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
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
• • 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
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • 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
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• Anthracyclines are a class of antibiotics derived from certain types of Streptomyces bacteria. Anthracyclines are often used as cancer therapeutics and function in part as nucleic acid intercalating agents and inhibitors of the DNA repair enzyme topoisomerase II, thereby damaging nucleic acids in cancer cells, preventing the cells from replicating.
• An anthracycline cancer therapeutic is doxorubicin, which is used to treat a variety of cancers including breast cancer, lung cancer, ovarian cancer, lymphoma, and leukemia.
• the 6-maleimidocaproyl hydrazone of doxorubicin (DOXO-EMCH) was originally synthesized to provide an acid-sensitive linker that could be used to prepare immunoconjugates of doxorubicin and monoclonal antibodies directed against tumor antigens (Willner et al., Bioconjugate Chem 4:521-527 (1993)).
• antibody disulfide bonds are reduced with dithiothreitol to form free thiol groups, which in turn react with the maleimide group of DOXO-EMCH to form a stable thioether bond.
• the doxorubicin-antibody conjugate is targeted to tumors containing the antigen recognized by the antibody.
• the conjugate Following antigen-antibody binding, the conjugate is internalized within the tumor cell and transported to lysosomes. In the acidic lysosomal environment, doxorubicin is released from the conjugate intracellularly by hydrolysis of the acid-sensitive hydrazone linker. Upon release, the doxorubicin reaches the cell nucleus and is able to kill the tumor cell.
• doxorubicin and DOXO-EMCH see, for example, U.S. Pat. Nos. 7,387,771 and 7,902,144 and U.S. patent application Ser. No. 12/619,161, each of which are incorporated in their entirety herein by reference.
• DOXO-EMCH A subsequent use of DOXO-EMCH was developed by reacting the molecule in vitro with the free thiol group (Cys-34) on human serum albumin (HSA) to form a stable thioether conjugate with this circulating protein (Kratz et al., J Med Chem 45:5523-5533 (2002)). Based on these results, it was hypothesized that intravenously-administered DOXO-EMCH would rapidly conjugate to HSA in vivo and that this macromolecular conjugate would preferentially accumulate in tumors due to an “enhanced permeability and retention” (EPR) intratumor effect (Maeda et al., J Control Release 65:271-284 (2000)).
• EPR enhanced permeability and retention
• DOXO-EMCH Acute and repeat-dose toxicology studies with DOXO-EMCH in mice, rats, and dogs identified no toxicity beyond that associated with doxorubicin, and showed that all three species had significantly higher tolerance for DOXO-EMCH compared to doxorubicin (Kratz et al., Hum Exp Toxicol 26:19-35 (2007)). Based on the favorable toxicology profile and positive results from animal tumor models, a Phase 1 clinical trial of DOXO-EMCH was conducted in 41 advanced cancer patients (Unger et al., Clin Cancer Res 13:4858-4866 (2007)). This trial found DOXO-EMCH to be safe for clinical use. In some cases, DOXO-EMCH induced tumor regression.
• DOXO-EMCH Due to the sensitivity of the acid-cleavable linker in DOXO-EMCH, it is desirable to have formulations that are stable in long-term storage and during reconstitution (of, e.g., previously lyophilized compositions) and administration.
• DOXO-EMCH when present in compositions, diluents and administration fluids used in current formulations, is stable only when kept at low temperatures. The need to maintain DOXO-EMCH at such temperatures presents a major problem in that it forces physicians to administer cold (4° C.) DOXO-EMCH compositions to patients. Maintaining DOXO-EMCH at low temperatures complicates its administration in that it requires DOXO-EMCH to be kept at 4° C. and diluted at 4° C. to prevent degradation that would render it unsuitable for patient use. Further, administration at 4° C. can be harmful to patients whose body temperature is significantly higher (37° C.).
• Lyophilization has been used to provide a stable formulation for many drugs.
• reconstitution of lyophilized DOXO-EMCH in a liquid that does not maintain stability at room temperature can result in rapid decomposition of DOXO-EMCH.
• Use of an inappropriate diluent to produce an injectable composition of DOXO-EMCH can lead to decreased stability and/or solubility. This decreased stability manifests itself in the cleavage of the linker between the doxorubicin and EMCH moieties, resulting in degradation of the DOXO-EMCH into two components: doxorubicin and linker-maleimide.
• anthracycline-EMCH e.g., DOXO-EMCH
• injectable compositions containing the same are required to solve these problems and to provide a suitable administration vehicle that can be used reasonably in treating patients both for clinical trials and commercially.
• the present invention provides reconstituted formulations and injectable compositions of anthracycline compounds.
• the invention provides a reconstituted formulation comprising an anthracycline compound and a reconstitution liquid, wherein the reconstituted formulation is prepared by reconstituting a lyophilized composition of the anthracycline compound in a reconstitution liquid comprising ethanol and water.
• An exemplary reconstituted formulation comprises DOXO-EMCH and a reconstitution liquid.
• the volume:volume ratio of ethanol:water in a reconstituted formulation of the invention is about 10:90 to about 90:10, for example, about 40:60 to about 60:40, about 45:55 to about 55:45, or about 50:50.
• the concentration of anthracycline compound in a reconstituted formulation is from about 1 mg/ml to about 30 mg/ml, for example, from about 5 mg/ml to about 25 mg/ml. In certain embodiments, the concentration of anthracycline compound in the reconstituted formulation is about 10 mg/ml.
• the invention also provides an injectable composition of an anthracycline compound comprising a reconstituted formulation (as described above) and Lactated Ringer's solution.
• An exemplary injectable composition comprises DOXO-EMCH, a reconstitution liquid, and Lactated Ringer's solution.
• the concentration of anthracycline compound in the injectable composition is from about 0.1 mg/ml to about 25 mg/ml, for example from about 7 mg/ml to about 17 mg/ml, from about 0.1 mg/ml to about 5 mg/ml, or from about 0.25 mg/ml to about 4.5 mg/ml. In certain embodiments, the concentration of the anthracycline compound in the injectable composition is about 2.4 mg/ml.
• the invention further provides a reconstituted formulation or injectable composition as described above for use in treating cancer in a patient.
• the cancer may be selected from the following non-limiting examples: a solid tumor cancer, breast cancer, lung cancer, endometrial cancer, ovarian cancer, pancreatic cancer, cancer of the adrenal cortex, non-Hodgkin's lymphoma, multiple myeloma, leukemia, Kaposi's sarcoma, Ewing's sarcoma, soft tissue sarcoma, nephroblastoma, glioblastoma, prostate cancer, liver cancer, bone cancer, chondrosarcoma, renal cancer, bladder cancer, and gastric cancer.
• the cancer is selected from: a solid tumor cancer and a soft tissue sarcoma.
• the pancreatic cancer is pancreatic ductal adenocarcinoma.
• the invention provides the use of a reconstituted formulation or injectable composition as described above in the manufacture and administration of a medicament for treating cancer in a patient.
• the cancer may be selected from the following non-limiting examples: a solid tumor cancer, breast cancer, lung cancer, endometrial cancer, ovarian cancer, pancreatic cancer, cancer of the adrenal cortex, non-Hodgkin's lymphoma, multiple myeloma, leukemia, Kaposi's sarcoma, Ewing's sarcoma, soft tissue sarcoma, nephroblastoma, glioblastoma, prostate cancer, liver cancer, bone cancer, chondrosarcoma, renal cancer, bladder cancer, and gastric cancer.
• the cancer is selected from: a solid tumor cancer and a soft tissue sarcoma.
• the pancreatic cancer is pancreatic ductal adenocarcinoma.
• the invention also provides a method for treating cancer in a patient, comprising administering (e.g., intravenously) a reconstituted formulation or injectable composition as described above to a patient.
• the cancer may be selected from the following non-limiting examples: a solid tumor cancer, breast cancer, lung cancer, endometrial cancer, ovarian cancer, pancreatic cancer, cancer of the adrenal cortex, non-Hodgkin's lymphoma, multiple myeloma, leukemia, Kaposi's sarcoma, Ewing's sarcoma, soft tissue sarcoma, nephroblastoma, glioblastoma, prostate cancer, liver cancer, bone cancer, chondrosarcoma, renal cancer, bladder cancer, and gastric cancer.
• the cancer is selected from: a solid tumor cancer and a soft tissue sarcoma.
• the pancreatic cancer is pancreatic ductal adenocarcinoma.
• the invention also provides a method of preparing a reconstituted formulation of an anthracycline compound (e.g., DOXO-EMCH), comprising reconstituting a lyophilized composition of the anthracycline compound in a reconstitution liquid comprising ethanol and water.
• an anthracycline compound e.g., DOXO-EMCH
• the volume:volume ratio of ethanol:water is about 10:90 to about 90:10, for example, about 40:60 to about 60:40, about 45:55 to about 55:45, or about 50:50.
• the concentration of the anthracycline compound in the reconstituted formulation useful in the method is from about 1 mg/ml to about 30 mg/ml, for example, from about 5 mg/ml to about 25 mg/ml. In certain embodiments, the concentration of the anthracycline compound in the reconstituted formulation is about 10 mg/ml.
• the invention provides a method of preparing an injectable composition of an anthracycline compound comprising diluting a reconstituted formulation as described above with Lactated Ringer's solution.
• the concentration of the anthracycline compound in the injectable composition is from about 1 mg/ml to about 25 mg/ml, for example, from about 1 mg/ml to about 5 mg/ml or from about 7 mg/ml to about 17 mg/ml.
• an “anthracycline” refers to a member of a class of antibiotics produced by Streptomyces peucetius and/or Streptomyces coerulubidus .
• exemplary anthracyclines include, but are not limited to, doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin, pirarubicin, zorubicin, aclarubicin, caminomycin, mitoxantrone, and ametantrone.
• Lactated Ringer's solution refers to a sterile solution of sodium chloride, potassium chloride, calcium chloride, and sodium lactate in sterile water for injection.
• a 100 mL of solution of LR generally contains between 285.0 mg and 315.0 mg of sodium, between 14.2 mg and 17.3 mg of potassium, between 4.90 mg and 6.00 mg of calcium, between 368.0 mg and 408.0 mg of chloride, and between 231.0 mg and 261.0 mg of lactate.
• LR does not contain antimicrobial agents.
• the pH of the Lactated Ringer's solution is generally between 6 and 8. Lactated Ringer's solution is described in USP 35, Official Monographs, pgs. 4564-4565 (2012), incorporated in its entirety herein by reference.
• patient and “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice and rats).
• livestock animals e.g., bovines, porcines
• companion animals e.g., canines, felines
• rodents e.g., mice and rats.
• pharmaceutically acceptable carrier refers to a non-toxic carrier that may be administered to a patient, together with an anthracycline compound of this invention, and which does not destroy the pharmacological activity of the anthracycline.
• excipient refers to an additive in a formulation or composition that is not a pharmaceutically active ingredient.
• the term “pharmaceutically effective amount” refers to an amount effective to treat cancer in a patient, e.g., effecting a beneficial and/or desirable alteration in the general health of a patient suffering from a disease (e.g., cancer).
• treating cancer includes, but is not limited to, killing cancer cells, preventing the growth of new cancer cells, causing tumor regression (a decrease in tumor size), causing a decrease in metastasis, improving vital functions of a patient, improving the well-being of the patient, decreasing pain, improving appetite, improving the patient's weight, and any combination thereof.
• a “pharmaceutically effective amount” also refers to the amount required to improve the clinical symptoms of a patient.
• the therapeutic methods or methods of treating cancer described herein are not to be interpreted or otherwise limited to “curing” cancer.
• anthracycline compound useful in the formulations, compositions, and methods of the invention are described by the following schematic, wherein the anthracycline compound comprises an anthracycline, a linker molecule, and at least one protein-binding molecule, and wherein “X” represents a cleavable bond.
• anthracyclines include, but are not limited to, doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin, pirarubicin, zorubicin, aclarubicin, caminomycin, mitoxantrone, and ametantrone.
• the anthracycline is doxorubicin.
• the cleavable bond (“X”) is an acid-cleavable bond.
• exemplary acid-cleavable bonds include, but are not limited to, acetal, ketal, imine, hydrazone, carboxylhydrazone or sulphonylhydrazone bonds, or cis-aconityl bonds or bonds containing a substituted or unsubstituted trityl group.
• the acid-cleavable bond is a hydrazone bond.
• the linkers of the present invention are organic molecules.
• Such linkers may comprise an aliphatic carbon chain and/or an aliphatic carbon ring with 1-12 carbon atoms, wherein any of the carbon atoms maybe substituted with an —OH or ⁇ O, and wherein any of the carbon atoms may be substituted for oxygen atoms where appropriate and chemically feasible.
• the aliphatic linker may comprise an alkyl chain comprising 1-12 carbon atoms, an alkenyl chain comprising 2-12 carbon atoms, or an alkynyl chain comprising 2-12 carbon atoms, wherein any of the carbon atoms maybe substituted with an —OH or ⁇ O, and wherein any of the carbon atoms may be substituted for oxygen atoms where appropriate and chemically feasible.
• the aliphatic linker is an alkyl chain comprising 1-12 carbon atoms, wherein any of the carbon atoms maybe substituted with an ⁇ O, where appropriate and chemically feasible.
• the aliphatic linker is an alkyl chain comprising 3-9, 4-8, or 5-7 carbon atoms, wherein any of the carbon atoms maybe substituted with an ⁇ O, where appropriate and chemically feasible.
• the aliphatic linker is an alkyl chain comprising 6 carbon atoms, wherein the carbon atom attached to the cleavable bond “X” is substituted with an ⁇ O.
• the cleavable bond (“X”) is enzyme-cleavable.
• exemplary enzyme-cleavable linkers include, but are not limited to, peptide linkers and linkers comprising one or more carbamate bonds.
• a peptide linker can comprise, for example, 1-5, 1-10, 1-15, 1-20, 1-25, 1-30, 1-35, 1-40, 2-5, 2-10, 2-15, 2-20, 2-25, 2-30, 2-35, or 2-40 amino acid residues.
• Exemplary peptide linkers include, but are not limited to, linkers comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residues.
• a peptide linker may be designed to be specifically cleavable by one or more proteases.
• the bond being cleaved is a peptide bond, an imide bond, or a carboxyl-hydrazone bond.
• Exemplary protein-binding groups include, but are not limited to, a maleimide group, a haloacetamide group, a haloacetate group, a pyridyldithio group, an N-hydroxysuccinimide ester group, and an isothiocyanate group.
• the protein-binding group is a maleimide group.
• Exemplary protein-binding groups also include a disulphide group, a vinylcarbonyl group, an aziridine group or an acetylene group.
• a disulphide group may be activated by a thionitrobenzoic acid (e.g. 5′-thio-2-nitrobenzoic acid) as the exchangeable group.
• a maleimide, pyridyldithio, or N-hydroxysuccinimide ester group can, where appropriate, be substituted by an alkyl group or by the above water-soluble groups.
• a protein-binding group possesses protein-binding properties, i.e., it binds covalently (“a covalent protein-binding group”) or noncovalently (“a noncovalent protein-binding group”), in a physiological environment, to particular amino acids on the surface of the protein.
• the maleimide group, the haloacetamide group, the haloacetate group, the pyridyldithio group, the disulphide group, the vinylcarbonyl group, the aziridine group, and/or the acetylene group preferably reacts with thiol (—SH) groups of cysteines, while the N-hydroxysuccinimide ester group and/or the isothiocyanate group preferably react with the amino group (—NH) of lysines, on the surface of a protein.
• the anthracycline compounds of the invention include any and all combinations of one or more anthracyclines, cleavable bonds, linkers, and protein-binding groups.
• Exemplary anthracycline compounds comprise an anthracycline, an acid-cleavable bond, an alkyl linker, and a covalent protein-binding group.
• the anthracycline compound comprises an anthracycline, a hydrazone bond as the acid-cleavable bond, an alkyl linker, and a maleimide group as the covalent protein-binding group.
• the anthracycline compound comprises an anthracycline, a hydrazone bond as the acid-cleavable bond, a 6-carbon alkyl linker wherein the carbon atom attached to the cleavable bond is substituted with an ⁇ O, and a maleimide group as the covalent protein-binding group (i.e., an anthracycline-EMCH molecule).
• DOXO-EMCH An exemplary compound of the present invention is DOXO-EMCH.
• DOXO-EMCH alone or in combination with any other term, refers to a compound as depicted by the following structure:
• DOXO-EMCH is also referred to as (E)-N′-(1-((2S,4S)-4-(4-amino-5-hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy-2,5,12-trihydroxy-7-methoxy-6,11-dioxo1,2,3,4,6,11-hexahydrotetracen-2-yl)-2-hydroxyethylidene)-6-(2,5-dioxo-2H-pyrrol-1(5H)yl)hexanehydrazide.HCl.
• the present invention is based on the surprising discoveries that lyophilized DOXO-EMCH is stable when reconstituted in a mixture of ethanol and water after lyophilization, and that it maintains its stability when subsequently diluted in Lactated Ringer's solution.
• Previous formulations of DOXO-EMCH comprised N-acetyltryptophan, phosphate buffer, and sucrose. Indeed, N-acetyltryptophan was thought to be required to maintain the stability of DOXO-EMCH. However, even in the presence of N-acetyltryptophan, the DOXO-EMCH in those previous formulations degraded at room temperature, resulting in the release of doxorubicin.
• doxorubicin in previous formulations, the imide bond between the doxorubicin and EMCH moieties undergoes acid-catalyzed hydrolysis.
• Administration of doxorubicin is undesirable because it is more toxic than DOXO-EMCH, especially at concentrations required for the effective treatment of cancer.
• DOXO-EMCH unlike DOXO-EMCH, doxorubicin is not targeted to tumors.
• anthracycline compounds e.g., anthracycline-EMCH
• individual components i.e., anthracycline and EMCH
• anthracyclines such as doxorubicin
• anthracyclines such as doxorubicin
• provision of anthracycline compound formulations which both reduce the amount of degradation of anthracycline compounds and allow for reconstitution and administration to patients at room temperature are particularly useful.
• the formulations and compositions of the invention stabilize DOXO-EMCH and produce low amounts of free doxorubicin as a degradation byproduct. This stability is observed even without N-acetyltryptophan. Even more surprisingly, the formulations and compositions of the present invention are stable at room temperature. Previous formulations and compositions were stable only at cold temperatures (e.g., 4° C.), making handling and administration difficult for clinicians. In addition, the required administration of DOXO-EMCH formulations at 4° C. was dangerous for the patients. The formulations and compositions of the present invention eliminate the need for cold handling of the drug product, and allow physicians to administer the drug product at room temperature.
• a reconstitution liquid of ethanol and water stabilized the DOXO-EMCH. It was also unexpected that a reconstitution liquid of ethanol and water would quickly solubilize the DOXO-EMCH (within less than a minute).
• lyophilized pharmaceutical formulations and compositions are not reconstituted in ethanol and water, likely because ethanol is potentially toxic and must be titrated carefully to avoid intoxication of the patient. Indeed, buffered saline solution is typically used as a reconstitution liquid due to its low toxicity.
• Lyophilization is often used as a means of storing compounds susceptible to degradation in solution. However, determining a proper liquid in which to reconstitute a lyophilized formulation is not always a straightforward endeavor. Use of an unsuitable reconstitution liquid can negatively impact a compound's stability or solubility, causing its aggregation or degradation.
• lyophilized DOXO-EMCH formulations had been reconstituted in a solution of 10 mM sodium phosphate in 5% D-(+)-glucose (pH 6.4) (Unger et al., Clin Cancer Res 13:4858-4866 (2007), Kratz et al., Hum Exp Toxicol 26:19-35 (2007)) or in sterile water for injection.
• the invention provides a reconstituted formulation comprising an anthracycline compound (e.g., DOXO-EMCH) and a reconstitution liquid comprising ethanol and water.
• the invention provides a reconstituted formulation consisting of an anthracycline compound (e.g., DOXO-EMCH) and a reconstitution liquid consisting of ethanol and water.
• the volume:volume ratio of ethanol:water for use as a reconstitution liquid may be from about 10:90 to about 90:10, e.g., about 10:90, about 20:80, about 30:70, about 40:60, about 41:59, about 42:58, about 43:57, about 44:56, about 45:55, about 46:54, about 47:53, about 48:52, about 49:51, about 50:50 (e.g., 50 ⁇ 5:50 ⁇ 5), about 51:49, about 52:48, about 53:47, about 54:46, about 55:45, about 56:44, about 57:43, about 58:42, about 59:41, about 60:40, about 70:30, about 80:20, about 90:10, or any intermediate ratio thereof.
• the concentration of anthracycline compound (e.g., DOXO-EMCH) useful in a reconstituted formulation of the invention is any amount of the anthracycline compound that remains stable in the composition.
• the concentration of the anthracycline compound may be from about 1 mg/ml to about 500 mg/ml in a reconstituted composition.
• the concentration of the anthracycline compound may be from about 1 mg/ml to about 100 mg/ml, from about 1 mg/ml to about 30 mg/ml, or from about 5 mg/ml to about 25 mg/ml.
• the concentration of the anthracycline compound may be 1.0 ⁇ 0.5 mg/ml, 2.0 ⁇ 0.5 mg/ml, 3.0 ⁇ 0.5 mg/ml, 4.0 ⁇ 0.5 mg/ml, 5.0 ⁇ 0.5 mg/ml, 6.0 ⁇ 0.5 mg/ml, 7.0 ⁇ 0.5 mg/ml, 8.0 ⁇ 0.5 mg/ml, 9.0 ⁇ 0.5 mg/ml, 10.0 ⁇ 0.5 mg/ml, 11.0 ⁇ 0.5 mg/ml, 12.0 ⁇ 0.5 mg/ml, 13.0 ⁇ 0.5 mg/ml, 14.0 ⁇ 0.5 mg/ml, 15.0 ⁇ 0.5 mg/ml, 16.0 ⁇ 0.5 mg/ml, 17.0 ⁇ 0.5 mg/ml, 18.0 ⁇ 0.5 mg/ml, 19.0 ⁇ 0.5 mg/ml, 20.0 ⁇ 0.5 mg/ml, 21.0 ⁇ 0.5 mg/ml, 22.0 ⁇ 0.5 mg/ml, 23.0 ⁇ 0.5 mg/ml 24.0 ⁇ 0.5 mg/ml, and 25.0 ⁇ 0.5 mg/ml.
• the concentration of the anthracycline compound may be from about 1.4 mg/ml to about 3.4 mg/ml, for example, 1.4 ⁇ 0.1 mg/ml, 1.5 ⁇ 0.1 mg/ml, 1.6 ⁇ 0.1 mg/ml, 1.7 ⁇ 0.1 mg/ml, 1.8 ⁇ 0.1 mg/ml, 1.9 ⁇ 0.1 mg/ml, 2.0 ⁇ 0.1 mg/ml, 2.1 ⁇ 0.1 mg/ml, 2.2 ⁇ 0.1 mg/ml, 2.3 ⁇ 0.1 mg/ml, 2.4 ⁇ 0.1 mg/ml, 2.5 ⁇ 0.1 mg/ml, 2.6 ⁇ 0.1 mg/ml, 2.7 ⁇ 0.1 mg/ml, 2.8 ⁇ 0.1 mg/ml, 2.9 ⁇ 0.1 mg/ml, 3.0 ⁇ 0.1 mg/ml, 3.1 ⁇ 0.1 mg/ml, 3.2 ⁇ 0.1 mg/ml, 3.3 ⁇ 0.1 mg/ml, or 3.4 ⁇ 0.1 mg/ml.
• one of skill in the art would know how to test for stability of the anthracycline compound (e.g.,
• a reconstituted formulation comprises less than 2.5% free doxorubicin, for example, at room temperature.
• the doxorubicin has been produced via degradation of DOXO-EMCH.
• a reconstituted formulation comprises less than 2.4%, less than 2.3%, less than 2.2%, less than 2.1%, less than 2.0%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, or less than 0.75% free doxorubicin at room temperature.
• the reconstituted formulation maintains these amounts of doxorubicin (i.e., no additional doxorubicin is formed via the degradation of DOXO-EMCH present in the reconstituted formulation) at room temperature for 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes or more after reconstitution of DOXO-EMCH.
• the reconstituted formulation maintains these amounts of doxorubicin (i.e., no additional doxorubicin is formed via the degradation of DOXO-EMCH present in the reconstituted formulation) at room temperature for 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12 hours or more after reconstitution of DOXO-EMCH.
• the reconstituted formulation comprises less than 0.75% free doxorubicin at room temperature 10 minutes after reconstitution of DOXO-EMCH, e.g., when the DOXO-EMCH is reconstituted to 10 mg/ml.
• the reconstituted formulation comprises less than 1.1% free doxorubicin at room temperature 20 minutes after reconstitution of DOXO-EMCH, e.g., when the DOXO-EMCH is reconstituted to 10 mg/ml.
• a reconstituted formulation comprises greater than 96.0% DOXO-EMCH, e.g., at room temperature.
• a reconstituted formulation comprises greater than 96.1%, greater than 96.2%, greater than 96.3%, greater than 96.4%, greater than 96.5%, greater than 96.6%, greater than 96.7%, greater than 96.8%, greater than 96.9%, greater than 97.0%, greater than 97.1%, greater than 97.2%, greater than 97.3%, greater than 97.4%, greater than 97.5%, greater than 97.6%, greater than 97.7%, greater than 97.8%, greater than 97.9%, greater than 98.0%, greater than 98.1%, greater than 98.2%, greater than 98.3%, greater than 98.4%, greater than 98.5%, greater than 98.6%, greater than 98.7%, greater than 98.8%, greater than 98.9%, greater than 99.0%
• the reconstituted formulation maintains these amounts of DOXO-EMCH (i.e., no additional DOXO-EMCH is degraded into, e.g., free doxorubicin) at room temperature for 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes or more after reconstitution of DOXO-EMCH.
• DOXO-EMCH i.e., no additional DOXO-EMCH is degraded into, e.g., free doxorubicin
• the reconstituted formulation maintains these amounts of DOXO-EMCH (i.e., no additional DOXO-EMCH is degraded into, e.g., free doxorubicin) at room temperature for 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12 hours or more after reconstitution of DOXO-EMCH.
• the reconstituted formulation comprises greater than 99.0% DOXO-EMCH at room temperature 10 minutes after reconstitution of DOXO-EMCH, e.g., when the DOXO-EMCH is reconstituted to 10 mg/ml.
• the reconstituted formulation comprises greater than 96.0% DOXO-EMCH at room temperature 10 minutes after reconstitution of DOXO-EMCH, e.g., when the DOXO-EMCH is reconstituted to 10 mg/ml.
• the reconstituted formulation comprises 100.0% DOXO-EMCH at room temperature 10 minutes after reconstitution of DOXO-EMCH, e.g., when the DOXO-EMCH is reconstituted to 10 mg/ml.
• the reconstituted formulation comprises greater than 96.2% DOXO-EMCH at room temperature 20 minutes after reconstitution of DOXO-EMCH, e.g., when the DOXO-EMCH is reconstituted to 10 mg/ml.
• the reconstituted formulation comprises greater than 98.2% DOXO-EMCH at room temperature 20 minutes after reconstitution of DOXO-EMCH, e.g., when the DOXO-EMCH is reconstituted to 10 mg/ml.
• a reconstitution liquid comprising ethanol and water may not in itself be suitable for parenteral administration (e.g., administration by injection).
• parenteral administration e.g., administration by injection
• injectable compositions comprising an anthracycline compound and a reconstitution liquid.
• Such injectable compositions also should be suitable for an intravenous administration solution in that they are stable at room temperature, maintain the stability of the reconstituted drug product, and are easy to administer.
• a survey of all oncology drug products described in the Physicians' Desk Reference indicates that 5% dextrose, 0.9% NaCl, or 5% plus 0.45% NaCl are recommended for all of the intravenously administered oncology products.
• the reconstituted formulations of the invention required Lactated Ringer's solution (LR) as the administration solution to create an injectable composition suitable for administration at room temperature.
• LR Lactated Ringer's solution
• the total amount of an anthracycline compound (e.g., DOXO-EMCH) in a composition to be injected in a patient is one that is suitable for that patient.
• an anthracycline compound e.g., DOXO-EMCH
• the amount of the anthracycline compound is a pharmaceutically effective amount. The skilled worker would be able to determine the amount of the anthracycline compound in a composition needed to treat a patient based on factors such as, for example, the age, weight, and physical condition of the patient.
• the concentration of anthracycline compound depends on its solubility in the intravenous administration solution and the volume of fluid that can be administered.
• the concentration of the anthracycline compound may be from about 0.1 mg/ml to about 50 mg/ml in the injectable composition.
• the concentration of the anthracycline compound may be from about 0.1 mg/ml to about 25 mg/ml, from about 7 mg/ml to about 17 mg/ml, from about 0.1 mg/ml to about 5 mg/ml, or from about 0.25 mg/ml to about 4.5 mg/ml.
• the concentration of anthracycline compound may be about 0.1 mg/ml, about 0.2 mg/ml, about 0.3 mg/ml, about 0.4 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml, about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1.0 mg/ml, about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 1.9 mg/ml, about 2.0 mg/ml, about 2.1 mg/ml, about 2.2 mg/ml, about 2.3 mg/ml, about 2.4 mg/ml, about 2.5 mg/ml, about 2.6 mg/ml, about 2.7 mg/ml, about 2.8 mg/ml, about 2.9 mg/ml, about 3.0 mg/ml, about
• the concentration of the anthracycline compound may be about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 11 mg/ml, about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml, about 20 mg/ml, about 21 mg/ml, about 22 mg/ml, about 23 mg/ml, about 24 mg/ml, about 25 mg/ml, about 26 mg/ml, about 27 mg/ml, about 28 mg/ml, about 29 mg/ml, or about 30 mg/ml.
• an injectable composition comprises less than 3.0% free doxorubicin, for example, at room temperature.
• the doxorubicin has been produced via degradation of DOXO-EMCH.
• an injectable composition comprises less than 2.9%, less than 2.8%, less than 2.7%, less than 2.6%, less than 2.5%, less than 2.4%, less than 2.3%, less than 2.2%, less than 2.1%, less than 2.0%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, or less than 0.75% free doxorubicin.
• the reconstituted formulation maintains these amounts of doxorubicin (i.e., no additional doxorubicin is formed via the degradation of DOXO-EMCH present in the reconstituted formulation) at room temperature for 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes or more after reconstitution of DOXO-EMCH.
• the reconstituted formulation maintains these amounts of doxorubicin (i.e., no additional doxorubicin is formed via the degradation of DOXO-EMCH present in the reconstituted formulation) at room temperature for 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12 hours or more after reconstitution of DOXO-EMCH.
• the injectable composition comprises less than 1.0% free doxorubicin or less than 1.1% free doxorubicin at room temperature 0 minutes after dilution, for example, in Lactated Ringer's solution and/or at a DOXO-EMCH concentration of 2.4 mg/ml.
• the injectable composition comprises less than 1.1% free doxorubicin or less than 1.3% free doxorubicin at room temperature 30 minutes after dilution, for example, in Lactated Ringer's solution and/or at a DOXO-EMCH concentration of 2.4 mg/ml. In particular embodiments, the injectable composition comprises less than 1.3% free doxorubicin or less than 1.5% free doxorubicin at room temperature 60 minutes after dilution, for example, in Lactated Ringer's solution and/or at a DOXO-EMCH concentration of 2.4 mg/ml.
• the injectable composition comprises less than 1.7% free doxorubicin at room temperature 90 minutes after dilution, for example, in Lactated Ringer's solution and/or at a DOXO-EMCH concentration of 2.4 mg/ml. In particular embodiments, the injectable composition comprises less than 1.9% free doxorubicin or less than 2.1% free doxorubicin at room temperature 120 minutes after dilution, for example, in Lactated Ringer's solution and/or at a DOXO-EMCH concentration of 2.4 mg/ml.
• the injectable composition comprises less than 2.3% free doxorubicin at room temperature 150 minutes after dilution, for example, in Lactated Ringer's solution and/or at a DOXO-EMCH concentration of 2.4 mg/ml. In particular embodiments, the injectable composition comprises less than 3.4% free doxorubicin at room temperature 240 minutes after dilution, for example, in Lactated Ringer's solution and/or at a DOXO-EMCH concentration of 2.4 mg/ml.
• an injectable composition comprises greater than 96.0% DOXO-EMCH, e.g., at room temperature and/or at 2.4 mg/ml of DOXO-EMCH and/or when diluted in Lactated Ringer's solution.
• an injectable composition comprises greater than 96.1%, greater than 96.2%, greater than 96.3%, greater than 96.4%, greater than 96.5%, greater than 96.6%, greater than 96.7%, greater than 96.8%, greater than 96.9%, greater than 97.0%, greater than 97.1%, greater than 97.2%, greater than 97.3%, greater than 97.4%, greater than 97.5%, greater than 97.6%, greater than 97.7%, greater than 97.8%, greater than 97.9%, greater than 98.0%, greater than 98.1%, greater than 98.2%, greater than 98.3%, greater than 98.4%, greater than 98.5%, greater than 98.6%, greater than 98.7%, greater than 98.8%, greater than 98.9%, greater than 99.0%, greater than 99.1%, greater than 99.2%, greater than 99.3%, greater than 99.4%, greater than 99.5%, greater than 99.6%,
• the reconstituted formulation maintains these amounts of DOXO-EMCH (i.e., no additional DOXO-EMCH is degraded into, e.g., free doxorubicin) at room temperature for 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes or more after reconstitution of DOXO-EMCH.
• DOXO-EMCH i.e., no additional DOXO-EMCH is degraded into, e.g., free doxorubicin
• the reconstituted formulation maintains these amounts of DOXO-EMCH (i.e., no additional DOXO-EMCH is degraded into, e.g., free doxorubicin) at room temperature for 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12 hours or more after reconstitution of DOXO-EMCH.
• DOXO-EMCH i.e., no additional DOXO-EMCH is degraded into, e.g., free doxorubicin
• excipients it may be beneficial to include one or more excipients in a reconstituted formulation or an injectable composition of the invention.
• One of skill in the art would appreciate that the choice of any one excipient may influence the choice of any other excipient. For example, the choice of a particular excipient may preclude the use of one or more additional excipients because the combination of excipients would produce undesirable effects.
• One of skill in the art would be able to empirically determine which excipients, if any, to include in the formulations or compositions of the invention.
• Excipients of the invention may include, but are not limited to, co-solvents, solubilizing agents, buffers, pH adjusting agents, bulking agents, surfactants, encapsulating agents, tonicity-adjusting agents, stabilizing agents, protectants, and viscosity modifiers.
• solubilizing agents may be useful for increasing the solubility of any of the components of the formulation or composition, including an anthracycline compound (e.g., DOXO-EMCH) or an excipient.
• anthracycline compound e.g., DOXO-EMCH
• the solubilizing agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary solubilizing agents that may be used in the formulations or compositions of the invention.
• solubilizing agents include, but are not limited to, ethyl alcohol, tert-butyl alcohol, polyethylene glycol, glycerol, methylparaben, propylparaben, polyethylene glycol, polyvinyl pyrrolidone, and any pharmaceutically acceptable salts and/or combinations thereof.
• the pH of the reconstituted formulations and injectable compositions of the invention may be any pH that provides desirable properties for the formulation or composition. Desirable properties may include, for example, anthracycline compound (e.g., DOXO-EMCH) stability, increased anthracycline compound retention as compared to compositions at other pHs, and improved filtration efficiency.
• the pH of the reconstituted formulations and injectable compositions of the invention may be from about 3.0 to about 9.0, e.g., from about 5.0 to about 7.0.
• the pH of the reconstituted formulations and injectable compositions of the invention may be 5.5 ⁇ 0.1, 5.6 ⁇ 0.1, 5.7 ⁇ 0.1, 5.8 ⁇ 0.1, 5.9 ⁇ 0.1, 6.0 ⁇ 0.1, 6.1 ⁇ 0.1, 6.2 ⁇ 0.1, 6.3 ⁇ 0.1, 6.4 ⁇ 0.1, or 6.5 ⁇ 0.1.
• a buffer may have a pKa of, for example, about 5.5, about 6.0, or about 6.5.
• a buffer may have a pKa of, for example, about 5.5, about 6.0, or about 6.5.
• Buffers are well known in the art. Accordingly, the buffers described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary buffers that may be used in the formulations or compositions of the invention.
• a buffer includes, but is not limited to, Tris, Tris HCl, potassium phosphate, sodium phosphate, sodium citrate, sodium ascorbate, combinations of sodium and potassium phosphate, Tris/Tris HCl, sodium bicarbonate, arginine phosphate, arginine hydrochloride, histidine hydrochloride, cacodylate, succinate, 2-(N-morpholino)ethanesulfonic acid (MES), maleate, bis-tris, phosphate, carbonate, and any pharmaceutically acceptable salts and/or combinations thereof.
• Tris Tris HCl, potassium phosphate, sodium phosphate, sodium citrate, sodium ascorbate, combinations of sodium and potassium phosphate, Tris/Tris HCl, sodium bicarbonate, arginine phosphate, arginine hydrochloride, histidine hydrochloride, cacodylate, succinate, 2-(N-morpholino)ethanesulfonic acid (MES), maleate, bis-tri
• pH-adjusting agent in the compositions of the invention. Modifying the pH of a formulation or composition may have beneficial effects on, for example, the stability or solubility of an anthracycline compound, or may be useful in making a formulation or composition suitable for parenteral administration.
• pH-adjusting agents are well known in the art. Accordingly, the pH-adjusting agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary pH-adjusting agents that may be used in the formulations or compositions of the invention. pH-adjusting agents may include, for example, acids and bases.
• a pH-adjusting agent includes, but is not limited to, acetic acid, hydrochloric acid, phosphoric acid, sodium hydroxide, sodium carbonate, and combinations thereof.
• a bulking agent in the reconstituted formulations and injectable compositions of the invention.
• Bulking agents are commonly used in lyophilized compositions to provide added volume to the composition and to aid visualization of the composition, especially in instances where the lyophilized pellet would otherwise be difficult to see. Bulking agents also may help prevent a blowout of the active component(s) of a pharmaceutical composition and/or to aid cryoprotection of the composition. Bulking agents are well known in the art. Accordingly, the bulking agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary bulking agents that may be used in the formulations or compositions of the invention.
• Exemplary bulking agents may include carbohydrates, monosaccharides, disaccharides, polysaccharides, sugar alcohols, amino acids, and sugar acids, and combinations thereof.
• Carbohydrate bulking agents include, but are not limited to, mono-, di-, or poly-carbohydrates, starches, aldoses, ketoses, amino sugars, glyceraldehyde, arabinose, lyxose, pentose, ribose, xylose, galactose, glucose, hexose, idose, mannose, talose, heptose, glucose, fructose, methyl a-D-glucopyranoside, maltose, lactone, sorbose, erythrose, threose, arabinose, allose, altrose, gulose, idose, talose, erythrulose, ribulose, xylulose, psicose, tag
• Sugar alcohol bulking agents include, but are not limited to, alditols, inositols, sorbitol, and mannitol.
• Amino acid bulking agents include, but are not limited to, glycine, histidine, and proline.
• Sugar acid bulking agents include, but are not limited to, aldonic acids, uronic acids, aldaric acids, gluconic acid, isoascorbic acid, ascorbic acid, glucaric acid, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, neuraminic acid, pectic acids, and alginic acid.
• surfactants in general, reduce the surface tension of a liquid composition. This may provide beneficial properties such as improved ease of filtration. Surfactants also may act as emulsifying agents and/or solubilizing agents. Surfactants are well known in the art. Accordingly, the surfactants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary surfactants that may be used in the formulations or compositions of the invention.
• Surfactants that may be included include, but are not limited to, sorbitan esters such as polysorbates (e.g., polysorbate 20 and polysorbate 80), lipopolysaccharides, polyethylene glycols (e.g., PEG 400 and PEG 3000), poloxamers (i.e., pluronics), ethylene oxides and polyethylene oxides (e.g., Triton X-100), saponins, phospholipids (e.g., lecithin), and combinations thereof.
• sorbitan esters such as polysorbates (e.g., polysorbate 20 and polysorbate 80), lipopolysaccharides, polyethylene glycols (e.g., PEG 400 and PEG 3000), poloxamers (i.e., pluronics), ethylene oxides and polyethylene oxides (e.g., Triton X-100), saponins, phospholipids (e.g., lecithin), and combinations thereof.
• an encapsulating agent in the reconstituted formulations and injectable compositions of the invention.
• Encapsulating agents can sequester molecules and help stabilize or solubilize them.
• Encapsulating agents are well known in the art. Accordingly, the encapsulating agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary encapsulating agents that may be used in the formulations or compositions of the invention.
• Encapsulating agents that may be included in compositions include, but are not limited to, dimethyl- ⁇ -cyclodextrin, hydroxyethyl- ⁇ -cyclodextrin, hydroxypropyl- ⁇ -cyclodextrin, and trimethyl- ⁇ -cyclodextrin, and combinations thereof.
• a tonicity-adjusting agent in the reconstituted formulations and injectable compositions of the invention.
• the tonicity of a liquid composition is an important consideration when administering the composition to a patient, for example, by parenteral administration.
• Tonicity-adjusting agents thus, may be used to help make a formulation or composition suitable for administration.
• Tonicity-adjusting agents are well known in the art. Accordingly, the tonicity-adjusting agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary tonicity-adjusting agents that may be used in the formulations or compositions of the invention.
• Tonicity-adjusting agents may be ionic or non-ionic and include, but are not limited to, inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols, and carbohydrates.
• Exemplary inorganic salts may include sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate.
• An exemplary amino acid is glycine.
• Exemplary sugars may include sugar alcohols such as glycerol, propylene glycol, glucose, sucrose, lactose, and mannitol.
• Stabilizing agents help increase the stability of an anthracycline compound in compositions of the invention. This may occur by, for example, reducing degradation or preventing aggregation of an anthracycline compound. Without wishing to be bound by theory, mechanisms for enhancing stability may include sequestration of the anthracycline compound from a solvent or inhibiting free radical oxidation of the anthracycline compound. Stabilizing agents are well known in the art. Accordingly, the stabilizing agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary stabilizing agents that may be used in the formulations or compositions of the invention. Stabilizing agents may include, but are not limited to, emulsifiers and surfactants.
• a protectant in the reconstituted formulations and injectable compositions of the invention.
• Protectants are agents that protect a pharmaceutically active ingredient (e.g., an anthracycline compound) from an undesirable condition (e.g., instability caused by freezing or lyophilization, or oxidation).
• Protectants can include, for example, cryoprotectants, lyoprotectants, and antioxidants.
• Cryoprotectants are useful in preventing loss of potency of an active pharmaceutical ingredient (e.g., an anthracycline compound) when a formulation is exposed to a temperature below its freezing point.
• cryoprotectant could be included in a reconstituted lyophilized formulation of the invention so that the formulation could be frozen before dilution for intravenous (IV) administration.
• Cryoprotectants are well known in the art. Accordingly, the cryoprotectants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary cryoprotectants that may be used in the formulations or compositions of the invention.
• Cryoprotectants include, but are not limited to, solvents, surfactants, encapsulating agents, stabilizing agents, viscosity modifiers, and combinations thereof.
• Cryoprotectants may include, for example, disaccharides (e.g., sucrose, lactose, maltose, and trehalose), polyols (e.g., glycerol, mannitol, sorbitol, and dulcitol), glycols (e.g., ethylene glycol, polyethylene glycol, propylene glycol).
• disaccharides e.g., sucrose, lactose, maltose, and trehalose
• polyols e.g., glycerol, mannitol, sorbitol, and dulcitol
• glycols e.g., ethylene glycol, polyethylene glycol, propylene glycol.
• Lyoprotectants are useful in stabilizing the components of a lyophilized formulation or composition.
• an anthracycline compound could be lyophilized with a lyoprotectant prior to reconstitution.
• Lyoprotectants are well known in the art. Accordingly, the lyoprotectants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary lyoprotectants that may be used in the formulations or compositions of the invention.
• Lyoprotectacts include, but are not limited to, solvents, surfactants, encapsulating agents, stabilizing agents, viscosity modifiers, and combinations thereof.
• Exemplary lyoprotectants may be, for example, sugars and polyols. Trehalose, sucrose, dextran, and hydroxypropyl-beta-cyclodextrin are non-limiting examples of lyoprotectants.
• Antioxidants are useful in preventing oxidation of the components of a formulation or composition. Oxidation may result in aggregation of a drug product or other detrimental effects to the purity of the drug product or its potency. Antioxidants are well known in the art. Accordingly, the antioxidants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary antioxidants that may be used in the formulations or compositions of the invention. Antioxidants may be, for example, sodium ascorbate, citrate, thiols, metabisulfite, and combinations thereof.
• Viscosity modifiers change the viscosity of liquid formulations or compositions of the invention. This may be beneficial because viscosity plays an important role in the ease with which a liquid composition is filtered.
• a reconstituted formulation of the invention may be filtered prior to lyophilization and reconstitution, or after reconstitution.
• Viscosity modifiers are well known in the art. Accordingly, the viscosity modifiers described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary viscosity modifiers that may be used in the formulations or compositions of the invention.
• Viscosity modifiers include solvents, solubilizing agents, surfactants, and encapsulating agents.
• Exemplary viscosity modifiers that may be included in compositions include, but are not limited to, N-acetyl-DL-tryptophan and N-acetyl-cysteine.
• doxorubicin is an intercalating agent as well as a topoisomerase II inhibitor, and preferentially kills rapidly dividing cells, such as tumor cells.
• DOXO-EMCH is an anthracycline compound that can be used to treat solid tumors as well as hematological malignancies. DOXO-EMCH acts by covalently binding to albumin wherein the free thiol of cysteine-34 of albumin binds the DOXO-EMCH maleimide via a Michael attack.
• DOXO-EMCH-albumin conjugate then circulates in the bloodstream until reaching a tumor, where the lower pH in the tumor results in cleavage of the hydrazone bond between doxorubicin and the EMCH moiety, thereby releasing the doxorubicin.
• the reconstituted formulations and injectable compositions of the invention may be administered for the treatment of various diseases or conditions, or used in the manufacture of a medicament for the treatment of various diseases or conditions.
• Exemplary cancers that may be treated include the following non-limiting examples: a solid tumor cancer, breast cancer, lung cancer, endometrial cancer, ovarian cancer, pancreatic cancer, cancer of the adrenal cortex, non-Hodgkin's lymphoma, multiple myeloma, leukemia, Kaposi's sarcoma, Ewing's sarcoma, soft tissue sarcoma, nephroblastoma, glioblastoma, prostate cancer, liver cancer, bone cancer, chondrosarcoma, renal cancer, bladder cancer, and gastric cancer.
• the cancer is selected from: a solid tumor cancer and a soft tissue sarcoma.
• the pancreatic cancer is pancreatic ductal adenocarcinoma.
• the injectable compositions of the invention are suitable for parenteral administration. These compositions may be administered, for example, intraperitoneally, intravenously, or intrathecally.
• a method of administering an anthracycline compound formulation or composition of the invention would depend on factors such as the age, weight, and physical condition of the patient being treated, and the disease or condition being treated. The skilled worker would, thus, be able to select a method of administration optimal for a patient on a case-by-case basis.
• a reconstituted formulation or injectable composition of the present invention may be used in the manufacture of a medicament for treating cancer.
• a reconstituted formulation of the invention can be prepared by reconstituting a lyophilized anthracycline compound composition in a reconstitution liquid comprising ethanol and water. Such reconstitution may comprise adding the reconstitution liquid and mixing, for example, by swirling or vortexing the mixture.
• the reconstituted formulation then can be made suitable for injection by mixing Lactated Ringer's solution with the formulation to create an injectable composition.
• an important factor of the reconstituted formulations and injectable compositions of the invention is the stability of the anthracycline compound present in the compositions.
• degradation of DOXO-EMCH into doxorubicin and EMCH is known to occur in solution.
• it is important to test for stability of an anthracycline compound of the invention for example, by HPLC. Stability can be measured by measuring the quantity of the anthracycline compound (e.g., DOXO-EMCH) or a degradation product thereof (e.g., doxorubicin) in a sample at two or more time points, and comparing the quantities.
• anthracycline e.g., doxorubicin
• an anthracycline compound of the invention e.g., DOXO-EMCH
• aggregation of the anthracycline compound also is indicative of instability.
• a composition is stable when the quantity of the undegraded anthracycline compound at a second time point is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the quantity of the undegraded anthracycline compound at the first time point.
• a composition is stable when the quantity of the undegraded anthracycline compound at a second time point is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the quantity of the undegraded anthracycline compound at the first time point.
• a reconstituted formulation or injectable composition is stable when stored at a temperature from about ⁇ 80° C. to about 30° C., for example when stored at a temperature from about ⁇ 80° C. to about ⁇ 70° C., from about ⁇ 25° C. to about ⁇ 15° C., from about 0° C. to about 10° C., from about 2° C. to about 8° C., from about 15° C. to about 30° C., or from about 20° C. to about 30° C.
• Exemplary temperatures include, but are not limited to, ⁇ 85.0 ⁇ 1.0° C., ⁇ 84.0 ⁇ 1.0° C., ⁇ 83.0 ⁇ 1.0° C., ⁇ 82.0 ⁇ 1.0° C., ⁇ 81.0 ⁇ 1.0° C., ⁇ 80.0 ⁇ 1.0° C., ⁇ 79.0 ⁇ 1.0° C., ⁇ 78.0 ⁇ 1.0° C., ⁇ 77.0 ⁇ 1.0° C., ⁇ 76.0 ⁇ 1.0° C., ⁇ 75.0 ⁇ 1.0° C., ⁇ 74.0 ⁇ 1.0° C., ⁇ 73.0 ⁇ 1.0° C., ⁇ 72.0 ⁇ 1.0° C., ⁇ 71.0 ⁇ 1.0° C., ⁇ 70.0 ⁇ 1.0° C., ⁇ 69.0 ⁇ 1.0° C., ⁇ 68.0 ⁇ 1.0° C., ⁇ 67.0 ⁇ 1.0° C., ⁇ 66.0 ⁇ 1.0° C., and ⁇ 65.0 ⁇ 1.0° C.
• Further exemplary temperatures include ⁇ 25.0 ⁇ 1.0° C., ⁇ 24.0 ⁇ 1.0° C., ⁇ 23.0 ⁇ 1.0° C., ⁇ 22.0 ⁇ 1.0° C., ⁇ 21.0 ⁇ 1.0° C., ⁇ 20.0 ⁇ 1.0° C., ⁇ 19.0 ⁇ 1.0° C., ⁇ 18.0 ⁇ 1.0° C., ⁇ 17.0 ⁇ 1.0° C., ⁇ 16.0 ⁇ 1.0° C., and ⁇ 15.0 ⁇ 1.0° C.
• Still further exemplary temperatures include 0.0 ⁇ 1.0° C., 0.5 ⁇ 1.0° C., 1.0 ⁇ 1.0° C., 1.5 ⁇ 1.0° C., 2.0 ⁇ 1.0° C., 2.5 ⁇ 1.0° C., 3.0 ⁇ 1.0° C., 3.5 ⁇ 1.0° C., 4.0 ⁇ 1.0° C., 4.5 ⁇ 1.0° C., 5.0 ⁇ 1.0° C., 5.5 ⁇ 1.0° C., 6.0 ⁇ 1.0° C., 6.5 ⁇ 1.0° C., 7.0 ⁇ 1.0° C., 7.5 ⁇ 1.0° C., and 8.0 ⁇ 1.0° C.
• Additional exemplary temperatures include 20.0 ⁇ 1.0° C., 21.0 ⁇ 1.0° C., 22.0 ⁇ 1.0° C., 23.0 ⁇ 1.0° C., 24.0 ⁇ 1.0° C., 25.0 ⁇ 1.0° C., 26.0 ⁇ 1.0° C., 27.0 ⁇ 1.0° C., 28.0 ⁇ 1.0° C., 29.0 ⁇ 1.0° C., and 30.0 ⁇ 1.0° C.
• a reconstituted formulation or injectable composition is stable when stored at a temperature from about ⁇ 80° C. to about 30° C., for example from about ⁇ 80° C. to about 0° C., from about ⁇ 80° C. to about ⁇ 10° C., or from about ⁇ 30° C. to about ⁇ 10° C.
• Exemplary temperatures include, but are not limited to, ⁇ 30.0 ⁇ 1.0° C., ⁇ 29.0 ⁇ 1.0° C., 28.0 ⁇ 1.0° C., ⁇ 27.0 ⁇ 1.0° C., ⁇ 26.0 ⁇ 1.0° C., ⁇ 25.0 ⁇ 1.0° C., ⁇ 24.0 ⁇ 1.0° C., ⁇ 23.0 ⁇ 1.0° C., ⁇ 22.0 ⁇ 1.0° C., ⁇ 21.0 ⁇ 1.0° C., ⁇ 20.0 ⁇ 1.0° C., ⁇ 19.0 ⁇ 1.0° C., ⁇ 18.0 ⁇ 1.0° C., ⁇ 17.0 ⁇ 1.0° C., ⁇ 16.0 ⁇ 1.0° C., ⁇ 15.0 ⁇ 1.0° C., ⁇ 14.0 ⁇ 1.0° C., ⁇ 13.0 ⁇ 1.0° C., ⁇ 12.0 ⁇ 1.0° C., ⁇ 11.0 ⁇ 1.0° C., and ⁇ 10.0 ⁇ 1.0° C.
• a reconstituted formulation or injectable composition is stable when stored at a temperature from about ⁇ 5° C. to about 10° C., or from about 2° C. to about 8° C.
• Exemplary temperatures include, but are not limited to, 2.0 ⁇ 1.0° C., 3.0 ⁇ 1.0° C., 4.0 ⁇ 1.0° C., 5.0 ⁇ 1.0° C., 6.0 ⁇ 1.0° C., 7.0 ⁇ 1.0° C., and 8.0 ⁇ 1.0° C.
• a reconstituted formulation or injectable composition is stable when stored at a temperature from about 10° C. to about 35° C., for example when stored at a temperature from about 15° C. to about 30° C., from about 18° C.
• Exemplary temperatures include, but are not limited to, about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., and about 30° C.
• a reversed-phase (RP) HPLC method for quantitation of DOXO-EMCH and various impurities and degradation products was utilized.
• Samples (1.0 ⁇ L injection volume) were assayed on an HPLC system fitted with a Phenomenex Gemini C18 column 100 ⁇ 2.0 mm, 3 ⁇ m at a flow rate of 0.3 mL/min.
• the mobile phase used was an acetate/acetonitrile gradient.
• the degradation product EMCH could not be detected using the above HPLC method, and was therefore determined using a liquid chromatography-mass spectrometry (LC-MS) method.
• LC-MS liquid chromatography-mass spectrometry
• IV bag solutions including Dextrose 5% in Water (D5W), Lactated Ringers (LR), and Sodium Chloride Solution 0.9% (Normal Saline; NS) were used.
• DOXO-EMCH active pharmaceutical ingredient (API) were dissolved in 50 mL of the IV bag solution for a concentration of ⁇ 2 mg/mL DOXO-EMCH API.
• the IV bag solutions containing API were then aliquoted in glass vials. The vials were stored at 25° C. in a stability chamber for the length of the experiment.
• Tables 8-10 below list the results for the IV bag study using 0.9% NaCl, D5W, and Lactated Ringer's Solution.
• the 0.9% NaCl and Lactated Ringer's solutions met the goal of less than 3% doxorubicin over 3-5 hours.
• the DOXO-EMCH was not completely soluble in 0.9% NaCl or Lactated Ringer's. The sample was opaque and precipitated material visibly settled over a 2-hour period. By contrast, DOXO-EMCH was completely soluble in ethanol:water and, surprisingly, went into solution quickly (e.g., within less than a minute).
• the samples were mixed well before dilution of each sample for HPLC assay, but non-homogeneity of these samples is reflected in the DOXO-EMCH recovery values for those two IV bag solutions.
• the D5W solution formed a clear red solution showing that DOXO-EMCH was completely soluble in D5W, but also had significant degradation of DOXO-EMCH and formation of doxorubicin over a 6 hour period (Table 9).
• the pH for D5W was the lowest of all the IV bag solutions tested.
• DOXO-EMCH Sterile Water for Injection
• LR Lactated Ringers Solution
• SWFI Sterile Water for Injection
• LR Lactated Ringers Solution
• SWFI 50% (v/v) ethanol/SWFI.
• the study evaluating solubility and stability of lyophilized DOXO-EMCH reconstituted in SWFI, LR, and 50% (v/v) ethanol/SWFI was conducted using a target DOXO-EMCH concentration of 10 mg/mL.
• a target DOXO-EMCH concentration 10 mg/mL.
• 20 mL of diluent was added to a vial containing 200 mg of lyophilized DOXO-EMCH, and the vial was gently swirled to dissolve the contents and obtain a homogeneous mixture.
• the sample was mixed and visually inspected for complete reconstitution to a clear solution for a) approximately 30 seconds, b) approximately 60 seconds, and c) 120 seconds, then up to a total of 10 minutes.
• the vial may have been intermittently swirled up to a maximum of 10 minutes.
• Duplicate vials were prepared for each study. Each vial was assessed 10 minutes and 20 minutes after the addition of reconstitution diluent for visual appearance of the reconstituted solution and sampled at these time points for determination of DOXO-EMCH content, doxorubicin content, and solution pH. If a given diluent was determined to provide acceptable solubilization and stability of the vial contents after 20 minutes at ambient conditions, the diluent proceeded directly to part 2 of the study.
• IV Admixture stability studies were designed to use the reconstitution diluent found suitable in part 1 following the procedure outlined below. Briefly, to prepare an IV Admixture solution containing approximately 2.4 mg/mL DOXO-EMCH, the required volume for reconstitution was withdrawn from the IV infusion solution bag intended for final administration. The required volume for reconstitution of the respective diluent was measured and transferred into a vial of lyophilized DOXO-EMCH. The vial of DOXO-EMCH and diluent was gently swirled to dissolve the vial contents and obtain a homogeneous mixture. The reconstituted solution of drug product was inspected to ensure that the drug was completely dissolved and that no foreign matter was present.
• the IV Admixture study was performed by first removing 3 vials of 200 mg lyophilized DOXO-EMCH drug product from storage at ⁇ 20° C. and allowing the vials to come to room temperature. Next, the volume required for reconstitution of three vials of DOXO-EMCH drug product (60 mL for a 10 mg/mL reconstituted drug product) was withdrawn from the infusion solution bag to be used for preparing the IV Admixture using a syringe. Each of the vials was reconstituted as described above. The required volume of the reconstitution diluent (20 mL) was drawn into an appropriate plastic syringe using a needle and injected through the stopper of each vial.
• the reconstituted vial was then swirled and visually inspected for complete reconstitution to form a clear, homogeneous solution.
• the time required to form a clear, homogeneous solution was recorded.
• the required volume (20.0 mL of a 10 mg/mL constituted solution) was transferred from each reconstituted vial into the 250 mL IV Admixture solution as described above.
• the required volume (20.0 mL of a 10 mg/mL constituted solution) was withdrawn using an appropriate plastic syringe and needle, the required volume of constituted drug product was transferred from each of three vials into the 250 mL IV Admixture using an appropriate plastic syringe and needle, and the contents of the IV Admixture were completely mixed to obtain a homogeneous mixture at a target concentration of approximately 2.4 mg/mL.
• the IV bag was weighed prior to injection of constituted drug product.
• the contents of the IV Admixture infusion bag were then analyzed at 0, 60, and 120, 150, and 240 minutes by removing 10.0 mL of the solution, diluting with chilled HPLC method diluent to achieve a target concentration of 0.24 mg/mL, and injecting into the HPLC. Duplicate injections were made for each time point. The pH of the IV Admixture and sample prepared for HPLC analysis were also measured at each time point. The empty IV bag was weighed at study completion to calculate the actual IV Admixture solution volume in each bag.
• the reconstituted solution was evaluated for color, clarity, and absence of insoluble material in the vial after every 30 seconds of mixing.
• the resulting IV Admixture was evaluated for color, clarity, and absence of any insoluble material immediately after mixing and at each analytical time point. The amount (area %) of the degradation product doxorubicin was determined at each time point.
• DOXO-EMCH was first tested for reconstitution in SWFI. Solutions appeared visually clear after mixing with SWFI for 90-120 seconds, although a few undissolved particles remained in the vial. Table 11 depicts the results of the study, which demonstrated average doxorubicin percentages of 1.54% and 1.27% for the two trials, respectively, at 20 minutes. By contrast, the average amount of doxorubicin at 20 minutes was lower (0.97% and 0.95% for two trials) when DOXO-EMCH was reconstituted in LR (Table 12). The solutions also appeared visually clear after mixing for 120 seconds. In both SWFI and LR, a peak slowly formed at RRT 1.02 (not shown); this peak has been shown to contain dimeric forms of DOXO-EMCH.
• DOXO-EMCH was tested for stability in LR after reconstitution in SWFI, LR, or 50:50 ethanol/water. Briefly, lyophilized DOXO-EMCH was reconstituted with each of the above diluents at a concentration of approximately 10 mg/mL, then further diluted in a 250 mL LR IV bag to a final concentration of approximately 2.4 mg/mL. The IV bag was maintained under ambient temperature for the duration of the study and sampled at the indicated time points. Degradation of DOXO-EMCH (measured by doxorubicin content of the solution) and pH were determined for each sample. Results for DOXO-EMCH drug product reconstituted in SWFI and diluted in LR are shown in Table 14. The doxorubicin content was about 2.3% at the two hour time point. The pH dropped by about 0.4 units (from about 6.1 to about 5.7) from zero to two hours.
• IV solutions of DOXO-EMCH in LR prepared with lyophilized DOXO-EMCH drug product reconstituted in any of the three diluents (SWFI, LR, and 50:50 ethanol/water) were relatively stable for two hours or more, based on the accumulation of the doxorubicin degradation product in the test solutions.
• the IV solution prepared using ethanol/water as reconstitution diluent showed less degradation (2.0% doxorubicin) at two hours than the solutions prepared using the other reconstitution diluents (2.3% and 2.4% doxorubicin respectively for SWFI and LR). Since the primary degradation pathway for DOXO-EMCH is cleavage of the acid-labile linker at low pH, the higher pH afforded by use of the ethanol/water diluent may explain its superior performance.

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