MXPA96003010A

MXPA96003010A - Pharmaceutical compositions

Info

Publication number: MXPA96003010A
Application number: MXPA/A/1996/003010A
Authority: MX
Inventors: Arvinte Tudor
Original assignee: Cibageigy Ag
Priority date: 1994-01-26
Filing date: 1996-07-25
Publication date: 1998-01-01

Abstract

The present invention provides a freeze-dried pharmaceutical composition, which comprises hirudin, potassium phosphate, and a sugar

Description

PHARMACEUTICAL COMPOSITIONS The present invention relates to compositions containing hirudin and in particular, to stable powder formulations. Hirudin, an anticoagulant that occurs naturally in leeches (Hirudo medicinalis), is not a single species of polypeptide, but a class of polypeptides that act equally consistently on at least four representatives designated as a variant of hirudin.

(HV1), variant of hirudin 2 (HV2) (cf Patent Application European Number 158,564) variant of hirudin 3 (PA) [cf.

TCP Application Number 86/03493] and "des- (Val) 2-hirudin" (sf European Patent Application Number 158,986). The variants differ in structure from one another by a number of amino acids (especially, the N-terminal sequence of the variant hirudin 1 is Val-Val-Tyr, that of the variant hirudin 2 and that of the variant hirudin 3 is Ile-Thr-Tyr and that of "des- (Val) 2-hirudin" is Thr-Tyr), but they have an accumulation of hydrophobic amino acids at the N-terminus and polar amino acids at the C-terminus, a tyrosine residue ( Tyr63) present as a sulphate monoester, three disulfide bridges, and anti-coagulant activity in common. In the past few years, cDNAs and synthetic genes that code for hirudin variants have been cloned and expressed in microbial hosts. Although the expression products lack the sulfate onoster group in Tyr63 - and therefore were designated as "desulfatohirudins" - they were found to exhibit approximately the same biological activity as natural sulfated hirudins. The desulfatohirudin variant, HV1, has been expressed in Escherichia coli (European Patent Applications Nos. 158,564, and 168,342) and in Saccharomvces cerevisiae (European Patent Applications Nos. 168,342, 200,655, 225,633, 252,854, and 341,215). In a similar manner, desulfatohirudin HV2 has been expressed in Escherichia coli (European Patent Application Number 158,564), and in Saccharomyces cerevisiae (European Patent Application Number 200,655, Application for TCP Number 86/01224) and des- (Val) 2-desulfatohirudin has been expressed in Escherichia coli (European Patent Application Number 158,986). According to the present invention, the term "hirudin" is intended to encompass hirudin, desulfatohirudin, a variant of hirudin, or a variant of desulfatohirudin, or a mutant thereof, respectively, described in the literature and in particular a desulphatohirudin compound or a mutant thereof that can be obtained from a strain of a transformed microorganism containing DNA encoding a desulfatohirudin or a mutant thereof. These desulfatohirudins are, for example, the variant of desulfatohirudina HV1, modified HV1 (a, b), HV2, modified HV2 (a, b, c), HV3, the variants of HV3, and des- (Val2) - desulfatohirudina. Preferred desulfatohirudins are those having the formula (SEQUENCE IDENTIFICATION NUMBER: 1): Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys 1 5 10 15 Glu Gly Ser Asn Val Cys Gly Gln Gly Asn Xaa Cys lie Leu Gly Ser 20 25 30 Asp Gly Glu Xaa Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Xaa Pro 35 40 45 Gln Ser Xaa Asn Asp Gly Asp Phe Glu Glu lie Pro Glu Xaa 50 55 60 (I) where: a) Xaa in 27, 36 and 47, are each Lys, Xaa in 51 is His, and Xaa in 62 is the peptide residue Glu-Tyr-Leu-Gln (HV1), or b) Xaa in 27 is lie or Glu and Xaa in 36, 47, 51 and 62 are as defined in a) (HV1 modified a), or c) Xaa in 36 is lie or Glu and Xaa in 27, 47, 51 and 62 are as defined in a) (modified HV1 a), or d) Xaa in 47 is lie or Glu, and Xaa in 27, 36, 51, and 62 are as defined in a) ( Modified HV1 a), or e) Xaa at 51 is Leu or Asp and Xaa at 27, 36, 47 and 62 are as defined in a) (modified HV1 a), or f) Xaa at 62 is selected from the group consisting of Glu-Tyr , Glu-Tyr-Leu, Glu-Asp-Leu-Gln, Glu-Glu-Leu-Gln, Glu-Tyr-Lys-Arg, Glu-Asp-Lys-Arg, Glu-Lys-Leu-Gln, Ser-Phe -Arg-Tyr, Trp-Glu-Leu-Arg, Glu-Tyr-Leu-Gln-Pro and Glu-Tyr-Leu-Gln-Arg and Xaa in 27, 36, 47, and 51 are as defined in a) (HV1 modified b), or having the formula (IDENTIFICATION OF SEQUENCE NUMBER: 2): Leu Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys 1 5 10 15 Glu Gly Ser Asn Val Cys Gly Gln Gly Asn Lys Cys He Leu Gly Ser 20 25 30 Asp Gly Glu Lys Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro 40 45 Gln Ser His Asn Asp Gly Asp Phe Glu Glu He Pro Glu Glu Tyr Leu 50 55 60 Gln 65 (ID or that have the formula (IDENTIFICATION OF SEQUENCE NUMBER: 3): He Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys 1 5 10 15 Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys He Leu Gly Ser 20 25 30 Asn Gly Lys Gly Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Xaa Pro 40 45 Glu Ser His Asn Asn Gly Asp Phe Glu Glu He Pro Glu Glu Xaa Leu 50 55 60 Gln 65 (III) where: a) Xaa in 47 is Asn and Xaa in 63 is Tyr (HV2), or b) Xaa in 47 is Lys, Arg or His, and Xaa in 63 is Tyr (modified HV2 a), or c) Xaa at 63 is Glu or Asp and Xaa at 47 is Asn (modified HV2 b), or having the formula (IDENTIFICATION OF SEQUENCE NUMBER: 4): Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys 1 5 10 15 Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys He Leu Gly Ser 20 25 30 Asn Gly Lys Gly Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Asn Pro 35 40 45 Glu Ser His Asn Asn Gly Asp Phe Glu Glu He Pro Glu Glu Tyr Leu 50 55 60 Gln 65 (IV) or having the formula (IDENTIFICATION OF SEQUENCE NUMBER: 5): He Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys 1 5 10 15 Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys He Leu Gly Ser 20 25 30 Gln Gly Lys Asp Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro 35 40 45 Gln Ser His Asn Gln Gly Asp Phe Glu Pro He Pro Glu Asp Ala Tyr 50 55 60 Asp Glu 65 (V) HV3 and HV3 variants that are characterized by a shortening of the primary structure by one or two amino acids in the N term, or by 18, 10, 9, 6, 4 or 2 amino acids in the term C. Particularly preferred desulphatohirudin compounds are those of formula I wherein the groups Xaa are as defined in a), or the compound of the formula III where Xaa in 47 is Lys, and Xaa in 63 is Tyr. The most preferred hirudin is desulfatohirudina HV1 having the formula I, wherein Xaa at 27, 36, and 47 are each Lys, Xaa at 51 is His, and Xaa at 62 is the peptide residue Glu-Tyr-Leu-Gln. The hirudins used in the present invention can be prepared synthetically, for example, chemically, or preferably by recombinant techniques, or by isolation from leeches. In accordance with the present invention, the term "mutant" refers to proteins (uteins) that exhibit a / ... (Ü Antithrombotic activity that differs from native hirudma or desulfatohirudin by single or multiple mutations (European Patent Applications Nos. 352,227 and 352.228). DNA encoding these mutants, which can be prepared by methods known in the art, for example, site-directed mutagenesis, is cloned and expressed in microbial hosts such as Escherichia coli and Saccharomyces cerevisiae. The hirudin compounds used in the invention may be in the free form, but also in the form of their salts. Since they contain a free amino group in various amino acid residues, the compounds may be in the form of acid addition salts. Suitable acid addition salts are in particular pharmacologically acceptable salts with conventional therapeutically acceptable acids. The representative inorganic acids are halohydric acids (such as hydrochloric acid), and also sulfuric acid, phosphoric acid, and pyrophosphoric acid. Representative organic acids are in particular arenesulfonic acids (such as benzenesulfonic or p-toluenesulfonic acid), or lower alkane sulphonic acids (such as methanesulfonic acid), as well as carboxylic acids such as acetic acid, lactic acid, palmitic acid, stearic acid , malic acid, tartaric acid, ascorbic acid, and citric acid. However, since the compound used in the invention also contains free carboxyl groups in various amino acid residues, whose carboxyl groups impart an acidic character to the entire peptide, may also be in the form of salts with inorganic or organic bases, for example, sodium, potassium, calcium, or magnesium salts, or also ammonium salts derived from ammonia, or a base containing pharmacologically acceptable organic nitrogen. However, since they contain both free carboxyl groups and free amino groups at the same time, they can also be in the form of internal salts. The pharmacologically acceptable salts are preferred. A problem in the development of a dosage form containing hirudins, is its poor stability in aqueous solutions and in powder form. Poor stability can be seen when hirudin is analyzed by chromatographic methods, such as HPLC of reverse phase (RP-HPLC). RP-HPLC method; A LiChroCART 125-4 column (Merck Lichrospher 100 RP-18 5μm) is used. Solvent A is 0.5 percent ammonium acetate in 5 acetonitrile / water (10:90), (volume: volume); solvent B is 0.5 percent ammonium acetate in acetonitrile / water (25:75). The elution is carried out at 45 ° C using a flow rate of 0.5 milliliters per minute. The binary elution is a linear gradient that starts at time zero lß 'with 23 percent of solvent B, and reaches 46 percent of solvent B after 24 minutes. After 2 minutes at 70 percent of solvent B, the column is equilibrated for 7 minutes to 23 percent of solvent B. A typical chromatogram of recombinant hirudin HV1 (CGP 39393) in water, using the RP-HPLC method (1 milligram / milliliter of hirudin). In Figure 1, the relative area of the main peak is 95.15 percent. Storage of hirudin in water at room temperature results in a increase of by-products over time. This is shown by a decrease in the area of the main peak, and an increase in the area of the small peaks. The changes that occur can be accelerated by the use of temperature stress experiments, that is, by storage at elevated temperatures.

Now we have discovered that potassium phosphate can be used to increase stability in hirudin. In accordance with the above, the present invention provides a freeze-dried pharmaceutical composition comprising hirudin, potassium phosphate, and a sugar. The composition of the invention can be produced by forming an aqueous solution of the ingredients, and then freeze-dried in a conventional manner. Potassium phosphate is preferably dipotassium acid phosphate. It can be used in the solution before freeze drying, in a molarity of 0.1 to 0.5, preferably 0.1 to 0.3. Suitable sugars include mannitol, trehalose, sucrose, sorbitol, fructose, glucose, maltose, lactose, and dextran. The preferred sugars are mannitol and trehalose. The amount of sugar in the solution before freeze drying can be such that it produces a concentration of 5 to 50 percent (w / v), and preferably 5 to 20 percent (w / v). The solution before freeze drying is preferably isotonic. The pH of the solution before drying by freezing may be from 4 to 10, preferably from 6 to 9, and more preferably from 6.5 to 8. If desired, a citrate buffer may be added to the solution before freeze drying, for example, by adding citric acid. The molarity of the citrate can be from 0.1 to 0.5, preferably from 0.1 to 0. 3. The concentration of hirudin in the solution before freeze drying can be from 0.1 to 500 milligrams / milliliter, preferably from 20 to 250 milligrams / milliliter. The freeze-dried product is stable for long periods of time without the need for refrigerated storage. In addition, after the product has been redissolved in water, the resulting solution is also stable for long periods, although the stability in solution is not as good as the stability of the freeze-dried powder. The solutions made by the redissolution of the freeze-dried product can be used in the production of standard ampoules, prefilled double-chamber syringes, or multi-administration systems. The solutions, of course, can also be used immediately for administration.

The invention is illustrated by the following Examples: EXAMPLE 1 Aqueous solutions of recombinant desulfatohirudin HV1 (CGP 39393 from Ciba-Geigy) are produced by dissolving: (a) water; (b) 70 parts by volume of a 5 percent trehalose solution and 30 parts by volume of a citric acid / K2HP04, 150mM mixture at a pH of 7.4; (c) 30 parts by volume of a 5 percent mannitol solution and 70 parts by volume of a 150mM citric acid / K2HP04 mixture at a pH of 7.4; and (d) 30 parts by volume of a 5 percent mannitol solution and 70 parts by weight of K2HP04, 150mM at a pH of 7.4. In each case, the concentration of hirudin is 30 milligrams / milliliter. The solutions are freeze-dried and stored at 46 ° C. At different times, samples are dissolved in water up to 1 milligram / milliliter of hirudin, and the main peak is measured by RP-HPLC. The results obtained are given in the following Table 1.

Table 1 It can be seen that the stability is maintained at a high level even when stored for long periods at 46 ° C.

EXAMPLE 2 Aqueous solutions of recombinant desulfatohirudin HVl (CGP 39393 from Ciba-Geigy) are made, dissolving it in different sugar / citrate / phosphate mixtures, as follows. In each case, 30 parts by volume of sugar are mixed with 70 parts by volume of citrate / phosphate. 5% mannitol: CKP K2HP04 100 M (1.74%); citric acid, 7mM (0.134%); mannitol, 82mM (1.5%); 4mM hirudin (0.3%). 10% sucrose: CKP K2HP04 100mM (1.74%); citric acid, 7mM (0.134%); sucrose, 87.6mM (3%); 4mM hirudin (0.3%). 10% trehalose: CKP K2HP04 100mM (1.74%); citric acid, 7mM (0.134%); trehalose, 87.6mM (3%); 4mM hirudin (0.3%).

The solutions are dried by freezing, and stored at different temperatures. After a certain storage time, a sample is redissolved in water to 1 milligram / milliliter of hirudin, and the main peak is measured by RP-HPLC. The results obtained are given in the following Table 2.

Table 2 You can see the improved stability when the powder is stored at different temperatures. At 26 ° C, which is probably slightly higher than normal environmental conditions, there is no noticeable degradation after 132 days.

LIST OF SEQUENCE GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Ciba-Geigy AG (B) STREET: Klybeckstrasse 141 (C) CITY: Basel (E) COUNTRY: Switzerland (F) POSTAL CODE: 4002 (G) TELEPHONE: 061 969 1111 (H) TELEFAX: 061 969 7976 (I) TELEX: 962991 (ii) TITLE OF THE INVENTION: Pharmaceutical Compositions iii) SEQUENCE NUMBER: 5 (iv) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Chemtext Version 1.50 IDENTIFICATION OF SEQUENCE NUMBER: 1 (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 63-66 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple?) TOPOLOGY: linear (ii) - TYPE OF MOLECULE: protein? < ? - (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys? 10 15 Glu Gly Ser Asn Val Cys Gly Gln Gly Asn Xaa Cys He Leu Gly Ser 20 25 30 0 Asp Gly Glu Xaa Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Xaa Pro 35 40 45 Gln Ser Xaa Asn Asp Gly Asp Phe Glu Glu He Pro Glu Xaa 50 55 60 IDENTIFICATION OF SEQUENCE NUMBER: 2 (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 65 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein go (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no Leu Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys 1 - . 1 - 5 10 15 Glu Gly Ser Asn Val Cys Gly Gln Gly Asn Lys Cys He Leu Gly Ser 20 25 30 Asp Gly Glu Lys Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Pro 35 40 45 Gln Ser His Asn Asp Gly Asp Phe Glu Glu He Pro Glu Glu Tyr Leu 50 55 60 Gln 5 65 IDENTIFICATION OF SEQUENCE NUMBER: 3 (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 65 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no He Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys 5 1 15 Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys He Leu Gly Ser 20 25 30 Asn Gly Lys Gly Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Xaa Pro 35 40 45 Glμ Ser tfis Asn Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Xaa Leu 50 55 60 Gln 65 IDENTIFICATION OF SEQUENCE NUMBER: 4 (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 65 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) "TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no Val Val Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys 1 5 10 15 • ~ Glu Gly Ser Asn Val Cys Gl and Lys G ly Asn Lys Cys He Leu Gly Ser 20 25 30 0 Asn Gly Lys Gly Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Asn Pro 35 40 45 Glu Ser His Asn Asn Gly Asp Phe Glu Glu He Pro Glu Glu Tyr Leu 50 55 60 Gln 5 65 IDENTIFICATION OF SEQUENCE NUMBER: 5 (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 66 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no He Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys Leu Cys 1 5 10 15 Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys He Leu Gly Ser 20 25 30 Gln Gly Lys Asp Asn Gln Cys Val Thr Gly Glu Gly Thr Pro Lys Prc 40 45 Gln Ser His Asn Gln Gly Asp Phe Glu Pro He Pro Glu Asp Ala Tyr 50 55 60 Asp Glu 65

Claims

1. A freeze-dried pharmaceutical composition which comprises hirudin, potassium phosphate, and a sugar.

2. A composition as claimed in claim 1, wherein the potassium phosphate is dipotassium acid phosphate.

3. A composition as claimed in claim 1 or 2, wherein the sugar is mannitol, trehalose, sucrose, sorbitol, fructose, glucose, maltose, lactose, or dextran.

4. A composition as claimed in any of the preceding claims, wherein the hirudin is a variant of desulfatohirudin or a mutant thereof.

5. A composition as claimed in any of the preceding claims, wherein the hirudin is desulfatohirudin HVl.

6. A composition as claimed in any of the preceding claims, which also contains a citrate.

7. A composition as claimed in any of the preceding claims, which is obtained by dissolving the ingredients in water, and then freezing the solution.

8. A composition as claimed in claim 7, wherein the pH of the solution before freeze drying is from 4 to 10. A composition as claimed in any of claims 7 to 9, wherein the concentration of hirudin in the solution before freeze drying is 0.1 to 500 milligrams / milliliter. 10. A composition as claimed in any of claims 7 to 11, wherein the solution, before freeze drying, is isotonic.