EA038010B1 - Stable succinic acid hexapeptide salts - Google Patents

Abstract

The invention relates to pharmacology and medicine, in particular to new stable succinic acid hexapeptide salts of general formula (I)where n=0.5 or 1, for preparation of pharmaceutical compositions suitable for the treatment of, in particular, dry eye syndrome.

Description

Scope of invention

The invention relates to the field of pharmacology and medicine, in particular to stable succinic acid salts of the hexapeptide H-Tyr-D-Ala-Gly-Phe-Leu-Arg-OH for use as a pharmaceutical substance in the composition of medicines.

Background Art

Known synthetic hexapeptide having the chemical formula

which corresponds to the amino acid sequence H-Tyr-D-Ala-Gly-Phe-Leu-Arg-OH, or in the one-letter designation YdAGFLR. The synthetic hexapeptide is a modification of the natural leu-enkephalin pentapeptide H-Tyr-Gly-Gly-Phe-Leu-OH and has deltaopioid receptor agonist activity. Due to the presence of an arginine residue in the molecule, the hexapeptide is a strong base (isoelectric point pH 9.6); therefore, its use as a pharmaceutical substance in the physiological pH range of 5.5-7.7 requires preliminary preparation of a salt with a suitable non-toxic acid.

The only known pharmaceutical substance based on this hexapeptide is a salt with two equivalents of acetic acid (diacetate) having the formula

and used for the production of drugs under the international non-proprietary name (INN) tyrosyl-D-alanyl-phenylalanyl-leucyl-arginine diacetate or the abbreviated trade name dalargin. The specified substance (hereinafter referred to as hexapeptide diacetate or dalargin) is included in commercially available drugs for the treatment of gastric ulcer and duodenal ulcer, pancreatitis and pancreatonecrosis. The use of hexapeptide diacetate (dalargin) in the composition of medicines is disclosed in the following patents of the Russian Federation (Nos.): 2032422, 2241488, 2198641, 2104717, 2306146, 2261721, 2432922, 2644285, 2405582, 2381034, 2323019, 2294746, 2254890,

2614105, 2488391, 2188675, 2594430, 2341282, 2396963, 2413528, 2270025, 2351334, 2343885, 2405534,

2363455, 2515550, 2180598, 2635083, 2436588, 2473325, 2144831, 2646569, 2200026, 2146530, 2155608,

2185176, 2139725, 2228762, 2185849, 2496493, 2366417, 2017488, 2167671, 2521199, 2045949, 2164153,

2181564, 2284192, 2290203, 2286793, 2366432, 2299065, 2429002, 2196603, 2142814, 2285522, 2203693,

2230549, 2180591, 2142736, 2113856, 2217139, 2266752, 2261722, 2362580, 2223741, 222814869, 2217186, 2266130, 2326661, 2299742, 2218896, 2416398, 2318503, 2430753, 2229699.22585

The main disadvantage of hexapeptide diacetate is its instability due to the presence of two acetic acid residues in its composition. Only one of the two equivalents of acetic acid participates in the formation of the hexapeptide salt and forms a strong ionic bond with the hexapeptide, while the second acetic acid residue forms an unstable solvate and is easily isolated with a slight increase in temperature and storage. Therefore, hexapeptide diacetate often smells of released acetic acid, which is indicated in the instructions for dalargin preparations. Hexapeptide diacetate is unstable in aqueous solution. The preparation of the solution for injection is accompanied by the hydrolysis of hexapeptide diacetate and the release of acetic acid, which causes irritation and soreness at the injection site, which is also indicated as a side effect by the instructions for dalargin preparations. The instability of hexapeptide diacetate makes it difficult to obtain and store a substance with the declared ratio of hexapeptide to acetic acid, therefore manufacturers indicate a wide range of permissible acetic acid content (from 7 to 14%) in the preparation, despite the declared exact molar ratio of 2: 1, which means that the content active substance

- 1 038010 (hexapeptide) in the composition of the substance varies widely from 86 to 93%. This is a significant drawback of the dalargin substance, since the inaccuracy of the composition directly affects the accuracy of the dosage of the drug. Thus, there is a need to create a stable pharmaceutical substance of the specified hexapeptide with a reproducible content of the hexapeptide in the substance.

The essence of the invention

The objective of the present invention is to provide a stable pharmaceutical substance of the hexapeptide H-Tyr-D-Ala-Gly-Phe-Leu-Arg-OH with a fixed reproducible content of the hexapeptide in the substance.

This problem is solved by using as a pharmaceutical substance succinic acid salts of the hexapeptide of the general formula (I)

where n = 0.5 or 1.

The compound of formula (I) can also be written using the three-letter amino acid designations as

H-Tyr-D-Ala-Gly-Phe-Leu-Arg-OH

О о Jn where η = 0.5 or 1.

The compound of formula (I) can also be written using single letter amino acid designations as

YdAGFLR

О ^NO Г ^ ОН о Jn where η = 0.5 or 1.

Succinic acid is a non-toxic pharmaceutically acceptable diacid, CAS No. 110-15-6, chemical formula HOOCCH2CH2COOH, capable of forming stable individual succinic acid salts of YdAGFLR hexapeptide at molar ratios of hexapeptide to succinic acid 1: 1 and 1: 0.5.

In one embodiment of the present invention, the compound of formula (I) is a salt formed with a hexapeptide base and succinic acid in a molar ratio of 1: 0.5 (n = 0.5), formula (II):

In another embodiment of the present invention, the compound of formula (I) is a salt formed with a hexapeptide base and succinic acid in a 1: 1 molar ratio (n = 1), formula (III):

In carrying out the present invention, the succinic acid salt of a peptide compound of the formula

- 2 038010 (II) can be obtained by mixing succinic acid with the base of the hexapeptide or its salts (trifluoroacetate or diacetate) in a molar ratio of 2: 1, followed by purification of the resulting compound.

In carrying out the present invention, the succinic acid salt of a peptide compound of formula (III) can be prepared by mixing a compound of formula (II) with succinic acid in a 1: 1 molar ratio, followed by purification of the resulting compound.

In carrying out the present invention, compound (II) can be used in the preparation of a pharmaceutical substance containing compound (II) and succinic acid in a molar ratio of 1: 0.01 to 1: 1. In the practice of the present invention, compounds (II) and (III) can be used as a mixture in a molar ratio of 0.05: 0.95 to 0.95: 0.05.

In the implementation of the present invention, the compound of general formula (I) is used in pharmaceutical compositions (drugs) as an active pharmaceutical ingredient. The pharmaceutical composition of the present invention comprises a compound of general formula (I) and a pharmaceutically acceptable excipient. In one embodiment of the present invention, the pharmaceutical composition comprises a compound of formula (II). In another embodiment, the pharmaceutical composition comprises a compound of formula (III).

The pharmaceutical compositions of the present invention are prepared by methods well known in the art and in accordance with recognized pharmaceutical procedures, for example, as described in the Remington Pharmaceutical Science Directory, 17th Edition, ed. Alfonso R. Gennaro, Mack Publishing Company, Easton, PA, 18th edition (Remington's Pharmaceutical Sciences, seventeenth edition, ed. Alfonso R. Gennaro, Mack Publishing Company, Easton, Pa., Eighteenth edition, 1990).

The pharmaceutical compositions of the present invention contain from 0.01 to 10 mg of a compound of general formula (I) in a unit dosage form. In one embodiment of the present invention, the unit dosage form is one ampoule of a lyophilisate of a compound of general formula (I) for preparing a solution. The specified solution can be used for intravenous, intramuscular, subcutaneous administration, as well as parenteral and external administration. In another embodiment, the unit dosage form is one ampoule of a solution of a compound of general formula (I) for intravenous, intramuscular and subcutaneous administration.

The term pharmaceutically acceptable excipient denotes substances of inorganic or organic origin used in the production process, manufacture of medicinal preparations to impart the necessary physicochemical properties to them. The list of excipients is indicated in the national pharmacopoeias (RF, Eur.Ph., Br.Ph., USP, JP) and reference books (Physician's Desk Reference, Vidal, Rote Liste, Fiedler Encyclopedia of Excipients, Japanese Pharmaceutical Excipients, Handbook of Pharmaceutical Excipients, Inactive Ingredients Guide's of the FDA). Non-exclusive examples of such adjuvants include gelatin, gum arabic, lactose, microcrystalline cellulose, starch, sodium starch glycolate, calcium hydrogen phosphate, magnesium stearate, talc, colloidal silicon dioxide and the like, stabilizers, wetting agents, emulsifiers, flavors, binders compounds, antiaggregants, lubricants, slip compounds, antiadhesive compounds, propellants, antimicrobial agents and antioxidants. Water for injection and purified water are the preferred pharmaceutically acceptable excipients of the present invention. In the range of therapeutically effective concentrations of the compounds of general formula (I), aqueous solutions are hypotonic, therefore, the auxiliary substance normalizing or increasing the tonicity of the solution is an important auxiliary component in the pharmaceutical compositions of the present invention. Non-exclusive examples of tonicity modifying agents are sodium chloride, magnesium chloride, mannitol, dextrose, and mixtures thereof. The content of the excipient in the pharmaceutical composition of the present invention can be from 0.100 to 99.999%.

In one embodiment of the present invention, the pharmaceutical composition is a stable aqueous solution for injection containing a compound of formula (II) and sodium chloride in the following ratios, wt%:

Compound of formula (II)

Sodium chloride Water for injection

0.01-1.00%

0.9% to 100%

In another embodiment of the present invention, the pharmaceutical composition is a stable aqueous solution for injection containing a compound of formula (III) and sodium chloride in the following ratios, wt%:

- 3 038010

Compound of formula (III) 0.01 - 1.00%

Sodium chloride 0.9%

Water for injection up to 100%

These compositions can be prepared industrially or just prior to administration to a patient in need thereof by dissolving the lyophilisate of compound (II) or (III) in saline. Additionally, the compositions may contain auxiliary substances that maintain the pH of the composition in the range from 5.0 to 8.0. Examples of such substances include, without limitation, succinic acid and other organic non-toxic acids, as well as organic and inorganic bases.

The technical result of the present invention is a high stability of the pharmaceutical substance in the form of succinic acid salt, in comparison with diacetate, which manifests itself as an increase in the shelf life of the pharmaceutical substance and the drug prepared on its basis when using the invention.

The following examples demonstrate the invention. The examples illustrate the invention and are not intended to limit the scope of the invention in any way.

Example 1.

The characteristics of compounds (II) and (III) in comparison with hexapeptide diacetate (dalargin) are presented in table. one.

Table 1

Characteristic Formula Share of hexapeptide yes,% Gross formula Like. weight, g / mol Dalargin CasHsiNgOe х 2НзССООН 85.8 C39H59N9O12 845.9 Compound (II) C35H51N9O8 x (HOOCC2H ₄ COOH) o, 5 92.5 Szunb ^ dOsh 784.9 Compound (III) C35H51N9O8 x HOOSS ₂ H ₄ COOH 86.0 C39H57N9O12 843.9

Tab. 1 shows that the compounds of the present invention differ in chemical structure, molecular weight and hexapeptide content of the compounds compared to the reference substance dalargin. Compound (II) contains 92.5% active hexapeptide, while hexapeptide diacetate only 85.8% (6.5% less).

FIG. 1 demonstrates clear differences in the characteristics of the ¹ H NMR spectra (A) of diacetate hexapeptide (dalargin) and (B) of compound (II). The ¹ H NMR spectrum of compound (II) in DMSO-d ₆ contains the signal of the CH ₂ CH ₂ group of succinic acid (singlet, chemical shift 2.30 ppm, -CH ₂ CH ₂ -), while the hexapeptide diacetate contains the signal of the CH ₃ group of acetic acid. acid (singlet, chemical shift 1.87 ppm, CH ₃ -).

Thus, the compounds of the present invention differ in chemical structure and properties from the reference substance dalargin.

Example 2.

Method for preparing compound (II).

To 0.730 kg of Boc2-Tyr-d-Ala-Gly-Phe-Leu-Arg-OH add 3.400 kg of cooled trifluoroacetic acid and stir until the precipitate is completely dissolved and a solution of trifluoroacetate H-Tyr-dAla-Gly-Phe-Leu-Arg- OH. The solvent is distilled off with a gradual increase in the bath temperature from 20 to 40 ° C and a residual pressure from 25 to 30 mbar. Diethyl ether is added to the oily precipitates obtained after evaporation and kept with stirring until a homogeneous precipitate forms, filtered, the precipitate is washed with diethyl ether and dried. The yield in terms of the original Boc2-TyrAla-Gly-Phe-Leu-Arg-OH is 98%. Next, 0.650 kg of H-Tyr-d-Ala-Gly-Phe-Leu-ArgOH trifluoroacetate is mixed with a 1% aqueous solution of acetonitrile until the precipitate is completely dissolved and applied to a chromatographic column (LPS-500, 70 μm) XK-23, washed with 20 l 3% ammonium succinate solution and collect the eluent. The eluent fraction containing the succinic acid salt of H-Tyr-d-Ala-Gly-Phe-Leu-ArgOH, compound (II) is evaporated at a bath temperature of 40-45 ° C and a pressure of 20 to 30 mbar. 2.50 L of purified water is added to the oily residue and stirred until complete dissolution, filtered, the filtrate is collected, frozen and dried in an LS-33 freeze dryer at a condenser temperature of -82 ° C and a residual pressure of no higher than 215 mTorr. The yield of compound (II) 78%.

NMR ¹ H (DMSO-d ₆ , δ-scale ppm): 0.85 (dd, 6H), 1.11 (d, 3H), 1.35-1.70 (br.s, 7H), 2.30 (s, 2H), 2.55 (m , 1H), 2.82 (m, 2H), 2.97 (m, 2H), 3.07 (m, 2H), 3.5 (m, 1H), 3.65 (m, 1H), 3.88 (m, 1H), 4.20 (m, 2H), 4.54 (m, 1H), 6.65 (d, 2H), 6.97 (d, 2H), 7.24 (m, 5H), 7.37 (m, 5H), 8.0-8.5 (br.s, 4H), 8.92 (br.s, 1H). Elemental analysis for C ₃₇ H ₅₄ N ₉ O ₁₀ (784.9): calculated% C 56.62; H 6.93; N 16.06; found% C 56.71; H 7.01; N 16.01.

- 4 038010

Example 3.

Method for preparing compound (III).

10.0 g of compound (II) is dissolved in purified water, 1.50 g of succinic acid is added and stirred until complete dissolution. The solution is frozen and dried in an LS33 freeze dryer at a condenser temperature of -82 ° C and a residual pressure not exceeding 215 mTorr. The yield of compound (III) is 98%. Elemental analysis for C39H ₅₇ N ₉ O ₁₂ (843.9): calculated% C 55.50; H 6.81; N 14.94; found% C 55.42; H 6.78; N 14.99.

Example 4.

Comparative studies of the stability of compounds.

Samples of compounds (II) and (III), as well as the reference substance dalargin (hexapeptide diacetate), in an amount of 50 mg each, were heated in vacuum at 100 ° C for 24 h. samples after heating were evaluated by ¹ H NMR (solvent D2O). FIG. 2 shows the change in the ratio of the signal of the residue of acetic (CH ₃ -group, 3H, singlet, chemical shift δ-scale 1.90 ppm) or succinic acid (-CH2CH2-, 4H, singlet, chemical shift δ-scale 2.50 ppm) to the reference signal of the amino acid residue leucine (2CH ₃ -, 6H, doublet, chemical shift 0.9 ppm) after heating for 24 hours. The results are presented in table. 2 as the loss of residual acid (acetic or succinic, respectively) in the samples after heating as a percentage of the initial content of the corresponding residues before heating.

table 2

Substance Losses of residual acid after heating,% Dalargin -67 Compound (II) -3 Compound (III) -4

Tab. 2 shows that heating dalargin for 24 hours led to the loss of 67% of the residual acetic acid, which corresponds to the conversion of hexapeptide diacetate with the declared ratio of hexapeptide to acetic acid 1: 2 to a compound with a new ratio of 1: 1.34, which indicates a loss of authenticity pharmaceutical substance. Heating compounds (II) and (III) under the same conditions led only to an insignificant loss of residues of succinic acid, respectively 3 and 4%, which did not affect the authenticity of pharmaceutical substances. Thus, the succinic acid salts of the hexapeptide of general formula (I) provide high stability of the pharmaceutical substances of the hesapeptide, which is much higher than the stability of the reference substance dalargin.

Example 5.

Tab. 3 shows a pharmaceutical composition of the present invention.

Table 3

Composition component Content, wt% Compound (II) 0.01-1.00 Sodium chloride 0.9 Water for injections up to 100

When preparing the composition, the lyophilisate of compound (II) is dissolved in saline in the proportions indicated in table. 3, and the resulting solution is dispensed into vials or ampoules under a nitrogen atmosphere.

Example 6.

Tab. 4 shows a pharmaceutical composition of the present invention.

Table 4

Composition component Content, wt% Compound (III) 0.01-1.00 Sodium chloride 0.9 Water for injections up to 100

When preparing the composition, the lyophilisate of compound (III) is dissolved in saline and the resulting solution is poured into ampoules.

Example 7.

Stability studies of pharmaceutical compositions.

Samples of pharmaceutical compositions (Table 5) were subjected to stability tests at 40 ° C and 75% relative humidity for 6 months. Stability was evaluated as the constancy of the composition of the composition in the range of 95-105% of the original.

- 5 038010

Table 5

Composition No. Ingredient content, wt% Compound (II) Compound (III) NaCl Water one 0.01 - 0.9 up to 100 2 0.10 - 0.9 up to 100 3 1.00 - 0.9 up to 100 4 - 0.01 0.9 up to 100 five - 0.10 0.9 up to 100 6 - 1.00 0.9 up to 100

Tests have shown that compositions Nos. 1-6 were stable under test conditions, and the content of hexapeptide and succinic acid residue in these compositions did not change according to the analysis.

Example 8.

Study of the effectiveness of the compounds of the present invention.

The efficacy of compounds (II) and (III) was studied in a dry eye model as described in Xiong C et al. A rabbit dry eye model induced by topical medication of a preservative benzalkonium chloride. Invest Ophthalmol Vis Sci. 2008, 49 (5): 1850-6. In this model, instilling a 0.1% solution of benzalkonium chloride (BC) into one rabbit eye twice a day induces dry eye syndrome, accompanied by a significant decrease in tear production in the Schrimmer test. Evaluation of the effectiveness of the compounds of the present invention was carried out in the Schrimmer test on the wetting of Whatman 41 filter paper, placed under the lower eyelid for 5 minutes. White rabbits were divided into groups (n = 8) and received twice a day (8:00 and 14:00 h) for 7 days in the right eye of nat. solution; 0.1% solution BH (control); pp 0.1% BC + 0.1% compound (II); solution 0.1% BC + 0.1% compound (III); solution 0.1% BC + pharmaceutical composition containing 0.1% of compound (II); or solution 0.1% BC + pharmaceutical composition containing 0.1% of compound (III). The Schrimmer test was performed on the 8th day. The test results are presented in table. 6 as mean ± mean error of wet track length in mm. Statistical processing of the results was carried out using the Student's t-test.

Table 6

Group Schrimmer test, mm Phys. rr 9.1 ± 0.7 0.1% solution BH (control) 4.8 ± 0.5 * 0.1% BH + 0.1% compound (II) 8.5 ± 0.5 * 0.1% BH + 0.1% compound (III) 7.5 ± 0.6 * 0.1% BC + 0.1% compound (II) + 0.9% NaCl 8.1 ± 0.7 * 0.1% BC + 0.1% compound (III) + 0.9% NaCl 7.8 ± 0.8 *

^ significant difference from control (p <0.05); ^ reliable difference from nat. solution (p <0.05)

The results of the Schrimmer test show that the administration of 0.1% HD causes reduced production of tear fluid (dry eye syndrome). Drip administration of solutions of compounds (II) and (III), as well as pharmaceutical compositions based on them, significantly improved the secretion of lacrimal fluid, impaired by the introduction of HD. Thus, compounds (II) and (III), individually and in pharmaceutical compositions, were effective in the treatment of dry eye syndrome when administered in the form of a solution.

Claims (3)

CLAIM 1. Compound of general formula (I) where n = 0.5 or 1. 2. The compound according to claim 1, characterized in that at n = 0.5 it has the formula (II) - 6 038010 SNHOO FIG. one 3. A compound according to claim 1, characterized in that for n = 1 it has the formula (III)