AU671881B2

AU671881B2 - Novel Process for the Preparation of Lyophilised Peptides

Info

Publication number: AU671881B2
Application number: AU55235/94A
Authority: AU
Inventors: Jurgen Engel; Thomas Reissmann; Dieter Sauerbier; Burkhard Wichert
Original assignee: Asta Medica GmbH
Current assignee: Zentaris IVF GmbH
Priority date: 1993-02-19
Filing date: 1994-02-17
Publication date: 1996-09-12
Also published as: HRP940117A2; JPH06271476A; DK0611572T3; DE4305225A1; NZ314707A; TW387812B; HU218281B; SK19594A3; FI940779A0; EP0947200A2; CZ31294A3; ZA941136B; NZ250906A; EP0947200A3; JP4033919B2; GR3034237T3; CN1109557C; AU671881C; SG46632A1; KR100372187B1

Abstract

Sterile freeze-dried cetrorelix acetate (a peptide described in EP299402) is used in the treatment of female infertility. Independent claims are also included for the following: (1) use of sterile freeze-dried cetrorelix acetate for protecting gonads against noxious agents that damage germ cells, e.g. radiation treatment and chemotherapy; (2) a composition comprising sterile freeze-dried cetrorelix acetate and optionally excipients for use in the treatment of female infertility; (3) a composition comprising sterile freeze-dried cetrorelix acetate and optionally excipients for protecting gonads against noxious agents that damage germ cells, e.g. radiation treatment and chemotherapy with cytostatic agents.

Description

_I I Novel Process for the Preparation of Lyophilised Peptides The present invention relates to a lyophilisate of a peptide with 3-15 amino acids and optionally one or several bulking agents, characterised in that 1 part by weight of peptide is dissolved in 100 10,000 parts by weight of acetic acid and then transferred to water, the solution so obtained being lyophilised.

According to a first embodiment of this invention, there is provided a lyophilisate of a peptide with 3-15 amino acids and optionally one or several bulking agents, characterised in that 1 part by weight of peptide is dissolved in 100 10,000 palcs by weight of acetic acid and then transferred to water, the solution so obtained being lyophilised.

According to a second embodiment of this invention, there is provided a method for the treatment or prophylaxis of female infertility in a female mammal requiring said treatment or prophylaxis, which method comprises administering to said mammal an effective amount of at least one lyophilisate according to the first embodiment.

According to a third embodiment of this invention, there is provided a method for gonad protection in a mammal requiring said protection, which method comprises administering to said mammal an effective amount of at least one lyophilisate according to the first embodiment.

Suitable peptides to which the invention can be applied include those which are the 20 subject of European Patent 229 402. Such peptides are of the formula:

X-R

1

-R

2

-R

3 -Ser-Tyr-R 6 -Leu-Arg-Pro-R 10

-NH

2 wherein X is an acyl or carbamoyl group; R 1 is D- or L-Pro, D- or L-A3-Pro, D-Phe, 4halo-D-Phe, D-Ser, D-Thr, D-Ala, 3-(1-naphthyl)-D-Ala or 3-(2-naphthyl)-D-Ala; R 2 is D-Phe or 4-halo-D-Phe; R 3 is D-Trp, D-Phe, 3-(3-pyridyl)-D-Ala, 3-(l-naphthyl)-D-Ala or 3-(2-naphthyl)-D-Ala; R 6 is D-Cit, D-Hci, alkyl substituted D-Cit or alkyl substituted D-Hci and R 10 is Gly or D-Ala.

Cetrorelix is a decapeptide with a terminal acid amide group that is used as an acetate salt. The synthesis and some pharmacological effects are described in European Patent Application 299 402. It should be possible to administer the active substance subcutaneously in a dose of 0.1 to 2 mg. Aqueous solutions of the decapeptide are unstable, autoclaving in the final container is not possible. With conventional sterilisation according to the prescribed conditions the decapeptide tends to decompose. To obtain an injectable solution it was therefore necessary to develop a lyophilisate.

The amount of active substance in the solution to be lyophilised is, however, so small that, in low active substance concentrations, only a loose fluff results on the glass wall of the ampoule after drying the solution free of auxiliary substances and this fluff is carried out of the vial with the stream of steam used for sterilisation purposes. It is therefore necessary to use a bulking agent that forms a stable cake. In high concentrations S this auxiliary substance can be dispensed with. The following auxiliary substances may S ,o be considered as bulking agents: hexitols, in particular mannitol, glucitol, sorbitol, such [n:\libvv100592:hrw __I L- ~I I L~-L I 1.

as D-sorbitol, dulcitol, allitol, altritol (for example D- and L-altritol), iditol (for example D- and L-iditol), their optically active forms and L-forms) as well as the corresponding racemates. Mannitol is used in particular, such as D-mannitol, L-mannitol, DL-mannitol, sorbitol and/or dulcitol and of these preferably D-mannitol. The hexitol used may also be mixtures of the hexitols named, for example mixtures of mannitol and sorbitol and/or dulcitol. Since dulcitol is less water soluble than, for example, mannitol, the dulcitol content in the aqueous solution should not exceed for example 3 percent by weight. Mannitol and sorbitol, on the other hand, can for example be mixed in any ratio.

Apart from hexitol it is also possible to add other, conventional pharmaceutical auxiliary substances, such as amino acids, such as alanine, glycine, lysine, phenylalanine, asparaginic acid, glutaminic acid, leucine, lactose, polyvinylpyrrolidone, glucose, fructose, albumin and equivalent bulking agents. Urea and sodium chloride may also be used as bulking agents. The total amount of such substances in the solution which is used for 0 0 0 0* 0.0 .IcC p s\ :b t

C

In:\libvv]00592:hrw L- ~I -I I I 2 freeze-drying, is for example 0 16.9 parts by weight, for example 0.1 7 parts by weight, based on 1 part by weight of cetrorelix. In the finished lyophilisate the total amount of such auxiliary substances may be up to 16.9 parts by weight, based on one part by weight of hexitol. In detail, the amount of such auxiliary substances depends on the amount of hexitol present and to such an extent that the total amount of hexitol and such other auxiliary substances in the finished lyophilisate may not be more than a maximum of 17 parts by weight, based on 1 part by weight of cetrorelix. If only 0.1 part by weight of hexitol is present in the lyophilisate, it is thus possible to have up to 16.9 parts by weight of other auxiliary substances; if, for example 8.5 parts by weight of hexitol are present, the amount of other auxiliary substances may for example be up to 8.5 parts by weight, based on 1 part by weight of cetrorelix.

It was, however, found during development work on the lyophilisate that the active substance behaves in a widely different and unpredictable manner during processing. The first batches gave good results, but it soon transpired that difficulties occurred during sterile filtration and faulty batches resulted.

It is known from the literature, for example from Powell, Pharmaceutical Research, 1258-1263 (8)15191; Dathe, M: Int. J. Peptide Protein Res. 344-349 (36) 1990; Szejtli, Pharmaceutical Technology International 16-22, 1991 that oligopeptides, particularly those with terminal acid amide function, tend to form gels. During sterile filtration this is apparent from the speed of filtration, indeed, the increased viscosity of Ssuch solutions can often already be detected organoleptlcally. A gelatinous layer remains on the sterile filter. It is then no longer possible to prepare a medicament with an exactly S defined active substance content.

Table 1 lists various results of the first 11 batches.

The active substance contents fluctuate between 100% and 36%.

Table 1: Cetrorelix acetate r a Batch Dosage Active substance content 1 100tg 100 2 500pig 100 3 500g 4 500.tg 36 500tg 100 6 500.tg 7 Img 8 1mg 100 9 2mg 100 2mg 11 2mg 100 ILbWI01669:JOC I--sl To avoid this gel formation, the literature lists the following addiuvas which may be tried out on an experimental basis: Organic solvents may be considered, for example acetonitrile, n-butanol, tertiary butanol, ethanol, isopropanol, octanol and benzyl alcohol. It is also possible to use salts and buffer solutions, such as acetate buffer, citrate buffer, sodium chloride, sodium phosphate, sodium EDTA, sodium bicarbonate, phosphate buffer, guanidine acetate, urea.

Polymers may also be used, such as gelatin, polyethylene glycol 600, hydroxyethyl starch, polyvinylpyrrolidone, polyvinyl alcohol. The use of amino acids, for example alanine, glycine, lysine, phenylalanine, asparaginic acid, glutaminic acid and leucine has also been described. Acids that were used were citric acid, caprylic acid, octanic acid, hydrochloric acid, sulfuric acid and acetic acid. Physiologically acceptable tensides that may be used are benzalkonium chloride, cetyl alcohol, bile acids, lecithins, polysorbates, Spans® and Pluronics®. Carbohydrates and cyclodextrins such as glucose, lactose, mannitol, saccharose, ca-, and y gyclodextrins, hydroxypropyl-a- and 3-cyclodextlins, hydroxyethyl cyclodextrins and methyl cyclodextrins have already been used. These auxiliary substances were tested as filtration supporting agents to prevent gel formation.

No satisfactory solution of the problem could, however, be found. Only acidification with acetic acid showed partial success. Here, too, it was, howel always necessary to accept high filtration losses. It was then surprisingly found that cetrorelix can be easily S 20 dissolved in 30% volume/volume acetic acid. The solution is then filled up to a final concentration of 3% cetrorelix with water for injection purposes and mannitol is added.

Although it is stated in the literature that the terminal amide group hydrolyses easily in acid medium, this was not found in the case of cetrorelix. Solutions prepared according to this method caused no difficulties during filtration. The correct amounts of active substance were always ~found. The filtration speed attains values that ensure satisfactory production sequences. A general process for sterile lyophilisation is described in pages 557 559 of Sucker, Fuchs and Speiser (Publishers) "Pharmazeutische Technologie" 2nd edition 1991, Thieme-Verlag, Stuttgart-New York. A further description of the lyophilisation process used is given in German published specification (DOS) 37 35 614. The lyophilisate is used in the treatment: of female sterility. One therapeutic process has hitherto consisted in stimulating follicle maturation using human menopause gonadotrophin and then triggering ovulation by administering human chorion gonadotrophin. The ovulation triggered thereby occurred 32 hours later. The ova collected thereby are available for in vitro fertilisation.

A disadvantage of this treatment with agonists is the fact that up to 10 follicles mature during the stimulation phase. This elevated follicle maturation leads io hormone level peaks in the LH. These peaks result in an early stage of follicle maturation and ovulation at an unpredicted point in time. This impaired ovulation occurs in about 25% of treated cases and is a disadvantage since the cycle that displays disturbed ovulation of this kind cannot be used for the collection of ova and the entire treatment has to be repeated about 1 month later.

ILibWO 1669:JOC I I ii I Another disadvantage of the conventional simulation treatment is the long treatment duration of 4 weeks which is needed to achieve satisfactory suppression. The agonists continue to display a hyperstimulation syndrome in 1-2% of cases in which the follicle cells hypertrophy. The risk of hyperstimulation is particularly great in the case of polycystic ovaries. The hyperstimulation syndrome is a severe side effect which can lead to fatalities.

It has now been found that the antagonist cetrorelix displays the following advantages in this particular treatment: Treatment with cetrorelix over 5 days is sufficient to achieve total suppression. The hyperstimulation syndrome cannot arise. In addition, less HMG is used in the 2nd phase of therapy, the ovulation triggering phase. This gives this in-vitro fertilisation treatment a not inconsiderable cost advantage. In-vitro fertilisation is, for example, used when a tube anomaly is present. To perform this treatment it is necessary to precisely monitor the cycle and to establish the time of ovulation as precisely as possible. This has hitherto only been achieved to a limited extent since preovulatory LH increase often occurred too early due to simulation with HMG/HCG, or was not maintained for a sufficiently long period.

Avoidance of this premature increase is, however, of critical importance for the success of the treatment in order to precisely determine the time of fertilisation. This reduces the physical and mental burden on the patient and makes optimum -use of hospital logistics.

To achieve this objective with great reliability it is necessary to suppress endogenous hormone production (LH-FSH, oestradiol) as completely as possible in -*Aer to simultaneously stimulate follicle maturation through administration ot .ogenous gonadotrophins (HMG/HCG) and to monitor the hormone status at any time. It is only when a sufficiently large number of follicles have been achieved having ap ,roximately the same degree of maturation, that ovulation is triggered by administering an HCG bolus injection.

Use of an antagonist makes treatment substantially more successful and safer for the patient. Another area of use of the cetrorelix lyophilisate according to the present invention is to protect the gonads in male patients. Male patients are pre-treated with cetrorelix lyophilisate and the activity of the gonads is reinforced. As a result, other harmful noxious agents, such as radiation therapy or treatment with cytostatics, have no or only a small possibility of affecting the sensitive tissue of the gonads.

SExample of the method of preparation: Approx. 1.5 litres of water for injection purposes are prepared in a suitable glass vessel. 210g water for injection purposes are prepared in another glass vessel and 91.17g acetic acid are added. The amount of cetrorelix acetate calculated (1.62 1.695g, depending on the content of the batch used) is dissolved in the prepared 30% acetic acid with stirring. This solution is transferred to the glass vessel with 1.5 litres of water for injection purposes, 82.2g mannitol are added, dissolved and made up to 3039g with water for injection purposes.

ILibWIO 1 59:JOC 4 ol 7 I I II II.

In-process checks: pH value: 2.5 Density: 1.009 1.017 g/cm 3 at Refractive index: 1.227 1.340 at 440nm and The solution is sterilised by filtration through an appropriate membrane filter (pore size 0.2|tm) under aseptic conditions. 100mL first runnings should be discarded. The filters should be sterilised with superheated steam. Cetrorelix freeze-dried solution should be protected from recontamination during storage. The solution is immediately filled into colourless injection bottles DIN 2R, hydrolytic class I under aseptic conditions and provided with sterile freeze-drying stoppers. The nominal filling amount is 1.0mL 2.026g.

The mL injection bottles were rinsed in an injection bottle washing machine, dried with hot air and sterilised. The cleaned freeze-drying stoppers were autoclaved. The closed injection bottles were transferred to a freeze-drying installation d frozen at a plate temperature of -40°C. Drying was carried out using a drying programme with a plate temperature of -40°C rising to +20 0 C. The installation is then flooded with sterile nitrogen, the bottles are closed in the installation and the stoppers secured with crimped caps.

The injection bottles are checked visually for "aulty closures and outer faults. Faulty injection bottles are removed and destroyed.

.Cetrorelix lyophilisate 1mg is a white, solid, freeze-dried cake in a colourless 2mL injection bottles which is closed with grey freeze drying stoppers and yellow flip-off 20 crimped caps.

ILibWO1659:JOC 6 of 7 -e

Claims

1. A lyophilisate of a peptide with 3-15 amino acids and optionally one or several bulking agents, characterised in that 1 part by weight of peptide is dissolved in 100 10,000 parts by weight of acetic acid and then transferred to water, the solution so obtained being lyophilised.

2. A lyophilisate according to claim 1 characterised in that the peptide is of the formula: X-R 1 -R 2 -R 3 -Ser-Tyr-R 6 -Leu-Arg-Pro-R 1 0 -NH 2 wherein X is an acyl or carbamoyl group; R 1 is D- or L-Pro, D- or L-A3-Pro, D-Phe, 4- halo-D-Phe, D-Ser, D-Thr, D-Ala, 3-(1-naphthyl)-D-Ala or 3-(2-naphthyl)-D-Ala; R 2 is D-Phe or 4-halo-D-Phe; R 3 is D-Trp, D-Phe, 3-(3-pyridyl)-D-Ala, 3-(1-naphthyl)-D-Ala or 3-(2-naphthyl)-D-Ala; R 6 is D-Cit, D-Hci, alkyl substituted D-Cit or alkyl substituted D-Hci and R 10 is Gly or D-Ala.

3. A lyophilisate according to claim 1 or claim 2, characterised in that the peptide is cetrorelix.

4. A lyophilisate according to any one of claims 1 to 3, characterised in that the bulking agent used is mannitol.

A lyophilisate of a peptide with 3-15 amino acids and optionally one or several bulking agents, substantially as hereinbefore described with reference to the example. 20

6. A method for the treatment or prophylaxis of female infertility in a female mammal requiring said treatment or prophylaxis, which method comprises administering to said mammal an effective amount of at least one lyophilisate according to any one of claims 2 to

7. A method for gonad protection in a mammal requiring said protection, which S 25 method comprises administering to said mammal an effective amount of at least one lyophilisate according to any one of claims 2 to Dated 15 July, 1996 ASTA Medica Aktiengesellschaft Patent Attorneys for the Applicant/Nominated Person 30 SPRUSON FERGUSON o*e [n:\libvvi00592:hrw I Y I Novel Process for the Preparation of Lyophilised Peptides Abstract A novel lyophilisate and method of preparation as well as the use of the lyophilisate to treat female infertility and for gonad protection. The lyophilisate is of a peptide with 3-15 amino acids and optionally one or several bulking agents, and is characterised in that 1 part by weight of peptide is dissolved in 100 000 parts by weight of acetic acid and then transferred to water, the solution so obtained being lyophilised. A suitable peptide is cetrorelix. *o e 0 S S S *o o *el oo (LibW1\01559:JOC i