Method for preparing pharmaceutical composition comprising peptide of lhrh antagonist

FIELD: peptides, pharmacy.

SUBSTANCE: invention relates to a new method for preparing a pharmaceutical composition for the parenteral administration in mammals that comprises salt of difficulty soluble peptides. Method involves treatment of the parent readily soluble acid-additive salt of the basic peptide of LHRH antagonist in the presence of suitable diluting agent with the mixed ion-exchange resin or mixture of acid and basic ion-exchange resins to form free basic peptide. Then ion-exchange resin is removed and free basic peptide is treated with inorganic or organic acid to form the final product, required acid-additive peptide salt followed by addition of suitable pharmaceutical vehicles and/or filling agents and removal of a diluting agent.

EFFECT: improved preparing method.

5 cl, 1 dwg, 1 ex

 

The technical field to which the invention relates.

The present invention relates to a new method of obtaining salts of the peptides, primarily insoluble salts of the peptides, and their use to obtain pharmaceutical compositions.

The level of technology

In the international application PCT/ER/03904 described obtaining insoluble peptide through the interaction of the basic peptide, dissolved in acetic acid, with an aqueous solution of salt of the acid with the subsequent precipitation of sparingly soluble acid additive salt of the peptide. For example, described receiving embonate of cetrorelix-LHRH antagonist.

In the published German application DE 4342092 A1 describes the obtaining of sparingly soluble salts peptide antagonists of LHRH containing amino acids, in particular, embonate of cetrorelix. The invention

The present invention is to develop a new method of obtaining salts of the peptides, in particular insoluble salts of peptides.

The object of the present invention is a method of obtaining salts of the peptides according to which the source of acid additive salt of the basic peptide is treated in the presence of a suitable diluent mixed ion exchanger or a mixture of acidic and basic ion exchangers with the formation of free basic peptide, and then an ion exchanger to remove the free basic peptide is treated with an inorganic or organic acid with the formation of the final product, the desired acid additive salt of the peptide, then the diluent is removed.

The object of the invention is a pharmaceutical composition containing at least one salt of a peptide obtained using the method according to the present invention, pharmaceutical excipients, carriers and/or excipients.

We propose a method to obtain pharmaceutical compositions corresponding to the invention, according to which pharmaceutical excipients, carriers and/or excipients add fully or partially before removing the diluent.

List of drawings and other materials

The figure presents the results of biological experiments 3107, illustrating the dependence of the average concentration of cetrorelix in the plasma of time after intramuscular (I/m) injection 60 mg of pamoate (embonate) cetrorelix (CET) male volunteers.

Information confirming the possibility of carrying out the invention

The term "basic peptide" in this context refers to poly(amino acids), as well as part of a structure within a large General structure, which contains essential amino acids such as arginine, pyridinoline or lysine, or l-terminal fragment of the peptide, or at least one main group.

Preferred peptides are antagonists of LHRH, so is e as anted, A-75998, ganirelix, Nal-Glu antagonist, cetrorelix, teverelix (entrelacs®), and antagonists according to the US patents 5942493 and DE 19911771.3, the contents of which are included in this description as a reference. Other peptides are abarelix, azalin, Detralex, remorseless (see Stoeckemann and Sandow, J. Cancer Res. Clin. Oncol., 1993, t, str) and RS-68439. The structure of the above-mentioned peptides presented in the following documents: Behre et al., browse GnRH Antagonists", proceedings of the 2nd world conference on ovulation induction, (GnRH antagonists: an overview, Proceedings of the 2-nd World Conference on Ovulation Induction), The Parthenon Publishing Group Ltd.; Kutscher et al., Angew. Chem., 1997, t, str.

As highlighted products used acid additive salts of the peptides preferably use salt in the form of soluble salts such as the acetate, hydrochloride, sulfate.

According to the method according to the present invention, the original salt of the peptide is completely or partially dissolved or suspended in the solvent. Then add the diluent. The solvent and the diluent may be the same or different. As solvent or diluent is used, for example, water, ethanol, methanol, propanol, isopropanol, butanol, acetone, diethylketone, methyl ethyl ketone, dimethylacetamide, dimethylformamide, N-organic, acetonitrile, pentane, hexane, heptane and mixtures thereof.

Preferred and are ethanol, isopropanol or acetone. Preferred also is the water content, which ranges from 1 to 60%, more preferably from 5 to 50%.

To the solution or suspension of the original peptide salt added mixed ion exchanger, and a mixture of acidic and basic ion exchangers. As an ion exchanger is used, for example, Amberlite®.

The quantity of ion exchanger are calculated according to the number of basic groups in the peptide. The amount determined by adding ion exchanger to a constant pH. For example, 1 g of cetrorelix required 10 g Amberlite MV-3.

The pH value of the solution of the grounds upon receipt of the grounds set in the range from 7.5 to 13 or depending on the salt of the active agent, primarily from the salt of a peptide containing the basic reactive amino acids, especially salts of LHRH antagonists (for example, cetrorelix, D-63153, abarelix, ganirelix, remoralize that may exist, for example, in the form of acetate), or depending on the active agent.

The temperature should not exceed 25-30°to avoid decomposition of the peptide. Duration of response when you obtain the free base is usually several minutes, e.g. 20 min in the case of acetate cetrorelix. The duration of reaction can be increased, for example, when listello up to 1 h in the case of embonate of cetrorelix. After reaching a constant value of pH of the reaction should be stopped, because it can be formed, for example, decomposition products due to the basicity of the solution.

At the end of the reaction medium to remove the ion exchanger. Removal can be accomplished by screening, filtration, centrifugation or filtration through a column.

Transparent or turbid solution of the free base of the peptide, which is unstable, it should be possible to quickly process the acid with the formation of the desired acid salt additive. Adding acid can be made in the form of a solid substance or in the form of a solution or suspension. Similarly, the solution of the free peptide can be added to the acid.

The duration of reaction is in the range from several minutes to several hours. For example, the duration of reaction in the formation of embonate of cetrorelix 1.5 hours Then the reaction mixture, which is usually transparent, sterilized by filtration. After that, the solvent can be removed, you get a pure salt of the peptide. In another case, before removing the solvent in the solution add excipient, additive or carrier. Excipients can be added as solids before sterilization by filtration or after sterilization filtration is by Finance in the form of sterilized by filtration of the solution.

As auxiliary substances are used, for example, mannitol, sorbitol, xylitol, soluble starches.

According to the present invention, the following salts can be obtained by adding the corresponding acids: acetate, adipate, ascorbate, alginate, benzoate, bansilalpet, bromide, carbonate, citrate, chloride, dibutyltin, digitaltruth, dickerhoff, dihexadecyl, fumarate, gluconate, glucuronate, glutamate, bicarbonate, hydrotartrate, hydrochloride, hydronitrate, iodide, lactate, alpha-lipoic acid, malate, maleate, malonate, pamoate (embonate), palmitate, phosphate, salicylate, stearate, succinate, sulfate, tartrate, tannat, oleate, ActiveState.

The present invention is illustrated in the following example, do not limit the claims of the invention.

Example 1

46,47 g D-20761 portions add to 1193 g of water and dissolved with stirring (solution 1). Then the solution 1 with stirring, diluted 3261 g 96%ethanol (solution 2). Solution 2 after dilution was filtered through a glass fiber filter for pre-treatment, and to the filtrate was added with stirring 390 g Amberlite MV-3 (mixed ion exchanger containing a strong acid Catino and aminoalkenes) (mixture 1). 316,8 g of mannitol dissolved under stirring in 1267 g of water (solution 3). After 15 min stirring measure the pH value poluchennogo the solution mixture of 1, and then pH was measured again after 5 min of additional stirring. Then the solution after reaching a pH of 12.5 is separated from Amberlite MV-3 using sieves with small holes (solution 4).

4162 g of the solution 4 is treated with stirring 5.34 g monowai acid. The resulting mixture was intensively stirred for another 1.5 h and then slightly turbid solution is filtered through a glass fiber filter.

The pH value of this solution is 8.4 (solution 5). The pH value was measured using a ground electrode, filled with a viscous electrolyte fluid. The pH should be considered as relative, as measured in a solution or suspension containing ethanol and therefore are characterized by a seemingly higher pH values.

3333 g of the solution 5 is sterilized by filtration in the reaction vessel, which set the room temperature, and 528 g of the solution 3 is sterilized by filtration at room temperature in a solution of 5 (solution 6).

A solution of 6 heated to 40°C, then a mixture of ethanol/water is evaporated in a vacuum to ≤1931 (suspension 1). A suspension of 1 embonate of cetrorelix cooled to room temperature and diluted by adding under stirring 3000 g of sterilized by filtration of water for injection (suspension 2). Then the prepared suspension is cooled to room temperature, spill the t in vials for injection volume of 10 ml (3.0 g in each bottle), equipped with nozzles for lyophilization, and transferred to the installation for lyophilization.

Vials for injection freeze when the temperature of the plate, installed approximately -40°C. Drying is performed with the use of the program is drying, at which the temperature of the plate increases from approximately -40°to 20°C. Installation for lyophilization filled with sterile nitrogen, the vials for injection in the installation, closed with rubber gaskets and afterwards.

After drying the closed vials for injection, sterilized by gamma radiation at 12 kGy (minimum value) to 15 kGy. The last operation is optional.

One vial for injection contains 140,07 mg of material, including 34,07 mg embonate of cetrorelix, which corresponds to 30 mg cetrorelix, and 106 mg of mannitol. The contents dissolved in 2 ml of water for injection. The resulting suspension can be entered intramuscular or subcutaneous means.

Biological tests

Obtained according to Example 1 lyophilisate (30 mg) embonate of cetrorelix (2:1), resuspending in 2 ml of water for injection, can then be introduced parenterally, preferably by subcutaneous (s/C) or intramuscular (I/m) methods.

It is shown that if p/introduction bioavailability embonate of cetrorelix (2:1) approximately 30-50% (100% to take the try bioavailability when administered intravenously acetate cetrorelix). The main part of the freeze-dried embonate of cetrorelix (2:1) causes minor so-called effect of irritability or lack of patients. The duration of action depends on the dose and the dose of 30-150 mg is 2-8 weeks or more. The freeze-dried embonate of cetrorelix (2:1) of the present invention is currently in phase I clinical trials in humans.

The drawing shows the concentration dependence of cetrorelix in the plasma depending on time (h) after the/m introduction volunteers 60 mg of freeze-dried embonate of cetrorelix (2:1), obtained according to Example 1. The effect of increased irritability (approximately 100 ng/ml) is not observed. The duration of action is more than 700 hours in 150 h after the introduction of a constant level in plasma of approximately 2 ng/ml Bioavailability of approximately 40%.

The use of salts of the peptides of the present invention are, for example, benign hypertrophy of the prostate (national Department of standardization), fibroids and endometriosis.

1. A method of obtaining a pharmaceutical composition for parenteral administration to mammals, characterized in that the source of easily soluble acid additive salt of a basic peptide-LHRH antagonist in the presence of a suitable solvent is treated with a mixed ion the exchanger or a mixture of acidic and basic ion exchangers with the formation of free basic peptide, then the ion exchanger is removed and the free basic peptide is treated with an inorganic or organic acid with the formation of the final product required sparingly soluble acid additive salt of the peptide, then add a suitable pharmaceutical carriers and/or excipients, and then remove the diluent.

2. The method according to claim 1, characterized in that as the source of salt peptide use salt cetrorelix, teverelix, abarelix, ganirelix, azalina, antide, A-75998, Detralex, remoralize, RS-68439.

3. The method according to claim 1 or 2, characterized in that the acid is chosen amborovy acid, stearic acid or salicylic acid.

4. The method according to one of claims 1 to 3, characterized in that the peptide used cetrorelix, and the acid - amborovy acid at a molar ratio of cetrorelix and monowai acid 2:1.

5. The method according to one of claims 1 to 4, characterized in that the diluent is removed by lyophilization.



 

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