity to contain the combined volume of the solutions, for example, about 20-50 mL, about 25-40 mL, or about 30-35 mL.
• the separate components are provided as a kit including 3 or more vials.
• At least one of the vials contains a vincristine solution containing, e.g., 1 mg/mL, 2 mg/mL, or 5 mg/mL vincristine sulfate in buffer containing, e.g., 100 or 200 mg/mL mannitol (other excipients that are pharmaceutically acceptable, and in which vincristine remains stable for extended periods, can also be used), and adjusted to pH 3.5 to 5.5, or preferably pH 4.5 to pH 4.7.
• One of the vials contains a solution comprising sphingomyelin and cholesterol liposomes suspended in a 300 mM citrate buffer at, e.g., pH 4.0.
• Another vial or vials contains an alkaline phosphate buffer (e.g., pH 9.0) such as dibasic sodium
• the ingredients for the constitution of VSLI are provided separately in three vials containing (i) vincristine sulfate USP (5 mg/5 mL), which is equivalent to 4.5 mg/5 mL vincristine free base, and 500 mg/5 mL mannitol; (ii)
• SPLI sphingomyelin/cholesterol liposomes injection
• sphingomyelin 29.5 mg/mL cholesterol, 33.6 mg/mL citric acid, 35.4 mg/mL sodium citrate, and not more than 0.1% ethanol; and (iii) Sodium phosphate injection (SPI) containing 355 mg/25 mL dibasic sodium phosphate and 225 mg/25 mL sodium chloride all prepared with water for injection.
• SPI Sodium phosphate injection
• the containers used in the methods of the invention are sterile and composed of any pharmaceutically acceptable substance (e.g., glass or plastic).
• any pharmaceutically acceptable substance e.g., glass or plastic.
• the components are constituted in a sterile vial with an average diameter of about 36.5 mm, and an average range of about 35.8 to about 37.3 mm.
• Suitable dry block heaters which provide a safe, dry, constant temperature source, are commercially available from a number of sources (e.g, Bibby Scientific Ltd, V& P Scientific, Inc., Fisher Scientific Inc., VWR Scientific, Thermolyne Inc.).
• Heat conductive inserts having one or more calibrated bores adapted to receive the container of constituting solution may be metal (e.g., anodized aluminum, copper) or other suitable heat conductive materials. Inserts containing bores of openings of the appropriate size can be readily obtained (e.g., V&P Scientific, Inc.), or manufactured using standard methods.
• the heat block contains openings that are between about 1-5%, or about 4.5%, 4.2%, 4.0%, 3.8%, 3.5%, 3.3%, 3.0%, 2.8%, 2.5%, 2.2%, 2.0%, 1.8%, 1.5%, 1.2% or 1.0% larger than the average length or diameter of the container containing the constituting solution.
• the heat block contains cylindrical openings. In one embodiment, the openings are between 37.2 to 37.8 mm in diameter, or between about 37.4 to 37.6 mm in diameter.
• the constituting solution is heated for about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes at about 75°C. In one embodiment, the constituting solution is heated for about 14 minutes in at heat block equilibrated at 75°C.
• the constituted VSLI may be admixed with a pharmaceutically acceptable diluent suitable for intravenous administration to the patient (e.g., dextrose, sodium chloride) which may be provided, for example in a pre-filled, sterile container (glass bottle, plastic bottle or plastic bag).
• a pharmaceutically acceptable diluent suitable for intravenous administration to the patient e.g., dextrose, sodium chloride
• the volume of the patients calculated dose is removed from an infusion bag and replaced with the calculated volume of the constituted VSLI solution into an infusion bag, for example, where the final volume of the infusion container will be 100 mL.
• the pharmaceutically acceptable diluents is of 5% Dextrose Injection or 0.9% Sodium Chloride Injection.
• the VSLI produced according to the methods described herein appears as a white to off-white, translucent suspension, essentially free of visible foreign matter and aggregates. Typically, greater than about 95%, about 96%>, about 97%, about 98%> or more of the vincristine sulfate is encapsulated in the liposomes.
• the VSLI produced according to the methods described herein contains total impurities of less than about 4.0%, 3.5%, 3.4%, 3.2%, 3.1%> or 3.0%>. In some embodiments, the VSLI contains less than about 2.0%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4% or 1.3% N- desformylvincristine.
• the VSLI produced according to the methods described herein has an average in vitro release rate (IVR) or in vivo release rate of at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% or about 85% within 72 hours.
• IVR in vitro release rate
• in vivo release rate of at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% or about 85% within 72 hours.
• the VSLI produced according to the methods described herein contains vincristine sulfate at about 0.1 mg/mL to about 0.5 mg/mL. In certain embodiments, the vincristine sulfate is present at about 0.15 mg/mL to about 0.2 mg/mL. In one embodiment, the vincristine sulfate is present at about 0.16 mg/mL. In one embodiment, the VSLI contains 5 mg vincristine sulfate, 500 mg mannitol, 73.5 mg sphingomyelin, 29.5 mg cholesterol, 36 mg sodium citrate, 38 mg citric acid, 355 mg sodium phosphate, and 225 mg sodium chloride. Dosage and Administration
• VSLI prepared according to the methods described herein can be used to treat any type of cancer including primary, relapsed and refractory cancers.
• the patient or subject treated with the VLSI may be a variety of animals, including humans, non-human primates, avian species, equine species, canine species, feline species, bovine species, swine, lagomorphs, rodents, and the like.
• the VSLI is used to treat cancers of the blood and lymphatic systems including, but not limited to, lymphomas, leukemias and myelomas.
• the VSLI is used to treat tumors including, but not limited to neuroblastomas and brain cancers.
• the VSLI can be used as a single agent or in combination with other
• chemotherapeutic agents such as cyclophosphamide, doxorubicin and/or prednisone.
• the VSLI is administered along with as cyclophosphamide, doxorubicin and prednisone as a liposomal CHOP formulation ("lipo-CHOP).
• the VSLI is co -administered with at least one additional anti-tumor agent.
• the additional anti-tumor agent is an anti-tumor monoclonal antibody, such as OncolyTM, RituxanTM, or BexxarTM.
• the additional anti-tumor agent is an antisense drugs or an anti-tumor vaccine.
• the VSLI is coadministered with a prophylactic or therapeutic treatment for neurotoxicity, such as gabapentin (NeurontinTM).
• the VSLI is prepared within about 24 hours of administration to the patient and is stored at room temperature (15°C to 30°C,) or refrigerated (2-8°C).
• the VSLI is administered to the patient systemically by intravenous delivery.
• the VSLI is administered by intravenous infusion over a period of, e.g., about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes or longer.
• the VSLI is administered periodically, e.g., once every 7-28 days. In certain embodiments, the VSLI is administered once every 3, 5, 7, 10, 14, 21 or 28 days. In one embodiment, the VSLI is administered by intravenous infusion every 14 days. In another embodiment, the VSLI is administered by intravenous infusion every 7 days. As used herein, each administration of VSLI is considered one "course" of treatment.
• the amount of VSLI administered per dose will depend on a number of factors, such as the medical history of the patient, the use of other therapies, and the nature of the disease (e.g., first line, relapsed or refractory cancer).
• the VSLI prepared according to the methods described herein is administered at a dosage of about 1.4 to about 2.4 mg/m .
• the VSLI is administered at a dose of about 1.5 mg/m , about 1.8 mg/m 2 , about 2.0 mg/m 2 , 2.1 mg/m 2 , 2.2 mg/m 2 , 2.3 mg/m 2 or 2.4 mg/m 2 (i.e., mg vincristine per m body surface area).
• the VSLI is administered at a dose of 2.25 mg/m by intravenous infusion over about 60 minutes once every 7 days.
• the dose of VSLI may be temporarily interrupted and/or reduced during treatment.
• the dosage of VSLI may be temporarily interrupted and/or reduced during treatment.
• administered to a patient portraying a Grade 3 peripheral neuropathy or persistent Grade 2 peripheral neuropathy may be discontinued for up to about 7 days, and then reduced to a dose of about 2mg/m upon recovery to Grade 1 or 2.
• the dosage administered to a patient portraying a persistent Grade 2 peripheral neuropathy, even after receiving a reduced dose may be discontinue for up to 7 days, and then reduced to dose of
• the dosage of VSLI is determined by calculating the body surface area (BSA) of the subject according to well-known methods. For example, according to Mosteller's formula in which the BSA equals the square root of product of the weight of the subject in kg times the height in cm divided by 3600.
• BSA body surface area
• the "normal" BSA in humans is generally taken to be 1.7 m but also depends on other factors including the age and gender of the individual. For example:
• the temperature profile of the VSLI vial solution was investigated during the heating with the dry block process and compared with that of the temperature profile observed when heating with a water bath following the approved label instructions for Marqibo ® (FDA/cder Reference ID: 3172211, August 2012).
• Temperature profile measurements were generated by recording the solution temperature inside the vial after being placed in the heating apparatus (i.e., dry block or water bath). Time zero was the point where the vial was placed in the heating apparatus.
• the following procedure was used: 1.
• the components, VSI and SCLI, were combined into a single SPI vial (Swiss Precision Instruments, Inc.).
• a digital thermocouple was inserted through the septum of the vial and held approximately 5 mm from the bottom surface of the vial and positioned in the middle of the liquid solution.
• the dry block heater was used with a block containing a 1.480 inch vial container hole.
• the block temperature was monitored by placing a thermometer into the block's thermometer hole, which is situated in proximity to the vial container hole.
• the block heater was set to a temperature of 75°C and allowed to warm until the block thermometer read 75 ⁇ 2°C.
• the heated block was equilibrated for a minimum of 15 minutes at 75 ⁇ 2°C.
• the vial was then inserted into the well of the 75 ⁇ 2°C equilibrated block for 14 minutes ⁇ 15 seconds, and then removed.
• the constituted vial was allowed to come to ambient temperature by placing in ambient conditions for about 60 minutes. The procedure was repeated twice using the same vial.
• the water bath was allowed to heat until the water temperature was 65 ⁇ 5°C.
• the water bath was equilibrated for a minimum of 15 minutes.
• the vial containing constituted VSLI was then inserted into the water bath for 10 minutes ⁇ 1 minute, and then removed.
• the constituted vials were allowed to come to ambient temperature.
• the results tabulated in Table 1 and Figure 1 show that the dry block heated the liquid vial contents at a mean rate of 3.26°C/minute as compared to the water bath, which heated the vial contents at a mean rate of 4.21°C/minute.
• the desired temperature of 65° ⁇ 5°C was achieved in 14 minutes in the dry block compared to the water bath which took 10 minutes. With both heating apparatuses, once the vial was removed from the heating source there was gradual cooling.
• encapsulation of vincristine is a thermodynamic process which is dependent on the overall exposure to temperatures that promote membrane encapsulation rather than the kinetics of achieving those conditions.
• the dry block and water bath can provide a thermodynamic profile that allows encapsulation of vincristine into the sphingomyelin cholesterol liposomes for the efficient preparation of VSLI.
• Thermometer with a diameter no greater than 7 mm, and accurate to ⁇ 1°C in the range of O°-100°C.
• the vial having a diameter closest to or equal to 1.41 inches (“vial 1"), the lower end of the allowed SPI vial diameter had a measured diameter of 1.4305 inches.
• the vial having a diameter closest to or equal to 1.47 inches (“vial 2"), the upper end of the allowed SPI vial diameter, was measured to have a diameter of 1.4545 inches.
• kits were constituted as described above and tested for Vincristine Sulfate, Related Compounds and particle size and distribution following adapted Vincristine sulfate injection USP methods.
• the two study kits were constituted using the above instructions, except that instead of the water bath, the Dri-Block heater was used with a block containing a 1.480 inch vial container hole.
• the block temperature was monitored by placing a thermometer into the block's thermometer hole which is situated in proximity to the vial container hole.
• the block heater was set to a temperature of 75°C and allowed to heat until the block thermometer read 75 ⁇ 2°C.
• the heated block was then equilibrated for a minimum of 15 minutes (time recorded). The conditions are noted in Table 3.
• Each vial was then inserted into the 75 ⁇ 2°C heated block for 14 minutes ⁇ 15 seconds (time recorded), and then removed and allowed to come to ambient temperature by placing the vial in ambient conditions over about 60 minutes.
• VSLI The constitution of VSLI was achieved using constitution vials having a diameter of 1.4305 and 1.4545 inches using the Dri- Block heater. These represented vials closest to the 1.41 inch lower limit and 1.47 inch upper limits allowed for vial diameters.
• the block temperature dropped no more than 1 degree due to temperature equilibration between the space gap between the block well diameter and either diameter of vials. This kinetics of the heat transfer did not affect the preparation of VSLI.
• the resulting constituted VSLI from both vials achieved greater than 97% vincristine encapsulation .
• the incubation using the Dri-Block resulted in an encapsulation efficiency averaging 2.175% free vincristine.
• No new or increased impurities were observed with the Dri-Block heating profile.
• the principle degradant, N-desformylvincrisine was observed at an average of 1.33%, with no other impurity greater than 0.574%, and total impurities of no more than 3.10%.
• the particle size distribution was consistent with VSLI specifications with an average diameter of 107.5 nm diameter and mean D 25 of 90.5 nm and D 0 of 138.5 nm, VSLI prepared with Dri-Block heating released approximately 84 percent of the vincristine by 72 hours by IVR analysis..
• VSLI constituted using the dry block heater equilibrated at 75 ⁇ 2° with an incubation period of 14 minutes ⁇ 15 seconds encapsulated greater than 99% of vincristine in the sphingomyelin-cholesterol liposomes, and no anomalies were recorded during this experiment.
• the data establish a dry block constitution temperature profile that leads to a product that efficiently encapsulates vincristine and demonstrates that the dry block heater can be substituted for a water bath in the constitution of VSLI.
• Dri-Block ® equipped with 1.476" ( ⁇ 0.004) diameter well and thermometer pocket.
• Thermometer with a diameter no greater than 7 mm, and accurate to ⁇ 1°C in the range of O°-100°C.
• Three Marqibo ® Kits were selected at random and constituted by heating in the dry block at three different times (13, 14 and 15 minutes, respectively) at 75°C.
• the constituted vials were tested for Total and Free Vincristine Sulfate, Related Compounds, particle size and distribution using adapted vincristine sulfate Injection USP methods.
• the study kits were constituted using the instructions as described above except that, instead of a water bath, the Dri-Block ® heater was used with a block containing a 1.476 inch ( ⁇ 0.004") vial container hole. The block temperature was monitored by placing a
• thermometer into the block's thermometer hole which is situated in proximity to the vial container hole.
• the block was set to a temperature of 75°C and allowed to heat until the thermometer in the block reads 75 ⁇ 2°C.
• the heated block was then equilibrated for a minimum of 15 minutes.
• the conditions were recorded in Table 9.
• Each vial was then inserted into the 75 ⁇ 2°C heated block for 13 minutes ⁇ 15 seconds, 14 minutes ⁇ 15 seconds, and 15 minutes ⁇ 15 seconds, respectively.
• the vials were then removed and placed at ambient temperature.
• the constituted vials were allowed to cool at ambient temperature for at least 60 minutes prior to testing.
• VSLI constituted with the substitution of the water bath with a Dri- Block equilibrated at 75 ⁇ 2° C with an incubation period of 13 to 15 minutes ( ⁇ 15 seconds) encapsulated vincristine efficiently and no anomalies were recorded during the conduct of the experiment.
• the data establishes a Dri-Block constitution temperature profile that leads to efficient VSLI constitution and shows that the Dri-block heater can be substituted for a water bath in the constitution of the VSLI.

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