Semisynthetic sulfamethoxazole with high antimetastatic activity and reduced hemorrhagic risk
(57) Abstract:The invention can be used in medicine for the treatment of cancer patients. Declared sulfamethoxazole as antimetastatic agents and pharmaceutical composition containing them. New connections are saying.m. 3200-40000 and the ratio of sulfate groups to the carboxyl of about 2.2-2.3. They are derived from K5 polysaccharide of Escherichia coli N-deacetylation of hydrazine and subsequent N-sulfation of the trimethylamine adduct/sulfuric anhydride and then O-sulfation adduct of pyridine/sulphur trioxide at 0oC for 0.25 to 2 h At O-sulfation ratio adduct/polysaccharide is equal to 5 (in equivalent proportions SO3/OH). Then the product N-sulfation. The new compounds possess anti-metastatic activity comparable to the activity of heparin, but have lower compared with him anticoagulation activity. In this regard, their use for the treatment of metastases will reduce hemorrhagic risk. 2 S. and 1 C.p. f-crystals, 1 table. The invention relates to medicine.The aim of the invention is the use of sulfamethoxazole as antimetastatic funds.Metastasis is the process involved is of Asadov, as well as migration and growth in the extravascular space. The mentioned phenomenon, in particular adhesion to vessel walls, appears to be regulated by endogenous polysaccharide heparan sulfate (HS). Some anticoagulants, among whom was heparin (NER), structurally similar to heparansulfate, were investigated as potential antimetastatic means (I. Viodavsky et. al. "Modulation of neovascularization and metastasis by species in heparin", in "Heparin and Related Polysaccharides" (D. A. Lane et al., Eds. ), Plenum Press, New York 1992, 317-327). Among these drugs heparin is particularly active antimetastatic, but its high anticoagulant activity implies hemorrhagic risk, so the search heparinoids substances having reduced anticoagulant activity, is of particular interest (D. J. Tyrrell et al.: "Therapeutic uses of heparin beyond its traditional role as an anticoagulant", TIPS 16, 198- 204, 1995).Engaging in this invention, we have found that some of the semi-synthetic heparansulfate belonging to the class of sulfamethoxazole (SAHS) (B. Casu et al.,: "Heparin-like compounds prepared by chemical modification of capsular polysaccharide from E. coli K5", Carbohydr. Res. 263, 271-284 (1994), unexpectedly have antimetastatic activity in vivo, comparable to the activity of heparin, even if the s found that only sulfamethoxazole derived from K5 polysaccharide of Escherichia coli by removal of acetyl residue and subsequent sulfation adduct sulfur trioxide/trimethylamine held at 0oC for 0.25 to 2 hours, and the ratio of the reagent and the polysaccharide (equivalents of SO3/equivalent-groups) equal to 5 (denoted as SAHS-B) and having a molecular mass component 5000-40000 have antimetastatic activity, comparable with the normal activity of heparin, while sulfamethoxazole obtained in other experimental conditions, have antimetastatic activity, significantly lower than the activity of heparin and SAHS-B.Moreover, we also found that the fraction SAHS-B having a molecular weight below 5000, retain significant antimetastatic activity (exceeding the relevant activity of heparins, low molecular weight).Therefore, a semisynthetic heparansulfate SAHS-B are represented as antimetastatic drugs low hemorrhagic risk.To this end SAHS-B should be made in the form of a pharmaceutical acceptable compositions, ispv in doses depending obviously on several factors, but usually ranging from 1 to 1000 mg SAHS-B from one to several times per day.As described B. Casu et al., 1994, loc. cit.; PCT/EP94/01660, sulfamethoxazole obtained from K5 polysaccharides, which is a component of the cell membrane strain Escherichia coli K5. In particular, the polysaccharide K5 selectively N-deacetylase (otscheplaut acetyl residue) and N - sulfation, and then On-sulfation, as shown in the following diagram, receiving sulfamethoxazole of various kinds, such as SAHS-B, SAHS-C, SAHS.< / BR>The first stage includes N-deacetylation by hydrazinolysis polysaccharide K5 (K5-PS). The resulting product (heparin, EN) N-sulfation adduct sulfur trioxide/trimethylamine (TMA/SO3), receiving sulfamonomethoxine (SAH). The numbers above the arrows indicate the order of the reaction temperature (oC), the ratio of reagent to the polysaccharide (equivalents of SO3/equivalents available hydroxyl groups) and duration of response (hours).Antimetastatic activity of sulfamethoxazole, heparin and other comparative polysaccharides determined using the method of colonization in light of melanoma cells B16B16 (N. Casella et al., Thromb. Haern. 73, 964 (1995)).
2(5%) in wet conditions and at a temperature of 37oC. the Cells divide twice a week, treating them with 0.25% trypsin/0.05% of ethylenediaminetetraacetic acid. Studied polysaccharides dissolved in physiological saline or phosphate (PBS) buffer properly and apply on the spot. Melanoma cells B16B16 diluted in PBS (105cells/0.1 ml/mouse) injected into the lateral tail vein of C57B16 mice with an average weight of 20 grams, with a final volume of 0.2 ml/mouse. Mice killed after 12-16 days after injection of cancer cells; the lungs were removed and fixed in a solution Buoin to calculate surface metastatic nodules that look like black education in the yellow box. Then determine the ratio of pulmonary nodules in treated and control mice. Each experiment is carried out at least five mice, often 8-10 mice. Percentage inhibition of metastases obtained in several experiments shown in C is aroundwhat, types a, b and C
Below is a description of the standard procedures for obtaining some sulfamethoxasole with different antimetastatic activity. Define the following product specifications: average molecular weight (gel filtration), the degree of sulfation (expressed as molar ratio of sulfate groups and carboxyl groups and defined conductometry) and the distribution of sulfate groups (defined 1H and13With NMR spectrometry), as described by Casu et al., 1994 (loc. cit.).
The K5 polysaccharide (100 mg) and hydrazine sulfate (138 mg) was dissolved in anhydrous hydrazine (1,38 mg) and incubated in a sealed tube under nitrogen atmosphere for 5 hours at 96oC. the Solution is dried in a rotary evaporator, the reaction product is dissolved in distilled water and pH adjusted to 4 with 37% HCL. the pH of the effluent is between distilled water and the solution deleteroute against distilled water for 3 days (3 2 l each laziness; the threshold bandwidth 14 000 D) and, finally, lyophilizer.Ib N-sulfation
The polysaccharide obtained as in 1a) (100 mg), dissolved in distilled water, the pH of the solution was adjusted to 9 by addition of solid sodium bicarbonate and the temperature was raised to 55oC. At this temperature without stirring the mixture, add 100 mg of adduct comprising trimethylamine/sulfuric anhydride (TMA/SO3). After 4 hours, add an equal quantity of the adduct and continue the reaction is generally within 24 hours. N - sulfated polysaccharide is allocated, as described above.1c) O-sulfation
The polysaccharide obtained as in 1b) (100 mg), dissolved in distilled water (20 ml) and the solution passed through a column of Amberlite IR-120 (H+at room temperature. The column is washed with another 20-ml portion of distilled water, collected eluate and their pH was adjusted to 5.5 with 10% tributylamine in ethanol (weight/about.) (3 ml). Excess tributylamine remove diethyl ether (40 ml) and lyophilizers.Thus obtained product (188,2 mg) was dissolved in anhydrous DMF (33 ml), add adduct of pyridine and sulfur trioxide (Py/SO3that number is listed below), dissolved in 15 ml of anhydrous dimethylformamide, and videris the Idov of sulfamethoxazole use different reaction temperature, the amount of sulfur adduct and the reaction periods. In particular, sulfamethoxasole type And receive at 0oC, using 460 mg of pyridine/SO3within 1 hour. Product (G1524-3; average molecular weight 11700; the molecular ratio of sulfate carboxyl groups is 1.8) shows antimetastatic activity, corresponding to 17.5% inhibition of metastasis for doses of 0.5 mg/mouse; in the same test comparative heparin shows a 97.5% inhibition, and heparan sulfate from porcine pancreas - 54.8 per cent.Sulfamethoxazole type To receive at 0oC using 765 mg sulfur adduct corresponding to the ratio of the adduct/polysaccharide (equivalent ratio SO3/OH), equal to 5, for 0.25 to 2 hours (preferably 1 hour), again subjecting the product of repeated N-sulfation, as described in 1b). Typical end product (G1669; the average molecular mass of 25 700; molar ratio of sulfate carboxyl groups is 2.2) shows antimetastatic activity (dose of 0.5 mg/mouse) corresponding to 92.7% of inhibition of metastasis.Sulfamethoxazole type To get at the 25oC for 1 hour, ispolzuya groups 2,8) shows antimetastatic activity (dose of 0.5 mg/mouse), corresponding to 8.8% of metastatic inhibition.Example 2
Antimetastatic activity of sulfamethoxazole type IN
Tests for anti-metastatic activity can be repeated for the SAHS-B, obtained as described in example 1 (product G1669), on the three doses (0.5, 0.2 and 0.1 mg/mouse). The corresponding inhibition of metastasis is 78.5 percent, 62.5 per cent and 20.5 per cent; at the same doses comparative inhibition by heparin was 95.5%, 91.3 percent and 80.3%, respectively.Example 3
Antimetastatic activity of sulfamethoxazole type IN
Test antimetastatic activity repeat for sulfamethoxazole type, obtained as described in example 1 (product 1669), relative to the dose component of 0.5 mg/mouse, getting inhibition equal to 98.5% of metastases. When the same dose comparative heparin shows inhibition equal to 98.5%, and "eversonpaladini" heparin with low molecular weight - inhibition equal 91,0%.Example 4
Obtaining and antimetastatic activity of fractions sulfamethoxazole type having different molecular weight
1) fractionary by gel-chromatography on Sephadex and distinguish the following three fractions, characterized by similar relations sulfate carboxyl groups (2,2 - 2,3) and different molecular weights: G1668a (average molecular mass of 38 200), G1668c1 (22 700) and G1668b1 (3 200). Corresponding antimetastatic activity (dose 0.5 mg/mouse) was similar for the three fractions (inhibition - 97-98%), and also similar to another nefrackzionirovannam drug SAHS-B (G1783), obtained as described in example 1. In this series of experiments, comparative heparin shows inhibition equal to 95-97%.Example 5
Antimetastatic activity SAHS-B with low-molecular-weight
Comparison with other natural and eversonpaladini glycosaminoglycans
It turned out that the antimetastatic activity of fractions G1668b1 having a low molecular weight (obtained as described in example 4), corresponds with 83.6% of metastatic inhibition. In the same test comparative heparin shows inhibition equal to 92.8 per cent, "eversonpaladini" heparan sulfate with low molecular weight (ssLMWHS) - 46,4% and dermatan sulfate 4,6-desulfation (DS4, 6S) - 65,32%.Example 6
Antimetastatic activity SAHS-B Drug SAHS-B (product G1668 received, in creatures is euromania metastasis. (In the same test comparative heparin shows 98.5% inhibition).Example 7
Antimetastatic activity of a small dose fractions SAHS-B with low-molecular-weight
Fraction G1668c1 having a low molecular weight described in example 4, at a dose of 0.1 mg/mouse, shows antimetastatic activity corresponding to the inhibition of 41.0% metastasis. (At the same dose nefrackzionirovannam comparative heparin provides inhibition equal to 91.1%).Example 8
Antimetastatic activity of a very small dose fractions SASH-B with low-molecular-weight
Fraction G1668c1 having a low molecular weight described in example 4, at a dose equal to 0.02 mg/mouse, shows antimetastatic activity corresponding to the inhibition of 24% of metastases. (At the same dose comparative heparin inhibited 30% of metastases).Example 9
Anticoagula activity SAHS-B
Anticoagula activity G1668, defined as the duration of APTT values in mice (intravenous injection of 0.5 mg/mouse; experiments with group comprising 4 mice) is > 300; 44,4 and 39.9, respectively, after 1, 2 and 4 hours, respectively, after injection. Relevant is laminariales, having a molecular weight of from 3200 to 40000 and the ratio of sulfate/carboxyl groups of about 2.2-2.3, derived from K5 polysaccharide of Escherichia coli N-deacetylation of hydrazine, the subsequent N-sulfation of the trimethylamine adduct/sulfuric anhydride, subsequent O-sulfation adduct pyridine/sulfur trioxide at a temperature of 0oC for 0.25 to 2 h and the ratio of adduct/polysaccharide (equivalent ratio SO3/OH), equal to 5, and a second N-sulfation as antimetastatic agents.2. The pharmaceutical composition exhibiting anti-metastatic activity comprising as an active agent sulfamethoxazole having a molecular weight of from 3200 to 40000 and the ratio of sulfate/carboxyl groups of about 2.2-2.3, derived from K5 polysaccharide of Escherichia coli N-deacetylation of hydrazine, the subsequent N-sulfation of the trimethylamine adduct/sulfuric anhydride, subsequent O-sulfation adduct pyridine/sulfur trioxide at a temperature of 0oC for 0.25 to 2 h and the ratio of adduct/polysaccharide (equivalent ratio SO3/OH), equal to 5, and a second N-sulfation.3. The composition according to p. 2, in which sulfamethoxazole has a degree sylvatrop equal to approximately 2.2).
< / BR>where X is O,
< / BR>R1indicatesor< / BR>R2and R3each, independently of one another, denotes H, A or benzyl;
A denotes alkyl with 1-6 C-atoms;
D denotes amidinopropane, aminomethyl, aminohydrocinnamic, 5-methyl-1,2,4-oxadiazolidine-3-yl or guanidinate;
r and s independently of one another denote 0, 1, 2, 3 or 4;
however, if necessary, free amino - or amidinopropane can be protected partially or fully protective for the amino function groups, as well as their enantiomers, diastereomers and physiologically acceptable salts
FIELD: medicine, thoracic surgery, anesthesiology.
SUBSTANCE: as non-narcotic medicinal preparation one should apply heparin to be introduced intratracheally at the dosage of 300-500 IU/kg, moreover, heparin should be introduced during the first 30 min after the operation is over. The present innovation enables to create prolonged anesthetizing effect in combination with prophylaxis of postoperational thrombohemorrhagic complications due to heparin capacity to be kept in the body due to its accumulation by mast cells at blockade of their fermentative activity followed by its gradual release into the blood.
EFFECT: higher efficiency.
1 cl, 1 ex, 3 tbl
FIELD: chemistry of natural compounds, polymers.
SUBSTANCE: invention proposes a method for preparing low-molecular heparins from high-molecular heparins by the enzymatic fermentative depolymerization using the enzyme complex from Streptomyces kurssanovii immobilized on silochrome in the weight ratio heparin : immobilized enzyme = 1:1. Then suspension is stirred, the immobilized preparation is separated by centrifugation, washed out and washing liquid is combined with supernatant followed by desalting on column with Sephadex 6-10 and the obtained product is lyophilized.
EFFECT: improved preparing method, reduced cost of method.