SU1028662A1 - Polypeptide having the effect of lowering calcium content of serum - Google Patents

Abstract

The novel polypeptide of the formula <IMAGE> and the pharmaceutically acceptable acid-addition salts thereof reduce the levels of serum calcium. The compound is built up from the amino acids and/or peptides shown in the formula in the amino acid sequence shown in the formula, and the peptide unit, which contains the <IMAGE> group, in which R is a reactive ester, is subjected to a ring-closure reaction at any desired step of the reaction.

Description

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ND The invention relates to the syntez of a new compound, a polypeptide that has the ability to reduce serum calcium, which can be used in biology and medicine. Human calcitonin is known, which has the ability to decrease serum calcium in serum tl. Human calcitonin is characterized by the disulfide bond, which is extremely unstable and easily decomposed or polymerized, and its stability as a pharmaceutical compound is low. The purpose of the invention is to expand the senal of the means of influencing a living organism. The goal is achieved by the proposed polypeptide of the formula. (CHOs1-Co-6iy-A $ rv-Leu-Ser-Trp-KNCHCO-Wel-Leu-Giy-Trp-Tyr-Trp-em-Agp-Phe-AS t - L | j $ -ptTt-Ry $ - rrp-phe-pro-clu-trp-ai -3ie- &amp; iy-v-Qi-.-vtQ-Mj - acetate with the ability to reduce serum calcium. In the proposed L-Cys polypeptide in the 1st and 7th -m positions are replaced with (-based acid: .HOOC (CH2) 5-CH-IIH.2 I., COOH carboxyl group in the LU position is bound to glycine to form a cyclic structure. The proposed polypeptide has a high chemical stability and 10 times the biological activity of human calcitonin. Since the proposed calcitonin has the same amino acid sequence, which is human from the 11th to the 32nd fragment}, it is immunologically identical with natural human calcintonin and, therefore, is physiologically safe and has no cross-immunity with swine or salmon calcitonin 2 The synthesis of the proposed compound can be carried out using the conventional method of synthesis of peptides C3, i.e.; an amino acid and / or a peptide of 2-4 amino acids is introduced into the condensation reaction to obtain a given amino acid sequence and a structural unit,. comprising the following amino acid sequence of the formula; i (CHzljCOOI iH-per-ASn-Leu-Ser-Trp-l HCHCo, where R is the reactive ester residue, is introduced into the ring formation reaction at any stage of the peptide unit design; and the protective group for the reactive group is removed at any stage of the reaction. can be converted into its salt by the addition of an acid or a complex. Protective groups for the synthesis of starting materials or intermediates are the usual protective groups used in the synthesis of peptides, and can be easily removed by hydrolysis, acid decomposition, reduction nor aminolysis or hydrazinolysis. The proposed polypeptide can be obtained as a free base or as its salt. The free base can be converted into its salt in the usual way. The free base can be converted into its pharmaceutically acceptable salt by reacting with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, or phosphoric acid, or with an organic acid, such as formic acid, acetic acid, propionic acid, glycolic acid, milk acid, pyruvic acid, oxalic acid, succinic acid, malic acid, citric acid, tartaric acid, benzoic acid, salicylic acid, lower alkanesulfonic acid, benzenesulfonic acid or toluenesulfonic acid. The following abbreviations are used in the description: VOSgTRt-butoxycarbonyl CBZ: benzyloxycarbonyl Bzl: benzyl Biz (C22): dichlorobenzyl OEt: ethyl ester OBzI: benzyl ester SertL serine LeurL leucine Val: L valine ArgrL arginine Ala: L alanine alanine lean : L glutamic acid Tyr: L tyrosine he: L-phenyl Nine FC: trifluoroacetic acid GA: cyclohexylamine GF: tetrahydrofuran COEt: ethyl acetate U5C: H-ethyl -N-dimethylaminopropylrodiimide) TSI: S-oxysucimimide: OHI; acetic acid HMPA: hexamethylphosphorotriamide DOS: tert-amyloxycarbonyl ClCbz: O-chlorobenzyloxycarbonyl Tor: tosyl Ovi; tert-butyl ester ONP: n-nitrophenyl ester OSu; fJ-oxysuccinimyl ester Asn: L asparagine TrpsL threonine PrO: L proline AsptL a 5 paraginic acid Gly; glycine Gln: L glutamine Hys: L histid Met: L methionic Jlu: L isoleucine TozOH: p-toluenesulfonic acid DCA: dicyclohexylamine DMF; Dimethylformamide DCC: dicyclohexylcarbodiimide OBT: 1-hydroxybenzotriaeol EtOH: ethanol BuON: butanol Example. - (CIS) 5G -eo-6ir-Asn-le "- $" r-Trp - NHCHCO-Het-L - &amp; 1y-Thr-Thr-tg p - §10 -AS p, - Phe-Asn- L y $ -Hij5-Trp-h е-Pr O and 1 tv-tg Q-3ie-b1 y-Ta:. - | С11 -А1й-ТгО - ЛН2- 2.0 g (0.6 mol 8os-tgr (Bzg) B2 e (ce) (Bze) -G, -Lsr (OBGg) -Pbe-Asn-L-ys cec & zJ-Pbe-H-Trgr (Bze) -P1ie - Pro-GEn-Trp (828 / -ABo-aee-G, e: / - vae-eev-lec-Pro-wHg is dissolved in 10 ml of THF. the mixture is stirred for 30 m at room temperature, the solution is concentrated in vacuo and the product is precipitated with ethyl acetate, the precipitate is dried over sodium hydroxide, the dry residue is dissolved in 10 ml of dimethylformamide and adjusted to pH 9 by addition of triethylamine with cooling water is added to the solution to form a precipitate. The precipitate is dried on PrjOj to give the product as a free base, 0g of g (0.71 mol) - (CH7), -CO-Cla-ASIbLeiJ-Ser (B2l) -Trp (Bzi) -NHCHC -hei-Ley-Gi-OH is dissolved in 5 ml of a mixture of dimethylformamide - N-methylpyrrolidone (1: 122 mg of OSu was added to the solution, then 146 mg of dicycohexylcarbodiimide dissolved in 3 ml of dimethylformamide, cooled, stirred overnight. The precipitate was separated and added to the solution. 100 mg of 1-hydroxybenzotriazole is added to the bodine base obtained by vieye, the mixture is stirred for 5 days at 30 ° C. Upon completion of the reaction, water is added to the mixture. The precipitate formed in this way is filtered off, washed with water, dried, yielding 2.6 g. I; - (cis) 5-I To-C.1y-A5P-1e-5e (B2: 1Ttgr (Wg BJHACSOL-1e11-S1y-tgr (Vg.1) -ddg-VgiS1g)) SSh-A &amp; p (OBz.lbPbe-ASr-LyS (ClCBz) -PheHHij5 -Tgr (vg1) -ReBe-Pro-clIv-tgp vgl -Ala-ale-th y-va 1-b ly-Ai Q-pro-BN 1 1.0 g of the crude product is treated with 25 ml of hydrogen fluoride, 0.1 g of methionine and 2 ml of anisole at -5 ° C for 60 min while cooling with dry ice - methanol, under reduced pressure in the presence of dimethylthioether. After distilling off hydrogen fluoride, the residue is washed with diethyl ether and the residue is collected decane This is repeated by repeating it three times, and dissolved in a mixture: 30 ml of acetic acid and 10 ml of water. Then the solution is passed through a Dowex 1x2 column, the acetate form (2.5-8 cm), washed with 200 ml of water and again Pass the eluate through an HP-20 column (2.5 x 7 cm). The 80% ethanol solution is passed through the column and after distillation of the eluate to remove ethanol, the solution is lyophilized, yielding 440 mg of a slick. 440 mg of this powder, dissolved in 0.01 M; aqueous ammonium acetate, is poured onto the top of a column filled with CM-cellulose (2.2 x 25 cm) and eluted with a linear gradient of 0.01-0.2 M ammonium acetate (each 750 ml) (pH 4.5). Every 10 g of the fraction is collected and the active fractions (fractions No. 67-70) are combined and dried by freezing to obtain an active powder. The lyophilisate is dissolved in 1 M acetic acid and chromatographed on a Sephadex LH-20 column (2.2 x 137 cm), eluting with 1 M AcOH 6 g of the eluate, fractionated, then the active fractions are collected (No. 28-37) and lyophilized. The lyophilisate is dissolved in the upper layer of the butanol: acetic acid: water mixture (4: 1: 5), and the solution is loaded onto a Sephadex G-25 column (2.7x52sM), which is packaged as a suspension with the lower layer of the mixture

solvent and then placed in the upper layer of the specified mixture of solvents. The elution is carried out with the same upper layer, fractionated with every b g fraction, the active fractions (5-14 tubes are collected and lyophilized. The powder thus obtained is re-chromatographed under the same conditions on Sephadex-6-2 and lyophilized. The lyophilized active powder solution are placed in 1 M acetic acid, poured onto a Sephadex LH-20 column (2.2x137 cm), eluted with 1 M acetic acid in 5 g fractions. The active fractions are combined and lyophilized, 29.7 mg product (1000 MRCU / mg) are obtained

R 0.82 (carrier; Merck cellulose, producer: n-butanol: acetic acid: water: pyridine 15: 3: 12: 10).

P 0.43 (carrier: Merck cellulose, producer: n-bu.thanol: acetic acid: water (upper layer)).

toL 69.6 (С 0.72, 1 M acetic acid).

Amino acid analysis: Lys 1.15 / 1 /, His 1.01 / 1 /, Asp 2.94 / 3 /, Trp 4.80 / 5 /, Ser l, 0 (5 / l / r Glu 2.14 / 2 /, Pro 1.96 / 2 /, Gly 4.0 / 4 /, Ala 2.00 / 2 /, Val 0.95 / 1 /. Met 0.87 / 1 /, Jie 0.96 / 1 / , Leu 1.84 / 2 /, Tyr 0.96 / 1 /, Phe3.18 / 3 /, α-amino acid of 1.02 / 1 /.

A sample for amino acid analysis is prepared as follows.

The proposed polypeptide is hydrolyzed with 6N hydrochloric acid (with the addition of a few drops of anisole at 110 ° C for 45 hours and dried under reduced pressure. The radioactive immune anapandaroid calcitonin is administered using labeled (Asu) - human calcitonin analog,

Materials and methods. Antibody preparation. 180 µg of synthetic () -LCT (an analogue of human calcitonin), dissolved in 1.0 m of a salt solution, emulsified in full Freund's stimulator (Kalbnokem) and injected subcutaneously with three rabbit sashs (2.6-3.0 kg) . Immune: Trace through an auxiliary injection of 2.4 and 2.4 weeks. On the 8th week of immunization, a serum is obtained from each rabbit, the obtained samples are tested and their ability to bind the labeled analogue of human calcitonin. Natural synthetic LCT (240325 mg) was dissolved in 1 ml of salt solution, emulsified in a full stimulator and injected subcutaneously into five female rabbits. An auxiliary subcutaneous injection is performed at 8–12 months, and the antibodies are checked for CT at 10th and 16th weeks. The LGT antibody preparations were supplied by 4-calbiochem and were used for comparison. Aiken Chemical Co., Ltd., goat standard rabbit anti-y-globulin serum was used.

Preparation of labeled LCT and LCT analog. LCT and LCT analog (2.55, 0 µg) methyl (1.5-3.3 m Ci) by the method of glucose oxidase with lactoperoxidase. The selection of the labeled peptide from the saline solution was carried out using gel filtration on a column (0.8x25 cm) with Sephadex G-15, eluted with 1N acetic acid. The labeled peptides are checked for chemical purity by additionally gel filtration on Sephadex G-50- (1.1x57 cm). Cellulose acetate electrophoresis (2x10 cm is carried out at a constant voltage of 20 V / ohm (2 mA) for 510 minutes using 0.01 M phosphate buffer pH 7.4. Labeled peptides obtained after gel filtration are stored at 4 ° C in acetic acid (1N solution) containing 5% bovine serum albumin. The specific activity is calculated based on the radioactivity of the peptide peak to the salt peak radioactivity which is separated by electrophoresis. Sol Na 1 commercial (Association of Radioisotopes, Japan).

Cultivation sample. Typical cultivation is carried out in plastic tubes (1x7 cm) by the two-step method of the double antibody technique. For initial culture, 0, .1 ml of standard synthetic LCT for serum and 0.1 ml of anti-LCT antibodies with appropriate dilution (usually; 1: 5000 was added to O, 1 m of diluent containing 0.037% disodium salt (Ethylenediaminetetraacetic acid 0, 5% bovine serum albumin and 0.14 sodium azide in saline phosphate buffer solution (0.01 M; pH 7, 0.15 M NaCl); The mixtures are cultured at 4 ° C for 2 seconds and then 0.1 ml is added LCT analogue sufficient to provide 10,000 cpm After 4 days after the initial culture, 0.1 ml goat serum is added. rabbit anti-globulin (1:40) and 0.1 ml of 0.5% normal rabbit serum, and after 24 hours the reaction mixtures were centrifuged at 3000 rpm for 30 minutes. Dilution of all materials used for testing was carried out diluent

The proposed sample is tested for calcium lowering activity in

Serum. The tests were carried out according to the following method.

The sample was adequately diluted with 0.1% and sodium acetate solution -0.1% albumin. Male Rats were individually intravenously administered with 0.2 NUI of the appropriate diluted solution. After 1 hour, all rats were sacrificed, blood was collected and the calcium content of the protein was determined; in each blood sample with atomic absorption c1 ёK1Р1photometry. -. :::. :

By comparison with the appropriate standard, the activity of calcitonin in the sample is determined.

Based on the tests carried out, it was established that the proposed

Compound - (Asu -) LCT (analog of human capcitonin) - has an activity (potential) of 100 units. MR, C / mg, while the known compound, human calcitonIN (LCT), has an activity of only 82 units. MR, C / mg, i.e. The proposed compound is more than 10 times more active.

Claims (3)

POLYPEPTIDE FORMULAS 1 .----- - (SDg) ₅ ----------- 1 -co-Ciy-Asn-ieO — ger — Tgr — VISN.C0 — Mti-leu -Giy ί-TGr-Tug-Trp-Glfl-ASp-Phe-ASn-LyS-Pbe -.HyS-TrWbe-PrO- 0in-Trp-Aia-7ie-Gly-Vai- Giy-Alo-pr chnh _z -4 acetate with the ability to reduce serum calcium. (56) 1. US Patent 3891614, CL. C 07 C 103/52, published. 1975. 2. G. Neupep, R. Franch i mon t> -Human calcitonin Radioimmunoassay informal .and Pathological conditions. - Europ. J. Clin, investigation, 1974,4, 213. 3. Schroeder E,. Lyubke K. Peptides. h [Ϊ.Μ., Mir, 1967, p. 116. SU ..,. 1028662 1028662.