CA2115943C - Novel process for the preparation of cetrorelix lyophilisate - Google Patents

Abstract

A novel lyophilisate and method of preparation as well as the use of the lyophilisate to treat female infertility and for gonad protection. A lyophilisate of a peptide with 3 - 15 amino acids and optionally one or several bulking agents is prepared by dissolving 1 part by weight of peptide in 100 - 10,000 parts by weight of acetic acid and then transferring to water, the solution so obtained being lyophilized. Cetrorelix is preferred as the peptide.

Description

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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 published European Patent Application 299 402.
It should be possible to administer the active substance subcutaneously in a dose of 0.1 to 20 mg. Aqueous solutions of the decapeptide are unstable, autoclaving in the final container is not possible. With conventional sterilization 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 lyophilized is, however, so small that, in low active substance concentrations, only a loose fluf:~ 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 sterilization purposes. It is therefore necessary to use a bulking agent that forms a stable cake. In high concentrations this auxiliary substance can be dispensed with. The following auxiliary substances may be considered as bulking agentss hexitols, in particular mannitol, glucitol, sorbitol, such as D-sorbitol, dulcitol, allitol, altritol (for example D- and L-altritol), iditol (for example D- and L-iditol), their optically active forms (D- and L-foams) 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 ~5 ratio.

21~~943 _ 2 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 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 an 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, M.F.;
Pharmaceutical research, 1258-1263 (8)1991; Dathe, M: Int. J.
Peptide Protein Res. 3~4-3~9 (36) 1990; Szejtli, J.:
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 such solutions can often already be detected organoleptically. ~ gelatinous layer remains on the sterile filter. Tt is then no longer possible to prepare a medicament pith an exactly defined active substance content.
Table 1 lists various results of the first 11 batches.
The active suLStance contents fluctuate between 100 and 36~.
Table 1: Cetrorelix acetate Batch Dosage active substance content 1 100 ug 100 2 500 ug 100 3 500 ug 90 4 500 ug $6 500 ug 100 6 500 ug 85 7 1 mg 80 8 1 mg 100 9 2 mg 100 2 mg 80 11 2 mg 100 21~ ~~~~~

To avoid this gel formation, the literature lists the following additives 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 ~DTA, sodium bicarbonate, phosphate buffer, guanidine acetate, urea.
polymers may also be used, such as gelatin, polyethylene glycol X00, hydraxyethyl starch, polyvinylpyrxalidone, polyvinyl alcohol. The use of amino acids, for examp:Le alanine, glycine, lysine, phenylalanine, asparaginic acid, g:Lutaminic acid and leucine has also been described.
Acids that were used were citric acid, caprylic acid, octanic acid, hydrochloric acid, sulphuric acid and acetic acid.
physiologically acceptable tensides that may be used are benzalDsonium chloride, cetyl alcohol, bile acids, lecithins, polysorbates, Spans{R~ said pluronicscR).
Carbohydrates and cyclodextrines such as glucose, lactose, mannitol, saccharose, alpha-, beta- and gamma gyclodextrins, hydroxypropyl-alpha- and beta-cyclodextrins, hydroxyethyl cyclodextrins and methyl cycladextrins have already been used.
These auxiliary substances were tested as filtration supporting agents to prevent gel formation.
ri1a satisfactory solution of, the problem could, however, be found. Only acidification with acetic acid showed partial success, Mere, too, it was, however, always necessary to accept high filtration losses. It was then surprisingly found that cetrorelix can be easily dissolved in 30~ volume/valume acetic acid. The solution is then filled up to a final concentration of 3~ cetrare.lix 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 describeo: in pages 557 - 559 of Sucker, Fuchs and Speiser (Publishers) "F~harmazeutisch~=_ 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 :ire the treatment of female sterility.
One therapeutic process hae; 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 trea..tment with agonists is the fact that up to 10 follicles mature during the stimulation phase. This elevated follicle maturat:ic>n leads to hormone level peaks in the LH. These peaks result in an early stage of follicle maturation and ovulation at an unpredi.eted point in time. This impaired ovulation occurs in about ~5% 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.
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 ri~~k of hyperstimulation is particularly great in the case of polyc~rstic ovaries. The hyperstimulat.ion syndrome is a severe side effect which can lead to fatalities.

- 5a -The present invention provides a lyophilisate of cetrorelix and optionally one or more bull~:ing agents. The lyophilisate is obtained by a process comprising dissolving 1 part per weight of cetrorelix in 100 to 10,000 parts by weight of acetic acid, diluting the cetrorelix/acetic acid solution with water, optionally, adding a bulking agent, and lyophilising the resulting solution.
The present invention also provides a process for the preparation of a sterile cetrorelix lyophilisate, in which cetrorelix is (Ac-D-Nal (2) 1, D-phe (4C1) z, D-PaL (3) 3, D-Cit6, D-Alal°) -LHRH, wherein cetrorelix is dissolved in aqueous acetic acid in the ratio 1:100-10,000 wt/wt, the solution 1S diluted with water, if required a wilder is added, and the :solution is sterile-filtered, filled into injection vials and :Lyophilised.
T'he cetrorelix being dissolved can be in the form of an acetate salt.
It has now been found that the antagonist cetrorelix displays the following advantages in this particular treatment:

_ 5 _ 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, aestradiol) as completely as possible in order to simultaneously stimulate follicle maturation through administration of exogenous 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 (4-6), having approximately 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.
Example of the method of preparatian~

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Approx. 1.5 litres of water for injection purposes are prepared in a suitable glass vessel. 210 g water for injection purposes are prepared in another glass vessel and 91.17 g acetic acid are added. The amount of cetrorelix acetate calculated (1.62 - 1.695 g, depending on the content of the batch used) is dissolved in the prepared 30~ acetic acid with stirring. This salution is transferred to the glass vessel with 1.5 litres of water for injection purposes, 82.2 g mannitol are added, dissolved and made up to 3039 g with water for injection purposes.
In-process checks:
pFI value : 2 . 5 - 3 . 0 Density: 1.009 - 1.017 g/cm3 at 20°C
Refractive index: 1.227 - 1.340 at 440 nm and 20°C
The solution is sterilised by filtration through an appropriate membrane filter (pore size 0.2 um) under aseptic conditions. 100 ml 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 2.0 ml = 2.026 g.
The 2 ml injection bottles were rinsed in an injection bottle washing machine, dried with hot air and sterilised. The cleaned freeze-drying stoppers were autoclaved. 'Phe closed injection bottles were transferred to a freeze-drying installation .and 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°C. rfhe installation is then flooded with sterile nitrogen, the bottles are closed in the installation and 'the stoppers secured with crimped caps.

_ g _ The injection bottles are checked visually for faulty closures and outer faults. Faulty injection bottles are removed and destroyed.
Cetrorelix lyophilisate 1 mg is a white, solid, freeze-dried cake in a colourless 2 ml injection bottle which is closed with grey freeze-drying stoppers and yellow flip-off crimped caps.

Claims (10)

1. A lyophilisate of cetrorelix and optionally one or more bulking agents, wherein the lyophilisate is obtained by a process comprising:
dissolving 1 part per weight of cetrorelix in 100 to 10,000 parts by weight of acetic acid;
diluting the cetrorelix/acetic acid solution with water;
optionally, adding a bulking agent; and lyophilising the resulting solution.

2. A lyophilisate according to claim 1, wherein the cetrorelix being dissolved is in the form of an acetate salt.

3. A lyophilisate according to claim 1 or 2, wherein the bulking agent is mannitol.

4. A lyophilisate according to claim 1, 2 or 3, wherein the solution is sterile-filtered before lyophilisation.

5. Use of the lyophilisate according to any one of claims 1 to 4, for the treatment of female infertility.

6. Use of the lyophilisate according to any one of claims 1 to 4, for gonad protection.

7. Use of the lyophilisate according to any one of claims 1 to 4, to prepare a medicament for the treatment of female infertility.

8. Use of the lyophilisate according to any one of claims 1 to 4, to prepare a medicament for gonad protection.

9. Process for the preparation of a sterile cetrorelix lyophilisate, in which cetrorelix is (Ac-D-Nal(2)1, D-phe (4Cl)2, D-PaL(3)3, D-Cit6, D-Ala10) -LHRH, wherein cetrorelix is dissolved in aqueous acetic acid in the ratio 1:100-10,000 wt/wt, the solution is diluted with water, if required a builder is added, and the solution is sterile-filtered, filled into injection vials and lyophilised.

10. The process according to claim 9, wherein the cetrorelix being dissolved is in the form of an acetate salt.