HK1173383A - Depot systems comprising glatiramer or a pharmacologically acceptable salt thereof - Google Patents

Abstract

The present invention provides long acting parenteral pharmaceutical compositions comprising a therapeutically effective amount of glatiramer. In particular, the present invention provides a long acting pharmaceutical composition comprising a therapeutically effective amount of glatiramer acetate in depot form suitable for administering at a medically acceptable location in a subject in need thereof. The depot form is suitable for subcutaneous or intramuscular implantation or injection.

Description

Depot systems comprising glatiramer or a pharmaceutically acceptable salt thereof

Technical Field

The present invention relates to long acting dosage forms of glatiramer acetate and other pharmaceutically acceptable salts of glatiramer acetate. Particularly preferred are depot systems (depot systems) and other implantable systems for the extended release of glatiramer acetate.

Background

Glatiramer acetate

Copolymer-1, also known as glatiramer acetate and is available under the trademark GelatimerSold, contains acetate salts of polypeptides containing L-glutamic acid, L-alanine, L-tyrosine, and L-lysine. The average molar fractions of the amino acids were 0.141, 0.427, 0.095 and 0.338, respectively, and the average molecular weight of copolymer-1 was 4,700 to 11,000 daltons. Chemically, glatiramer acetate is a designated L-glutamic acid polymer and has L-alanine, L-lysine and L-tyrosine, acetic acid (salt). The structural formula is as follows:

(Glu,Ala,Lys,Tyr)xCH₃COOH(C₅H₉NO₄_C₃H₇NO₂_C₆H₁₄N₂O₂_C₉H₁₁NO₃)xC₂H₄O₂[CAS-147245-92-9]about the ratio Glu₁₄Ala₄₃Tyr₁₀Lyz₃₄x(CH₃COOH)₂₀。Is clear and clear,A colorless to yellowish, sterile, non-pyrogenic solution for subcutaneous injection. Contains glatiramer acetate 20mg and mannitol 40mg per ml. The pH of the solution ranges from about 5.5 to 7.0.

Mechanism of action

Glatiramer acetate is a random polymer (average molecular mass of 6.4kD) comprising 4 amino acids found in myelin basic protein. The mechanism of action of glatiramer acetate is unknown, although some important immunological properties of this copolymer have emerged. Administration of copolymer-1 will move the T cell population from pro-inflammatory Th1 cells to regulatory Th2 cells, which suppress the inflammatory response (FDA)A label). In view of its similarity to myelin basic protein, copolymer-1 can also serve as a bait to transfer an autoimmune response relative to myelin. However, at least in the early stages of treatment, the integrity of the blood-brain barrier is not significantly affected by copolymer-1.

Copolymer-1 is a non-autoantigen that has been shown to inhibit Experimental Allergic Encephalomyelitis (EAE) induced by various encephalitogenic factors, including Mouse Spinal Cord Homogenate (MSCH), including myelin antigens such as Myelin Basic Protein (MBP) (Sela M et al, Bull Inst Pasteur (1990) 88303-. EAE is an accepted model of multiple sclerosis.

Copolymer-1 has been shown to be active when injected subcutaneously, intraperitoneally, intravenously, or intramuscularly (Teitelbaum D et al, Eur J Immunol (1971) 1242. cndot. 248; Teitelbaum D et al, Eur J Immunol (1973) 3273. cndot. 279). In phase III clinical trials, daily subcutaneous injection of copolymer-1 was found to slow the progression of disability and reduce the recurrence rate of exacerbation-remission multiple sclerosis (Johnson KP, Neurology (1995)165-70;www.copaxone.com). In totalMer-1 treatment is currently limited to daily subcutaneous administration. Treatment by ingestion or inhalation of copolymer-1 is disclosed in US 6,214,791, but these routes of administration have not been shown to achieve clinical efficacy in human patients.

Therapeutic effect

Evidence supporting the effectiveness of glatiramer acetate in reducing the frequency of relapses in patients with relapsing-remitting multiple sclerosis (RR MS) comes from two placebo-controlled trials, both using a dose of glatiramer acetate of 20 mg/day. No other doses or dose regimens were studied in placebo-controlled trials of RR MS (www.copaxone.com). Comparative trials of approved 20mg and 40mg doses showed no significant difference in efficacy between these doses (The 9006 trial; CohenJA et al, Neurology (2007) 68939-944). Various clinical trials of glatiramer acetate are in progress. These clinical trials included studies with higher doses of glatiramer acetate (40 mg-FORTE study); studies performed on patients with clinically isolated syndrome (PreCISe study); and many combination and induction protocols in which glatiramer acetate is administered with or after another active product.

Side effects

At present, all the specifically recognized treatments for multiple sclerosis involve self-injection of the active substance. Frequently observed injection site problems include irritation, hypersensitivity, inflammation, pain and even necrosis (in the case of interferon 1 β treatment) and low levels of patient compliance.

Side effects typically include lumps at the injection site (injection site reactions), pain, fever, and chills. The nature of these side effects is generally mild. Occasionally, a response occurred minutes after injection, with flushing, shortness of breath, anxiety, and rapid heart beat. These side effects subside within 30 minutes. Over time, visible indentations may develop at the injection site due to local destruction of adipose tissue, known as subcutaneous lipoatrophy. Thus, an alternative administration method is desired.

According to FDA prescription labeling, more serious side effects of glatiramer acetate have been reported, including serious side effects on the cardiovascular system, the digestive system (including liver), the hematopoietic and lymphatic systems, the musculoskeletal system, the nervous system, the respiratory system, the special senses (especially the eye), the urogenital system of the body; metabolic and nutritional disorders have also been reported; however, the link between glatiramer acetate and these adverse effects has not been established explicitly (FDA)A label).

Medicine storage system (depot system)

The parenteral route by Intravenous (IV), Intramuscular (IM), or Subcutaneous (SC) injection is the most common and effective form of delivering smaller and larger molecular weight drugs. However, the pain, discomfort and inconvenience resulting from needle sticks make this drug delivery modality the least preferred by patients. Thus, any drug delivery technique that can minimize the total number of injections is preferred. In practice, the frequency of drug administration can be reduced by the use of injectable depot formulations that release the drug in a slow but predictable manner, thus improving compliance. For most drugs, depending on the dose, the injection frequency can be reduced: from once daily to once or twice or even longer (6 months). In addition to improving patient comfort, less frequent injections of the drug properties of the drug storage formulation can moderate the plasma concentration-time curve by eliminating peaks and troughs. Such a relaxation of the plasma distribution is likely to not only improve the therapeutic effect in most cases, but also to reduce any unwanted events such as immunogenicity and the like (often accompanied by larger molecular weight drugs).

Microparticles, implants and gels are the most common forms of biodegradable polymer devices used in practice for prolonged release of drugs in the body. The microparticles are suspended in an aqueous medium immediately prior to injection and can be loaded with up to 40% solids in suspension. The implant/stick type is delivered to SC/IM tissue by means of a special needle in a dry state without the need for an aqueous medium. This feature of the rod/implant facilitates delivery of higher quality dosage forms and drug content. In addition, in the rod/implant, the problem of the initial burst is minimized due to the much smaller area of the implant compared to the microparticles. In addition to biodegradable systems, there are non-biodegradable implants and infusion pumps that can be worn outside the body. Non-biodegradable implants require access by a physician: not only for implanting the devices into SC/IM tissue, but also for removing them after the drug release period.

Injectable compositions comprising microparticulate formulations are particularly prone to problems. Microparticle suspensions may contain up to 40% solids, as compared to 0.5-5% solids in other types of injectable suspensions. In addition, microparticles used in injectable drug storage products have a size up to about 250 μm (60-100 μm on average) compared to the particle size of less than 5 μm suggested for IM or SC administration. Higher concentrations of solids, and larger solid particle sizes, require larger size needles (about 18-21 gauge) to inject. In summary, although smaller and uncomfortable needles are rarely used, patients prefer to administer dosage forms less often than daily drug injections with smaller needles.

Biodegradable polyesters of polylactic acid (PLA) and copolymers of lactide and glycolide, known as poly (lactide-glycolide) (PLGA), are the most common polymers used in biodegradable dosage forms. PLA is a hydrophobic molecule and PLGA degrades faster than PLA because of the presence of the more hydrophilic glycolide groups. These biocompatible polymers undergo random non-enzymatic hydrolytic cleavage of ester linkages to form lactic acid and glycolic acid, which are normal metabolic compounds in the body. Absorbable sutures, clips, and implants were the earliest applications of these polymers. In 1970, the southern research Institute developed the first synthetic absorbable sutureThe first patent describing the use of PLGA polymers in sustained release dosage forms appeared in 1973 (US 3,773,919).

Today, PLGA polymers are commercially available from a number of suppliers: alkermes (Medisorb Polymers), Absorbable Polymers International [ formerly BirminghamPermers (a division of Durect) ], Purac and Boehringer Ingelheim. In addition to PLGA and PLA, natural cellulosic polymers such as starch, starch derivatives, dextran, and non-PLGA synthetic polymers have also been developed as biodegradable polymers in the above systems.

Currently, long acting dosage forms of glatiramer acetate are not available. This is a great unmet medical need as these dosage forms would be extremely beneficial to many patients, especially those with neurological symptoms or physical disabilities.

Disclosure of Invention

The present invention provides long acting parenteral pharmaceutical compositions comprising a therapeutically effective amount of a pharmaceutically acceptable salt of glatiramer, e.g., glatiramer acetate. In particular, the present invention provides long acting pharmaceutical compositions comprising a therapeutically effective amount of a depot form (depot form) glatiramer salt suitable for parenteral administration to a subject in need thereof at a medically acceptable site. The present invention further provides a method for the treatment of multiple sclerosis comprising the parenteral administration or implantation of a composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of glatiramer, preferably glatiramer acetate.

Unexpectedly, it has now been found that long acting pharmaceutical compositions according to the principles of the present invention can provide the same or better therapeutic efficacy relative to commercially available daily injectable dosage forms, with reduced incidence and/or severity of side effects at local and/or systemic levels.

According to some embodiments, glatiramer acetate comprises acetate salts of L-alanine, L-glutamic acid, L-lysine, and L-tyrosine in a molar ratio of about 0.14 glutamic acid, about 0.43 alanine, about 0.10 tyrosine, and about 0.33 lysine.

According to other embodiments, glatiramer acetate or other pharmaceutically acceptable salts of glatiramer comprise from about 15 to about 100 amino acids.

According to certain embodiments, the implantable depot is suitable for subcutaneous or intramuscular implantation.

According to an alternative embodiment, the long acting parenteral pharmaceutical composition comprises a pharmaceutically acceptable biodegradable or non-biodegradable carrier for a glatiramer salt, such as glatiramer acetate.

According to some embodiments, the carrier is selected from the group consisting of PLGA, PLA, PGA, polycaprolactone, polyhydroxybutyrate, polyorthoesters, polyalkaneanhydrides, gelatin, collagen, oxidized cellulose, and polyphosphazene. Each possibility represents a separate embodiment of the invention.

According to a specific embodiment, the long acting pharmaceutical composition of the present invention has the form of microparticles prepared by a water-in-oil-in-water double emulsification process (water-in-oil-in-water emulsification process). In a presently preferred embodiment, the long acting pharmaceutical composition of the present invention comprises: an internal aqueous phase comprising a therapeutically effective amount of a pharmaceutically acceptable salt of glatiramer; a water-immiscible polymeric phase comprising a carrier selected from biodegradable and non-biodegradable polymers; and an external aqueous phase. In other presently preferred embodiments, the water immiscible polymer phase comprises a biodegradable polymer selected from PLA and PLGA. Each possibility represents a separate embodiment of the invention. In further embodiments, the external aqueous phase comprises a surfactant selected from polyvinyl alcohol (PVA), polysorbates, polyethylene oxide-polypropylene oxide block copolymers, and cellulose esters. Each possibility represents a separate embodiment of the invention.

The present invention encompasses the use of glatiramer acetate or any other pharmaceutically acceptable salt of glatiramer in depot form suitable for implantation into an individual in need thereof for the treatment of multiple sclerosis.

The invention further encompasses the use of an implantable depot of glatiramer acetate which is suitable for providing prolonged release or prolonged action of glatiramer in a subject.

Within the scope of the present invention are pharmaceutically acceptable salts of glatiramer in depot form suitable for use in treating multiple sclerosis or providing prolonged release or prolonged action of glatiramer in a subject.

The invention also encompasses the combination of glatiramer acetate with at least one additional drug, preferably an immunosuppressive agent, especially fingolimod.

According to some embodiments, the long acting pharmaceutical composition is suitable for a dosing schedule of once weekly to once every 6 months.

According to a particular embodiment, the composition is suitable for administration from once every 2 weeks to once a month.

According to some embodiments, the long acting composition comprises a dose of 20-750mg glatiramer acetate per injection.

Specific examples of long acting compositions would include biodegradable or non-biodegradable microspheres, implants of any suitable geometry, implantable rods, implantable capsules, implantable rings, extended release gels, and erodible matrices. Each possibility represents a separate embodiment of the invention.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Drawings

FIG. 1 release of glatiramer acetate from PLGA microparticle formulation MPG-02-07 in PBS and at 37 ℃. The data presented are normalized to a standard peptide solution stored under the same conditions.

FIG. 2 release of glatiramer acetate from PLGA microparticle formulations MPG-05R, 08-11 and the tocopherol succinate of glatiramer (1:1) in PBS and at 37 ℃. The data presented are normalized to a standard peptide solution stored under the same conditions.

FIG. 3 release of glatiramer acetate from PLGA microparticle formulation MPG-12-15 in PBS and at 37 ℃. The data presented are normalized to a standard peptide solution stored under the same conditions.

FIG. 4 release of glatiramer acetate from PLGA microparticle formulations MPG-14SU-1 and MPG-15SU-1 in PBS in vitro at 37 ℃ and pH 7.4.

FIG. 5 release of glatiramer acetate from PLGA microparticle formulations MPG-14SU-2 and MPG-15SU-2 in PBS in vitro at 37 ℃ and pH 7.4.

Detailed Description

The present invention provides long acting parenteral pharmaceutical formulations of pharmaceutically acceptable salts of glatiramer, preferably glatiramer acetate, which can provide the same or better therapeutic efficacy compared to daily injections, thus resulting in improved patient compliance. In addition to providing the same therapeutic effect, long-acting injections or implants can also reduce glatiramer side effects (local and/or systemic) originating from frequent injections.

According to a first aspect, the present invention provides a long acting parenteral pharmaceutical composition comprising a therapeutically effective amount of glatiramer acetate or any other pharmaceutically acceptable salt of glatiramer. As used herein, the term "parenteral" refers to a route selected from Subcutaneous (SC), Intravenous (IV), Intramuscular (IM), Intradermal (ID), Intraperitoneal (IP), and the like. Each possibility represents a separate embodiment of the invention. As used herein, the term "therapeutically effective amount" is used to define the amount of copolymer that will achieve the goal of alleviating the symptoms of multiple sclerosis. Suitable dosages include, but are not limited to, 20-750mg per dosage form. It will be understood, however, that the amount of copolymer administered will be determined by a physician, in the light of various parameters, including the chosen route of administration, age, weight, and severity of the patient's symptoms. According to various embodiments of the invention, the therapeutically effective amount of the at least one copolymer is from about 1mg to about 500mg per day. Alternatively, the above therapeutically effective amount of the at least one copolymer is from about 20mg to about 100mg per day.

In another aspect, the present invention provides a long acting pharmaceutical composition comprising a therapeutically effective amount of glatiramer acetate or any other pharmaceutically acceptable salt of glatiramer in depot form suitable for administration to a subject in need thereof at a medically acceptable location. As used herein, the term "long-acting" refers to a composition that provides for the long-term, sustained or extended release of glatiramer salt to the general systemic circulation of the subject or to the site of local action of the subject. The term may further refer to a composition that provides a long-term, sustained or extended duration of action (pharmacokinetics) of the glatiramer salt in a subject. In particular, the long acting pharmaceutical compositions of the present invention provide a dosing regimen ranging from once a week to once every 6 months. According to a more presently preferred embodiment, the dosing regimen ranges from once a week, twice a month (approximately once every two weeks) to once a month. Depending on the duration of action desired, each depot or implantable device of the invention will typically contain from about 20 to 750mg of active ingredient designed to be released over a time period ranging from two weeks to several months.

In some embodiments, depot formulations of the invention include, but are not limited to: a suspension of glatiramer or a pharmaceutically acceptable salt thereof in an aqueous, oil or wax phase; insoluble polymer electrolyte complexes (polyelectrolytes) of glatiramer or a pharmaceutically acceptable salt thereof; an "in situ" gel-forming matrix based on a combination of a water-miscible solvent and glatiramer or a pharmaceutically acceptable salt thereof; and biodegradable polymer microparticles incorporating glatiramer or a pharmaceutically acceptable salt thereof. Each possibility represents a separate embodiment of the invention. In particular, the compositions of the invention are in the form of injectable microparticles in which glatiramer or a pharmaceutically acceptable salt thereof is entrapped in a biodegradable or non-biodegradable carrier. The microparticle composition of the present invention may comprise an aqueous-in-oil-in-aqueous double emulsion. Within the scope of the present invention, a microparticle composition comprises: an internal aqueous phase comprising glatiramer or any pharmaceutically acceptable salt thereof; an oil or water immiscible phase comprising a biodegradable or non-biodegradable polymer; and an external aqueous phase. The external aqueous phase may further comprise a surfactant, preferably polyvinyl alcohol (PVA), polysorbate, polyoxyethylene-polyoxypropylene block copolymer or cellulose ester. The terms "oil phase" and "water immiscible phase" may be used interchangeably herein.

The invention further provides a method of treating multiple sclerosis by parenteral administration to a subject in need thereof of a long acting pharmaceutical composition comprising a therapeutically effective amount of glatiramer acetate or any other pharmaceutically acceptable salt of glatiramer. Within the scope of the present invention is a method of treating multiple sclerosis by administering glatiramer acetate or any other pharmaceutically acceptable salt of glatiramer in depot form to an individual in need thereof. As used herein, the term "treatment" refers to the inhibition or alleviation of symptoms after the onset of multiple sclerosis. Common symptoms after the onset of multiple sclerosis include, but are not limited to, a reduction or loss of vision, fall-offs and unbalanced gait, slurred speech, and urinary frequency and incontinence. In addition, multiple sclerosis can cause mood changes and depression, muscle spasms and severe paralysis. The "subject" to which the drug is administered is a mammal, preferably but not limited to a human. As used herein, the term "multiple sclerosis" refers to an autoimmune disease of the central nervous system that is associated with one or more of the symptoms described above.

As used herein, the term "glatiramer acetate" refers to the compound previously referred to as copolymer 1, under the trade name thereofIs sold and consists of acetate salts of synthetic polypeptides, comprising 4 naturally occurring amino acids: l-glutamic acid, L-alanine, L-tyrosine, and L-lysine at average molar fractions of 0.141, 0.427, 0.095, and 0.338, respectively. In thatThe average molecular weight of the medium-sized glatiramer acetate is 4,700-11,000 daltons (FDA)Label) and the number of amino acids ranges from about 15 to about 100 amino acids. The term also refers to chemical derivatives and analogs of the compounds. Typically, the compounds are prepared and characterized as described in any one of the following U.S. patent nos.: 5,981,589, 6,054,430, 6,342,476, 6,362,161, 6,620,847, and 6,939,539, the entire contents of each of which are incorporated herein by reference.

In some embodiments, the composition may comprise any other pharmaceutically acceptable salt of glatiramer, including, but not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, hydroiodide, acetate, nitrate, propionate, decanoate, octanoate, acrylate, formate, isobutyrate, decanoate, heptanoate, propiolate, oxalate, malonate, succinate, tocopherol succinate, suberate, sebacate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methoxinate, dihydrogenate, dihydrogenphosphate, acetate, nitrate, propionate, decanoate, octanoate, dihydrogenate, phthalates, terephthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, beta-hydroxybutyrate, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-2-sulfonates, p-toluenesulfonates, mandelates and the like. Each possibility represents a separate embodiment of the invention.

The copolymer may be prepared by any procedure available to those skilled in the art. For example, the copolymer can be prepared under condensation conditions with the desired molar ratio of amino acids in solution, or by a solid phase synthesis procedure. The condensation conditions include appropriate temperature, pH, and solvent conditions for condensing the carboxyl group of one amino acid with the amino group of another amino acid to form a peptide bond. Condensing agents, such as dicyclohexylcarbodiimide, may be used to promote the formation of peptide bonds.

Protecting groups may be used to protect functional groups, such as side chain moieties and some amino or carboxyl groups, from undesirable side reactions. The process disclosed in U.S. patent No. 3,849,550, the entire contents of which are incorporated herein by reference, can be used to prepare the copolymers of the present invention. For example, tyrosine N-carboxy anhydride, alanine, γ -benzylglutamic acid and N, ε -trifluoroacetyl-lysine are polymerized in anhydrous dioxane at ambient temperature and with diethylamine as an initiator. The gamma-carboxyl group of glutamic acid can be deblocked by hydrogen bromide in glacial acetic acid. The trifluoroacetyl group was removed from lysine by 1 mole piperidine. It will be readily apparent to those skilled in the art that the process can be adjusted to produce peptides and polypeptides containing the desired amino acid, i.e., three of the four amino acids in copolymer 1, by selectively eliminating reactions involving any of glutamic acid, alanine, tyrosine, or lysine. U.S. patent nos. 6,620,847, 6,362,161, 6,342,476, 6,054,430, 6,048,898 and 5,981,589, the entire contents of which are incorporated herein by reference, disclose improved processes for the preparation of glatiramer acetate (Cop-1). For the purposes of this application, the terms "ambient temperature" and "room temperature" generally refer to temperatures of from about 20 ℃ to about 26 ℃.

The molecular weight of the copolymer can be adjusted during polypeptide synthesis or after the polymer has been prepared. In order to regulate molecular weight during polypeptide synthesis, the synthesis conditions or the amount of amino acids are adjusted such that synthesis is stopped when the polypeptide reaches approximately the desired length. After synthesis, polypeptides having the desired molecular weight can be obtained by any available size selection procedure, such as chromatography of the polypeptide with molecular weight fractionation columns (molecular weight sizing columns) or gels, and collection of the desired molecular weight range. The polypeptide may also be partially hydrolyzed to remove high molecular weight species, for example, by acid or enzyme hydrolysis, and then purified to remove the acid or enzyme.

In one embodiment, a copolymer having a desired molecular weight can be prepared by a method comprising reacting a protected polypeptide and hydrobromic acid to form a trifluoroacetyl-polypeptide having a desired molecular weight distribution. The reaction is carried out for a time and at a temperature predetermined by the test reaction or reactions. The time and temperature are varied during the test reaction and the molecular weight range of a given batch of test polypeptide is determined. Test conditions that provide the optimal molecular weight range for the batch of polypeptides were used for the above batches. Thus, trifluoroacetyl-polypeptides having a desired molecular weight distribution can be prepared by a process which comprises reacting a protected polypeptide with hydrobromic acid for a time and at a temperature which is predetermined by a test reaction. The trifluoroacetyl-polypeptide having the desired molecular weight distribution is then further treated with an aqueous piperidine solution to form a deprotected polypeptide having the desired molecular weight.

In a preferred embodiment, a test sample from a given lot of a protected polypeptide is reacted with hydrobromic acid at a temperature of about 20-28 ℃ for about 10-50 hours. The optimum conditions for the batch were determined by running several test reactions. For example, in one embodiment, the protected polypeptide and hydrobromic acid are reacted for about 17 hours at a temperature of about 26 ℃.

In certain embodiments, the dosage forms include, but are not limited to, biodegradable injectable drug depot systems (depot systems) such as PLGA-based injectable drug depot systems, non-PLGA-based injectable drug depot systems, and injectable biodegradable gels or dispersions. Each possibility represents a separate embodiment of the inventionThe method is as follows. As used herein, the term "biodegradable" refers to a composition that erodes or degrades over time on its surface, resulting at least in part from contact with substances present in the surrounding tissue fluid, or through cellular action. In particular, the biodegradable component is a polymer such as, but not limited to, a lactic acid based polymer such as a polylactide, e.g., poly (D, L-lactide), i.e., PLA; glycolic acid based polymers such as Polyglycolide (PGA), e.g. from DurectPoly (D, L-lactide-glycolide) i.e. PLGA (from Boehringer)RG-504、RG-502、RG-504H、RG-502H、RG-504S、RG-502S, from Durect) (ii) a Polycaprolactone such as poly (e-caprolactone), PCL (from Durect)) (ii) a A polyanhydride; poly (sebacic acid) SA; poly (ricinoleic acid) RA; poly (fumaric acid), FA; poly (fatty acid dimer), FAD; poly (terephthalic acid), TA; poly (isophthalic acid), IPA; poly (p { carboxyphenoxy } methane), CPM; poly (p { carboxyphenoxy } propane) CPP; poly (p { carboxyphenoxy } hexane) CPH; polyamines, polyurethanes, polyesteramides, polyorthoesters { CHDM: cis/trans-cyclohexyldimethanol, HD: l, 6-hexanediol, DETOU: (3, 9-diethylene-2, 4,8, 10-tetraoxaspiro undecane) }; polydioxanone; polyhydroxybutyrate; polyalkylene oxalates (polyalkylene oxides); a polyamide; a polyester amide; a polyurethane; a polyacetal; polyketal; a polycarbonate; a poly (orthocarbonate); a polysiloxane; polyphosphazene; a succinic acid ester; hyaluronic acid; poly (malic acid); poly (amino acids); polyhydroxyvalerate; polyalkylene succinates (polyalkylene succinates); polyvinylpyrrolidone; polystyrene; synthesizing a cellulose ester; polyacrylic acid; poly (butyric acid); triblock copolymers (PLGA-PEG-PLGA), triblock copolymers (PEG-PLGA-PEG), poly (N-isopropylacrylamide) (PNIPAAm), poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) triblock copolymers (PEO-PPO-PEO), poly (valeric acid); polyethylene glycol; polyhydroxyalkylcellulose; chitin (chitin); chitosan; polyorthoesters and copolymers, terpolymers; lipids such as cholesterol, lecithin; poly (glutamic acid-ethyl glutamate) and the like, or mixtures thereof.

In some embodiments, the compositions of the present invention comprise a biodegradable polymer selected from, but not limited to, PLGA, PLA, PGA, polycaprolactone, polyhydroxybutyrate, polyorthoesters, polyalkaneanhydrides, gelatin, collagen, oxidized cellulose, polyphosphazene, and the like. Each possibility represents a separate embodiment.

The presently preferred biodegradable polymers are lactic acid based polymers, more preferably polylactide, or poly (D, L-lactide-glycolide), i.e. PLGA. Preferably, the biodegradable polymer is present in an amount of about 10% to about 98% w/w of the composition. The lactic acid-based polymer has a monomer ratio of lactic acid to glycolic acid of 100:0 to about 0:100, preferably 100:0 to about 10:90, and has an average molecular weight of about 1,000 to 200,000 daltons. It should be understood, however, that the amount of biodegradable polymer is determined by parameters such as the duration of the application, etc.

The compositions of the present invention may further comprise one or more pharmaceutical excipients selected from, but not limited to, co-surfactants, solvents/co-solvents, water-immiscible solvents, water-miscible solvents, oily components, hydrophilic solvents, emulsifiers, preservatives, antioxidants, antifoaming agents, stabilizers, buffers, pH adjusting agents, osmotic agents, channeling agents, osmotic agents, or any other excipient known in the art. Suitable co-surfactants include, but are not limited to, polyethylene glycol, polyoxyethylene-polyoxypropylene block copolymers (known as "poloxamers"), polyglycerol fatty acid esters such as decaglycerol monolaurate (decaglycerol monolaurate) and decaglycerol monomyristate (decaglycerol monomyrisate), sorbitan fatty acid esters such as sorbitan monostearate, polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monooleate (tween), polyethylene glycol fatty acid esters such as polyoxyethylene monostearate, polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether, polyoxyethylene castor oil and hardened castor oil such as polyoxyethylene hardened castor oil and the like or mixtures thereof. Each possibility represents a separate embodiment of the invention. Suitable solvents/co-solvents include, but are not limited to, alcohols, glyceryl triacetate, dimethyl isosorbide, glycofurol (propylene carbonate), water, dimethylacetamide, and the like, or mixtures thereof. Each possibility represents a separate embodiment of the invention. Suitable antifoams include, but are not limited to, silicone emulsions or sorbitan sesquioleate. Suitable stabilizers for preventing or reducing deterioration of ingredients in the compositions of the present invention include, but are not limited to, antioxidants such as glycine, alpha-tocopherol or ascorbic acid, BHA, BHT, and the like, or mixtures thereof. Each possibility represents a separate embodiment of the invention. Suitable tonicity adjusting agents include, but are not limited to, mannitol, sodium chloride, and dextrose. Each possibility represents a separate embodiment of the invention. Suitable buffers include, but are not limited to, acetate, phosphate, and citrate (with suitable cations). Each possibility represents a separate embodiment of the invention.

The compositions of the present invention may be prepared by any means known in the art. It is currently preferred to incorporate glatiramer or a salt copolymer thereof into a colloidal delivery system, e.g., biodegradable microparticles, to facilitate release retardation by diffusion through the polymer wall of the particle and by polymer degradation in aqueous media or biological fluids in the body. The compositions of the invention may be prepared by a process known as "double emulsification" and in the form of injectable microparticles. Briefly, a concentrated solution of the water-soluble copolymer is dispersed in a solution of the biodegradable or non-biodegradable polymer in a water-immiscible volatile organic solvent (e.g., methylene chloride, chloroform, etc.). The "water-in-oil" (w/o) emulsion thus obtained is then dispersed in a continuous external aqueous phase containing a surfactant (e.g., polyvinyl alcohol-PVA, polysorbates, polyethylene oxide-polypropylene oxide block copolymers, cellulose esters, etc.) to form "water-in-oil-in-water (w/o/w) double emulsion" droplets. After evaporation of the organic solvent, the microparticles are solidified and collected by filtration or centrifugation. The collected Microparticles (MP) were washed with purified water to eliminate most of the surfactant and unbound peptide, and then centrifuged again. The washed MPs were collected and freeze-dried without additives or with the addition of a cryoprotectant (mannitol) to facilitate their subsequent reconstitution.

The particle size of the "water-in-oil-in-water (w/o/w) double emulsion" can be determined by various parameters, including but not limited to the amount of applied force during this step, the mixing speed, the surfactant type and concentration, and the like. Suitable particle sizes are about 1 to 100 μm.

The drug storage system of the present invention includes any form known to those skilled in the art. Suitable forms include, but are not limited to, biodegradable or non-biodegradable microspheres, implantable rods, implantable capsules, and implantable rings. Each possibility represents a separate embodiment of the invention. Additionally included are extended release gel reservoirs and erodible matrices. Each possibility represents a separate embodiment of the invention. Suitable implantable systems are described, for example, in US 2008/0063687, the entire contents of which are hereby incorporated by referenceIncorporated herein. As is known in the art, suitable micro-extruders such as those described, for example, inhttp://www.randcastle.com/prodinfo.htmlThe micro-extruder described in (1) to prepare implantable rods.

In accordance with the principles of the present invention, the long acting pharmaceutical compositions of the present invention may provide the same or better therapeutic efficacy relative to commercially available daily injectable dosage forms, and have a reduced incidence of side effects and a reduced severity of side effects at a local and/or systemic level. In some embodiments, the compositions of the invention can provide extended release or prolonged action of glatiramer in a subject as compared to an immediate release formulation of approximately the same dose of glatiramer acetate.

The present invention encompasses combination therapies of glatiramer acetate or any other pharmaceutically acceptable salt of glatiramer with at least one other active agent. Active agents within the scope of the present invention include, but are not limited to, interferons, such as pegylated or non-pegylated alpha-interferons, or beta-interferons, such as interferon beta-1 a or interferon beta-1 b, or tau-interferon; immunosuppressive agents with alternative antiproliferative/antitumor activity, such as mitoxantrone (mitoxantrone), methotrexate, azathioprine (azathioprine), cyclophosphamide, or steroids such as methylprednisolone, prednisone or dexamethasone, or steroid secreting agents such as ACTH; adenosine deaminase inhibitors, such as cladribine (cladribine); IV immunoglobulin G monoclonal antibodies against various T cell surface markers (e.g., as disclosed in Neurology,1998, May50(5): 1273-81), e.g., natalizumabOr alemtuzumab (alemtuzumab); TH2 promotes cytokines, such as IL-4, IL-10, or compounds that inhibit TH1 from promoting cytokine expression, such as phosphodiesterase inhibitors, e.g., pentoxifylline (pentoxifylline); antispasmodics including baclofen (baclofen), diazepam (diazepam), piracetam, dantrolene (dantrolene), lamotrigine (lamotrigine), riluzole (R) (I)rifluzole), tizanidine, clonidine, beta blockers, cyproheptadine, oxyphennaramine, or cannabinoids; AMPA glutamate receptor antagonists, e.g., 2, 3-dihydroxy-6-nitro-7-sulfamoylbenzo (f) quinoxaline, [1,2,3,4, -tetrahydro-7-morpholinyl-2, 3-dioxo-6- (trifluoromethyl) quinoxalin-i-yl]Methylphosphonate, 1- (4-aminophenyl) -4-methyl-7, 8-methylene-dioxo-5H-2, 3-benzodiazepine (1- (4-aminophenyl) -4-methyl-7,8-methylene-dioxy-5H-2, 3-benzodiazepine), or (-)1- (4-aminophenyl) -4-methyl-7, 8-methylene-dioxo-4, 5-dihydro-3-methylcarbamoyl-2, 3-benzodiazepine; antagonists of inhibitors of VCAM-1 expression or its ligands, e.g. antagonists of the alpha 4 beta l integrin VLA-4 and/or alpha-4-beta-7 integrin, e.g. natalizumabAnti-macrophage migration inhibitory factor (Anti-MIF); xii) cathepsin S inhibitors; xiii) mTor inhibitors. Each possibility represents a separate embodiment of the invention. One other active agent that is presently preferred is FTY720 (2-amino-2- [2- (4-octylphenyl) ethyl)]Propane-1, 3-diol; fingolimod), which is an immunosuppressive agent.

The following examples serve to more fully illustrate certain embodiments of the invention. They should in no way be construed, however, as limiting the scope of the invention. Numerous variations and modifications of the principles disclosed herein will be readily apparent to those skilled in the art without departing from the scope of the invention.

Examples

Example 1: general preparation method

Injectable long-acting particles based on PLGA

Microparticles were prepared by a solvent extraction/evaporation method (single emulsion). A50: 50 dichloromethane/ethanol solution containing 250mg PLGA and 200mg glatiramer acetate was slowly poured into an aqueous solution (200ml) containing 2% PVA and emulsified at 25 ℃ using a mechanical stirrer (300 rpm). The organic solvent was evaporated for 2h with stirring (100 rpm). The microparticles thus formed were collected by centrifugation and washed with distilled water to remove excess emulsifier. The final suspension was then freeze-dried to obtain a fine powder.

Injectable drug depot based on polycaprolactone: (depot) Granules

Microparticles were prepared by a solvent extraction/evaporation method (single emulsion). A70: 30 dichloromethane/acetone solution containing 500mg of polycaprolactone and 200mg of glatiramer acetate was slowly poured into an aqueous solution (200ml) containing 2% PVA, 1% Tween 80, and then emulsified at 25 ℃ using a mechanical stirrer (500 rpm). The organic solvent was evaporated under stirring (300rpm) for 4 h. The microparticles formed were collected by centrifugation and washed with distilled water to remove excess emulsifier. The final suspension was then freeze-dried to obtain a fine powder.

PLGA-based implant rod

PLGA-based biodegradable rod-shaped implants 20mm in length and 2mm in diameter were prepared by a solvent extraction/evaporation method. A50: 50 dichloromethane/ethanol solution containing 250mg PLGA and 200mg glatiramer acetate was slowly poured into a specially prepared rod-shaped mold. The organic solvent was evaporated in a vacuum oven at room temperature for 12 hours. Alternatively, the rod-shaped implants were prepared by extruding a mixture of 250mg of PLGA and 200mg of glatiramer at 85-90 ℃ using a screw extruder (Microtruder Rancaster RCP-0250 or similar extruder) with a die diameter of 0.8 or 1.0 mm.

Example 2: analytical method-measurement of glatiramer acetate

Device

Spectrophotometer

Analytical balance, accurately weighing to 0.01mg

Materials and reagents

Glatiramer acetate 83% as a reference standard

2,4, 6-trinitrobenzenesulfonic acid (TNBS, picrylsulfonic acid (170.5 mM)), 5% in MeOH

0.1M Borate buffer pH9.3 (sodium tetraborate decahydrate MW 381.37)

Purified water

0.5, 1.0, 2.0 and 7.0mL volumetric pipettes

A wide variety of glassware.

Preparation of

Glatiramer stock solution 400μPreparation of g/mL

4.8mg of glatiramer acetate (potency 83%, as basis for reference standard) are weighed into a 10ml volumetric flask. Approximately 7ml of 0.1M borate buffer was added to dissolve glatiramer acetate in the ultrasonic bath. The solution was further diluted with 0.1M borate buffer to obtain a glatiramer stock solution 400 μ g/ml (as a basis).

Preparation of 0.25% TNBS working solution

Prior to use, a 5% stock solution of TNBS was diluted with water (20-fold; e.g., 50. mu.l and 950. mu.l water) to obtain a 0.25% TNBS working solution.

Standard preparation of calibration Curve

8 glatiramer calibration standard solutions (cSTD; 4ml each) were prepared according to Table 1.

TABLE 1 Standard solution of glatiramer acetate

Optical density measurement

1.0ml of each glatiramer calibration standard solution, sample (in duplicate) and reagent blank (0.1M borate buffer) were transferred to a 1.5ml polypropylene centrifuge tube to which 50. mu.l of a 0.25% TNBS working solution was added. The solution was mixed well and kept at room temperature for 30 minutes. The optical densities of each of the obtained solutions were read at 420nm and 700nm and the difference in these densities was calculated to avoid errors in light scattering due to the colloidal system. A calibration curve for the selected concentration range was calculated.

Acceptance criteria

The difference between the results of duplicate sample preparations was NMT 5% as calculated by the following equation:

where Rspl1 is the result obtained from sample 1 and Rspl2 is the result obtained from sample 2.

Example 3: preparation of PLGA microparticles loaded with glatiramer acetate

External (continuous) aqueous phase: 30ml of 0.75% NaCl solution in purified water, further containing 0.5% partially hydrolyzed (87-89%) polyvinyl alcohol (PVA) as surfactant, 0.2% polysorbate-80 (Tween-80) for MPG-10 and 2% PVA for blank MP formulation.

Inner aqueous phase (for peptide solution): 150-. The glatiramer acetate was dissolved in water using an ultrasonic bath.

Organic polymer solution (oil phase): 165-300mg of PLGA in 2-5mL of dichloromethane. Optionally, the counter ion is further dissolved or dispersed in the organic phase.

Preparation procedure

Water-in-oil (w/o) emulsion preparation: directly mixing an internal aqueous phase comprising dissolved glatiramer acetate and an aqueous phase contained in CH in a test tube₂Cl₂The oil phase of the PLGA solution of (1). The mixture was shaken well and treated with an ultrasonic indenter (titanium tip, 120 watts maximum power, 10-15% operating power, 3-5 cycles of 5 seconds each). Optionally, cooling is performed with ice or ice water to avoid boiling of the dichloromethane.

Double emulsion (w/o/w) preparation: the w/o emulsion of glatiramer acetate solution in a polymeric PLGA organic solution thus obtained was further treated with a high shear mixer (mini mixer, VDI-12, 10mm diameter shaft, and larger mixer, OMNI-1100, 18mm diameter shaft) at various speeds for 30-120 seconds.

Solvent elimination: the open beaker with the double emulsion so formed was placed on a magnetic plate stirrer and stirred in a fume hood at room temperature for 3-4 hours until all the dichloromethane had evaporated and the microparticles had solidified.

Centrifugation of microparticles: the suspension of solidified microparticles was centrifuged at 2000-.

Washing of microparticles: the settled microparticles according to the above procedure were suspended in 10ml of purified water using a vortex and ultrasonic bath and then shaken or sonicated for 2-3 minutes. The suspension of microparticles was centrifuged again at 2000-.

And (3) freeze drying: the washed pellet of microparticles was resuspended in 3-5ml purified water or 5% mannitol, transferred to a 10ml pre-weighed glass vial, frozen using a freeze-drying plate set at-37 to-43 ℃, and then freeze-dried (primary drying at-20 ℃ and 0.05 bar vacuum for 16-48 hours, final drying at +20 ℃ and 0.025 bar for 12-16 hours). After freeze-drying the vials were weighed, closed with a brominated butyl rubber stopper, and then stored under refrigerator storage conditions until use.

Estimation of particle size: using light field and phase contrast microscopy (Leutz Orthoplan)^TMGermany) with 40x and 10x objective lenses and a bench scale micrometer with a range of 1-1000 μm.

All microparticle formulations were prepared using an aqueous phase containing 0.75% sodium chloride to increase the external osmotic pressure and improve the incorporation of water-soluble charged drugs. Blank microparticles were obtained with 2% PVA as surfactant (first experiment), while 0.5% PVA was used for the preparation of all peptide-loaded formulations.

The compositions and parameters of the preparation process are listed in tables 2-5.

TABLE 2 PLGA microparticles for sustained release of Glatiramer Acetate (GA) (formulations 1-4)

A VWR VDI-12 high shear mixer from IKA, Germany with a smaller diameter of the stator (12mm axis) and a speed range of 8-30,000rpm was set at position #5 (approximately 24,000 rpm). A short treatment (30 seconds) of an approximately 10% PLGA solution in dichloromethane in 2% PVA phase was used to prepare blank MP samples, which resulted in smooth spherical microparticles with a relatively broad size distribution (10-50 μm). Due to foaming, further treatment is carried out at lower concentrations of surfactant. The homogenization time (1 or 2 minute treatment) was also extended to obtain a narrower size distribution.

Due to the presence of the internal aqueous phase in the double emulsion, all microparticles prepared with glatiramer peptide had visible inclusions and pore marks on the MP surface or within the particles when observed under an optical microscope.

TABLE 3 PLGA microparticles for sustained release of Glatiramer Acetate (GA) (formulations 5-7)

The resulting microparticles loaded with glatiramer acetate were centrifuged, the particles were resuspended in purified water, washed and centrifuged repeatedly. The supernatants and (in some cases) the wash water were analyzed for glatiramer acetate content. The centrifuged pellet is resuspended in purified water or 5% mannitol solution and then freeze-dried.

Table 4: PLGA microparticles (formulation 05R, 08-011 and tocopherol succinate of glatiramer) for sustained release of Glatiramer Acetate (GA)

Tocopherol succinate (MW 530, one COOH equivalent, 265 Dalton) and glatiramer acetate (MW4,700-11,000, one NH) were prepared by suspending an aqueous solution of glatiramer in dichloromethane containing an equimolar amount of previously dissolved tocopheryl succinate, with the aid of an ultrasonic head for 60 seconds (6X10 seconds) and ice cooling₂Equivalent, — 693 daltons) of an equimolar complex (salt). Collecting the water-insoluble material thus formed after evaporation of the organic solvent and waterThe product, washed with purified water and with dry ethanol, was then used for further studies without additional purification.

TABLE 5 PLGA microparticles for sustained release of Glatiramer Acetate (GA) (formulation MPG-12-15)

Freeze drying

After centrifugation and washing, the microparticle preparation is freeze-dried "as is" after resuspension of the sediment in purified water, or in some cases by means of addition of a cryoprotectant (resuspending the sediment in a 5% mannitol solution). The samples were frozen at-37 to-43 ℃ for 1 hour using freeze dryer plates, then freeze dried at 0.050 mbar and-20 ℃ for 24-48 hours using a freeze dryer "Alpha 2-4 LSC" (Christ, Germany), and finally dried at 0.025 mbar and +20 ℃ for 10-16 hours. In both resuspension procedures, the lyophilized product can be easily reconstituted. The use of mannitol leads to an easily reconstitutable product compared to a formulation without cryoprotectant, but such a composition contains a significant amount of ballast material and thus requires more complex calculations to determine the true concentration of the active substance.

Example 4: in vitro release of glatiramer acetate from PLGA microparticles

Device

20ml vial

Multi-point magnetic stirrer

Constant temperature box

Pipette with a suction tube

UV-Vis spectrophotometer Shimadzu 1601

Reagent and plastic/glassware

Test sample

Formulations MPG-02, 03, 04, 05R, 06, 07, 12, 13, 14, and 15-50mg of dried freeze-dried microparticles.

Formulations MPG-08, 09, 10, and 11: corresponding to an amount of 50mg of dried microparticles, were lyophilized with 5% mannitol.

Control glatiramer acetate solution 20-50 μ g/mL (as a basis), in PBS with 0.05% sodium azide)

Temperature: 37 ℃ C

To evaluate the release of incorporated glatiramer acetate from biodegradable PLGA microparticles (various formulations) loaded with glatiramer acetate, the following process has been employed.

The process is described as follows: 20ml of PBS (0.01M phosphate, 0.05% NaN)₃) ph7.4 was added to each vial. The vial was placed at 37 ℃ and stirred with a small magnet. 600 μ l of the sample was centrifuged at 10,000g for 5 minutes. Mu.l of the supernatant was transferred to a 1.5ml microtube, followed by the addition of 500. mu.l of 0.1M borate buffer (2-fold dilution) and 50. mu.l of TNBS. The obtained composition was mixed vigorously and kept on the bench for 30 minutes. The analysis was performed using the TNBS method.

Will be washed with 500. mu.l fresh PBS (containing NaN)₃) The re-suspended remaining settled particles were returned to the vial. In an additional release process (2.5% for each time point), the correct calculation of the release of glatiramer acetate was performed.

The release of the incorporated glatiramer acetate was carried out in a tightly closed 20ml glass vial using a 37 ℃ incubator equipped with a multi-point magnetic stirrer. Phosphate Buffered Saline (PBS) (ph 7.4) was used as the release medium.

The release of glatiramer acetate was tested over a period of 10-32 days.

The equation for the calibration curve in the range of 1-200. mu.g/ml (Shimadzu UV-1601) was calculated as:

OD=0.035+0.0132*C (r²=0.9985)

in which OD-optical density (difference at 420 and 700 nm)

Concentration of C-glatiramer acetate base, μ g/ml

The results of peptide release for formulations MPG01-MPG07 are shown in FIG. 1. In formulation MPG-05, which is based on a low molecular weight PLGA polymer with acidic end groups (Resomer RG 502H) and no hydrophobic counter ions, the fastest release (1-10 days, 40%) of incorporated glatiramer acetate was obtained. Neutral polymer RG 502 with a relatively small amount of tocopheryl succinic acid as the counterion (MPG-03) also exhibited significant release (2-12 days, — 30%), but had a lower absolute release value. Formulations containing higher amounts of counter-ion show inhibition of drug release. Without being bound by any theory or mechanism of action, this may be due to the high hydrophobicity of the formed complex. In addition, the preparation of microparticles comprising DCP or DMPG is accompanied by the formation of aggregates and a broader particle size distribution.

The use of a larger and more powerful high shear mixer OMNI GLH (shaft diameter 20mm, 5000-. Increasing the amount of organic solvent (MPG-02) reduces the incorporation of the peptide into the microparticles. Without being bound to any theory or mechanism of action, this may be due to an increase in the intermediate o/w/o double emulsion droplet size. Similarly, the use of polysorbate as a non-ionic surfactant also negatively affected drug loading (MPG-10, with 0.2% tween-80). The addition of hydrophobic counter ions (tocopheryl succinic acid, dimyristoyl phosphatidylglycerol DMPG, dicetyl phosphate DCP) significantly hindered the release of peptides from the polymeric microparticles compared to formulations without counter ions (MPG-05, MPG-05R). Without being bound by any theory or mechanism of action, the addition of a hydrophobic counter ion may provide microparticles with compromised properties (MPG-06).

The chemical structure of the polymer used shows a controlled release compared to the molecular weight of PLGAA greater impact on performance. The Resomers RG 502H and RG 502(MW about 17,000 daltons) have very similar diffusion coefficients, but the main factor determining the release of the included peptides from the polymer matrix is the multi-point ionic interaction between the positively charged Lys moiety of glatiramer acetate and the carboxylic acid end groups in the PLGA polymer. Neutral propertyRG 502 exhibits a lower binding capacity even in the presence of counterions (MPG-02, 03), while having a higher molecular weight of neutralityRG 503 showed better binding but very slow release (MPG-10, 11).

For such small-scale batches, repeated release experiments from the same formulations (MPG-05 and MPG-05R) prepared separately showed reasonably similar behavior and good reproducibility. Glatiramer acetate andthe formulation of RG 502H showed similar burst effect (-30%), good initial peptide binding and rapid drug release (fig. 2).

Formulations of equimolar complexes (salts) of tocopheryl succinate have high binding and very low water solubility (-5 μ g/ml). Without being bound by any theory or mechanism of action, this may result from ionic crosslinking of the polyamine molecules of the diacid (tocopheryl succinic acid) and the polymer. The release of polymer from this salt in PBS is extremely slow. For polymer microparticles, when tocopherol succinate is incorporated into a PLGA matrix, only part of this diacid can interact with the polymer and higher amounts of tocopherol succinate are needed for complete release inhibition. Therefore, the release rate can be adjusted by the ratio of glatiramer and PLGA. The amount of organic solvent used may also be of importance (but to a lesser extent).

Formulations 12-15 based on drugs andwith different proportions of polymersRG 502H, indicating that the above ratios play an important role in controlling the initial burst effect, the level of binding and the release rate. The modulation of the amount of PLGA and peptide and the addition of a hydrophobic counter ion (e.g. tocopherol succinate) facilitates the preparation of a microparticle formulation (MPG-12-15) with high binding, low initial burst and reasonable release rate (fig. 3).

Example 5: scale up by scaling

Freeze-dried sample of glatiramer acetate microparticle preparation

The formulation of MPG-14 SU-1-MPG-014 was prepared using a larger reaction vessel and a larger homogenizer (OMNI GLH) at low speed.

A total of-13 vials; each vial contains approximately 235mg of the lyophilized formulation, with a total glatiramer acetate content of-18.2 mg per vial, equivalent to-75 μ g/mg of the lyophilized formulation.

The formulation of MPG-15 SU-1-MPG-015 was prepared using a larger reaction vessel and a larger homogenizer at low speed (OMNI GLH).

A total of-10 vials; approximately 145mg of the lyophilized formulation per vial diet, with a total glatiramer acetate content of-14.9 mg per vial, equivalent to-100 μ g per mg of lyophilized formulation.

The formulation of MPG-14 SU-2-MPG-014 was prepared using the same reaction vessel, the same homogenizer (VDI 12) and the same parameters, and the process was repeated several times. The composition is washed thoroughly to reduce initial burst.

A total of-12 vials; each vial contains approximately 88mg of the lyophilized formulation, with a total glatiramer acetate content of-6.3 mg per vial, equal to-72 μ g per mg of the lyophilized formulation.

The formulation of MPG-15 SU-2-MPG-015 was prepared using the same reaction vessel, the same homogenizer (VDI 12) and the same parameters, and the process was repeated several times. The composition is washed thoroughly to reduce initial burst.

A total of-12 vials; each vial contains approximately 55mg of the lyophilized formulation, with a total glatiramer acetate content of-5.6 mg per vial, equal to-100 μ g per mg of the lyophilized formulation.

All freeze-dried samples were stored in a refrigerator at +4 ℃ and reconstituted prior to use.

The ratio between the formulation and the diluent (glucose solution) is at least 1:5, preferably 1:10 and higher. Vigorous shaking was performed before administering the reconstituted sample. The release profiles of these formulations are shown in figures 4 and 5.

Thus, the incorporation of highly water-soluble peptides of glatiramer acetate into biodegradable polymer microparticles was demonstrated. The microparticles show good binding of the polymer, reasonable drug loading and a reduced initial release burst, which can be adjusted by using different compositions and manufacturing processes. Prepared at 37 ℃ in a stirred aqueous medium (phosphate buffered saline, pH7.4)502H PLGA microparticles loaded with glatiramer acetate may provide in vitro release of incorporated peptide at a rate of 3-5% per day for a period of 10-15 days.

Example 6: experimental Autoimmune Encephalomyelitis (EAE) model

Experimental Autoimmune Encephalomyelitis (EAE) is an inflammatory autoimmune demyelinating disease that can be induced in experimental animals by injection of myelin basic protein. The above diseases have become standard experimental models for the study of clinical and experimental autoimmune diseases. Indeed, many articles (e.g., Abramsky et al, J neuroimiumnol (1982)21 and Bolton et al, J neurolsi (1982) 56147) note that the similarity of chronic relapsing EAE in animals and multiple sclerosis in humans is particularly implicated in the value of EAE for studying autoimmune demyelinating diseases such as multiple sclerosis. Thus, the EAE assay model was used to establish the activity of the formulations of the invention relative to multiple sclerosis. The above test was performed according to the following procedure.

12.5 μ g Myelin Basic Protein (MBP) (prepared from guinea pig spinal cord) in complete Freund's adjuvant was injected into the soles of female Lewis rats. The formulations of the present invention were administered to experimental animals by injection at various doses once a week/two weeks/month. The control formulation was administered to certain other experimental animals. Animals were then weighed and scored daily for symptoms of EAE on a scale of 0 to 3 (0= no change; 1= relaxant tail; 2= hind limb disability; and 3= transiliac and transiliac paralysis/moribundity). If a score of 3 is reached, the animal is sacrificed.

Example 7: in vivo studies using EAE model

To determine the effect of the formulations of the invention on a murine model of MS, Experimental Autoimmune Encephalomyelitis (EAE) was induced. Prior to EAE immunization, the vaccine was administered with a composition containing 0.87% calcium and 1ng of 1,25- (OH)₂D₃Purified dietary supplement of (Vit D) 25-hydroxyvitamin D₃-1 alpha-hydroxylase knockout mice (1 alpha-OH KO) for two to three weeks. By subcutaneous immunization of 6 to 10 week old mice with 200 μ g of oligodendrocyte glycoprotein (MOG) relative to myelin_35-55) To induce EAE.

Peptides were synthesized by standard 9-fluorenyl-methoxy-carbonyl chemistry. The peptide was dissolved in Freund's complete adjuvant (CFA; Sigma) containing 4mg/ml of heat-inactivated Mycobacterium tuberculosis H837 a.

Mice were examined daily for clinical signs of EAE using the following scoring system: 0, no signs; 1, lameness tail; 2, weakness of hind limbs; 3, hind limb paralysis; 4, paralysis of the forelimbs; 5, dying or death.

For mice developing clinical signs of EAE and scoring ≧ 2, various doses of the formulations of the present invention were administered for treatment by injection, where injection was weekly/biweekly/monthly. The control group was treated with placebo or with the Gold standard protocol for glatiramer acetate [ e.g. PNAS,2005, vol.102, No.52, 19045-. Mice were then weighed and scored daily for symptoms of EAE. Statistical analysis was performed using a two-tailed fisher's correct probability test for incidence and an unpaired t-test for all other measurements. A value of P <0.05 was considered statistically significant.

While the invention has been particularly described, it will be apparent to those skilled in the art that many changes and modifications may be made. Accordingly, the invention is not limited to the specifically described embodiments, and the scope and concept of the invention will be more readily apparent by reference to the claims.

Claims (41)

1. A long acting parenteral pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of glatiramer.

2. A long acting pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of glatiramer in depot form suitable for implantation at a medically acceptable location in a subject in need thereof.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutically acceptable salt of glatiramer is glatiramer acetate.

4. The pharmaceutical composition according to claim 1 or 2, suitable for subcutaneous or intramuscular implantation.

5. The pharmaceutical composition according to claim 1 or 2, wherein the glatiramer comprises L-alanine, L-glutamic acid, L-lysine, and L-tyrosine in a molar ratio of about 0.14 glutamic acid, about 0.43 alanine, about 0.10 tyrosine and about 0.33 lysine.

6. The pharmaceutical composition according to claim 1 or 2, wherein the glatiramer comprises about 15 to about 100 amino acids.

7. The pharmaceutical composition according to claim 1 or 2, wherein the dose of the pharmaceutically acceptable salt of glatiramer is in the range of about 20 to about 750 mg.

8. The pharmaceutical composition of claim 1 or 2, further comprising a pharmaceutically acceptable biodegradable or non-biodegradable carrier.

9. The pharmaceutical composition of claim 8, wherein the carrier is selected from the group consisting of PLGA, PLA, PGA, polycaprolactone, polyhydroxybutyrate, polyorthoesters, polyalkaneanhydrides, gelatin, collagen, oxidized cellulose, and polyphosphazene.

10. The pharmaceutical composition according to claim 1 or 2, in the form of microparticles prepared by an aqueous-in-oil-in-aqueous double emulsification process.

11. The pharmaceutical composition according to claim 1 or 2, comprising: an internal aqueous phase comprising a therapeutically effective amount of a pharmaceutically acceptable salt of glatiramer; a water-immiscible polymeric phase comprising a biodegradable or non-biodegradable polymer; and an external aqueous phase.

12. The pharmaceutical composition of claim 11, wherein the water-immiscible polymer phase comprises a biodegradable polymer selected from PLA and PLGA.

13. The pharmaceutical composition of claim 11, wherein the external aqueous phase comprises a surfactant selected from polyvinyl alcohol (PVA), polysorbates, polyethylene oxide-polypropylene oxide block copolymers, and cellulose esters.

14. The pharmaceutical composition according to claim 1 or 2, suitable for a dosing schedule of once weekly to once every 6 months.

15. The pharmaceutical composition according to claim 14, suitable for a dosing schedule of once every 2 weeks to once a month.

16. The pharmaceutical composition according to claim 1 or 2, having the form: biodegradable microspheres, non-biodegradable microspheres, implants of any suitable geometry, implantable rods, implantable capsules, implantable rings, or extended release gels or erodible matrices.

17. The pharmaceutical composition according to claim 1 or 2, wherein the composition provides the same or better therapeutic effect, at a local and/or systemic level, a reduced incidence of side effects and/or a reduced severity of side effects, relative to a commercially available daily injectable dosage form of glatiramer acetate.

18. The pharmaceutical composition according to claim 17, wherein the composition provides an extended release or prolonged action of glatiramer in a subject as compared to an immediate release dosage form of glatiramer acetate at about the same dose.

19. A method for treating multiple sclerosis, wherein the long-acting pharmaceutical composition of claim 1 is administered parenterally.

20. A method for treating multiple sclerosis comprising the steps of: implanting a long-acting pharmaceutical composition according to claim 2 into a subject in need thereof.

21. The method according to claim 19 or 20, comprising the steps of: the pharmaceutical composition is administered subcutaneously or implanted intramuscularly.

22. The method of claim 19 or 20, wherein the pharmaceutical composition comprises glatiramer acetate.

23. The method of claim 22, wherein the glatiramer acetate comprises L-alanine, L-glutamic acid, L-lysine, and L-tyrosine in a molar ratio of about 0.14 glutamic acid, about 0.43 alanine, about 0.10 tyrosine, and about 0.33 lysine.

24. The method of claim 23, wherein the glatiramer comprises about 15 to about 100 amino acids.

25. The method according to claim 19 or 20, wherein the pharmaceutical composition comprises a pharmaceutically acceptable salt of glatiramer at a dose ranging from about 20 to about 750 mg.

26. The method of claim 19 or 20, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable biodegradable or non-biodegradable carrier.

27. The method of claim 26, wherein the carrier is selected from the group consisting of PLGA, PLA, PGA, polycaprolactone, polyhydroxybutyrate, polyorthoesters, polyalkaneanhydrides, gelatin, collagen, oxidized cellulose, and polyphosphazene.

28. The method of claim 19 or 20, wherein the pharmaceutical composition is in the form of microparticles prepared by an aqueous-in-oil-in-aqueous double emulsification process.

29. The method of claim 19 or 20, wherein the pharmaceutical composition comprises: an internal aqueous phase comprising a therapeutically effective amount of a pharmaceutically acceptable salt of glatiramer; a water-immiscible polymeric phase comprising a biodegradable or non-biodegradable polymer; and an external aqueous phase.

30. The method of claim 29, wherein the water-immiscible polymer phase comprises a biodegradable polymer selected from PLA and PLGA.

31. The method of claim 29, wherein the external aqueous phase comprises a surfactant selected from polyvinyl alcohol (PVA), polysorbates, polyethylene oxide-polypropylene oxide block copolymers, and cellulose esters.

32. The method of claim 19 or 20, wherein the administering or implanting step is performed on a dosing schedule of once weekly to once every 6 months.

33. The method of claim 32, wherein the dosing schedule is from once every 2 weeks to once a month.

34. The method of claim 19 or 20, wherein the pharmaceutical composition is in the form of: biodegradable microspheres, non-biodegradable microspheres, implants of any suitable geometry, implantable rods, implantable capsules, or implantable rings, extended release gels, or erodible matrices.

35. The method of claim 19 or 20, wherein the pharmaceutical composition provides the same or better therapeutic effect, reduced incidence of side effects and reduced severity of side effects at local and/or systemic level relative to commercially available daily injectable dosage forms.

36. The method according to claim 35, wherein the pharmaceutical composition provides an extended release or prolonged action of glatiramer in a subject as compared to an immediate release dosage form of glatiramer acetate at about the same dose.

37. Use of a pharmaceutically acceptable salt of glatiramer in depot form suitable for implantation into an individual in need thereof for the treatment of multiple sclerosis.

38. Use of an implantable depot according to claim 37 for providing prolonged release or prolonged action of glatiramer in a subject.

39. The use according to any one of claims 37 to 38, wherein the pharmaceutically acceptable salt of glatiramer is glatiramer acetate.

40. A pharmaceutically acceptable salt of glatiramer in depot form suitable for use in the treatment of multiple sclerosis.

41. A pharmaceutically acceptable salt of glatiramer in depot form suitable for use in providing prolonged release or prolonged action of glatiramer in a subject.