Title of Invention

'PHARMACEUTICAL FORM OD ADMINISTRATION FOR PEPTIDES METHODS FOR ITS PRODUCTION AND USE'

Abstract A pharmaceutical form of administration suitable for parenteral administration, which contains peptides prone to aggregation in dissolved or dispersed form, characterized in that the peptides are present in the form of their acetate, gluconate, glucuronate, tectate, citrate, aseorbate, benzoate or phosphate salts and in that these forms of administration addrtfortafty contain one of the acids just mentioned as free acids and, if appropriate, further additives and excipients from the class consisting of the acids, surface-active substances, polymers, lipids or sugars.
Full Text WO 01/87265 PCT/EP01/05555
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Pharmaceutical form of administration for peptides, methods for its production and use
The invention relates to novel pharmaceutical forms for the parenteral administration of peptides which are prone to aggregation, in particular of LHRH analogs or LHRH antagonists and agonists, and methods for their production and use.
It is known from EP 0 299 402 to employ pharma-ceutically active decapeptides such as SB-030, SB-075 (cetrorelix) and SB-088 in the form of their pharmaceutically acceptable, nontoxic acid addition salts such as hydrochlorides, hydrobromides, sulfates, phosphates, fumarates, gluconates, tannates, maleates, acetates, citrates, benzoates, succinates, alginates, pamoates, ascorbates and tartrates, etc.
A lyophilized peptide or protein preparation is further known from JP 06321800-A which contains gluconate salts as stabilizers. In an example, the solution contains 2.5% magnesium gluconate, with vasopressin, LHRH and insulin, inter alia, being described as active compound.
It is known from the literature, inter alia from Powell, M.F., Pharmaceutical Research, 1258-1263(8) 1991; Dathe, M. , Int. J. Peptide Protein Res. ¦ 344-349(36) 1990, and Szejtli, J. Pharmaceutical Technology International 16-22, 1991, that oligopeptides, namely particularly those having a terminal acid amide function, are prone to gel formation.
In EP 0 611 572, a preparation process for a lyophilizate of a peptide having 3-15 amino acids is described, according to which 100-10 000 parts by weight of the peptide are dissolved in acetic acid and treated with bulking substances such as mannitol, and subsequently lyophilized in order to obtain a sterile-
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filtered lyophilizate of the peptide and to avoid gel formation.
In DE A 195 42 873, pharmaceutical forms of administration of complicated composition in the form of microparticles are described, according to which an ABA triblock copolymer is used whose A block is a polymer of milk and glycolic acid and whose B ¦ polymer is a polyethylene glycol chain together with an additive from the group consisting of the serum proteins, polyamino acids, cyclodextrins, cyclodextrin derivatives, saccharides, amino sugars, amino acids, detergents or carboxylic acids, and mixtures of these substances. The microparticles described should also continuously release the polypeptide over a relatively long period after inclusion of small or aggregation-sensitive amounts of polypeptide.
In DD 141 996, the preparation of pharmaceutical forms of native LHRH is described, which are stable over a relatively long period and comply with the requirements for a parenterally administrable preparation. The key point here is the improvement of the storability of these preparations (page 2, lines 19-23). No statement is made about the filterability of the solutions. Moreover, for the improvement of the storability, buffer substances (also acetic acid) are also employed in order to establish a pH range of pH 3.5 - 6.5. The problem of preparing sterile lyophilizates from gel-forming peptide salts is not solved there. In EP 0 175 506, an aqueous solution of the peptide is treated with 1N acetic acid and then lyophilized in order to obtain the acetate salt of the peptide. The subject of this application is thus the synthesis of the peptide salts.
However, it has been shown that in the case of the known acetate salts of the peptides which are prone to aggregation, such as, for example, the LHRH antagonists, the preparation of sterile solutions for
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parenteral administration is indeed possible by means of filtration, especially at high concentrations, but aggregates can form shortly before injection after the dissolution of the lyophilizate. The aggregates then lead to a concentration-dependent lowering of the bioavailability from a peptide concentration of 0.5 mg/ml.
The problem mentioned occurs not only in the case of injection solutions which are administered for the purpose of rapid release of active compound, but is also observed in the case of injection preparations which exhibit delayed release. Thus peptides, incorporated in matrices which are intended to control the release of active compound, exhibit an undesirably low release on account of their proneness to aggregation. Thus the bioavailability is lowered here as well.
Starting from the fact that the preferred administration of pharmaceutically active peptides such as LHRH agonists and antagonists, for example antarelix and cetrorelix, is the parenteral pharmaceutical form, there was a need for the preparation of stable injection preparations having acceptable bioavailability, which can be conveniently prepared, sterile-filtered and formulated. This applies in particular to injection preparations in the form of reconstituted lyophilizates of soluble peptide salts and to microparticles, microcapsules or implants. This is all the more of importance in consideration of the versatile areas of application of LHRH antagonists, which are becoming more and more known.
A wider selection of parenterally injectable, in particular subcutaneously injectable, stable peptide solutions is desirable with respect to the rapidly growing indication areas of this class of substance.
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Pharmaceutical admininstration from suitable for parenteral administration, which contain peptides prone to aggregation in dissolved or dispersed form, have now been developed which are distinguished in that the peptides are present in the form of their acetate, gluconate, glucoronate, lactate, citrate, ascorbate, benzoate or phosphate salts and that these administration form can additionally contain one of the acids just mentioned as free acids, and, if appropriate, further additives and excipients from the class consisting of the acids, surface-active substances, polymers, lipids or sugars.
These pharmaceutical administration forms can be present in dissolved or dispersed form in water or in aqueous solvent mixtures.
According to a further embodiment of the invention, the pharmaceutical administration forms can also be present in dissolved or dispersed form in a physiologically tolerable oil, preferably medium-chain triglycerides (neutral oils, Miglyol®) or castor oil, sesame oil, cottonseed oil, corn oil, groundnut oil, olive oil, or in mixtures of such oils.
The peptides used are the LHRH antagonists antide, A-75998, ganirelix and Nal-Glu antagonist, but in particular cetrorelix and antarelix and the antagonists according to the patents US 5,942,493 and DE 19911771.3.
Acids employed in the excipient function are gluconic acid, glucuronic acid, galacturonic acid, glucaric acid, citric acid, ascorbic acid and amino acids.
It is thus possible to suppress the aggregation of the peptide and thus to fulfill the requirements of a preparation having good bioavailability, thus to enrich the pharmaceutical wealth and to do so with an efficient pharmaceutical technology.
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It has further surprisingly been found that by the addition of gluconic, glucuronic, citric, lactic or ascorbic acid the stability of various cetrorelix salts can moreover be considerably improved.
According to the invention, the preparation and formulation of sterile-filtered, stable preparations is thus possible without problems.
It is additionally advantageous to add suitable excipients. These excipients can be acids, surface-active substances, polymers, lipids or sugars. Examples of acids are gluconic acid, glucuronic acid, galacturonic acid, glucaric acid, lactic, citric acid, ascorbic acid and amino acids. Surface-active substances which can be employed are polyethylene glycol 12-(hydroxy)stearate (Solutol®) , polyoxyethylene ricinoleate (Cremophor®) , polysorbates, poloxamers, phospholipids, lecithins or benzalkonium chloride. Suitable polymers are albumins, polyethylene glycols, cellulose derivatives, starch derivatives or polyvinylpyrrolidone. Examples of sugars are cyclodextrins or cyclodextrin derivatives. "Chaotropic" substances such as urea can also be used as additives or excipients.
The area of use of the preparations according to the invention in "particular lies in the prevention and therapy of all sex hormone-dependent conditions and diseases which can be influenced by LHRH analogues, i.e. LHRH agonists and LHRH antagonists. The following are to be emphasized here:
Benign prostate hyperplasia, prostate carcinoma, precocious puberty, hirsutism, endometrial hyperplasia and its concomitant symptoms, endometrial carcinoma, in vitro fertilization (IVF/COS/ART), contraception, premenstrual syndrome (PMS), uterine myomatosis, breast cancer, tubal obstruction (PTO), ovarian cancer, uterine carcinoma. Particularly preferred LHRH
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antagonists for the composition according to the invention are the following substances:
cetrorelix, antarelix, antide, A-75998, ganirelix, the Nal-Glu antagonist and LHRH antagonists according to the patents US 5,942,493 and DE 19911771.3.
Example 1
By means of polarization microscopy, aggregation
investigations were carried out on solutions of various
cetrorelix salts without or with addition of
excipients.
In a polarization light microscope having crossed
polarizers, aggregated peptide solutions show images
which are very similar to those of liquid-crystalline
structures. In contrast to this, aggregate-free peptide
solutions produce no such effects.
Table 1: Influence of a gluconic acid addition on the aggregation behavior of cetrorelix acetate solutions..

Thus the addition of gluconic acid brings about an improvement in the stability of cetrorelix acetate solutions by delaying or preventing the aggregation.
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Further experiments concentrate on cetrorelix gluconate without or with addition of gluconic acid. The most important results are summarized in Table 2.
Table 2: Aggregation behavior of various solutions which were prepared from bulk cetrorelix gluconate product.

Cetrorelix gluconate thus offers advantages in comparison to the acetate salt. The addition of gluconic acid increases the storability of cetrorelix gluconate solutions.
Moreover, the stabilizing influence of glucuronic acid on cetrorelix acetate solutions and, as a further salt, also cetrorelix glucuronate was tested for its aggregation behavior. The results are summarized in Table 3.
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Table 3: Aggregation behavior of various concentrated solutions of cetrorelix acetate and cetrorelix glucuronate without or with addition of glucuronic acid.

Significant improvements with respect to the aggregation stability of cetrorelix solutions can also be obtained by the replacement of the acetate salt by a glucuronate salt, similarly to the gluconate salt. By the addition of glucuronic acid to cetrorelix glucuronate solutions, the aggregation stability of these solutions can be even further improved.

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Table 4: Aggregation-free period in days of cetrorelix acetate solutions after addition of 10% of a-cyclodextrin, 2 0% of hydroxypropyl-(3-cyclodextrin or 20% of y-cyclodextrin.

The aggregation stability of cetrorelix acetate solutions can be significantly improved by the addition of hydroxypropyl-ß-cyclodextrin and particularly of y-cyclodextrin.
Table 5: Aggregation-free period in days of 2.5 mg/ml cetrorelix gluconate solutions after addition of a-cyclodextrin, hydroxypropyl-ß-cyclodextrin or Y-cyclodextrin.

The aggregation stability of cetrorelix gluconate solutions can also be significantly improved by the
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addition of hydroxypropyl-ß-cyclodextrin or of y-cyclodextrin.
Table 6: Aggregation-free period in days of cetrorelix acetate solutions with addition of polyvinylpyrrolidone (Kollidon® 12 PF or 17 PF)

The aggregation stability of cetrorelix acetate solutions can also be significantly improved by the addition of various types of polyvinylpyrrolidone.
Table 7: Aggregation behavior of cetrorelix solutions with addition of various excipients assessed by means of polarization microscopy and according to the visual image (appearance).

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Example 2
Cetrorelix bulk product is dissolved in 30% strength acetic acid in a concentration of 10 mg/ml and diluted with an aqueous solution of the additives to a final concentration of 1 mg/ml of peptide in 3% acetic acid. This solution is then sterilized and lyophilized (5 mg per vial).
After reconstruction of these lyophilizates, the solutions (2.5 mg/ml of cetrorelix) are investigated for aggregate formation and release behavior in the following tests:
polarization microscopy (pol. mic): days without aggregation.
o filterability in %:
Cetrorelix solutions are prepared according to a
standardized process and filtered through 0.22 urn or 0.45 urn filters by means of centrifugation. The concentration of cetrorelix in the filtrate is determined by HPLC and indicated as a % value, based on the starting concentration before filtration (filterability in %).
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in-vitro release abbreviated form (RRS, release in Ringer's solution):
% released after 1 h and after 6 h.
The in-vitro release behavior is determined in a flow-through process using Ringer's solution as a medium at 37°C. The concentration measurement is carried out by HPLC. Cetrorelix samples, corresponding to 10 mg of cetrorelix base, are weighed into the flow-through cell, mixed with 4 ml of water and stirred for 10 min. After addition of 6 ml of Ringer's solution to the sample, Ringer's solution is pumped through the flow-through cell while stirring uniformly with a flow of 0.5 ml/min.
rat animal experiment: cetrorelix residual content in the muscle in % of the administered dose 168 h after injection.
Some cetrorelix acetate lyophilizate batches prepared and the corresponding test results of 2.5 mg/ml cetrorelix acetate solutions prepared therefrom are listed in Table 8a.

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From the examples mentioned, it is evident that with a large number of the excipients from various substance groups tested (surface-active substances, acids, amino acids, polymers, sugars, sugar alcohols, cyclodextrins, preservatives), individually or with mixtures of these excipients, stabilizing effects can be achieved in vitro (polarization microscopy, filterability, in vitro release) and in vivo. This lowered tendency to aggregation and thus improved in vitro active compound release also leads in the rat experiment to improved bioavailabilitv of the peptide compound and thus to reduced residual contents in the rat muscle.
Further in vitro and in vivo data of batches containing various cetrorelix salts without or with addition of stabilizing excipients are listed in Table 8b below.

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Example 3
Cetrorelix formulations which are less prone/slower to aggregate (better filterability/polarization microscopy) and exhibit more rapid in-vitro release in Ringer's solution stand out in the rat muscle experiment due to their lower cetrorelix residual content after 168 h. A higher bioavailability is expected of such formulations.
Some results of rat muscle experiments have already been listed in Tables 8a and 8b.
In the further rat muscle experiments shown in Table 9, in addition to the residual content in the muscle the cetrorelix content in the plasma was further determined. Also on the basis of these data, we the stabilizing influence of the excipients tested clear. Moreover, it was possible by the replacement of the acetate salt by other salt forms of cetrorelix to achieve an improved bioavailability and, accompanying this, a reduced residual amount in the rat muscle experiment.

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WE CLAIM

1. A pharmaceutical form of administration suitable
for parenteral administration, which contains
peptides prone to aggregation in dissolved or
dispersed form, characterized in that the peptides
are present in the form of their acetate,
gluconate, glucuronate, lactate, citrate,
ascorbate, benzoate or phosphate salts and in that
these forms of administration additionally contain
one of the acids just mentioned as free acids and,
if appropriate, further additives and excipients
from the class consisting of the acids, surface-
active substances, polymers, lipids or sugars.
2. The pharmaceutical form of administration for
parenteral administration as claimed in claim 1,
the form of administration being present in
dissolved or dispersed form in water or in aqueous
solvent mixtures.
3. The pharmaceutical form of administration for
parenteral administration as claimed in claim 1,
the form of administration being present in
dissolved or dispersed form in a physiologically
tolerable oil, preferably medium-chain
triglycerides {neutral oils, . Miglyol®) or castor
oil, sesame oil, cottonseed oil, corn oil,
groundnut oil, olive oil or in mixtures of such
oils.
4. The pharmaceutical form of administration for
parenteral administration comprising peptides
prone to aggregation as claimed in claim 1 to 3,
Wherethe peptides are the LHRH
antagonists antide, A-75998, ganirelix and Nal-Glu
antagonist, but in particular cetrorelix,
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5. The pharmaceutical form of , administration as
claimed in claim 1 to 4, Wherein the
acids employed in the excipient function are
gluconic acid, glucuronic acid, galacturonic acid,
glucaric acid, citric acid, ascorbic acid and
amino acids.
6. The pharmaceutical form of administration; as
claimed in claim 1 to 4, Where in the
surface-active substances employed are
polyethylene glycol 12- (hydroxy)stearate
(Solutol®) , polyoxyethylene ricinoleate
(Cremophor®) , polysorbates, poloxamers,
phospholipids, lecitins or in the form of
preservatives, such as, for example, benzalkonium
chloride or phenylmercuric acetate.
7. The pharmaceutical form of administration as
claimed in claim 1 to 4, Wherein the
polymers employed are albumins, polyethylene
glycols, cellulose derivatives, starch derivatives
or polyvinylpyrrolides.
8. The pharmaceutical form of, administration as claimed in claim 1 to 4, Wherein the sugars employed are cyclodextrins or its derivatives, and sugar alcohols.
9. The pharmaceutical form of administration as claimed in claim 1 to 4, Where in that urea or other chaotropic substances are employed as excipient.
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10. The pharmaceutical form of administration for
parenteral administration as claimed in claim 1, 2
and 4, characterized in that the peptide salts of
acetic acid, gluconic acid, glucuronic acid,
lactic acid, citric acid or of ascorbic acid are
present in solutions in a concentration of higher
than 0.5 mg/ml.
11. The pharmaceutical form of administration as
claimed in claim 1 to 9, Where in the
release of active compound is delayed by the use
of polymers, preferably of homo- or copolymers of
lactic and glycolic acid and in that the peptides-
are present as acetate, gluconate, glucuronate,
lactate, citrate, ascorbate, benzoate or phosphate
salts, and, if appropriate, further excipients
according to claim 5-9 are employed.
12. The pharmaceutical form of administration for
parenteral administration as claimed in claim 1,
2, 4 and 10, Wherin the peptides
employed are cetrorelix, antarelix and the
antagonists according to the patents US 5,942,493
and DE 19911771.3 in solutions in a concentration
of higher than 0.5 mg/ml.
13. The pharmaceutical form of administration as
claimed in claim 1 to 9 and 11, Whre in
the release of active compound is delayed by
the use of polymers, preferably of homo- or
copolymers of lactic and glycolic acid, where the
peptides antide, A-75998, ganirelix and Nal-Glu
antagonist, but in particular cetrorelix,
antarelix and the antagonists are
present in the form of their acetate, gluconate, glucuronate, lactate, citrate, ascorbate, benzoate
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or phosphate salts and, if appropriate, further excipients can be contained.
14 A process for the production of a pharmaceutical form of administration, as claimed in claim 1 and 4, Where in, by double decomposition of peptide salts with acetic acid, glucuronic acid, gluconic acid, lactic acid, citric acid or ascorbic acid, the corresponding salts are prepared in a stoichiometric ratio, dissolved in water for injection, mixed, if appropriate, with excipients as claimed in claims 5 - 9, then sterile-filtered, dispensed into injection vials and lyophi1ized.
15. A process for the production of a pharmaceutical form of administration as claimed in claim 1 and
4, Wherein, by double decomposition of peptide ""salts with acetic acid, glucuronic acid, gluconic acid, lactic acid, citric acid or ascorbic acid, the corresponding salts are prepared, in a stoichiometric ratio, these salts are incorporated in a manner known per se into delayed-release microparticles tides of homo- or copolymers of lactic and glycolic acid and these microparticles are suspended in a physiologically tolerable medium for injection purposes.

16A novel pharmaceutical form of administration as claimed in one or more of the preceding claims for parenteral administration in sex hormone-dependent, benign and malignant diseases.

17. A novel pharmaceutical compositions
as claimed in one or more of the preceding claims for parenteral administration in sex hormone-
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dependent, benign and malignant diseases, in particular in: benign prostate hyperplasia, prostate carcinoma, precocious puberty, hirsutism, endometrial hyperplasia and their concomitant symptoms, endometrial carcinoma, in vitro fertilization (IVF/COS/ART), contraception, premenstrual syndrome (PMS), uterine myomatosis, breast cancer, tubal obstruction (PTO), ovarian cancer and uterine carcinoma.
A pharmaceutical form of administration suitable for parenteral administration, which contains peptides prone to aggregation in dissolved or dispersed form, characterized in that the peptides are present in the form of their acetate, gluconate, glucuronate, tectate, citrate, aseorbate, benzoate or phosphate salts and in that these forms of administration addrtfortafty contain one of the acids just mentioned as free acids and, if appropriate, further additives and excipients from the class consisting of the acids, surface-active substances, polymers, lipids or sugars.

Documents:


Patent Number 203622
Indian Patent Application Number IN/PCT/2002/01328/KOL
PG Journal Number 11/2007
Publication Date 16-Mar-2007
Grant Date 16-Mar-2007
Date of Filing 24-Oct-2002
Name of Patentee ZENTARIS GMBH
Applicant Address Weismullerstrasse 45 , 60314 Frankfurt/Main,
Inventors:
# Inventor's Name Inventor's Address
1 BAUER HORST, Rohrenstrasse 12a, 91217 Herbruck,
2 DAMM MICHAEL Dleburger Strasse 106, 63322 Rodermark,
3 SARLIKIOTIS WERNER So. Dima 31, GR 19002 Peania,
PCT International Classification Number A6IK 9/08
PCT International Application Number PCT/EP01/05555
PCT International Filing date 2001-05-16
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 10024451.3 2000-05-18 Germany