AU775214B2 - Copolymer-1 improvements in compositions of copolymers - Google Patents

Description

S&FRef: 359455D2

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Yeda Research and Development Co. Ltd.

P O Box 76 100 Rehovot Israel Eliezer Konfino, Michael Sela, Dvora Teitelbaum, Ruth Amon Spruson Ferguson St Martins Tower,Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Copolymer-1 Improvements in Compositions of Copolymers Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c Copolymer-1I Improvements in Compositions of Copolymers Background of the Invention COPOlYmer- is a synthetic polypept-ade- analog of myel~n basic protein (M13p) which is a na tura component* of the 0 myelin sheath )t has been Suggest-ed as a poi-entaal therapeutic agent- for: multliple scICUi-, H L: .1 I 11n:1unol I 39V3 -1 :212, and J3. Neurol SC.! 19' 31-133) AI]rfe-nc~ ctitC hr: i d1?C 1icOrporat-ed( by, 1ic- er.ence al then 1 ii coo yme r I as anj immu not he-apy~ fo-, 1 t .i ul e SC (2ro1 st-ems from obsei--vat ons first made st-he 1.950' LIla myeli n components such as !FMP prevent- or- arr-est ex-pe rimen ta I auLtoi~mrune encephalIomyec 2 t s M AEL E, LA] disease resemvbl ing multitile sci ero ;thtcne induced an suscepti ble animals Copol yme r- was devel oped by Drs. Selae Arnon, and Lhe 3 co-workers at the Weizmrn Institute (Rehovot, israel.) It was sho% rn to suppress EAFE (Eur. 3. Inununol. [19721) .25 1:242; U.S. Patent No. 3,8,q9,550). More recently, copolymer- 1 was shown to be beneficial. for patients with the exacerbating- remitting form of multiple sclerosis

(W

Engi. J. Med. (1987] 317:40O8). Patienlts treated with daily injections of copoly-mer-1 had fewer exacerbations, and smaller increases in their disability status than th control patients.

Copolymer. 1 is a mixture of poly-peptides composed of alanine, glut-amic acid, lysine, and ty)rosine in a molar ratio of approximately 6:2:5:1, respectively. It is synthesised by chemically polymerising the four amino acids forming products with average molecular weights of 23,000 daltons Patent No. 3,849,550).

It is an object of the present invention to provide an improved composition of copolymer-1.

Summary of the Invention There is herein disclosed a composition of copolymer-1 substantially free of species of copolymer-1 having a molecular weight of over 40 kilodaltons (KDa).

There is herein disclosed a copolymer-1 having over 75% of its molar fraction within the molecular weight range from about 2 KDa to about 20 KDa.

In addition, there is disclosed a copolymer-1 having an average molecular weight of about 4 to about 8.6 KDa.

Moreover, there is disclosed to a pharmaceutical composition and a method for the treatment of multiple sclerosis, using the above-discussed copolymer-1.

According to a first embodiment of the invention, there is provided a method of manufacturing copolymer-1 of a predetermined molecular weight profile, comprising the steps of: selecting a predetermined molecular weight profile, reacting protected copolymer-1 with hydrobromic acid to form trifluoroacetyl 20 copolymer-1 having the predetermined molecular weight profile, wherein said reaction takes place for an extended time and at a temperature predetermined by test reaction, and treating said trifluoroacetyl copolymer-1 having the predetermined molecular weight profile with aqueous piperidine solution to form copolymer-1 having the S* predetermined molecular weight profile.

According to a second embodiment of the invention, there is provided a method of "i manufacturing trifluoroacetyl copolymer-1 having a predetermined molecular weight S°profile, comprising the steps of: S selecting a predetermined molecular weight profile, and then reacting protected copolymer- with hydrobromic acid for an extended time and at a S..reacting protected copolymer-1 with hydrobromic acid for an extended time and at a

S°

[I:\DayLib\LIBFF I S965spec.doc:gcc 2a temperature predetermined by test reaction to provide trifluoroacetyl copolymer-1 having the predetermined molecular weight profile.

Brief Description of the Drawings Figure 1 displays the molecular weight distribution of three batches of copolymer-1, showing the proportion of species with molecular weight above 40 KDa. Figure 2 shows similar data relating to the molar fraction.

Detailed Description of the Invention The present invention relates to a composition of copolymer-1 substantially free of species of copolymer-1 having a molecular weight of over 40 kilodaltons (KDa).

10 Preferably, the composition contains less than 5% of species of copolymer-1 having a molecular weight of g *o o* *o o*o *oo *oo •oo o*o [I:\DayLib\LIB FF jI 5965spec.doc:gcc KDa or more. More preferably, the comoosition contains less than 2.5% of species of copolymer-1 having a molecular weight of 40 KDa, or more.

The invention further relates to a copolymer-1 having over 75% of its molar fraction within the molecular weight range from about 2 KDa to about 20 KDa.

In addition, the invention relates to a copolymer-1 having an average molecular weight of about 4 to about 8.6 KDa. In particular, the invention relates to a copolymer-l having an average molecular weicht of about to about 8 KDa and a copolymer-1 having an average molecular weight of about 6.25 to about 8.4 KDa Copolvmer- according to the present invention, may be prepared by methods known in the art, for example the process disclosed in U.S. Patent 3,849,550, wherein the N-carboxyanhydrides of tyrosine, alanine, y-benzyl 20 glutamate and E-N-trifluoro-acetyllysine are polymerised at ambient temperature in anhydrous dioxane with diethylamine as initiator. The deblocking of the ycarboxyl group of the glutamic acid is effected by hydrogen bromide in glacial acetic acid and is followed by the removal of the trifluoroacetyl groups from the lysine residues by 1M piperidine. For the purposes of t: he application, the terms "ambient temperature" and "room temperature" should be understood to mean a temperature ranging from about 20 to about 26 OC.

The copolymer-i with the required molecular weight profile can be obtained either by methods known per se.

Such methods include chromatography of copolymer-l containing high molecular weight species and collecting the fractions without the undesired species or by partial acid or enzymatic hydrolysis to remove the high molecular weight species with subsequent purification by dialysis 4 or ultrafiltration. A further method to obtain copolymer-i with the desired molecular weight profile is by preparing the desired species while the amino acids are still protected and then obtain the correct species directly upon removing the protection Th comositions of the present invention may be formulated by conventional methods known in the art. Preferably che composition is lyophilized and formed into an aqueous solution suitable for sub-cutaneous injection Alternatively, copolymer-l may be formulated in any of the forms known in the art for preparing oral, nasal buccal, or rectal formulations of peptide drugs.

:T.ypically, copolymer-l is administered daily to oaieon suffering from multiple sclerosis at a dosage of SThe invention will be exemplified but not necessarily limited by the following Examples.

EXAMPLE 1

."C

ChromatooraDhic method of rearation of low-toxicity Scooolvmer- Two batches of copolymer-1 were prepared according to the methods known in the art, for examole, U.S. Patent No. 3,849,550.

One batch was then subjected to chromatographic o separation, as described below.

A column for gel filtration, FRACTOGEL TSK HW55 (600 x 26mm) was prepared in a Superformance 26 Merck cartridge according to the manufacturer's instructions. The column was equilibrated with water and acetone solution was injected for total volume determination. The column was equilibrated with 0.2M ammonium acetate buffer pH 30 ml copolymer-l samples 2 0mg/ml, in 0.2M ammonium acetate pH 5.0) were loaded on the column and fractions were collected every 10 minutes. A fraction having an average molecular weight of 7-8 KDa was isolated between 120-130 minutes (Batch

A).

Molecular Weight Analysis UV absorbance at 275 nm was determined in a UVIKON 810 spectrophotometer. Samples were diluted to obtain a UV absorbance lower than 1 Absorption Unit. The molecular distribution of the 2 batches was determined on a calibrated gel filtration column (Superose 12) Copolmer-l batch A was found to have an averE:e molecular weight of 7-8 KDa. 2.5- of this batch-had a molecular weight above 32 KDa, but no copolymer- secies Sresent in this batch had a molecular weight of over KDa.

The other batch of copolymer-1 which was not subjected to chromatography, had an average molecular weight of 12 KDa. 2.5% of the batch had a molecular weight above 20 42KDa and 5% of the total coPolymer-1 species in this batch had a molecular weight of over 40 KDa.

EXAMPLE 2 e eToxicity Analysis A: In Vivo Three batches of copolymer- having an average molecular s weight of 7.3 and 8.4 KDa (less than 2.5% copolymer-i species over 40KDa) and 22KDa (more than 5% copolymer-1 species over 40KDa) were subjected to the toxicity test described below. In each case 5 mice were used in each experimental group.

Method Copolymer- was dissolved in distilled water to yield a solution of 2mg/ml of the active ingredient. Each mouse was injected with 0.5mnl of the test solution into the lateral tail vein. Mice were observed for mortality and relevant clinical signs over a 48 hour period.

Observations were recorded 10 minutes, 24 hours and 48 hours post-injection. If, at the end of 48 hours, all the animals were alive and no adverse signs had been observed, then the batch was designated "non-toxic" If however, one or more of the mice had died or had shown adverse signs, then the batch was designated "toxic" The batches with the average molecular weight of 7.3 and 8.4 KDa were both designated "non-toxic", whereas in the batch with the average molecular weight of 22KDa, 3 out of 5 mice had died at the end of 48 hours, and it was consequently designated "toxic".

15 B: In Vitro RBL Deranulat ion test I. Introduction Histamine (or serotonin) release from basophile is an in vitro model for immediate hypersensitivity. The Rat 20 Basophilic Leukemia cell line (RBL-2H 3 was developed and characterized as a highly sensitive, uniform, easy to maintain in culture and reproducible system

(E.L.

Basumian, C. Isersky, M.G. Petrino and R.P. Siraganian Eur. J. Immunol. 11, 317 (1981)). The physiological stimulus for histamine release involves binding of the antigen to membrane-bound IgE molecules, resulting in the latter's cross-linking and the consequent triggering of an intricate biochemical cascade. Beside these physiological, immunoglobulin-mediated triggers, degranulation can be induced by different non-IgEmediated stimuli. Among these are various peptides and synthetic polymers, e.g. polylysine Siraganian.

Trends in Pharmacological Sciences, October 432 (1983)) The RBL degranulation test is, therefore, used in order to screen out those batches of copolymer-i which evoke substantial degranulation and thus might elicit undesirable local and/or systemic side effects.

II. Principle of the test method Rat Basophilic Leukemia cells (RBL-2H,), are loaded with ('H]-serotonin, followed by incubation with 100 pg of the copolymer-l to be tested. Batches of copolymer-l which induce non-specific degranulation, release [(H]-serotonin into the medium. The radioactivity in the medium is counted by a scintillation counter and the total radiolabeled serotonin incorporated into the cells is determined in the pelleted cells. Percent degranulation is calculated as the percentage of serotonin released o.r of the total inco-rporated.

I

TII Results Four batches of copolymer-l, with average molecular 15 weight between 6,250-14,500 were analyzed for both of the species with molecular weight over 40KDa and for degranulation of RBL's. Results are summarized in the following table.

Average of species with Serotonin 20 M.W (Daltons) M.W. over 40KDa Release 6,250 2.5 12.4 7,300 2.5 21.0 13,000 5 66.9 14,500 5 67.8 As can be seen, when the of high molecular weight species is low the release of serotonin, indicative of toxicity, is low, and vice versa.

EXAMPLE 3 Preparation of Trifluoroacetvl -Copolymer-1 Protected copolymer-l is prepared as described by Teitelbaum et al. Eur. J. Immun. Vol. 1 p. 242 (1971) from the N-carboxyanhydrides of tyrosine (18g), alanine (50g), y-benzyl glutamate (35g) and trifluoroacetyllysine (839) dissolved in 3.5 liters of dioxane.

The polymerization process is initiated by the addition of 0.01 0.02% diethylamine. The reaction mixture is stirred at room temperature for 24 hours and then poured into 10 liters ater. The product (protected copolymer- 1) is filtered, washed with water and dried. The removal of the gaimna-benzyl blocking groups from the glutamate residue is carried out by treating the protected copolvmer-1 with 33% hydrobromic acid in glacial acetic acia at room temperature for 6-12 hours with stirrino.

The product is poured into excess water, filtered, washec and dried, yielding the trifluoroacecyl-copolymer-1.

EXAMPLE 4 prenaration of Trifluoroacetvl-Conolymerl Protected copolymer-l is prepared as described by Teitelbaum et al. Eur. J. Immun. Vol. 1 p. 242 (1971) from the N-carboxyanhydrides of tyrosine (18g) alanine (50g), 7-benzyl glutamate (35g) and trifluoroacetyllysine (83g) dissolved in 3.5 liters of dioxane.

The polymerization process is initiated by the addition of 0.01 0.02% diethylamine. The reaction mixture is 25 stirred at room temperature for 24 hours and then Doured Sinto 10 liters water. The product (protected copolymer- 1) is filtered, washed with water and dried.

Protected copolymer-1 is treated with 33% HBr in acetic acid which removes the omega benzyl protecting group from the 5-carboxylate of.the glutamate residue and cleaves the polymer to smaller polypeptides. The time needed for obtaining copolymer-l of molecular weight 7,000±2,000 Da depends on the reaction temperature and the size of protected copolymer-l. At temperatures of between 28 0 C a test reaction is performed on every batch at different time periods for example, from 10-50 hours.

The results concerning the molecular weights of these small scale reactions are calculated and a curve of molecular weight against time is drawn. The time needed for obtaining molecular weight 7,000±2,000 Da is calculated from the curve and performed on larger scale reaction. On average, working at 26 0 C the time period is 17 hours. The product is poured into excess water, filtered, washed and dried, yielding the trifluoroacetvl .copolymer-1.

Preparation of low-toxicity conolvmer-2 of trifluoroacetyl-copolymer-1 are dispersed in 1 liter of water to which 100g piperidine are added. The mixture is stirred for 24 hours at room temoerature and filtered. The solution of crude copolvmer-1 is 15 distributed into dialysis bags and dialyzed at 10 0 -20 0

C

against water until a pH 8 is attained. It is then dialyzed against about 0.3% acetic acid and again water until a pH 5.5-6.0 is obtained. This solution is then concentrated and lyophilized to dryness.

Claims (9)

1. A method of manufacturing copolymer-1 of a predetermined molecular weight profile, comprising the steps of: selecting a predetermined molecular weight profile, s reacting protected copolymer-1 with hydrobromic acid to form trifluoroacetyl copolymer-1 having the predetermined molecular weight profile, wherein said reaction takes place for an extended time and at a temperature predetermined by test reaction, and treating said trifluoroacetyl copolymer-1 having the predetermined molecular weight profile with aqueous piperidine solution to form copolymer-1 having the predetermined molecular weight profile.

2. The method of claim 1, wherein said protected copolymer-1 is reacted with hydrobromic acid for about 10-50 hours at a temperature of about 20-28 0 C.

3. The method of claim 2, wherein said protected copolymer-1 is reacted with hydrobromic acid for about 17 hours at a temperature of about 26°C.

4. A method of manufacturing trifluoroacetyl copolymer-1 having a predetermined molecular weight profile, comprising the steps of: selecting a predetermined molecular weight profile, and then reacting protected copolymer-1 with hydrobromic acid for an extended time and at a temperature predetermined by test reaction to provide trifluoroacetyl copolymer- 20 1 having the predetermined molecular weight profile. oboe

5. The method of claim 4, wherein said protected copolymer-1 is reacted with hydrobromic acid for about 10-50 hours at a temperature of about 20-28 0 C.

6. The method of claim 5, wherein said protected copolymer-1 is reacted with hydrobromic acid for about 17 hours at a temperature of about 26°C. 25

7. A method of manufacturing copolymer-1 of a pre-determined molecular weight profile, substantially as herein described with reference to any one of the examples.

8. A method of manufacturing trifluoroacetyl copolymer-1, substantially as hereinbefore described with reference to any one of the examples. 30

9. Trifluoroacetyl copolymer-1 prepared in accordance with the method of any one of claims 4-6 or 8. []\DayLib\LIBFF] 5965spec.doc:gcc I1I Copolymer-1I prepared in accordance with the method of any one of claims 1, 3 or 7. Dated 17 November, 2003 Yeda Research and Development Co. Ltd Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 0 0@S.*S 0 000000 S 060 0 0 bOO S S* OS. .00 Os. 5000 0:510: S0SO a S 0*~0 0 SeeS *00005 S 400 0 0* Of 0 S. St *00000 S 000005 00 O 04.0 505000 O 4 I \I H'IIRF F]I