AU652361B2 - Aqueous pharmaceutical formulations of erythropoietin and the use thereof - Google Patents

Abstract

Aqueous pharmaceutical formulations of proteins with erythropoietin action, especially of natural human and recombinant human erythropoietin (EPO), are described. Highly purified EPO without foreign proteins or other conventional stabilisers retains at least about 78 % of its original activity at room temperature for at least 1 year in a physiologically tolerated aqueous phosphate buffer which contains a physiologically tolerated alkali metal halide but no other stabilising additives and has a pH of 6 to 8. The products in this aqueous pharmaceutical formulation are particularly suitable for subcutaneous or intramuscular administration.

Description

Form COMMON~WEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION,

(ORIGINAL)

Class Int, Class.

Application Number: Lodged: 9 rCemplete Specification Lodged: "C Accepted: Published: R* ority r 74 &plated Art *.-Name of Applicant Sr *;"Address of Applicant *AculInventor Address for Service BEHRINGWERKE AKTIENGESELLSCHAFT D-355fl Marburg, Federal Republic of Germany DIETER BRAZEL, BERNHARD SIEBOLD, THCI!1AS BRUNE ]OROTHEE KRUNWIER and WATERMARK PATENT TRADEMARK ATTORNEYS.

LOCKED BAG NO. 5, HAWTHORN, VICTORIA 3122, AUSTRALIA Complete Specification for the invention entitled: AQUEOUS PHARMACEUTICAL FORMULATIONS OF ERYTHROPOTETIN AND THE USE THEREOF The following statement is a full description of this invention, including the best method of performing it known to :-us II r BEHRINGWERKE AKTIENGESELLSCHAFT HOE 90/B 021 Ma 787 Dr. LP/VO Description Aqueous pharmaceutical formulations of erythropoietin and the use thereof The invention relates to aqueous pharmaceutical formulations of purified erythropoietin, in particular of human native and of recombinant human erythropoietin (rh EPO).

The formulations of the invention without foreign proteins, sugars, amino acids or other customary stabili- Szers retain at least about 78% of their original 10 activity for at least one year at a temperature of 4-8 C.

Erythropoietin (EPO) is a glycoprotein with 166 amino acids, 3 glycosylation sites at amino-acid positions 24, 38 and 83 and a molecular weight of about 34,000. EPO can either be isolated from natural sources such as human urine for example, Miyake et al., J. Biol. Chem., vol. 252 (1977), 5558-5564) or be prepared by genetic engineering processes for example, EP-A 0,148,605 and 0,267,678). Aqueous solutions of erythropoietin are unstable at temperatures from about 3°C to rocin tempera- 20 ture.

Patients with kidney failure are unable to form EPO and thus suffer from anemia. Attempts to compensate for this undersupply of EPO by administering EPO and to reduce the symptoms of anemia have already been successful. Further clinical uses comprise administration of hEPO for iatrogenic anemia after chemotherapy or radiotherapy of malignant diseases.

A single dose of EPO amounts to only a few microgrammes.

Because of the short half-life after i.v. administration, physiological plasma levels can best be achieved by subcutaneous injection.

The pharmaceutical formulations of erythropoietin

I!

b, r V 2 hitherto proposed or available contain added detergents and/or proteins, sugars or polyalcohols, which are intended, on the one hand, to stabilize EPO and, on the other hand, to prevent adsorption of EPO onto the inner wall of the storage container the ampoule) (EP-B-0,178,576; EP-A-0,178,665; G. Krystal et al., Blood vol. 67 (1986), 1, 71 79). Subcutaneous or i.m.

administration of EPO stabilized in this way results in local inflammation with the formation of granulomas.

Highly pure EPO prepared by known processes (EP-A-0,236,059; US-A 4,667,159; PCT/US 86/01342 WO 86/07594)) lost more than 25% of its activity within one week on storage at 24 0 C. EP-B-0,178,576 also discloses a solution of human erythropoietin, which has e* 15 been reductively methylated with 1 4 C-formaldehyde, in PBS; cf. Experiment 1 and 2.

The object on which the invention is based is to provide aqueous pharmaceutical formulations of purified erythropoietin which are free of the customary stabilizing 20 additive and nevertheless have adequate stability at temperatures from about 3°C to room temperature and are suitable in particular for subcutaneous or intramuscular administration.

It has been found, surprisingly, that purified erythro- 25 poietin (about 99% pure), especially purified native or recombinant human erythropoietin, is stable for a lengthy period at temperatures from 4 to 8"C in a physiologically tolerated aqueous phosphate buffer of pH 6 to 8 which contains a physiologically tolerated alkali metal halide, but otherwise no stabilizing additives, in sealed test tubes or glass ampoules.

Hence the invention relates to aqueous pharmaceutical formulations of purified erythropoietin in a physiologically tolerated aqueous phosphate buffer of pH 6 to 8 which contains a physiologically tolerated alkali metal halide, but otherwise no stabilizing additives. A buffer i 3 composed of 50 mM sodium phosphate, 100 mM NaCI, pH 7.8, is particularly preferred. The concentration of erythropoietin in the aqueous buffer solution is 50 1000 pg per ml of buffer solution.

Examples of physiologically tolerated aqueous phosphate buffers are sodium phosphate buffer and potassium phosphate buffer, preferably sodium phosphate buffer.

Examples of physiologically tolerated alkali metal halides are sodium chloride and potassium chloride, preferably sodium chloride. A preferred phosphate buffer is a buffer containing 50 mM sodium phosphate, 100 mM NaC1, pH 7.8. This buffer is also called PBS for brevity.

The aqueous solutions of erythropoietin in PBS are very well suited for subcutaneous or intramuscular administration.

The invention additionally relates to the use of the aqueous pharmaceutical formulations for preparing injection products for subcutaneous or intramuscular administration. Subcutaneous administration is particularly 20 preferred because, by contrast with the known formulations stabilized with proteins, it causes no irritation or inflammatory -pain. This has emerged from clinical trials.

S" The examples illustrate the invention.

S* *i, Example 1 Purification of rh EPO The purification of rh EPO started from serum-containing or serum-free medium which was conditioned by rh EPOproducing animal cells by the process described in EP-A 0,267,678, page 6, line 45, to page 9, line 3. The process comprises clarification, concentration and dialysis of the culture medium, 4 ion exchange chromatography, preparative reverse phase HPLC and gel filtration chromatography.

For the gel filtration chromatography, the column was equilibrated with PBS, i.e. 50 mM sodium phosphate buffer, 100 mM NaCI, pH 7.8. With this buffer solution, rh EPO was eluted in a single symmetrical peak (measurement of the eluate at 280 nm). The resulting rh EPO was at least 99% pure according to SDS-PAGE.

S. 10 Dilution The combined rh EPO fractions from the gel filtration step contained 0.1 0.8 mg of EPO per ml and were dispensed in single doses of 100 pg/ml using the sodium *I phosphate/sodium chloride buffer (PBS) pH 7,8.

Example 2 Stability testing 0* The stability of the PBS solution of rh EPO obtained as in Example 1 was tested after storage of the single doses in sterile glass tubes, comparing with various stabilizers. The activity of rh EPO was measured by the incorporation of 3 T into mouse spleen cells treated with phenylhydrazine in a conventional manner. The data shown in the Table which follows are related to the initial activity of the employed rh EPO 100%.

i

I

Storage at 4 8 0

C

Stabilizer Amount of stabilizer (wt./wt. relative to EPO) based on 100 pg EPO/mi PBS activity af ter 12 months PBS alone PBS sorbitol PBS glycerol 10 PBS HaemaccelR 5000 6150 100 78.5 51.

0 68 *0 S S *5

C.

C. 09 0 gee...

S

C.

S 9

C

ft..

Storage at 37 0 C (accelerated test) Stabilizer Amount of stabili zer Activity 1 2 after months 3 6 PBS alone PBS sorbitol PBS HaemaccelR 5000 100 853.3 33.5 42 39 10.7 14.8 11.2 9.9 5.1 0 6.0 5.1 g*

S

C g S. Note: HaemacceIR is a degraded gelatin The surprisingly. better stabilization of rh EPO in PBS (p1H 7.8) without further addition of a stabilizer is evident from the Tables.

e.g.

C *S *5 C S5 C C S

SC

Claims (6)

1. An aqueousApharmaceutical formulation ofA purified erythropoietin in a physiologically tolerated aqueous buffer solution of pH 6 to 8 which contains a physiologically tolerated alkali metal phosphate and alkali metal halide, but otherwise no stabili- zing additives.

2. A formulation as claimed in claim 1, wherein the aqueous buffer solution is composed of 50 mM sodium 10 phosphate, 100 mM NaC1, pH 7.8.

3. A formulation as claimed in claim 1 or 2, wherein S.the erythropoietin is human native or recombinant erythropoietin.

4. A formulation as claimed in any of claims 1 to 3, wherein the concentration of erythropoietin is 1000 pg per ml of buffer solution.

5. The use of an aqueous formulation of purified erythropoietin as claimed in any of claims 1 to 4 for preparing injection products. 20

6. A process for preparing a stable pharmaceutical formulation of erythropoietin as claimed in claim .4 1, which comprises dissolving erythropoietin with a purity of t 99% in a physiologically tolerated aqueous buffer solution of pH 6 to 8, said buffer solution containing a physiologically tolerated alkali metal phosphate and alkali metal halide, but otherwise no stabilizing additives. DATED this 29th day of April 1991. BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS S"THE ATRIUM" ©290 BURWOOD ROAD ,B -HAWTHORN. VIC. 3122. 1' l. a