AMINO ACIDS

This application claims priority of U.S. Provisional Application No. 62/198,845, filed July 30, 2015, and U.S. Provisional Application No. 62/203,814, filed August 11, 2015, the entire contents of each of which are hereby incorporated by reference herein.

Throughout this application various publications, published patent applications, and patents are referenced. The disclosures of these documents in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Background

Laquinimod Laquinimod is a compound which has been shown to be effective in the acute experimental autoimmune encephalomyelitis (aEAE) model (U.S. Patent No. 6,077,851). Its chemical name is N-ethyl-N- phenyl-1, 2-dihydro-4-hydroxy-5-chloro-l-methyl-2-oxoquinoline-3- carboxamide, and its Chemical Registry number is 248281-84-7. The processes of synthesis of laquinimod and the preparation of its sodium salt are disclosed in U.S. Patent No. 6,077,851. An additional process of synthesis of laquinimod is disclosed in U.S Patent No. 6, 875, 869.

Pharmaceutical compositions comprising laquinimod sodium are disclosed in, e.g., U.S. Patent No. 7, 989, 473 and PCT International Application Publication No. WO 2005/074899.

Laquinimod sodium has high oral bioavailability and has been suggested as an oral formulation for the treatment of Multiple Sclerosis (MS) . (Polman, 2005 and Sandberg-Wollheim, 2005) . Studies have also shown that laquinimod can reduce development of active MRI lesions in relapsing MS. (Polman 2005).

Glatiramer Acetate Glatiramer acetate (GA) , also known as Copolymer-1, has been shown to be effective in treating multiple sclerosis (MS)

(Lampert, 1978). Daily subcutaneous injections of glatiramer acetate (20 mg/inj ection) reduce relapse rates, progression of disability, appearance of new lesions by magnetic resonance imaging (MRI) , (Johnson, 1995) and appearance of "black holes"

(Filippi, 2001) .

COPAXONE® is the brand name for a formulation containing glatiramer acetate as the active ingredient. Glatiramer acetate is approved for reducing the frequency of relapses in relapsing- remitting multiple sclerosis (RRMS) . Glatiramer acetate consists of the acetate salts of synthetic polypeptides containing four naturally occurring amino acids: L-glutamic acid, L-alanine, L- tyrosine, and L-lysine with an average molar fraction in COPAXONE® of 0.141, 0.427, 0.095 and 0.338, respectively. In COPAXONE®, the average molecular weight of the glatiramer acetate is 4,700-11,000 daltons. Chemically, glatiramer acetate is designated L-glutamic acid polymer with L-alanine, L-lysine and L-tyrosine, acetate (salt). Its structural formula is: (Glu, Ala, Lys, Tyr) XACH3COOH

(C5H9N04AC3H7N02AC6H14N202AC9H11N03) PAPC2H402

CAS - 147245-92-9.

The recommended dosing schedule of COPAXONE® for relapsing- remitting multiple sclerosis is 20 mg per day or 40 mg three times per week injected subcutaneously (Physician's Desk Reference; see also U.S. Patent Nos . 3,849,550; 5,800,808; 5,858,964, 5,981,589; 6,048,898; 6,054,430; 6,214,791; 6,342,476; 6,362,161; and 8,399,413, all of which are hereby incorporated by reference) . Combination of Laquinimod and Glatiramer Acetate

Laquinimod as an add-on therapy to or in combination with GA for treating multiple sclerosis was described in U.S. Application Publication No. 2013-0029916. Stable formulations comprising laquinimod and GA have not been reported.

Summary of the Invention

The subject invention provides a stable pharmaceutical composition comprising a therapeutically effective amount of laquinimod, a therapeutically effective amount of glatiramer acetate related drug substance, and an amount of an amino acid.

The subject invention also provides a composition comprising a therapeutically effective amount of laquinimod and an amount of an amino acid.

The subject invention also provides a composition comprising a therapeutically effective amount of glatiramer acetate related drug substance and an amount of an amino acid.

The subject invention also provides a process for making a stable pharmaceutical liquid form comprising a therapeutically effective amount of laquinimod or pharmaceutically acceptable salt thereof, a therapeutically effective amount of glatiramer acetate related drug substance, and an amount of an amino acid comprising: a) obtaining the laquinimod or pharmaceutically acceptable salt thereof, the glatiramer acetate related drug substance, and the amino acid, and b) forming the liquid form of the laquinimod or pharmaceutically acceptable salt thereof, the glatiramer acetate related drug substance, and the amino acid.

The subject invention also provides a process for making a liquid form comprising a therapeutically effective amount of glatiramer acetate related drug substance and an amount of an amino acid comprising: a) obtaining the glatiramer acetate related drug substance and the amino acid, and b) forming the liquid form of the glatiramer acetate related drug substance and the amino acid.

The subject invention also provides a process for making a liquid form comprising a therapeutically effective amount of laquinimod or pharmaceutically acceptable salt thereof and an amount of an amino acid comprising: a) obtaining the laquinimod or pharmaceutically acceptable salt thereof and the amino acid, and b) forming the liquid form of the laquinimod or pharmaceutically acceptable salt thereof and the amino acid.

The subject invention also provides a stable pharmaceutical composition comprising a therapeutically effective amount of laquinimod or pharmaceutically acceptable salt thereof, a therapeutically effective amount of glatiramer acetate related drug substance, and an amount of an amino acid, prepared by the processes described herein.

The subject invention also provides a sealed package comprising the compositions described herein. The subject invention also provides a method for treating a subject afflicted with a form of autoimmune disease comprising administering to the subject a composition as described herein so as to thereby treat the subject.

In one embodiment, the autoimmune disease is multiple sclerosis. In one embodiment, the multiple sclerosis is relapsing multiple sclerosis. In one embodiment, the relapsing multiple sclerosis is relapsing-remitting multiple sclerosis.

The subject invention also provides a method for i) treating a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, and/or ii) alleviating a symptom of multiple sclerosis in a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, comprising administering to the subject a composition as described herein so as to thereby treat and/or alleviate the symptom of multiple sclerosis in the subject.

The subject invention also provides a method for providing neuroprotection to a subject in need thereof comprising administering to the subject a composition as described herein so as to thereby provide neuroprotection to the subject. The subject invention also provides use of a composition as described herein for treating a subject afflicted with an autoimmune disease. The subject invention also provides use of a composition as described herein for i) treating a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, and/or ii) alleviating a symptom of multiple sclerosis in a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome.

The subject invention also provides use of a composition as described herein for providing neuroprotection to a subject in need thereof. The subject invention also provides use of a composition as described herein in the manufacture of a medicament for treating a subject afflicted with an autoimmune disease.

The subject invention also provides use of a composition as described herein in the manufacture of a medicament for i) treating a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, and/or ii) alleviating a symptom of multiple sclerosis in a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome.

The subject invention also provides use of a composition as described herein in the manufacture of a medicament for providing neuroprotection to a subject in need thereof.

The subject invention also provides a composition as described herein for use in a) treating a subject afflicted with an autoimmune disease, b) i) treating a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, and/or ii) alleviating a symptom of multiple sclerosis in a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, and/or c) providing neuroprotection to a subject in need thereof. The subject invention also provides a pharmaceutical oral unit dosage form of a therapeutically effective amount of laquinimod or pharmaceutically acceptable salt thereof, a therapeutically effective amount of glatiramer acetate related drug substance, and an amount of an amino acid.

The subject invention also provides a package comprising: a) one or more unit doses, each such unit dose comprising i) a therapeutically effective amount of laquinimod or pharmaceutically acceptable salt thereof, ii) a therapeutically effective amount of glatiramer acetate related drug substance, and iii) an amount of an amino acid, and b) instructions for use of the one or more unit doses for i) treating a subject afflicted with an autoimmune disease, ii) treating a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, and/or alleviating a symptom of multiple sclerosis in a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, and/or iii) providing neuroprotection to a subject in need thereof.

Brief Description of the Drawings

Figure 1: Figure 1 is an illustration of diverse nanoparticles aimed to be used in drug delivery of nanodrugs .

Figure 2: Figure 2 is an illustration of the Ubbelohde

Viscometer .

Figure 3 shows the gelation (on the y-axis) in relation to the concentration of L-lysine (mg/mL) (on the x-axis) . Figure 4 : Figures 4Ά and 4B show the gelation (on the y-axis) in relation to the concentration of amino acids glycine, lysine, proline, histidine, and alanine (mg/mL) (on the x-axis) .

Figure 5 shows the least squares analysis of the gelation of GA-LAQ formulation with L-lysine and pure water (actual gelation on the y-axis; gelation predicted on the x-axis) .

Figure 6 is a laquinimod leverage plot with gelation leverage residuals on the y-axis and laquinimod leverage on the x-axis.

Figure 7 : Figure 7 is a GA leverage plot with gelation leverage residuals on the y-axis and GA leverage on the x-axis.

Detailed Description of the Invention

The subject invention provides a stable pharmaceutical composition comprising a therapeutically effective amount of laquinimod, a therapeutically effective an amount of glatiramer acetate related drug substance, and an amount of an amino acid.

The subject invention also provides a composition comprising a therapeutically effective amount of laquinimod and an amount of an amino acid.

The subject invention also provides a composition comprising a therapeutically effective amount of glatiramer acetate related drug substance and an amount of an amino acid.

In one embodiment, the composition is in liquid form. In one embodiment, the composition is a lyophilized powder. In one embodiment, the composition is an aqueous solution. In one embodiment, the composition is clear. In one embodiment, the composition is isotonic.

In one embodiment, the laquinimod is a pharmaceutically acceptable salt of laquinimod. In another embodiment, the pharmaceutically acceptable salt of laquinimod is potassium salt, lithium salt, sodium salt or calcium salt. In another embodiment, the pharmaceutically acceptable salt of laquinimod is laquinimod sodium

In one embodiment, the laquinimod is laquinimod acid.

In one embodiment, the therapeutically effective amount of laquinimod is less than 0.6 mg. In another embodiment, the therapeutically effective amount of laquinimod is 0.1-40.0 mg. In another embodiment, the therapeutically effective amount of laquinimod is 0.25mg - 1.5mg. In another embodiment, the therapeutically effective amount of laquinimod is 0.1 mg. In another embodiment, the therapeutically effective amount of laquinimod is 0.25 mg. In another embodiment, the therapeutically effective amount of laquinimod is 0.3 mg. In another embodiment, the therapeutically effective amount of laquinimod is 0.5 mg. In another embodiment, the therapeutically effective amount of laquinimod is 0.6 mg. In another embodiment, the therapeutically effective amount of laquinimod is 1.0 mg. In yet another embodiment, the therapeutically effective amount of laquinimod is 1.2 mg. In another embodiment, the therapeutically effective amount of laquinimod is 1.5 mg. In another embodiment, the therapeutically effective amount of laquinimod is 2.0 mg.

In one embodiment, the therapeutically effective amount of laquinimod has a concentration of 0.1-3 mg/mL. In one embodiment, the therapeutically effective amount of laquinimod has a concentration of 0.7 mg/mL. In one embodiment, the therapeutically effective amount of laquinimod has a concentration of 0.8 mg/mL. In one embodiment, the therapeutically effective amount of laquinimod has a concentration of 0.9 mg/mL. In one embodiment, the therapeutically effective amount of laquinimod has a concentration of 1 mg/mL. In one embodiment, the therapeutically effective amount of laquinimod has a concentration of 1.1 mg/mL. In one embodiment, the therapeutically effective amount of laquinimod has a concentration of 1.25 mg/mL. In one embodiment, the therapeutically effective amount of laquinimod has a concentration of 1.5 mg/mL. In one embodiment, the therapeutically effective amount of laquinimod has a concentration of 2 mg/mL. In one embodiment, the therapeutically effective amount of laquinimod has a concentration of 3 mg/mL.

In one embodiment, the glatiramer acetate related drug substance is glatiramer acetate.

In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance is 0.1-1000 mg. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance is 50-150 mg. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance is 0.1-70 mg. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance is 10-80 mg. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance is 1 mg. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance is 5 mg. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance is 15 mg. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance is 20 mg. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance is 30 mg. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance is 40 mg. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance is 50 mg. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance is 100 mg. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance has a concentration of 5-40 mg/mL. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance has a concentration of 5 mg/mL. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance has a concentration of 10 mg/mL. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance has a concentration of 30 mg/mL. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance has a concentration of 35 mg/mL. In one embodiment, the therapeutically effective amount of glatiramer acetate related drug substance has a concentration of 40 mg/mL.

In one embodiment, the composition is in a unit dose. In one embodiment, the unit dose comprises 0.1 mg, 0.25 mg, 0.3 mg, 0.5 mg, 0.6 mg, 1.0 mg, 1.2 mg, 1.5 mg, or 2.0 mg of laquinimod and 1 mg, 5 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, or 100 mg of glatiramer acetate related drug substance.

In one embodiment, the unit dose has a volume of 0.5-5mL. In one embodiment, the unit dose has a volume of 1.0 mL. In another embodiment, the unit dose has a volume of 1.5 mL. In another embodiment, the unit dose has a volume of 2 mL. In another embodiment, the unit dose has a volume of 3 mL. In another embodiment, the unit dose has a volume of 4 mL. In another embodiment, the unit dose has a volume of 5 mL. In one embodiment, the unit dose is in a syringe. In one embodiment, the unit dose is in a pre-filled syringe for administration. In another embodiment, the unit dose is in a vial. In another embodiment, the unit dose is in an ampule. In another embodiment, the unit dose is in a cartridge. In another embodiment, the unit dose is in an infusion.

In one embodiment, the amino acid is selected from lysine, glycine, proline, alanine, or histidine.

In one embodiment, the amino acid is lysine. In one embodiment, the amino acid is glycine. In one embodiment, the amino acid is proline. In one embodiment, the amino acid is alanine. In one embodiment, the amino acid is histidine.

In one embodiment, the amino acid has a concentration of 0.5-22 mg/mL. In one embodiment, the amino acid has a concentration of 0.5-5 mg/mL. In one embodiment, the amino acid has a concentration of 0.5-3.5 mg/mL. In one embodiment, the amino acid has a concentration of 0.7-1.8 mg/mL. In one embodiment, the amino acid has a concentration of 0.5 mg/mL. In another embodiment, the amino acid has a concentration of 0.6 mg/mL. In another embodiment, the amino acid has a concentration of 0.7 mg/mL. In another embodiment, the amino acid has a concentration of 0.8 mg/mL. In another embodiment, the amino acid has a concentration of 0.9 mg/mL. In another embodiment, the amino acid has a concentration of 1 mg/mL. In another embodiment, the amino acid has a concentration of 1.1 mg/mL. In another embodiment, the amino acid has a concentration of 1.2 mg/mL. In another embodiment, the amino acid has a concentration of 1.3 mg/mL. In another embodiment, the amino acid has a concentration of 1.4 mg/mL. In another embodiment, the amino acid has a concentration of 1.5 mg/mL. In another embodiment, the amino acid has a concentration of 2 mg/mL. In another embodiment, the amino acid has a concentration of 2.25 mg/mL. In another embodiment, the amino acid has a concentration of 2.5 mg/mL. In another embodiment, the amino acid has a concentration of 3 mg/mL. In another embodiment, the amino acid has a concentration of 3.5 mg/mL. In another embodiment, the amino acid has a concentration of 4 mg/mL. In another embodiment, the amino acid has a concentration of 5 mg/mL.

In one embodiment, the concentration ratio of lysine to laquinimod (Lys:Laq) is from about 0.4mg/mL: Img/mL to about 3.2mg/mL : Img/mL .

In one embodiment, the concentration ratio of glycine to laquinimod (Gly:Laq) is from about 0.5mg/mL : lrng/mL to about 4. Img/mL: Img/mL. In one embodiment, the concentration ratio of proline to laquinimod (Pro:Laq) is from about 1.5mg/mL : Img/mL to about 2.2mg/mL: Img/mL.

In one embodiment, the concentration ratio of alanine to laquinimod (Ala:Laq) is from about 0.6mg/mL: Img/mL to about 0.7mg/mL: Img/mL.

In one embodiment, the concentration ratio of histidine to laquinimod (His:Laq) is from about 1.2mg/mL: Img/mL to about 2.6mg/mL: Img/mL.

In one embodiment, the composition further comprises a tonicity agent. In one embodiment, the tonicity agent is mannitol, sodium chloride, or trehalose.

In one embodiment, the tonicity agent is mannitol. In one embodiment, the mannitol has a concentration of 7-50 mg/mL. In one embodiment, the mannitol has a concentration of 7 mg/mL. In one embodiment, the mannitol has a concentration of 20 mg/mL. In one embodiment, the mannitol has a concentration of 25 mg/mL. In one embodiment, the mannitol has a concentration of 30 mg/mL. In one embodiment, the mannitol has a concentration of 35 mg/mL. In one embodiment, the mannitol has a concentration of 37.5 mg/mL. In one embodiment, the mannitol has a concentration of 40 mg/mL. In one embodiment, the mannitol has a concentration of 43 mg/mL. In one embodiment, the mannitol has a concentration of 45 mg/mL. In one embodiment, the mannitol has a concentration of 50 mg/mL.

In one embodiment, the tonicity agent is sodium chloride. In one embodiment, the sodium chloride has a concentration of 5-10 mg/mL In one embodiment, the sodium chloride has a concentration of 5 mg/mL. In one embodiment, the sodium chloride has a concentration of 6 mg/mL. In one embodiment, the sodium chloride has a concentration of 7 mg/mL. In one embodiment, the sodium chloride has a concentration of 8 mg/mL. In one embodiment, the sodium chloride has a concentration of 9 mg/mL. In one embodiment, the sodium chloride has a concentration of 10 mg/mL.

In one embodiment, the composition further comprises a buffer. In one embodiment, the buffer is histidine, phosphate-buffered saline, phosphate salt, hydrocarbonate, or acetate. In one embodiment, the phosphate salt is a sodium salt, potassium salt, or sodium-potassium salt. In one embodiment, the hydrocarbonate is sodium bicarbonate. In one embodiment, the acetate is sodium acetate .

In one embodiment, the buffer has a concentration of 0.1-0.5 mol/L. In one embodiment, the buffer has a concentration of 0.1 mol/L. In another embodiment, the buffer has a concentration of 0.5 mol/L.

In one embodiment, the composition is prepared for periodic administration. In one embodiment, the periodic administration continues for more than 30 days. In another embodiment, the periodic administration continues for more than 42 days. In another embodiment, the periodic administration continues for 6 months or more.

In one embodiment, the composition is prepared for administration once daily. In another embodiment, the composition is prepared for administration more often than once daily. In one embodiment, the composition is prepared for administration three times per week. In another embodiment, the composition is prepared for administration less often than once daily. In one embodiment, the composition is prepared for administration by subcutaneous injection. In another embodiment, the composition is prepared for administration through an intravenous, intraperitoneal, intramuscular, intranasal, buccal, vaginal, rectal, intraocular, intrathecal, topical, oral, or intradermal route.

The subject invention also provides a process for making a stable pharmaceutical liquid form comprising a therapeutically effective amount of laquinimod or pharmaceutically acceptable salt thereof, a therapeutically effective amount of glatiramer acetate related drug substance, and an amount of an amino acid comprising: a) obtaining the laquinimod or pharmaceutically acceptable salt thereof, the glatiramer acetate related drug substance, and the amino acid, and b) forming the liquid form of the laquinimod or pharmaceutically acceptable salt thereof, the glatiramer acetate related drug substance, and the amino acid.

The subject invention also provides a process for making a liquid form comprising a therapeutically effective amount of glatiramer acetate related drug substance and an amount of an amino acid comprising: a) obtaining the glatiramer acetate related drug substance and the amino acid, and b) forming the liquid form of the glatiramer acetate related drug substance and the amino acid.

In one embodiment, the glatiramer acetate related drug substance is glatiramer acetate.

The subject invention also provides a process for making a liquid form comprising a therapeutically effective amount of laquinimod or pharmaceutically acceptable salt thereof and an amount of an amino acid comprising: a) obtaining the laquinimod or pharmaceutically acceptable salt thereof and the amino acid, and b) forming the liquid form of the laquinimod or pharmaceutically acceptable salt thereof and the amino acid.

In one embodiment, the process comprises forming a liquid form of the glatiramer acetate related drug substance and the amino acid prior to forming the liquid form of the laquinimod or pharmaceutically acceptable salt thereof, the glatiramer acetate related drug substance, and the amino acid.

In one embodiment, the process comprises forming a liquid form of the laquinimod or pharmaceutically acceptable salt thereof and the amino acid prior to forming the liquid form of the laquinimod or pharmaceutically acceptable salt thereof, the glatiramer acetate related drug substance, and the amino acid.

In one embodiment, the process comprises forming a liquid form of the glatiramer acetate related drug substance prior to forming the liquid form of the glatiramer acetate related drug substance and the amino acid. In another embodiment, the process comprises forming a liquid form of the amino acid prior to forming the liquid form of the glatiramer acetate related drug substance and the amino acid.

In one embodiment, the process comprises forming a liquid form of the laquinimod or pharmaceutically acceptable salt thereof prior to forming the liquid form of the laquinimod or pharmaceutically acceptable salt thereof and the amino acid. In another embodiment, the process comprises forming a liquid form of the amino acid prior to forming the liquid form of the laquinimod or pharmaceutically acceptable salt thereof and the amino acid.

In one embodiment, the liquid form is formed at stirring. In one embodiment, the process comprises passing the liquid form through a cellulose acetate syringe filter prior to forming a liquid form of the laquinimod or pharmaceutically acceptable salt thereof, the glatiramer acetate related drug substance, and the amino acid. In another embodiment, the process comprises storing the liquid form prior to forming a liquid form of the laquinimod or pharmaceutically acceptable salt thereof, the glatiramer acetate related drug substance, and the amino acid. In another embodiment, the liquid form is stored at 2-8 °C. In one embodiment, the liquid form is clear.

The subject invention also provides a stable pharmaceutical composition comprising a therapeutically effective amount of laquinimod or pharmaceutically acceptable salt thereof, a therapeutically effective amount of glatiramer acetate related drug substance, and an amount of an amino acid, prepared by the processes described herein.

In one embodiment, the glatiramer acetate related drug substance is glatiramer acetate.

The subject invention also provides a sealed package comprising the compositions described herein.

In one embodiment, the sealed package after storage at 25°C and at a relative humidity (RH) of 60% for at least 6 months is free of gelation. In one embodiment, the sealed package after storage at 25 °C and at a relative humidity (RH) of 60% for at least 12 months is free of gelation. In one embodiment, gelation is evaluated using the Ubbelohde tube method.

In one embodiment, the sealed package is a syringe. In one embodiment, the sealed package is a pre-filled syringe for administration. In another embodiment, the sealed package is a vial In another embodiment, the sealed package is an ampule. In another embodiment, the sealed package is a cartridge. In another embodiment, the sealed package is an infusion. The subject invention also provides a method for treating a subject afflicted with a form of autoimmune disease comprising administering to the subject a composition as described herein so as to thereby treat the subject.

The subject invention also provides a method for i) treating a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, and/or ii) alleviating a symptom of multiple sclerosis in a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, comprising administering to the subject a composition as described herein so as to thereby treat and/or alleviate the symptom of multiple sclerosis in the subject. The subject invention also provides a method for providing neuroprotection to a subject in need thereof comprising administering to the subject a composition as described herein so as to thereby provide neuroprotection to the subject.

The subject invention also provides use of a composition as described herein for treating a subject afflicted with an autoimmune disease.

The subject invention also provides use of a composition as described herein for i) treating a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, and/or ii) alleviating a symptom of multiple sclerosis in a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome.

The subject invention also provides use of a composition as described herein for providing neuroprotection to a subject in need thereof.

The subject invention also provides use of a composition as described herein in the manufacture of a medicament for treating a subject afflicted with an autoimmune disease.

The subject invention also provides use of a composition as described herein in the manufacture of a medicament for i) treating a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, and/or ii) alleviating a symptom of multiple sclerosis in a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome. The subject invention also provides use of a composition as described herein in the manufacture of a medicament for providing neuroprotection to a subject in need thereof. The subject invention also provides a composition as described herein for use in a) treating a subject afflicted with an autoimmune disease, b) i) treating a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, and/or ii) alleviating a symptom of multiple sclerosis in a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, and/or c) providing neuroprotection to a subject in need thereof.

The subject invention also provides a pharmaceutical oral unit dosage form of a therapeutically effective amount of laquinimod or pharmaceutically acceptable salt thereof, a therapeutically effective amount of glatiramer acetate related drug substance, and an amount of an amino acid.

The subject invention also provides a package comprising: a) one or more unit doses, each such unit dose comprising i) a therapeutically effective amount of laquinimod or pharmaceutically acceptable salt thereof, ii) a therapeutically effective amount of glatiramer acetate related drug substance, and iii) an amount of an amino acid, and b) instructions for use of the one or more unit doses for i) treating a subject afflicted with an autoimmune disease, ii) treating a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, and/or alleviating a symptom of multiple sclerosis in a subject afflicted with multiple sclerosis or presenting a clinically isolated syndrome, and/or iii) providing neuroprotection to a subject in need thereof .

For the foregoing embodiments, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Glatiramer Acetate

Glatiramer acetate mixtures, compositions, the process for the manufacture thereof, the use thereof for treatment of various conditions, and the corresponding dosages and regimens are described in, e.g., PCT International Application Publication Nos. WO 1998/30227, WO 2000/05250, WO 2000/18794, WO 2004/103297, WO 2006/029393, WO 2006/029411, WO 2006/083608, WO 2006/089164, WO 2006/116602. WO 2009/070298, WO 2011/022063, WO 2012/051106, WO 2003/048735, and WO 2011/008274, U.S. Patent Application Publication Nos . 2011-0230413 and 2008-027526, and U.S. Patent Nos. 8,399,413, 8,008,258 and 7,556,767, each of which is hereby incorporated by reference in its entireties into this application.

Laquinimod Laquinimod mixtures, compositions, and the process for the manufacture thereof are described in, e.g., U.S. Patent No. 6,077,851, U.S. Patent no. 7,884,208, U.S. Patent No. 7,989,473, U.S. Patent No. 8,178,127, U.S. Application Publication No. 2010- 0055072, U.S. Application Publication No. 2012-0010238, and U.S. Application Publication No. 2012-0010239, each of which is hereby incorporated by reference in its entireties into this application .

A pharmaceutically acceptable salt of laquinimod as used in this application includes lithium, sodium, potassium, magnesium, calcium, manganese, copper, zinc, aluminum and iron. Salt formulations of laquinimod and the process for preparing the same are described, e.g., in U.S. Patent No. 7,589,208 and PCT International Application Publication No. WO 2005/074899, which are hereby incorporated by reference into this application. Laquinimod can be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral administration. Laquinimod can be administered alone but is generally mixed with a pharmaceutically acceptable carrier, and co-administered in the form of a tablet or capsule, liposome, or as an agglomerated powder. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar.

Capsule or tablets can be formulated and can be made easy to swallow or chew; other solid forms include granules and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents (disintegrants ) , coloring agents, flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, microcrystalline cellulose and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn starch, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, povidone, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, sodium benzoate, sodium acetate, sodium chloride, stearic acid, sodium stearyl fumarate, talc and the like. Disintegrants include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, croscarmellose sodium, sodium starch glycolate and the like.

Specific examples of the techniques, pharmaceutically acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described, e.g., in U.S. Patent Application Publication No. 2005/0192315, PCT International Application Publication Nos. WO 2005/074899, WO 2007/047863, and WO/2007/146248, each of which is hereby incorporated by reference into this application.

General techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modern Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol. 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). These references in their entireties are hereby incorporated by reference into this application.

A dosage unit may comprise a single compound or mixtures of compounds thereof. A dosage unit can be prepared for oral dosage forms, such as tablets, capsules, pills, powders, and granules. Terms

As used herein, and unless stated otherwise, each of the following terms shall have the definition set forth below.

As used herein, "laquinimod" means laquinimod acid or a pharmaceutically acceptable salt thereof, as well as derivatives as laquinimod such as deuterium enriched laquinimod, and salts thereof .

A "salt" is salt of the instant compounds which have been modified by making acid or base salts of the compounds. The term "pharmaceutically acceptable salt" in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. A pharmaceutically acceptable salt of laquinimod as used in this application includes lithium, sodium, potassium, magnesium, calcium, manganese, copper, zinc, aluminum and iron. Salt formulations of laquinimod and the process for preparing the same are described, e.g., in U.S. Patent No. 7,589,208 and PCT International Application Publication No. WO 2005/074899, which are hereby incorporated by reference into this application . As used herein, "about" in the context of a numerical value or range means ±10% of the numerical value or range recited or claimed.

An "amount" or "dose" of laquinimod as measured in milligrams refers to the milligrams of laquinimod acid present in a preparation, regardless of the form of the preparation. The term "stable pharmaceutical composition" as used herein in connection with the composition according to the invention denotes a composition, which preserves the physical stability/integrity and/or chemical stability/integrity of the active pharmaceutical ingredient during storage. Furthermore, "stable pharmaceutical composition" is characterized by being free of gelation at 25°C/60%RH after at least 6 months.

As used herein, the term "glatiramer acetate related drug substance" (GARDS) is intended to include polypeptides with a predetermined sequence as well as mixtures of polypeptides assembled from the four amino acids glutamic acid (E) , alanine (A), lysine (K), and tyrosine (Y) ; from any three of the amino acids Y, E, A and K, i.e. YAK, YEK, YEA or EAK; or from three of the amino acids Y, E, A and K and a fourth amino acid. Examples of glatiramer acetate related polypeptides are disclosed in U.S. Patents 6,514,938 Al, 7,299,172 B2, 7,560,100 and 7,655,221 B2 and U.S. Patent Application Publication No. US 2009-0191173 Al, the disclosures of which are hereby incorporated by reference in their entireties. Glatiramer acetate related substances include glatiramoids . As used herein, a "glatiramer acetate related drug substance or drug product" is a glatiramer acetate related drug substance or a glatiramer acetate related drug product. As used herein a "glatiramoid" is a complex mixture of synthetic proteins and polypeptides of varying sizes assembled from four naturally occurring amino acids: L-glutamic acid, L-alanine, L- lysine, and L-tyrosine, in a defined molar ratio. Examples of glatiramoids include glatiramer acetate drug substance (e.g. Copaxone ^® ) as well as glatiramoids other than Copaxone ^® , e.g. GA- Natco .

As used herein, a "clear" solution, formulation or form is a solution, formulation or form which appears clear or transparent by visible inspection. A turbid solution is not expected to appear clear to the eye.

As used herein, "treating" encompasses, e.g., inducing inhibition, regression, or stasis of a disease, disorder or condition, or ameliorating or alleviating a symptom of a disease, disorder or condition. "Ameliorating" or "alleviating" a condition or state as used herein shall mean to relieve or lessen the symptoms of that condition or state. "Inhibition" of disease progression or disease complication in a subject as used herein means preventing or reducing the disease progression and/or disease complication in the subject.

As used herein, "effective" as in an amount effective to achieve an end, i.e., "therapeutically effective amount", means the quantity of a component that is sufficient to yield an indicated therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this disclosure. For example, an amount effective to treat a subject afflicted with a form of multiple sclerosis. The specific effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal being treated, the duration of the treatment, the nature of concurrent therapy (if any) , and the specific formulations employed and the structure of the compounds or its derivatives . "Administering to the subject" means the giving of, dispensing of, or application of medicines, drugs, or remedies to a subject to relieve, cure, or reduce the symptoms associated with a condition, e.g., a pathological condition. A "symptom" associated with multiple sclerosis includes any clinical or laboratory manifestation associated with multiple sclerosis and is not limited to what the subject can feel or observe .

As used herein, "a subject afflicted with multiple sclerosis" means a subject who was has been affirmatively clinically diagnosed to have multiple sclerosis which includes relapsing multiple sclerosis, relapsing-remitting multiple sclerosis, and Secondary Progressive multiple sclerosis.

As used herein, "pharmaceutically acceptable carrier" refers to a carrier or excipient that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. It can be a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the subject. "Pharmaceutically acceptable carrier" includes "fillers", which fill out the size of a tablet or capsule, making it practical to produce and convenient for the consumer to use. By increasing the bulk volume, the fillers make it possible for the final product to have the proper volume for patient handling. "Pharmaceutically acceptable carrier" also includes "lubricants", which prevent ingredients from clumping together and from sticking to the tablet punches or capsule filling machine. Lubricants also ensure that tablet formation and ejection can occur with low friction between the solid and die wall.

As used herein, a composition that is "free" of gelation means that the composition maintains its visual clarity and viscosity as measured using the Ubbelohde tube method, preferably after storage at 25°C and at a relative humidity (RH) of 60% for at least 6 months.

It is understood that where a parameter range is provided, all integers within that range, and tenths and hundredth thereof, are also provided by the invention. For example, "0.15-0.35%" includes 0.15%, 0.16%, 0.17% etc. up to 0.35%.

This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

Experimental Details

Example 1: 1.53 mq/mL laquinimod sodium (Laq-Na) and 40mg/mL glatiramer acetate (GA) with 40 mg/mL mannitol 4.6mg of laquinimod sodium (Laq-Na) was dissolved in 3.0425g of and aqueous solution containing 40mg/mL glatiramer acetate (GA) with 40 mg/mL mannitol as a tonicity agent.

Dissolution was achieved by effective stirring for 60 min at room temperature (RT) until a transparent solution containing 40 mg/mL GA and 1.53 mg/mL Laq-Na is formed (pH 5.89 at 22°C, osmolality 0.309 osmol/kg) .

A gel was observed after 2 days.

Example 2: 4.63 mg/mL Laq-Na and 30 mg/mL GA

18.5mg of Laq-Na was dissolved in 4ml of purified water (PW) under effective stirring at RT for 150 min. 120mg of GA was added at effective stirring to give a transparent solution containing 30mg/mL GA and 4.63mg/mL Laq-Na (pH 6.92).

A gel was observed.

Example 3: 3 mg/mL Laq-Na and 30 mg/mL GA with 9.0 mg/mL sodium chloride (NaCl) 123.4mg of Laq-Na was dissolved in 41.121g of PW at effective stirring with a magnet stirrer at RT. The solution looks transparent after 3 minutes (pH 7.45). 372.2 mg of sodium chloride (NaCl) was added as a tonicity agent (9 mg/mL) to ensure preparation of an isotonic or almost isotonic solution (0.290- 0.300 osmol/kg) . The solution was stirred for a few minutes at RT until the solution was transparent.

732.9mg of GA was added to 24.4g of the obtained solution containing 3mg Laq-Na/mL and about 9mg/mL NaCl. The solution was stirred at RT for one hour effectively but carefully, avoiding formation of air bubbles until the solution looked quite transparent (pH 5.9).

Following overnight storage at RT, a white gel was observed.

Example 4: 1 mg/mL Laq-Na and 30 mg/mL GA with about 8.1 mg/mL NaCl

60.1mg of Laq-Na was dissolved in 60.001g of PW under effective stirring at RT on a magnet stirrer. After 2 minutes the solution (Solution A) looked transparent.

44.5mg of NaCl was added to 5.5055g of Solution A as a tonicity agent and the solution was stirred for a few minutes at RT to get a transparent solution.

601.2mg of GA DS (30 mg/mL GA) was added to 20.018g (20 iriL) of the obtained solution containing lmg/mL Laq-Na (osmolality 0.01- 0.03 osmol/kg) and about 8. lmg/mL NaCl. The solution was stirred at RT for one hour effectively but carefully, to avoid formation of air bubbles. The solution looked quite transparent, was not viscous and had an osmolality 0.274 osmol/kg.

After 2 days at RT, the solution became more turbid and more viscous, finally becoming a gel, a bit more transparent than in Example 5.

Example 5A: 1 mg/mL Laq-Na and 30 mg/mL GA 34.685g of 0.05M potassium-phosphate buffer (pH 7.4) was added to 61 mg of Laq-Na. Concentration of Laq-Na was 1.017 mg/mL and osmolality was 0.113-0.114 osmol/kg.

602.5 mg of GA was added at stirring to 20 mL (20.019 g) of the transparent solution containing 1.017 mg/mL Laq-Na in 0.05 M buffer mixture (without NaCl) . Transparent solution was formed (osmolality 0.1135 - hypotonic).

Solution became almost transparent gels within a week at RT and at 2-8° C. Example 5B: 1 mg/mL Laq-Na and 30 mg/mL GA with 5.3 mg/mL NaCl

26.3183g of 0.05M potassium-phosphate buffer was added to 61 mg of Laq-Na. Concentration of Laq-Na was 1.017 mg/mL and osmolality was 0.113-0.114 osmol/kg.

48mg of NaCl (5.32 mg/mL NaCl) was added to 9 mL of the transparent solution containing 1.017 mg/mL Laq-Na in 0.05 M buffer mixture with stirring until a transparent solution was obtained.

233mg of GA was added to 7.78 g of this isosmotic solution (osmolality 0.289 osmol/kg) with stirring at RT for 60 min, giving the transparent solution containing both 1 mg/mL Laq-Na and 30 mg/mL GA.

Solution became almost transparent gels within a week at RT and at 2-8° C.

Example 6: 3 mg/mL Laq-Na and 30 mg/mL GA with 5.0 mg/mL NaCl 73.11mg of Laq-Na (3.0 mg/mL Laq-Na) was added to 24.35g of sodium phosphate buffer (pH 7.1). 54.4 mg of NaCl (5 mg/mL NaCl) was added to 10.8g of the solution. Osmolality of the resulting solution was 0.306 osmol/kg.

180.2mg of GA (30 mg/mL GA) was added to 6 mL of the resulting solution (pH 7.10) . At 21°C, gel formed.

Example 7: 3 mg/mL Laq-Na and 30 mg/mL GA with 9.0 mq/mL NaCl

123.4mg of Laq-Na (3 mg/mL Laq-Na) was added to PW at stirring at RT. Dissolution was achieved in 4 min. 372.3mg of NaCl was added to the solution at stirring at RT. Dissolution was achieved in 3 min. 732mg GA (about 30 mg/mL) was added to 24.4g of the obtained solution at stirring at RT. Dissolution was achieved in 60 min.

Overnight, a white gel formed.

Example 8: 3 mg/mL Laq-Na and 10.7-40 mg/mL GA with 5.6 mg/mL NaCl

180.3mg of Laq-Na was added to 60mL of 0.05 potassium-phosphate buffer (pH 7.1) with gradual addition of GA from 10.7 mg/mL (slightly turbid solution) to 40 mg/mL (rather turbid solution) .

The solution turned into gel very rapidly. Example 9: 2.2 mq/mL Laq-Na and 5.0 mg/mL GA with 9.07 mg/mL NaCl

101. lmg of GA (4.998 mg/mL GA) and 43.6mg Laq-Na (2.1555 mg/mL Laq-Na) was added to 20.227mL of PW. 183.4mg of NaCl (9.067 mg/mL) was also added (pH 6.09).

A turbid solution was observed. Example 10: 1 mg/mL laquinimod acid (Lag-acid) and 30 mg/mL GA with 1.33 mg/mL L-lysine and meglumine

1.0203g of meglumine solution was diluted by 3.5534g of PW and then 8mg of Laq-acid was added (Solution A) .

1.341g of the L-lysine stock solution containing 7.3 mg/mL in PW was added at stirring at RT to 3.6131g of the GA stock solution containing lOmg/mL in PW. To this clear solution, 2.2742g of the preliminary prepared Solution A of Laq-acid in meglumine was added at stirring. The obtained sample contained 5 mg/mL GA, 1.35 mg/mL lysine and 1 mg/mL Laq-acid. At first the obtained sample was not so turbid as having a GA concentration of 5mg/mL. However a rapid growth of turbidity was observed, followed by gel-formation.

Example 11: 3 mg/mL Laq-Na and 40 mg/mL GA with PEG-400 402.5mg of GA DS was added to 9.0048g of 0.05M phosphate buffer (pH 7.1), 30.2mg of Laq-Na, and 395.3mg of PEG-400. Additional phosphate buffer was added to bring the total volume to 10 mL (616.8 mg) .

In 3 days both solutions containing 3 mg/mL of Laq-Na and 40 mg/mL of GA (kept at RT and 2-8 °C) became viscous white gels.

Example 12: 2 mg/mL Laq-Na and 5 mg/mL GA

A transparent stock Solution A containing 10 mg/mL of GA-DS in PW was prepared by stirring 206.5mg of GA DS in 20.644g of PW at RT for 30 min (pH 5.90 at 22 °C) . A transparent stock Solution B containing 4 mg/mL of Laq-Na in PW was prepared by stirring 166. lmg of Laq-Na in 41.5173g of PW for 10 min at RT (pH 8.86 at 22°C) .

5.1663g of stock Solution A was mixed with 5.1575g of stock Solution B. In 20 minutes of stirring at RT for a transparent solution (pH 6.08 at 21°C) containing 2 mg/mL Laq-Na and 5 mg/mL of GA-DS, 59. lmg of NaCl was added as a tonicity agent while stirring at RT.

The first crystals of NaCl added resulted in a slightly turbid solution; however, after 5 minutes of stirring it became more transparent (pH 6.01 at 22°C) .

After stirring overnight (for 16h at RT) the solution looked transparent (pH 6.08 at 21°C) .

After storage at RT and 2-8 °C for week a solution started to become a gel: at first - a thin gel, however after 2 weeks it became a thick gel Example 13: 0.7 mg/rtL Lag-acid and 30 mg/mL GA

A saturated solution of laquinimod acid (3 mg/mL) was prepared by dissolving of 4.2283 g of Laq-acid in 0.1 M PBS (pH 7.4, 700 mL) for 3.5h at RT. After decantation of the suspension the clear upper part was filtered through a paper (Whatman) filter. This clear, filtered solution contained about 1.5 mg/mL of laquinimod acid (Solution A) .

60mg/mL of glatiramer acetate in 0.1 M PBS (600 mL) was solubilized at RT for 3.5 h (Solution B) . Equal volumes of Solutions A and B were mixed with stirring at RT and filtered through 0.2μπι polyethersulphone Parker filter 500cm ² . The yield of the filtered solution containing 30mg/mL GA and 0.7mg/mL laquinimod was 420mL.

The solution stored at RT and at 2-8 °C underwent slow gelation and growth of turbidity.

Example 14: 1.1 mg/mL Laq-Na and 32 mg/mL GA with 43.3 mg/mL mannitol and 1.4 mg/mL Lysine

36g of GA DS was weighed (on the basis of the dry powder, 4% of water in the DS batch was taken into account) . Purified water was added to bring the total GA solution weight to 1029.4g and stirring was performed for 30 min at RT until a nearly transparent, lump-free Solution A was obtained.

Mannitol powder (48 g) was added as a tonicity agent to Solution A with stirring at RT and afterwards stirring for 10 more minutes to obtain a lump-free Solution B.

Lysine-HCl (1.6249 g) was dissolved in 22.3596g of purified water (pH 5.60) and added to the GA solution at stirring. The obtained solution after stirring for 10 more minutes at RT was clear and free from visible particles, and was filtered through a 0.2 μιτι cellulose acetate Sartorius syringe (with 17 cm ² surface area) filter 1 (Solution C) . 2.028g of Laq-Na was dissolved in 68.8187g of PW for 20 min until a clear solution of Laq-Na (pH 9.0) was obtained, which was then filtered through a similar 0.2 pm cellulose acetate Sartorius syringe filter 2 (Solution D) . The filtered Solution C (GA + mannitol + lysine+ PW) (60 g) was sampled for investigational purposes and 60 g of the filtered Solution D (Laq-Na) were added dropwise with stirring to the remaining 993 g of Solution C. The resulting product was filtered through a similar 0.2 urn cellulose acetate Sartorius syringe filter 3. About 0.8 L was the final filtered product was obtained and manually filled into 1 cc USP type I glass pre-filled syringes (PFS) closed by brombutyl rubber stoppers, and into 10 cc USP type I glass clear vials (filling volume 5 cc) closed by brombutyl rubber stoppers . Syringe functional testing was performed for the PFS samples and the gliding and breakout force was similar to the values for the PFS filled with the marketed drug Copaxone 20 mg/mL (breakout force 2 N, gliding force 1 N after 20 days of storage) , which proved the absence of gelation. Assay testing for both components corresponded to a concentration of 32.4 mg/mL glatiramer acetate and 1.1 mg/mL of laquinimod. Assay of both ingredients remained practically the same after storage at 5°Cand 25°C/60%RH, and molecular weight distribution values were similar to those for Copaxone marketed drug products. The DP solution in vials stored at 25°C for 6 months and at 2-8 °C for 12 months and there were no essential changes in visual clarity and viscosity (measured using Ubbelohde tube method) , which proved absence of gelation.

Example 15: Comparison of Formulations Using Various Amino Acids 15.2889g of GA was weighed and dissolved with stirring for 30 min in 252.4 g of PW at RT prior to addition of 19.6 g of mannitol with stirring which went on for 10 minutes more followed by addition of 167.3g of PW was added to wash out carefully the powder on the walls of the compounding vessel and stirring was completed after 5 more minutes. A clear solution essentially free of visible particles was obtained and contained 36.4 mg/mL GA and 46.7 mg/mL mannitol. The solution was filtered through a 0.2 μπι cellulose acetate Sartorius syringe filter and stored at 2-8 °C before usage (pH 5.72) (Solution A).

221.9mg of Laq-Na was weighed and dissolved in 12.6951g of PW with stirring for 15 minutes at RT to give a clear solution containing 17.48 mg/mL of Laq-Na which was filtered through the similar filter and stored at 2-8 °C before usage (pH 9.05) (Solution B) .

10.4 mg of amino acid proline was weighed and PW was added with stirring to bring the total weight to 246.1 mg. The stirring continued for 10 minutes at RT until complete dissolution of the amino acid. Then, 10.7722 g of Solution A was added at stirring prior to the addition by drops of 0.7 mL by pipette (however also weighed afterwards) of Solution B.

Very similar amounts of Solutions A and B were added to the equimolar solutions of other amino acids (histidine, alanine, lysine, glycine) . In case of a control no amino acid was used with the similar amounts of PW, and solutions A and B. The obtained samples containing GA (about 33.4 mg/mL), mannitol (about 42.8 mg/mL) and 1.07-1.09 mg/mL of Laq-Na were clear, except for the sample with histidine, which was almost clear.

Example 16: Comparison of Scaled-up Formulations Using Various Amino Acids

For the scale-up of the formulations, in order to compare viscosity values at storage at 2-8 °C using Ubelohde tube, proportional quantities of each amino acid dissolved in PW were used, as well as of Solutions A and B to prepare not less than 30 g of the sample.

Usage of Ubbelohde tubes for evaluation of kinematic viscosity is based on measurements of time required to the tested liquid in the tube to pass the capillary inside from the upper mark to the lower mark. This old reproducible method has been mostly replaced by all kind of widespread Brookfield devices. However, stirring that occurs in Brookfield devices breaks the gel structure. In contrast, in an Ubbelohde tube, only slight partial disturbance of gel layers can take place. Therefore, we have found this method very useful for the systems inclined to gel-formation not only to evaluate the viscosity of the GA-Laq formulations, but also to observe the gelation process - in case of the presence of gel structure even where the product seems a liquid, decrease of the time results for each consecutive measurement proves the gel- formation.

Applying the Ubbelohde method, samples containing alanine (Sample A) and lysine (Sample B) stored in the Ubbelohde tubes at for 24 m at 2-8 °C was not accompanied by gel-formation, while a sample without amino acid (Sample C) showed slow gelation. Samples with glycine (Samples D) and proline (Sample E) differed not so much from the control - Sample C (in the amino acid concentrations used), while the histidine sample (Sample F) showed accelerated gel-formation in comparison to Sample C. Example 17:

10.21g of GA was weighed and dissolved at stirring for 60 min in 280.28g of Water for Injection at RT prior to addition of 13. lg of mannitol at stirring which went on for 20 minutes more. A clear solution essentially free of visible particles was obtained containing 35.14 mg/mL GA and 43.15 mg/mL mannitol. The solution was filtered through a 0.2 pm cellulose acetate Sartorius syringe filter and stored at 2-8°C before usage (pH 5.75) (Solution A).

224g of Laq-Na was weighed and dissolved in 12.815g of Water for Injection at stirring for 15 minutes at RT to give a clear solution containing 17.48mg/mL of Laq-Na which was filtered through the similar filter and stored at 2-8 °C before usage (pH 9.06) (Solution B) .

33mg of histidine was solubilized in 275 mg of Water for Injection after shaking for a minute at RT and 10.868g of a clear Solution A was added to a rather turbid aqueous suspension of the histidine. The clear solution was obtained after stirring at RT.

0.716mg of clear Solution B was added drop by drop at stirring at RT resulting into a clear solution containing 34 mg/itiL of GA, 42 mg/itiL of mannitol, 1.11 mg/itiL of Laq-Na and 2.93 mg/mL of histidine {Solution C) . After stirring overnight at RT the solution remained clear and its samples remained clear both at RT and 5°C.

To 6.015 g of Solution C additional amounts of Laq-Na were added drop by drop at stirring at RT. The solutions obtained were clear at 2-8°C and at RT at Laq-Na concentrations 1.28, 1.49, 1.75 mg/mL. The addition of Laq-Na up to 2.04 mg/mL resulted in a clear solution containing 32.1 mg/mL GA, 39.6 mg/mL Mannitol and 2.75 mg/mL histidine, which after stirring overnight at RT became a bit turbid (and remained as such afterwards) and thixotropic: a liquid at shaking or stirring which without shaking or stirring became a weak gel, and such transitions could occur for many days of observation.

Histidine concentration lower than 1.33 mg/mL could not solubilize additional portions of Laq-Na - 1.23, 1.39, 1/54 mg/mL - all of them gave thixotropic products with increased turbidity and increased ability to gel-formation.

Similar results (higher Laq-Na solubilization effect (higher Laq- Na dose incorporated in a clear product) at increase of amino acid concentration) was observed for other amino acids:

With increase of glycine from 0.6mg/mL to 4.2mg/mL, it was possible to incorporate 1.51 mg/mL of Laq-Na instead of 1.05- 1.07mg/mL.

With increase of proline from 0.9mg/mL to 2.45mg/mL, it was possible to incorporate 1.55 mg/mL of Laq-Na instead of 1.06- 1.09mg/mL. With increase of lysine from 1.39 mg/mL to 3.38 mg/mL, it was possible to incorporate 1.88 mg/mL of Laq-Na instead of 1.06- 1.09mg/mL.

Table 1 below summarizes that experiments on fixed-dose formations of laquinimod and glatiramer acetate.

Table 1.

Mannitol nice

filterability

+ See

example

1.04 29.9 PW 36.5 0.8 mg/mL 6 m clear , Less Lys mg/mL Lysine after 6.5 m a

Mannitol bit turbid

1.01 30 PW 36,6 0.8 mg/mL Worse than Less Lysine mg/mL Lysine # 54, 5 m

Mannitol clear, then

slow

turbidity

growth and

gelation

1.13 6 PW 7.3 0.55 Lysine Clear, no Low GA, mg/mL gelation at Low Lys, Mannitol storage very

hypotonic!

0.97 32 PW Mannitol 1.53 clear For

41mg/m mg/mL viscosity

L Lysine monitoring

1.04 29 PW Mannitol 1.53 clear reproducibil

40 mg/mL ity mg/mL Lysine

1.04 29 PW Mannitol 0.85 Clear, a bit Less Lysine,

37.2 mg/mL Worse than lower GA mg/mL Lysine # 57-58 at cone, storage higher Laq- Na cone.

1.06 29 PW Mannitol 3.38 Clear, More Lys,

37.2 mg/mL Worse than less PW mg/mL Lysine # 54,

1.05 29 PW Mannitol 1.58 clear

37.2 mg/mL

mg/mL Lysine

1.1 5.0 PW Mannitol 2.78 clear Lower GA,

37.6 mg/mL higher Lys mg/mL Lysine

2.0 4.7 PW Mannitol 4.20 Almost Higher Laq,

36.7 mg/mL clear, gets lower GA, mg/mL Lysine turbid highest Lys

1.6 5.0 PW Mannitol 1.8 mg/mL A bit turbid,

38.2mg/ L-Lysine slow growth

mL of turbidity

2.76 30 PW Mannitol 1.33 Very turbid High Laq

40 mg/mL L- cone mg/mL Lysine

2.15 30 PW Mannitol 1.34 turbid Another

40 mg/mL L- variant of mg/mL Lysine adding excipients

2.42 34 PW Mannitol 2.12 Very turbid Tween

41 mg/mL 80+Lys to mg/mL Lysine dissolve

Methods : For the gelation process study, several modern sophisticated methods were used: ultracentrifugation for fractionation of GA- LAQ mixture prior to chromatographic methods such as HPLC-SEC, LC-MS; cryo-transmission electron microscopy (Cryo-TEM) ; circular dichroism; amino acid analysis.

For selection of better formulations, viscosity testing using a Ubbelohhde viscometer (tube size 1) was performed.

Osmolality was evaluated using Osmomat 030-D.

In addition, for the characterization of the best scaled-up formulation comprising GA LAQ (1.1 mg/itiL) , L-lysine (1.4 mg/mL) and mannitol (43 mg/mL) as a tonicity agent, HPLC-SEC (size- exclusion chromatography) was used to evaluate the GA assay and molecular-weight distribution, while HPLC was used for LAQ assay. Clarity was evaluated using a 2100 AN turbidimeter. Functional tests for the drug product filled in pre-filled syringes were performed to evaluate Breakout and Gliding forces.

Discussion :

Developing formulations containing laquinimod and GA is particularly difficult because laquinimod and GA have different pH optimums in terms of stability and solubility and because of undesired effects, mainly gelation or the formation of turbid formulations in the presence of various buffers, solvents and surface active agents . GA requires a pH not exceeding 7.0, preferably 5.5-6.2, to limit degradation. Laquinimod, on the other hand, is more soluble and stable in alkaline media. In addition, the presence of laquinimod even at low concentrations (0.7-2.0 mg/mL) with GA has been found to cause gelation of GA and/or gelation of the final formulations Experiments show that fixed-dose GA-laquinimod formulations containing 5-40 mg/mL of GA and 0.7-5 mg/mL of laquinimod were not transparent liquid forms, and more or less rapidly turned into gels. The higher the concentration of GA or laquinimod, the more significant the growth of turbidity, the greater the viscosity, and the faster the rate of gelation was observed. The method and order of mixing of components were also important factors.

Numerous studies of various buffers, solvents and surface-active agents with various forms of laquinimod (acid and salts) have consistently led to viscous solutions that gradually form gels, or lead to sedimentation or to rapid formation of turbid formulations.

These studies show that use of various buffers was not preferable to the use of purified water. The use of various buffers caused turbidity of GA, or turbidity and/or gelation of the final formulations. In particular, phosphate salt buffers (sodium, potassium, and sodium-potassium salts) , acetate, and hydrocarbonate all providing pH of 5.5-8 in numerous experiments, were found to cause turbidity in GA solutions of 5-6 mg/mL, both at 5°C and 25°C/60% relative humidity storage.

A special study was performed in order to explain the reason for gelation of a great number of various formulations containing both actives - GA and laquinimod. Results of the study showed that gel formation was attributable to:

1. Reassembly of the GA polypeptides in the presence of laquinimod molecules, accompanied by appearance of hydrophobic nuclei and formation of fibrous structures through non-covalent forces, forming a three-dimensional gel network.

2. The hydrophobic nature of the interaction between GA and laquinimod.

3. Tyrosine residues playing a key role in the gel formation process. 4. The solution and GA-LAQ supernatant solution adopt an ex- helical structure, while the sedimented gel-like fraction of GA-LAQ has the more ordered β- sheet conformation . The study also showed that kinetics of the gelation of GA-LAQ strongly correlated with elevation of temperature, prolongation of incubation time and, in particular, with the concentration of LAQ in the tested mixtures.

Surprisingly, as disclosed herein, the inclusion of amino acids in formulations of laquinimod and GA eliminates gelation of the final product .

Good manufacturing results of a liquid clear fixed-dose GA- laquinimod formulation with no gel formation with their maximal possible concentrations - about 30 mg/mL of GA and 1-1.3 mg/mL Laq-Na - were achieved using amino acids (lysine, proline, alanine, glycine, histidine) . The process is highly reproducible and technology permits industrial manufacturing of the clear and stable drug product involving sterile filtration stages. Formulations with lysine or alanine did not undergo gel formation during storage at 2-8 °C for more than two years. Similar results were obtained with other amino acids .

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