HK1183229B - A sustained release formulation of methotrexate as a disease-modifying antirheumatic drug (dmard) and an anti-cancer agent - Google Patents

Abstract

Disclosed is are formulations comprising a multivesicular liposome and MTX, the administration of which results in a Cmax of MTX between 5% and 50% of the Cmax of an immediate release dosage form of MTX, the duration of which lasts from about 1 to about 30 days. Also disclosed are methods of treating autoimmune diseases and cancer by administering these formulations of MTX.

Description

Sustained release formulations of methotrexate as Disease Modifying Antirheumatic (DMARD) and anticancer agents

RELATED APPLICATIONS

Priority OF U.S. provisional patent application serial No. 61/334,546 entitled "SUSTAINED RELEASE EFFECTIVERATION OF METHERATE ASTRATE AS A DISEASE-MODIFYING ANTIREMATIC DRUG (DMARD) AND AN ANTI-CANCER AGENT (SUSTAINED release formulation OF METHOTREXATE as a disease MODIFYING antirheumatic DRUG (DMARD) and an anticancer agent)" filed on 13.5.2010 by Garcia et al, the entire contents OF which are incorporated herein by reference, and U.S. priority OF U.S. application serial No. 12/914,944 entitled "SUSTAINED RELEASE ORLATION OF METHOTRETEE AS A DISEASE-MODIFYING ANTIREMATIC DRUG (DMA) AND AN ANTI-CANCER AGENT (SUSTAINED release formulation OF METHOTREXATE as a disease MODIFYING antirheumatic DRUG (DMARD) and an anticancer agent)" filed on 28.10.2010 by Garcia et al, the entire contents OF which are incorporated herein by reference.

Technical Field

The present application relates to multivesicular liposome (MVL) formulations of Methotrexate (MTX) that minimize the side effects of MTX while maintaining or improving efficacy.

Background

MTX is the major disease-modifying antirheumatic drug (disease-modifying antirheumatic drug) (DMARD) for Rheumatoid Arthritis (RA) and has demonstrated efficacy in other autoimmune diseases. MTX has also been used to treat certain cancers and cancer disorders. Cancer is a leading cause of death in the united states. Despite the tremendous efforts to find new ways to treat cancer, primary treatment options remain surgery, chemotherapy, and radiation therapy, alone or in combination.

Over 500,000 patients worldwide were treated with immediate release MTX formulations (Sweierkot and szechinski, Pharmacol Reports,58,473-. Since MTX causes significant plasma concentration-related side effects (nausea, vomiting, abdominal discomfort, taste disturbances, anorexia, dyspepsia, and diarrhea) after each administration, it is usually performed once a week on saturday, allowing the patient to recover through the working week. The side effects are mainly caused by high C_maxCaused by (C)_maxIs a pharmacokinetic term that refers to the maximum plasma concentration of a drug. At doses that are generally effective, up to 80% of patients experience these side effects, and up to 35% of patients eventually discontinue use of MTX, even though it provides excellent efficacy in most patients (see r.j. mckendry et al, J Rheumatol20:1850 (1993)). Although many side effects of MTXThe use appears to be gastrointestinal, but some of them (e.g. nausea and vomiting) can be Central Nervous System (CNS) side effects, so subcutaneous rather than oral administration does not necessarily ameliorate the side effects.

Recent recommendations for the use of MTX in RA suggest that oral administration of MTX should begin at 10-15 mg/week with a step up to 20-30 mg/week. It is known that oral administration of high doses (> 15 mg) results in lower bioavailability than subcutaneous administration. This is due to the active absorption of MTX in the small intestine, which process can saturate at higher doses (Stamp et al, Biomed Pharmacother 60678-687 (2006)). Thus, parenteral administration may be used in situations where an inappropriate clinical response or oral administration is not tolerated.

For RA, the efficacy of MTX is independent of the maximum plasma concentration (Cmax) of MTX, but directly related to its plasma area under the curve ("AUC") (Hiraga et al, Mod rheumato 14:135 (2004)). The AUC of MTX correlates well with the intracellular levels of MTX-polyglutamate, the latter of which is believed to provide anti-inflammatory effects (Hornung et al, J.Rheumatotol, 35,1709-1715 (2008)). The standard AUC for an oral immediate release dosage form of MTX is 2466 mcg/L-hr (Hoekstra et al, J Rheumatotol, 31645-48 (2004)). The standard AUC for a subcutaneous immediate release dosage form of MTX was 3786 mcg/L-hr. The same document (Id.)

Because orally administered MTX is rapidly absorbed (t)_max1-3 hours) and rapidly cleared (t)_1/28-10 hours), it is difficult to control C with the conventional dosage form_max。t_maxIs the time when the maximum plasma concentration is reached after administration of the drug, i.e., when the rate of absorption equals the rate of clearance. Buccal and subcutaneous administration of C with MTX in a conventional immediate release dosage form_maxSimilarly (Hoekstra et al, J Rheumatotol, 31645-.

Importantly, it has been observed that MTX-preserving C_maxLow results in a reduction of the incidence of side effects of MTX to extremely low levels. It has been found that if C is_maxIf the concentration is kept below 0.16. mu. mol/l, the incidence of side effects is significantly reduced(Shoda et al, Mod. Rheumatotol, 17:311-316, 2007). Providing MTX low C_maxIn addition to therapeutically effective AUC (plasma concentration) over an extended period of time, the sustained release formulations of (a) will provide important benefits.

Embodiments of the present application provide these important benefits. Fast dissolving (instant) MTX MVL formulations provide formulations with low C_maxThe sustained release formulation of MTX and therapeutically effective AUC of (a) which reduces the incidence of side effects to extremely low levels.

The fast dissolving MTX MVL formulation also provided the AUC necessary to maintain the efficacy of MTX. Thus, the sustained release MTX MVL formulations described in this application achieve the AUC necessary for the onset of DMARD by MTX while providing a sufficiently low C_maxSo as to reduce the incidence of side effects of MTX use to very low levels.

In particular, MVL for delivery of MTX is defined herein as liposomes containing multiple non-concentric cavities within each liposome particle, similar to a "foamy" matrix. Such particles, unlike multilamellar vesicles (MLVs), also known as multilamellar liposomes, enclose (surround, enclose) concentric aqueous compartments. Another unique particle is a unilamellar vesicle (ULV), also known as a unilamellar liposome, which encloses a single internal aqueous compartment.

Disclosure of Invention

Embodiments of the present invention provide formulations comprising multivesicular liposomes (multivesicular liposomes) comprising an amount of methotrexate, wherein administration of a single dose of the formulation to a subject in need thereof results in the C of the methotrexate_maxC in immediate release form of methotrexate_maxAnd wherein the duration of said methotrexate in the subject is from about 1 to about 30 days. In some embodiments, the multivesicular liposome further comprises a salt of methotrexate. In some embodiments, the salt is a sodium salt. In other embodiments, the salt is a potassium salt. In further embodiments, the salt comprisesOrganic bases are used as counter ions (counterions). In some embodiments, the duration of methotrexate in a subject is from about 1 day to about 7 days.

In other embodiments, the multivesicular liposomes further comprise folate (folic acid). In some embodiments, the multivesicular liposome further comprises cholesterol, one or more phospholipids or one or more salts thereof, and one or more triglycerides. In certain embodiments, the phospholipid is a phosphatidylcholine, a phosphatidylglycerol or salt thereof, or a combination of these. In other embodiments, the phosphatidylglycerol is DPPG (dipalmitoylphosphatidylglycerol or 1, 2-dipalmitoyl-sn-glycero-3-phosphoryl-rac- (1-glycerol)). In a further embodiment, the phosphatidyl choline is DEPC (erucylphosphatidyl choline or 1, 2-erucyl-sn-glycero-3-phosphocholine (phosphocholine)). In other embodiments, the multivesicular liposome further comprises DOPC (dioleoylphosphocholine or 1, 2-dioleoyl-sn-glycero-3-phosphocholine). In another embodiment, the triglyceride is triolein (triolein), tricaprylin, or a combination of the two. In additional embodiments, the multivesicular liposome further comprises lysine.

In some embodiments, the duration of the methotrexate plasma levels is about 1 day. In other embodiments, the duration of the methotrexate plasma levels is about 2 days. In another embodiment, the duration of the methotrexate plasma levels is about 3 days. In another embodiment, the duration of the methotrexate plasma levels is about 4 days. In another embodiment, the duration of the methotrexate plasma levels is about 5 days. In another embodiment, the duration of the methotrexate plasma levels is about 6 days. In further embodiments, the duration of the methotrexate plasma levels is about 7 days.

In another embodiment, the formulation is stable for at least two years.

In some embodiments, the formulation may be delivered by a needle gauge of 27-31G.

Another embodiment provides a method for treating an autoimmune disease comprising administering to a subject in need thereof a fast-dissolving (i.e., instant) MTX-MVL formulation. In some embodiments, the autoimmune disease is rheumatoid arthritis. In other embodiments, the autoimmune disease is psoriasis. In another embodiment, the autoimmune disease is lupus. In another embodiment, the autoimmune disease is scleroderma. In another embodiment, the autoimmune disease is Sjogren's syndrome. In other embodiments, the autoimmune disease is Goodpasture's syndrome. In some embodiments, the autoimmune disease is Wegener's granulomatosis. In another embodiment, the autoimmune disease is polymyalgia rheumatica. In another embodiment, the autoimmune disease is Guillain-Barre syndrome (Guillain-Barre syndrome). In additional embodiments, the autoimmune disease is crohn's disease.

In some embodiments, the multivesicular liposome further comprises a sodium or potassium salt of methotrexate. In additional embodiments, the multivesicular liposome further comprises lysine.

In some embodiments, the multivesicular liposome further comprises a folate. In other embodiments, the folate is administered. In further embodiments, folate encapsulated in MVL without MTX is administered.

Also provided is a method, the multivesicular liposome further comprising cholesterol, a phospholipid or one or more salts thereof, and tricaprylin. In some embodiments, the multivesicular liposome further comprises DOPC and triolein. In additional embodiments, the multivesicular liposomes further comprise DEPC. In other embodiments, the multivesicular liposome further comprises lysine.

In some embodiments, the duration of the methotrexate plasma levels is about 1 day. In other embodiments, the duration of the methotrexate plasma levels is about 2 days. In another embodiment, the duration of the methotrexate plasma levels is about 3 days. In another embodiment, the duration of the methotrexate plasma levels is about 4 days. In another embodiment, the duration of the methotrexate plasma levels is about 5 days. In another embodiment, the duration of the methotrexate plasma levels is about 6 days. In further embodiments, the duration of the methotrexate plasma levels is about 7 days.

In other embodiments, administration of the formulation comprises injecting the subject with the MTX-MVL formulation described herein. In some embodiments, the injection is subcutaneous. In other embodiments, the injection is intramuscular. In some embodiments, the injection is intradermal. In further embodiments, the injection is intratumoral. In other embodiments, the injection is intraspinal. In one embodiment, the injection is administered every 5 to 7 days. In another embodiment, the injection is administered once a week.

Another embodiment provides a method for treating cancer comprising administering a fast-dissolving MTX-MVL formulation, wherein the formulation is administered no more than every two days and no less than every four days to a subject in need thereof. In some embodiments, the cancer is a solid tumor cancer. In other embodiments, the cancer is a gestational trophoblastic tumor, breast cancer, head and neck cancer, or lung cancer. In additional embodiments, the cancer is a gestational trophoblastic tumor (malignant hydodena destruens), choriocarcinoma, or hydatidiform mole). In some embodiments, the cancer is a hematologic cancer. In additional embodiments, the hematologic cancer is acute lymphoblastic leukemia, non-hodgkin's lymphoma, mycosis fungoides, or osteosarcoma.

In some embodiments, the multivesicular liposome further comprises a folate. In other embodiments, the folate is administered. In additional embodiments, folate encapsulated in MVL without MTX is administered.

In some embodiments, the multivesicular liposome further comprises a sodium or potassium salt of methotrexate. In other embodiments, the multivesicular liposome further comprises cholesterol, a phospholipid or one or more salts thereof, and tricaprylin. In additional embodiments, the multivesicular liposome further comprises DOPC. In another embodiment, the multivesicular liposome further comprises lysine.

In some embodiments, administration of the formulation comprises injecting the formulation into the subject. In other embodiments, the injection is subcutaneous. In another embodiment, the injection is given every 2 days.

Another embodiment provides a fast dissolving MTX-MVL formulation that is substantially free of cyclodextrin compounds, and preferably free of cyclodextrin compounds.

Drawings

Figure 1 is a graph showing plasma concentrations following subcutaneous injection of an example of MTX control solution and a fast-dissolving MVL MTX formulation.

Fig. 2 is a variation of fig. 1 in which the y-axis is varied, showing plasma concentrations following subcutaneous injection of an example of MTX control solution and a fast-dissolving MVLMTX formulation.

Figure 3 is a graph showing the percentage of degradants of formulation 1 over time.

Detailed Description

Embodiments of the present invention provide formulations comprising multivesicular liposomes containing a substantial amount of methotrexate, wherein administration of a single dose of said formulation to a subject in need thereof results in C of said methotrexate_maxIn immediate release dosage forms of methotrexate C_maxAnd wherein the duration of said methotrexate in the subject is from about 1 day to 50%For about 30 days.

Embodiments of the invention also provide methods for treating an autoimmune disease comprising administering to a subject in need thereof a formulation described herein.

Embodiments of the present invention further provide methods for treating cancer comprising administering a formulation described herein, wherein the formulation is administered to a subject in need thereof no more than every 2 days and no less than every 4 days.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar to those described herein can be used in the practice or testing of the present application, suitable methods and materials are described below. All publications, patent applications, patents, and other documents mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

In particular, the term AUC refers to the plasma area under the curve of the plasma concentration versus time plot; term C_maxIs a pharmacokinetic term and refers to the maximum plasma concentration of the drug; term t_maxMeans to arrive at C_maxThe time of (d); term C_maxAssociated side effects refer to side effects associated with plasma concentrations, which include, but are not limited to, nausea, vomiting, abdominal discomfort, taste disturbances, anorexia, dyspepsia, and diarrhea; the term MTX refers to methotrexate and its salts and lysates as discussed below; term t_1/2Is the time it takes for plasma to clear half of the MTX; the term MVL refers to multivesicular liposomes; the term DMARD refers to disease-modifying antirheumatic drugs; the term "duration of MTX plasma levels" refers to the time between administration and the cumulative plasma AUC reaching 90% of the AUC of the cumulative AUC over an infinite time period; the term "immediate release dosage form" refers to a dosage form that does not contain a substance that delays the release of the drug; the term "drug holiday" is when a drug is administered to a patient to substantially eliminate the patient's plasmaThe term "cyclodextrin compound" refers to one or more compounds selected from the group consisting of α, β, and gamma cyclodextrin or derivatives thereof one of these derivatives includes 2-hydroxypropyl- β -cyclodextrin, the other derivatives include sulfobutyl ether α -, β -, or gamma-cyclodextrin.

Autoimmune diseases result from the body's overactive immune response to substances and tissues that are normally present in the body. The immune system mishandles a certain part of the body as a pathogen and attacks it. This may be limited to certain organs or to specific tissues involving different locations. Typically, treatment of autoimmune diseases is performed in conjunction with immunosuppressive drug therapy that reduces the immune response. The term "autoimmune disease" as used herein includes, but is not limited to, crohn's disease, dermatomyositis, type 1 diabetes, goodpasture's syndrome, graves 'disease, guillain barre syndrome, hashimoto's disease, idiopathic thrombocytopenic purpura, lupus, mixed connective tissue disorders, myasthenia gravis, narcolepsy, pemphigus vulgaris, pernicious anemia, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, sjogren's syndrome, temporal arteritis, ulcerative colitis, vasculitis, and wegener's granulomatosis.

One embodiment of the present application provides MTX MVL formulations comprising MTX and salts thereof for the treatment of autoimmune diseases, particularly RA and psoriasis. Fast dissolving MTX MVL formulations have immediate release dosage form C typical at MTX_maxBetween 5% and 50% of C of MTX_max. In immediate release dosage forms, MTX has a very high and very early C_maxAnd then rapidly drops below the limit of quantitation ("LOQ") within 24 hours. As discussed above, administration of the fast-dissolving (ready-to-use) MTX MVL formulation maintains the C of MTX_maxLower, it reduces the incidence of side effects of MTX use to very low levels. For the treatment of autoimmune diseases, C is preferred_maxIt is maintained at 0.16. mu. mol/L or less than 0.16. mu. mol/L. The instant MTX MVL formulations of the present application provide a sustained AUC at therapeutic concentrations of MTX,wherein the duration is from about 1 day to about 30 days. Preferably, the duration is from about 1 to about 7 days. For the treatment of autoimmune diseases, the duration is preferably 1 to 5 days.

MTX has also been used to treat certain cancers and cancer disorders. The instant MTX MVL formulations can be used to treat solid tumor cancers. As discussed herein, these cancers include, but are not limited to, certain types of gestational trophoblastic tumors, including malignant hydatidiform mole, choriocarcinoma, and hydatidiform mole, as well as breast, head and neck, and lung cancers. The instant MTX MVL formulations can also be used to treat hematogenous cancer. As discussed herein, these cancers include, but are not limited to, acute lymphoblastic leukemia, non-hodgkin's lymphoma, mycosis fungoides, and osteosarcoma. The instant MTX MVL formulations can also be used for direct delivery to a tumor (e.g., intraspinal delivery to cerebrospinal fluid or by intratumoral delivery). In some embodiments, the formulations and methods of the present embodiments relate to the treatment of meningeal leukemia by intrathecal injection.

As discussed above, for the treatment of autoimmune diseases, the MTX MVL formulations of the present application have a C in an immediate release dosage form of MTX_maxBetween 5% and 50% of plasma C_max. For systemic treatment of cancer, significantly higher C is often used relative to autoimmune diseases_max. However, administration in an immediate release dosage form of MTX reduces C relative to administration of the same dose of MTX_maxReduced side effects can be achieved. For systemic treatment of cancer, the MTX MVL formulations of the present application also provide a sustained exposure to the therapeutic AUC of MTX, wherein the duration is from about 1 to about 30 days. In some embodiments, the duration is from about 1 to about 7 days. Some cancers can be treated locally. For example, meningeal leukemia is treated with 1mg/mL solution by intrathecal injection of MTX at a dose of 12 mg/square meter (15 mg maximum) every 2-5 days. Another example would be one or more tumors that can receive intratumoral injections. In these topical therapies, plasma concentrations are irrelevant. Rather, local concentration is important. The instant MVL MTX formulations are useful in these topical treatments as they provide sustained local levelsA medicine is provided. This provides the necessary efficacy and reduces the number of injections, thus providing patient and caregiver convenience.

For autoimmune diseases, in addition to providing C_maxPlasma C as immediate release dosage form of MTX_maxBetween 5% and 50% of MTX MVL formulation, unexpectedly, the instant MTX MVL formulation provides the same or better efficacy than the immediate release dosage form at the same MTX dose. Potency of MTX and C of MTX_maxRegardless of whether it is directly related to AUC. Thus, the MVL formulations of the present application have AUC comparable to or better than the immediate release dosage form of MTX as described above. Preferably, the fast dissolving MTX MVL formulation provides the same AUC as a subcutaneous immediate release dosage form of MTX. The AUC may be greater if the reduced side effects allow for higher doses. In addition, the instant formulations and methods of treatment using the formulations may be substantially free of (no) cyclodextrin, and preferably free of cyclodextrin. Unexpectedly, the instant formulations and methods have achieved the advantageous effects described herein without the use of cyclodextrin compounds. For example, the fast dissolving formulations and methods achieve long durations without the MTX being complexed with the cyclodextrin compound.

In addition, the instant formulations and methods of treatment using the formulations can be substantially free of cyclodextrin, and preferably free of cyclodextrin. Kim et al (Cancer Chemother. Pharmacol.33:303-306 (1994); U.S. Pat. No. 5,759,573) have previously reported the release of MTX from MVL formulations containing MTX complexed with hydroxypropyl- β -cyclodextrin following subcutaneous administration to rodents. The formation of the inclusion complex (see col.4: 27-34; col.5: 14-19) results in a decrease in the release rate. Unexpectedly, the instant formulations and methods have achieved the advantageous effects described herein without the use of cyclodextrin compounds. For example, the fast dissolving formulations and methods achieve long duration times without the MTX being complexed with the cyclodextrin compound.

Methotrexate (MTX)

Embodiments of the present invention utilize MTX and its salts, hydrates, and solutes, whether crystalline or amorphous, encapsulated in MVLs. Such salts include sodium, potassium, lysine, arginine, and other pharmaceutically acceptable mono-or divalent base addition salts. The term "MTX" includes any and all such forms as used herein. The instant MTX MVL formulations and methods exhibit their desirable properties without the need for MTX to complex with cyclodextrins or derivatives thereof.

Multivesicular liposomes

The formulations of the present embodiments employ MTX-encapsulated multivesicular liposomes (MTX-MVL) which encapsulate and provide the above-described modulated and sustained release of MTX. MVL was prepared by the following method.

A "water-in-oil" emulsion containing MTX is formed from two immiscible phases, a lipid phase and a first aqueous phase. The lipid phase is composed of at least one amphiphilic lipid and at least one neutral lipid in a volatile organic solvent. The term "amphipathic lipid" refers to a molecule having a hydrophilic "head" group and a hydrophobic "tail" group, and may have the ability to form a membrane. As used herein, amphiphilic lipids include those having a net negative charge, a net positive charge, and zwitterionic lipids (no net charge at their isoelectric point). The term "neutral lipid" refers to an oil or fat that does not have the ability to form vesicles itself, and lacks charged or hydrophilic "head" groups. Examples of neutral lipids include, but are not limited to, glycerol esters, ethylene glycol esters, tocopherol esters, sterol esters lacking a charged or hydrophilic "head" group, and alkanes and squalene.

The amphiphilic lipid is selected from various lipids having a hydrophobic region and a hydrophilic region in the same molecule. Suitable amphiphilic lipids are zwitterionic phospholipids, including phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, and lysophosphatidylcholine. Also suitable are anionic amphiphilic phospholipids, such as phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and cardiolipin. Also suitable are cationic amphiphilic lipids such as acyltrimethylammonium propanes, diacyldimethylammonium propanes, stearamides, and the like. Preferred amphipathic lipids include Dioleoylphosphatidylcholine (DOPC), erucylphosphatidylcholine (DEPC), and Dipalmitoylphosphatidylglycerol (DPPG). Certain embodiments of the amphiphilic lipids of the instant MTX MVL for use in the treatment of autoimmune diseases such as rheumatoid arthritis, as well as cancer, include DOPC and DEPC in combination with DPPG.

Suitable neutral lipids are triglycerides, propylene glycol esters, ethylene glycol esters, and squalene. Examples of triglycerides useful in the instant formulations and methods are Triolein (TO), tripalmitin, trimyristin, trilinolein, tributyrin, trihexanoic acid, tricaprylin, and tricaprin. The fatty chains in the triglycerides useful in this application may be identical, or not (mixed chain triglycerides), including completely different. The propylene glycol esters can each be mixed diesters of caprylic acid and capric acid. In particular, neutral lipids, e.g., triolein, can be increased to a percentage range of about 0.01% to about 20% in order to reduce C_maxThe advantages offered by these formulations, particularly in the treatment of autoimmune diseases, are still maintained. In particular, the percentage of triolein ranges from about 1% to 2% of the neutral lipid used in the instant formulation, such as tricaprylin, taking into account the molar ratio. In addition, cholesterol or phytosterols are contained in the fast dissolving MVLs.

Many types of volatile organic solvents may be used in this application, including ethers, esters, halogenated ethers, hydrocarbons, halogenated hydrocarbons, or freons. For example, diethyl ether, chloroform, dichloromethane, tetrahydrofuran, ethyl acetate, and any combination thereof are suitable for use in preparing the formulation.

Optionally, but highly desirably, other components are contained in the lipid phase. Among these are antioxidants, antimicrobial preservatives, cholesterol or phytosterols.

The first aqueous phase comprises MTX, a base, and an osmotic agent (e.g., sodium chloride, sucrose, glucose, fructose, or a mixture thereof). The lipid phase and the first aqueous phase are mixed by mechanical agitation, such as by using a rotating or vibrating knife, shaking, extrusion through a baffle structure or perforated tube, or by sonication to produce a water-in-oil emulsion. Thus, the MTX of the present application is directly included in the first step of MVL production.

The water-in-oil emulsion is then dispersed into the second aqueous phase by the method described above to form solvent globules suspended in the second aqueous phase to form a water-in-oil-in-water emulsion. The term "solvent globule" refers to a microscopic spherical droplet of organic solvent in which a plurality of smaller droplets of aqueous solution are suspended. Thus, the resulting solvent pellet contains a plurality of aqueous droplets in which the MTX is dissolved. The second aqueous phase may contain additional components such as glucose, sucrose, and/or lysine.

The volatile organic solvent is then removed from the pellet, for example by surface evaporation of the suspension. When the solvent is substantially or completely evaporated, MVL is formed. Gases that may be used for evaporation include nitrogen, argon, helium, oxygen, hydrogen, and carbon dioxide. Alternatively, the volatile solvent may be removed by sparging, rotary evaporation, diafiltration, or by means of a solvent selective membrane.

In some embodiments, the range of lipids in the fast-dissolving MTX MVL formulation may be as follows: cholesterol may be from about 5 to about 80 mM; the phosphatidylcholine can be from about 5 to about 50 mM; the tricaprylin may be from about 5 to about 80 mM; the triolein can be from about 0 to about 8 mM; and phosphatidylglycerol may be from about 0 to about 15 mM. DPPG is optional, as other charged lipids are used. For the treatment of cancer, a longer duration than RA is preferred. This can be achieved by increasing the amount of triglycerides, by increasing the ratio of long chain to short chain triglycerides, by using longer chain phosphatidylcholines or by increasing the cholesterol concentration.

Methods for preparing instant MVL formulations can also be found in U.S. publication No. 2007-0235889 to Hartouian et al, and WO99/25319 to Hartouian et al (PCT/US 98/24261), the entire contents of which are incorporated herein by reference.

Folate (Folic acid)

In further embodiments, folic acid, folinic acid (leucovorin), active enantiomers of either, or salts thereof (collectively referred to as folate) may be used in combination with MTX to reduce MTX-related side effects, particularly in view of the gastrointestinal tract, liver, and blood cells (Prey and Paul, Brit j. dermalto, 160: 622-. The dose and dosing frequency of the folate can vary from about 1 to about 27.5 mg/week (Oritz et al, j. rheumatol,25(1998) 36-43)). Typical doses may be from about 2.5 to about 5 mg/week or from about 1 to about 2 mg/day (Hoekstra et al, Ann Rheumdis,62:423-426 (2003)).

Fast dissolving MTX-MVL formulations can use folate to reduce C_maxAssociated side effects such as nausea and vomiting using the sustained release properties of MVL, and side effects that can be reduced by folate. The folate may be included in the fast dissolving formulation, for example, in one or more of the following ways. First, the folate can be in an external solution (non-encapsulated). Second, folic acid can be contained in the same first aqueous solution as MTX and, thus, will be released at about the same rate as MTX after administration. Third, folate and MTX can be formulated in heterovesicular liposomes (see Kim, U.S. patent No. 5,422,120,1995, 6.6). Fourth, folate and MTX can be contained in separate populations of MVL particles. Finally, folate and MTX can be included in any of the above combinations.

In a fourth case, these individual populations (formulations) may have the same lipid composition providing substantially the same release rate, or they may have different lipid compositions providing different release rates, in order to optimize the side effects, insufficient effects, of folate. The separate populations may be combined in one container, separate containers, or two-chamber containers to mix the two just prior to or during administration. In the case of two separate containers, the two are administered at the same or different times.

Methods of administration

The MTX formulations discussed above may be administered weekly by injection, more particularly by subcutaneous, intradermal, or intramuscular injection, based on systemic use including treatment of autoimmune diseases such as RA. In the treatment of cancer, they may also be delivered locally (e.g., intraspinally or intratumorally). For RA, the term "therapeutically effective" when applied to the compositions herein means that MTX present in the first aqueous phase within the MVL is released in a manner sufficient to achieve an effective AUC for MTX, such an AUC level being about 1000-. Higher levels will be used for systemic treatment of cancer. When local delivery is used to treat cancer, the amount delivered can be significantly reduced. The precise dosage will vary depending on patient factors such as age, sex, general condition, etc. These factors can be readily taken into account by those skilled in the art and used to establish effective therapeutic concentrations without undue experimentation.

For treatment of autoimmune diseases, it is preferred that the fast dissolving MTX MVL formulation will be injected subcutaneously 1 time per week. In one embodiment, the duration of MTX is from about 1 to about 5 days. Thus, the fast-dissolving MTX MVL formulation provided about 2 days of drug holiday. This holiday reduces the side effects associated with MTX.

For the treatment of cancer, no more than a fast-dissolving MTX MVL formulation will be injected every 2 days, and in another embodiment, every 7 days subcutaneously. The treatment will be repeated for several courses. In order to reduce side effects due to the use of large doses in cancer, patients may also be given "leucovorin rescue" (Link MP, Goorin AM, Miser AW, et al.; N.Eng.J.of Med.1986;314(No.25): 1600-1606), or allowed a 7-10 day rest period between treatments. However, due to the low C with MVL_maxThe release profile will reduce the need for leucovorin or resting period.

For the treatment of cancer, in adults, the conversion to 37mg/m for a body weight of 100mg/kg is²Giving a human dosage form guidance ratio of 1: 30. In fact, depending on age and size, the conversion factor varies between 1:20 and 1: 40.

For the treatment of autoimmune diseases such as RA, new patients will receive a dose of a fast dissolving MTX MVL formulation with a total amount of MTX of 2-5mg, titrated to 2-5mg per week up to 25-35mg per week. Such administration is useful to provide delivery of MTX over a period of several hours to several days, for example, up to about 5 days. Such MTX MVL formulations have reduced side effects, as listed above, relative to immediate release injectable or oral formulations, maintain AUC for subcutaneous injection of immediate release MTX formulations, and have increased C relative to current immediate release oral MTX administration_maxEqual or better efficacy as judged by the American College of Rheumatology rating system. See Felson et al, Arthritis&Rheumatism,Vol.38,No.6,June 1995。

For the treatment of cancer, MVL encapsulated MTX formulations are administered in the same manner as autoimmune diseases, but at higher doses. Again, based on systemic use in cancer treatment, the MTX formulations discussed above may be administered weekly by injection, more particularly by subcutaneous, intradermal, or intramuscular injection. They may also be delivered locally (e.g., intraspinally or intratumorally).

Non-limiting disclosure and incorporation by reference

While certain therapeutic agents, compositions and methods have been specifically described in accordance with certain embodiments, the following examples are intended to be illustrative of the compositions and methods of the present invention, and are not intended to be limiting thereof. Each reference, etc., cited in this application is hereby incorporated by reference in its entirety.

Examples

The fast-dissolving MVL MTX formulation was prepared in a manner similar to that reported by Kim et al (Biochim Biophys Acta,728(1983) 339-348). An aqueous solution containing MTX, sodium hydroxide and sucrose is emulsified with a chloroform solution containing DOPC or DEPC, DPPG, tricaprylin and/or triolein, and cholesterol, resulting in a water-in-oil (W/O) emulsion. The W/O emulsion is then emulsified in a second aqueous solution containing lysine and sucrose to produce a W/O/W emulsion. The W/O/W emulsion was then stirred at 37 ℃ under a nitrogen stream to remove the chloroform by evaporation. The resulting pellet was centrifuged, and the supernatant was replaced with physiological saline. After washing, the particles were diluted into physiological saline to obtain a product of about 50% of the bulk particle volume (PPV). PPV is the fraction of MVL particles by volume of the total formulation.

The compositions of the solutions used to prepare the four formulations by the above method are listed in table 1 below. The resulting particles had an intermediate particle size (mean diameters) of 13-15 microns and a potency of about 25 mg/mL.

Particle size distribution, d as used in Table 1 below₁₀Means that less than 10% of the particles have the diameter defined in the table. d₅₀Meaning that less than 50% of the particles have the diameter defined in the table. d₉₀Means that less than 90% of the particles have the diameter defined in the table. "span" is defined as the passage of d₁₀Minus d₉₀In combination with d₅₀Divided by the result.

As used in table 1, "Free% (% Free)" refers to the percentage of MTX outside the MVL, and PPV refers to the bulk particle volume.

TABLE 1

The final composition of formulation 1 will have a final composition approximately as given in the table below.

TABLE 2

Components	Preparation 1
		Methotrexate API	55.0mM（25mg/mL）
DOPC	14.52mM
		DPPG	3.08mM
Cholesterol	22mM
		Tricaprylin	22mM
NaOH	Adjusted to pH 7.4 (about 131 mM)
		Sucrose	38.7mM
NaCl	69.3mM

A Pharmacokinetic (PK) study was performed in rats comparing four different formulations of MVL encapsulated MTX to a dose level of 10mg/kg for MTX solution control. Male Sprague Langlie rats weighing 280-320g were injected subcutaneously with one of MTX solution or MVL encapsulated MTX formulation. Injections were made into the left hind limb of each rat using a 1mL syringe fitted with a 25G needle. Each treatment group contained 3 rats. Assuming an average weight of 300g for the rats, appropriate dilution with sterile saline solution was performed to provide MTX at a dose of 10mg/kg per rat. Rats were numbered by predose weight and then randomized into different treatment groups.

Liquid-liquid extraction (LLE) HPLC was performed using the Agilent1200Series system produced by Agilent Technologies, Inc., Santa Clara, Calif., at headquarters, and serum MTX analysis was performed to determine MTX in serum. A volume of 100. mu.L of rat serum was mixed with 300. mu.L of acetonitrile containing 1.1% formic acid (w/v) in a 1.5mL Eppendorf tube. Deproteinization was performed by vortexing for 1 min, followed by centrifugation at 13,000g for 5 min. All supernatants were transferred to 1.5mL microcentrifuge tubes with screw caps. Acetonitrile was removed by mixing 500 μ L of chloroform with the supernatant. The tube was vortexed for 1 minute and then centrifuged at 13,000g for 3 minutes. The 40 u L volume of the upper aqueous portion collected into 100 u L vial insert for HPLC sample injection.

The HPLC system was equipped with a detection device at 302nm and 2.1mmX250mmSupelcosil LC-18-S with a 5 μm particle size column. The mobile phase consisted of 102mM sodium phosphate (dibasic), 32.5mM citric acid, and 10% (v/v) acetonitrile (pH about 6.0). The flow rate was 0.6mL/min, the column temperature was 35 ℃ and the injection volume was 20. mu.L. MTX standard curves were prepared by adding 6 different concentrations of MTX (22.7-727.1 ng/mL) to rat serum containing no MTX. The same sample preparation method above was used to extract MTX in each added standard.

As shown in FIG. 1, the methotrexate solution control reached very high and very early plasma C_maxE.g., rapidly falling below LOQ within 24 hours. For clarity, FIG. 2 shows the same data with the y-axis scale changed, with the MTX solution truncatedC of (A)_max. All groups receiving MVL encapsulated MTX formulations successfully demonstrated sustained release. That is, unexpectedly, they result in an acceptable duration for reducing side effects, and an acceptable C_max. These PK results demonstrate that the release rate can be modulated by varying the lipid composition of MVL encapsulated MTX formulations. Thus, alteration of the lipid composition of the fast dissolving formulation may include altering the total amount of amphipathic lipids as well as neutral lipids, addition or removal of specific lipids (e.g., addition of TO), and replacement of one lipid for another (e.g., DEPC versus DOPC). Examples of how triglyceride levels can be varied are provided in Willis, U.S. patent No. 5,891,467, published 6.4.1999. Willis does not necessarily foreshadow the advantages of this embodiment. It has been previously shown that increasing the chain length of phosphatidylcholine provides increased potency of the encapsulated compound (see Ye et al, U.S. patent No. 6,171,613, published 2001, 1/9). Ye does not indicate the advantages of the present embodiment. For MTX, the data in fig. 1 and 2 demonstrate that increasing the chain length improves the duration.

Figures 1 and 2 show that formulation 1 released most of the drug and the maximum serum concentration (C) within the first 48 hours_max) About 1.5 umol/L. Reducing the amount of lipid in the MVL formulation by half, formulation 4 gave a similar release profile, except that it shifted to the left, giving a shorter duration and an earlier arrival at C_maxTime of (d). Addition of 2% TO TO formulation 3 significantly reduced C_maxAnd greatly extends the duration. Replacing DOPC of formulation 1 with DEPC of formulation 2 gave similar and even more profound effects, resulting in a release profile with about zero order release rate. By using these or similar methods, the PK profile obtained with the fast-dissolving MVL MTX formulation can be altered.

Additional PK studies were performed as above for formulations 1 and 2, including some studies at reduced dose MTX. PK parameters from these studies are summarized below. As can be seen, C is relative to MTX immediate release solution_maxIs significantly reduced. C_avgIs the mean plasma concentration over a period of 7 days. Thus, unexpectedly, the formulation has a remarkable effectLong duration and low C_maxThus, side effects will be low.

Table 3 additional PK studies for formulations 1 and 2

Stability of

In addition, the stability of the instant MTX MVL formulations is available to industry standards. Stability data for formulation 1 of runs 1-3 are shown in tables 4-6 below. The properties evaluated include drug content, percent unencapsulated drug (free%), material associated with the RP-HPLC method of the pharmacopoeia, bulk particle volume (PPV) evaluated in a manner similar to a hematocrit meter, appearance of visual evaluation, and particle size evaluated by laser light scattering. No significant change was observed over 12 months at refrigeration temperature (5 ℃). Also, no significant change was observed over 3 months at 25 ℃.

TABLE 4 MVL-MTX during storage at Normal and elevated temperatures (batch #1, formulation 1)

TABLE 5 MVL-MTX during storage at ambient temperature (batch #2, formulation 1)

TABLE 6 MVL-MTX during storage at ambient temperature (batch #3, formulation 1)

For the related substances, an increase of impurities B and C was detected, with the limits of the european pharmacopoeia of 0.3 and 0.5%, respectively. Impurities B and C are known impurities in the European Pharmacopoeia (EP) and are the only known impurities considered as degradants. Impurity B is (2S) -2- [ [4- [ [ (2, 4-diaminopterin-6-yl) methyl ] amino ] benzoyl ] amino ] glutaric acid (4-aminopropionic acid, aminopterin). Impurity C is (2S) -2- [ [4- [ [ (2-amino-4-oxo-1, 4-dihydroxypterin-6-yl) methyl ] methylamino ] benzoyl ] amino ] glutaric acid (N-methylfolate, methylpterin). Two years of storage is a pharmaceutically accepted shelf life of viability. By extrapolating the existing data (see fig. 3), formulation 1 will likely have impurities B and C well below the limiting levels at 2 years. Thus, the instant MTX MVL formulation will be stable for at least two years. The instant embodiments include instant MTX MVL formulations having a measured stability, as well as methods of administering these formulations for autoimmune diseases and cancers as discussed above.

Perfusion property

For general injection, especially subcutaneous injection, which is self-administered by patients with chronic conditions such as RA, it is desirable to administer drugs with needles having a small diameter (or high gauge (large gauge) needles). Such needles allow for minimal or no painful injections. 27-31G gauge needles are considered narrow and preferred. Many sustained release injectates cannot be delivered through such narrow needles due to the use of high viscosity (e.g., in situ gel formulations) or hard and/or relatively large particles (e.g., polymeric microparticles). It has been determined that using such a narrow needle, a fast dissolving MTX MVL formulation is easily administrable. The product characteristics of formulation 1 were unaffected after passing through the 27-31G needle (see table 7). These data were generated by using the needle attached to a 1mL syringe and representing the formulation at about 2 mL/min. The ability to use 27-31G needles provides characteristics to this delivery technique and is likely due to the low viscosity, small particle size, and elastic nature of the particles. The instant embodiments include instant MTX MVL formulations having a determined perfusability, as well as methods of administering such formulations for autoimmune diseases and cancer as discussed above.

TABLE 7 perfusability of formulation 1

All documents cited herein are incorporated by reference herein in their entirety. Where publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the ordinary and customary meaning as understood by those skilled in the art and are not to be limited to the specific or intended meaning unless otherwise specified herein.

The terms and phrases used in this application, and variations thereof, particularly in the appended claims, should be construed to be open ended, as opposed to limiting, unless otherwise expressly stated. As an example above, the term "comprising" should be understood to mean "including without limitation," "including without limitation," and the like; the term "comprising" as used herein is synonymous with "comprising," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; the term "having" should be interpreted as "having at least"; the term "comprising" should be interpreted as "including, but not limited to"; the term "example" is used to provide illustrative examples of the items discussed, and is not an exhaustive or limiting list thereof; adjectives such as "known," "normal," "standard," and terms of similar meaning should not be construed as limiting the item described to a given time period or item available at a given time, but instead should be read to encompass known, normal, or standard technologies that may be available or known at any time, whether presently or in the future; and the use of words such as "preferably," "preferred," "desired," or "desires" and words of similar import should not be construed as implying that a particular property is critical, essential, or even important to the structure or function of the invention, but rather are used merely to emphasize alternative or additional features that may or may not be utilized in a particular embodiment of the invention. Likewise, a group of items linked with the conjunction "and" should not be read as requiring that each and every one of those items in the group be present, but rather should be read as "and/or" unless expressly stated otherwise. Similarly, a group of items linked with the conjunction "or" should not be read as requiring mutual exclusivity between the groups, but rather should be read as "and/or" unless expressly stated otherwise.

With respect to any plural and/or singular terms used in the plural herein, those having skill in the art may translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. Various singular/plural variations may be explicitly given herein for clarity.

It will be further understood by those within the art that if a specific value is intended to be introduced in a claim recitation, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should typically be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific value is explicitly recited in the claims at the time of introduction, those skilled in the art will understand that such recitation should typically be interpreted to mean at least the recited value (e.g., the bare recitation of "two recitations," without other modifiers, typically means at least two recitations, or two or more recitations). Further, in those instances where a convention analogous to "A, B, or at least one of C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the construction (e.g., a system having "A, B, or at least one of C" would include, but not be limited to, a system having only A, only B, only C, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that two or more alternative terms, given in virtually any disjunctive word and/or phrase in the specification, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "a or B" will be understood to include the possibility of a "or" B "or" a and B ".

All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Claims (45)

1. A formulation comprising multivesicular liposomes containing an amount of methotrexate, NaOH, sucrose and NaCl, wherein the amount of methotrexate is 55.0mM, the amount of NaOH is 131mM, the amount of sucrose is 38.7mM and the amount of NaCl is 69.3mM, the lipid component of the multivesicular liposomes is DPPG, DOPC, tricaprylin and cholesterol, and the amount of DPPG is 3.08mM, the amount of DOPC is 14.52mM, the amount of tricaprylin is 22mM, and the amount of cholesterol is 22mM, and wherein administration of a single dose of the formulation to a subject in need thereof results in blood of the methotrexateSlurry C_maxPlasma C in immediate release form of methotrexate_maxFrom 5% to 50%, wherein said formulation is free of cyclodextrin, and wherein the duration of said methotrexate in said subject is from 1 to 30 days.

2. A formulation as in claim 1, wherein the multivesicular liposome further comprises a mono-or divalent basic addition salt of methotrexate.

3. The formulation of claim 2, wherein the basic addition salt is a sodium salt.

4. The formulation of claim 2, wherein the basic addition salt is a potassium salt.

5. The formulation of claim 2, wherein the basic addition salt comprises an organic base as a counterion.

6. A formulation as in claim 1, wherein the duration of methotrexate in the subject is 1 to 7 days.

7. The formulation of claim 1, wherein the multivesicular liposome further comprises a folate.

8. The formulation of claim 1, wherein the multivesicular liposome further comprises lysine.

9. A formulation as in claim 1, wherein the duration of the methotrexate plasma levels is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

10. The formulation of claim 1, wherein the formulation is stable for at least two years.

11. The formulation of claim 1, wherein the formulation can be delivered through a 27-31G gauge needle.

12. Use of a formulation according to any one of claims 1 to 11 in the manufacture of a medicament for the treatment of an autoimmune disease.

13. Use according to claim 12, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, psoriasis, lupus, scleroderma, sjogren's syndrome, goodpasture's syndrome, wegener's granulomatosis, polymyalgia rheumatica, guillain barre syndrome and crohn's disease.

14. The use according to claim 12, wherein the autoimmune disease is rheumatoid arthritis.

15. The use of claim 12, wherein the autoimmune disease is lupus.

16. The use of claim 12, wherein the autoimmune disease is crohn's disease.

17. The use of claim 12, wherein the multivesicular liposome further comprises a sodium or potassium salt of methotrexate.

18. The use of claim 12, wherein the multivesicular liposome further comprises a folate.

19. The use of claim 12, further comprising administering folate.

20. The use of claim 12, further comprising administering a folate encapsulated in an MVL that does not comprise MTX.

21. The use of claim 12, wherein the multivesicular liposome further comprises lysine.

22. The use of claim 12, wherein the duration of the methotrexate plasma levels is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

23. The use of claim 12, wherein administration of the formulation comprises injection of the subject with the formulation.

24. The use of claim 23, wherein the injection is subcutaneous.

25. The use of claim 23, wherein the injection is intramuscular.

26. The use of claim 23, wherein the injection is intradermal.

27. The use of claim 23, wherein the injection is intraspinal.

28. The use of claim 23, wherein the injection is administered every 5 to 7 days.

29. The use of claim 28, wherein the injection is administered once per week.

30. Use of a formulation according to any one of claims 1 to 11 in the manufacture of a medicament for the treatment of cancer, wherein the formulation is not more frequently administered every 2 days to a subject in need thereof.

31. The use of claim 30, wherein the cancer is a solid tumor cancer.

32. The use of claim 31, wherein the cancer is gestational trophoblastic tumor, breast cancer, head and neck cancer, or lung cancer.

33. The use of claim 32, wherein said gestational trophoblastic tumor is choriocarcinoma, or hydatidiform mole.

34. The use of claim 33, wherein said gestational trophoblastic tumor is a malignant hydatidiform mole.

35. The use of claim 30, wherein the cancer is a haematological cancer.

36. The use of claim 35, wherein the hematological cancer is acute lymphoblastic leukemia, non-hodgkin's lymphoma, mycosis fungoides, or osteosarcoma.

37. The use of claim 30, wherein the multivesicular liposome further comprises a sodium or potassium salt of methotrexate.

38. The use of claim 30, wherein the multivesicular liposome further comprises a folate.

39. The use of claim 30, further comprising administering folate.

40. The use of claim 30, further comprising administering a folate encapsulated in an MVL that does not comprise MTX.

41. The use of claim 30, wherein the multivesicular liposome further comprises lysine.

42. The use of claim 30, wherein administration of the formulation comprises injection of the subject with the formulation.

43. The use of claim 42, wherein the injection is subcutaneous.

44. The use of claim 42, wherein the injection is intratumoral.

45. The use of claim 43, wherein the injection is administered every 2 days.