JPH05504785A - Novel heparin derivative - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Novel heparin derivative Background and gist of the invention The present invention provides a pharmaceutical solution for heparin preparations currently on the market and used in anticoagulant therapy. This invention relates to novel heparin derivatives with modified properties. The novel derivatives of the present invention Anti-thrombin activity and its effects on factor Xa and platelet factor 4 (PF) The relationship between activities is different from that of hepasin. In other words, a large increase in PF4 Strong affinity includes anti-thrombin and thrombin catalyzed by heparin. This is accompanied by a decrease in activity for reactions between molecules.

Similarly, the interaction between antithrombin ■ and factor Xa catalyzed by heparin itself The reaction shows much higher sensitivity for the new derivatives of the invention. Derivation of the invention The body responds to reactions activated by thrombin and factor Xa, and to PF. The anti-thrombotic effect of heparin is different from that of heparin. Thrombosis can be prevented without the associated risk of bleeding. Toro/ It must be kept in mind that excessive effects of bin inhibition may lead to hemorrhagic activity. No. The novel derivatives of the present invention are capable of binding to PF, which is activated by factor Xa. The reaction catalyzed by thrombin can be catalyzed at low concentrations, and the reaction catalyzed by thrombin can be compared. Because it can only be catalyzed at temperatures as high as 21 degrees, this compound has been shown to be effective against arterial and venous thrombosis. It is suitable as a drug for treating.

The novel heparin derivatives and salts thereof of the present invention are obtained as follows: (1) N-hydrogen substituted or unsubstituted by a lower alkyl group Drocarbyl-pyrrolidin-2-one or N-hydrocarbyl-piperidine- 2-one and its heterocycle 10~, -S- and -NH- A heterocyclic system selected from derivatives blocked by a terror atom or a hetero group. with the quaternary ammonium salt of heparin dissolved in an organic solvent, or in an aprotic solvent. in a concentrated solution of the compound at room temperature or slightly higher temperature. Alkylating (etherifying) agents derived from hydrocarbons having carbon atoms of (2) The obtained reaction product is converted into an organic or inorganic base in an aqueous solution. Therefore, it is treated at high temperature, (3) Isolate the heparin derivative obtained in free form or as an alkali salt; and as desired (4) converting the resulting compound into free form or an alkali salt, or otherwise salts of metals or organic bases.

Novel heparin derivatives of the present invention having the above pharmaceutical properties different from those of heparin The body also has a modified chemical structure and a different molecular weight; The structure has not yet been completely defined.

However, the following matters have been confirmed regarding the derivatives of the present invention: (a) most of them are depolymerized; The starting product is composed of a mixture of polymerized (depolymerized) products. It has a moderate to low molecular weight compared to the have molecular weights that lie within a narrow range (they are the same), and this average Dislodged fragments are minimal.

(b) Compared to the starting product, the derivatives of the invention have iduronic acid type saccharide units. The ratio of glucuronic acid type units to glucuronic acid type units is significantly reduced.

Chemical structure related to heparin found in the novel derivatives of the present invention Other modifications include certain types of heparin esters obtained by alkaline treatment. Modifications already described for heparin fragments, such as desulfation (des ulfation) and the presence of unsaturated monosaccharide (monosic) units. .

In addition to the above-mentioned chemical tests, the novel derivatives of the invention are suitable for spectral measurements, especially for nuclear magnetism. It can be identified by gas resonance (NMR), and specifically, according to this, the present invention Differentiate the novel derivatives of Ming from heparin fragments already described in the literature. be able to.

selected from commonly used alkyl and other agents, i.e. derived from lower hydrocarbons. with other alkyl agents such as lower alkyl halides such as ethyl bromide. It is commonly used with dimethylsulfonate or methylformamide. By treating the quaternary ammonium salt of heparin in a solvent containing A heparin ester as described in No. 501095 is obtained. This S If the tell form is subjected to alkali treatment as included in the second step of the operation of the present invention, Different types of heparin fragments are obtained, which can be verified by NMR analysis. Can be done.

The novel low molecular weight heparin derivatives described in the Examples are composed of novel products. This is mainly due to high frequencies (400 to 500 MHz for proton analysis; C13 In the analysis, 100-12100-l25. : NMR spectrum measurement data at However, in NMR measurement, the transmission of FT (Fourier transform) is Using a unified one-dimensional measurement and using recent two-dimensional methods (H-H and C-H correlations) and, in some cases, if complex products such as those mentioned above need to be studied. was confirmed by new methods (eg TOC3Y=total correlation spectral measurements).

By integrating the results obtained by the above method, we found the following: l) The present invention "Novel Oral Molecular Heparin" is an oligosaccharide that is sulfated at both the N and ○ sites. A complex of 16 callides, reflecting their heterogeneity with the starting heparin. It is. Its basic constituents IdoA and G1cnA, which are differently sulfated and a secondary configuration (essentially GlcA) together give rise to different sequential combinations. ing.

2) Compared to the starting heparin, the product that is the object of the present invention has rdoA/Glc The N ratio is noticeably lower and this reduction can be detected by NMR. Therefore , anomaly of the typical IdoA sequence (2SO-)-GlcNSO, ) (6S○3) Signal at one site (102 and 99.5 ppm+ for carbon; 5.18 and 5.32 ppm) prove to be dramatically reduced. this Such conversions cannot be detected with known low molecular weight heparins, or even if they can be detected, their The extent is very small. Extremely strong/gnal of novel unsaturated monosaccharide units (see point 4).

3) Another characteristic fact is that the relationship between the nitrogen S○3H group and the GIcN residue is It is the emergence of new/gnals that are compatible only in relation to the For carbons correlated to 373 and 368 pp++ respectively for roton 54.2 and 53.3 ppm).

This property, which can be detected by 2-D NMR, is similar to that of known low molecular weight heparin samples. In practice, this is not acceptable.

4) Usually, most of the above oligosaccharides have a NMR (147 for C-13) and 172 ppm signal, 6 ppm doublet for protons) An unsaturated monosaocalide unit with 3 substitutions in the 1-2 positions as shown It's finished. These signals are absent for other known low molecular weight heparins; or has decreased significantly.

Thus, it is possible that the We must conclude that this is not a simple esterification, but a more complex chemical reaction. Must be.

Therefore, based on these findings, the heparin-like products of the present invention are novel and already It can be assumed that the heparin fragments are different from those described in the literature. can. For example, the products of the invention can be obtained by alkaline treatment of heparin esters. Unlike the fragment described in European Patent No. 0302034, This fragment is capable of forming complexes with copper ions that play an angiogenic role. It is suitable. Similarly, a well-defined heparin ester with a U.S. patent obtained by treatment with Na0H (0,10,5N) at ℃ to 60℃ The heparin fragments described in No. 4.440.926 are the products of the invention. is not the same as

The above properties of the novel heparin derivative of the present invention are demonstrated by the experiments described below. The experiment is described in the illustrative examples under the code name: PE This was carried out for the product abbreviated as . In this experiment, unfractionated heparin (UFH) and low molecular weight heparin derivatives (CY216 = Fraxi parine”)) is used as a comparative product. Both of these amphibiotic acids It is commercially available for the treatment of thrombosis.

Brief description of the drawing Figure 1 shows the in vitro anticoagulant activity of PE expressed in thrombin time with UFH and It is shown in comparison with CY216. The data obtained are 7- for each test product. This is the average value of 10 tests.

Figure 2 shows the effect of PE on thrombin time after intravenous administration. The in vivo anticoagulant activity is shown in comparison to UFH and CY216. This de Data are the average values of experiments performed with 5 rabbits for each test product.

Figure 3 shows <11 of PE based on anti-FXa activity! X vivo anticoagulant activity (subcutaneous Post-dose elimination kinetics) compared to UFH and CY216. This de Data are average values of experiments performed with 5 rabbits for each test product. .

Figure 4 shows the ex vivo activity of PFH after intravenous administration of PE. and CY216.

1. Materials and methods for in vitro anticoagulant activity on reconstituted human plasma Test product (solubilization and a degree) The following products were tested: One heparin derivative PE -Unfractionated heparin (UFH) One low molecular weight hepawan derivative (CY216-FraxiparineR) test student The products were dissolved in sterile saline and tested at concentrations of 0.5-12.5 μg/xQ.

parameter ■ Thrombin (indicating anticoagulant activity) 2, anti-FXa activity (anticoagulant activity) It is clear from Figure 1 that the knee PE is 125 μg/R. The concentration of Q indicates anticoagulant activity.

-The anticoagulant activity of PE is much inferior to that of unfractionated henokulin UFH ( Thrombin time increases at approximately 4-5 times higher concentrations than UFH). With CY216 No significant differences were observed.

2 Anti-FXa activity Preliminary test data in Table 1 shows that - PE is 3μg IRQ It shows anti-FXa activity from the concentration of .

- Anti-FXa activity is clearly inferior to heparin. Furthermore, this activity is It should also be noted that it is inferior to CY216. But in this case The observed differences are less pronounced compared to UFH.

Table 1 This data for PE compared to unfractionated heparin and CY216 is shown for each test generation. It is the average of three experiments performed for each a degree on one of the objects.

The anticoagulant activity of a new heparin derivative was investigated by an ex vivo method using rabbit plasma. was evaluated. The term rex vivoJ refers to means an in vitro measurement of blood. The specific tests are as follows.

(a) Arterial blood after rapid porous intravenous administration (i, v, ) of the test product. thrombin time (b) Anti-FX in arterial blood after rapid subcutaneous administration of test product Cs, c, a activity This method is very sensitive and allows the kinetics of disappearance of the test product to be monitored after subcutaneous administration. - is an indicator of the ``biological efficacy'' of the test product.

The test product was dissolved in sterile saline solution and administered by intravenous route at 0.86 mg/kg. In addition, 1 to 2 mg/kg was rapidly administered by subcutaneous route.

In this test, porous test products were administered at various times (2-5 Thrombin time of arterial blood samples taken from rabbits at -10-20-30-40 minutes) Evaluate the time.

(b) Anti-FXa activity Anti-FXa activity was measured (by a chromogenic test that is sensitive at very low concentrations of the compound). The presence of the test product in the circulatory system can be monitored for very long periods of time by can be done. Samples of arterial blood (from the central ear artery) were administered subcutaneously at various times. (Open at 1-2-3-4-5-6-7-8).

Results (a) Toro/bin time The following matters are clear from Figure 2.

-PE showed low anticoagulant activity at 0.86zg/kg i,v; This supports the in vitro data described above.

--The anticoagulant activity of PE is clearly inferior to that of UFH, but it is significantly different from that of CY216. There is no difference in authorship.

(b) Anti-FXa activity Figure 3 shows the following: -P E (after subcutaneous administration of 1 mg/kg) was administered at twice the dose, i.e. 2 It remains in the circulation for a longer time than UFH administered at 1 g/kgs, c.

Therefore, the elimination kinetics of the heparin derivative PE is slower than that of UFH, but are similar.

The experiments shown below showed that the novel heparin derivative P of the present invention against human 1-PF To assess the affinity of E, i.e. the “binding properties” of these products for PE4, Ta.

The ex-vivo method used allows for The kinetics of disappearance of PF, was monitored.

PF, previously administered heparin or glycosaminoglycans (GAGs) It is well known that τ significantly enhances its kinetics.

Ce1la et al., “Human platelet factor 4 and sulfate proteins in rabbits.” "Interaction of rotamine with glycosaminoglycans", Eur, J, Cl1n , Invest, , Mo1. 17. pp, 548-554 (1987 No way.

Therefore, a compound that can bind to PF should greatly prolong its presence in the bloodstream. be. This effect is proportional to the amount of injected GAG and its affinity for PF. do.

The test product was dissolved in sterile saline solution and tested at 0.86 mg/kg i,v. Administration of the compound was acute (intravenous porous) at a dose of 0.86 mg/kg. Ta. Arterial blood samples were taken from the central ear artery after 2-3 minutes (baseline sample). Five minutes after the first porous injection, a second hole of purified human PF4 was added. (30 μg/kg).

15.25.5.10.20, 30 minutes after the administration of PF, the su/pull was collected. . These were centrifuged and stored for RIA testing (commercially available) against human PF. An evaluation was conducted using the following kit.

Results As shown in Figure 4, the obtained data indicate that PE is platelet. It has an affinity for factor PF4, gradually disappears from the bloodstream, and reaches a low level approximately 30 minutes after administration. reaches a new value.

・The affinity of PE for PF is much lower than that of UFH (approximately 60%) The kinetics of disappearance of the complex with , PF, is much faster than that with UFH; Similar to that by CY216.

4. In vivo antithrombotic activity in rabbit arterial thrombosis model The novel heparin derivative PE was tested in viv according to the experiments described.

The antithrombotic activity was evaluated. The aim was to develop an acute model of rabbit carotid arterial thrombosis. Prevents arterial thrombus formation in the blood vessels, subsequent endothelial damage and reduction in vessel diameter The purpose was to test the efficacy of the product in doing so.

The test product was dissolved in sterile saline and given a JIf range of 1.2 to 5 B/kg. Administration was by the lv route.

Model description The patient was anesthetized with Nembutal (30 mg/kg i, V, porous) and injected through tracheotomy. Experiments were conducted using rabbits that were forced to breathe. Continuous administration of anesthesia and drugs For this purpose, a tube was inserted into the great cadaveric vein and into the oscilloscope.

Systemic blood pressure was monitored and the fighting artery was secured with a flowmeter and snare. The system has stabilized At that time, a stenosis was performed. After a suitable interval (not less than 20 minutes), use surgical forceps. mechanical injury was induced and stenosis was performed on the injured blood vessel. For blood vessel occlusion The decrease in flow was monitored until a corresponding value of 0 was reached. control vertebral movement Pulses were prepared similarly. The test product was then applied at 1 v for a total of 8 minutes and injected. Mechanical damage was performed 2 minutes later. flow until at least 1 hour after the last treatment. was continuously monitored. Animals were treated with overdose anesthesia at the end of the experiment.

Results As shown in Table 2, the obtained data indicate that PE It is effective in completely preventing the formation of blood clots.

The effect is evident at a dose of 6 mg/kg i,v.

・The antithrombotic efficacy of PE is lower than that of unfractionated heparin UFH (lower than 1 or 2 mg/kg). (approximately 5% ), the pharmaceutical efficacy of PE is similar to CY216.

Table 2 Number of unfractionated Hepa I77 treated animals in a model of rabbit arterial stenosis UFH1,2mg/kg 2/12 CY 216 6 mg/kg 2/12 The experiment described below was performed using rabbit venous blood. Using a venous A-plane technique by occluding the engineered vena cava of PE in an embolism model there was. In this method, the main cause is due to changes in blood flow at the junction of the vena cava and the left renal vein. Nifibrinous thrombus formation is induced.

That is, the model of this experiment was designed to investigate the activity of anticoagulants, especially the antithrombotic potential of heparin (administration and post-impact).

The test product was dissolved in sterile physiological saline at a concentration ranging from 0.5 to 3 mg/kg i, ν. The following doses were administered intravenously (15 minutes before bleed induction).

Exam description Experiments were conducted using a male CD-Co BSS 5y) with a body weight of 175 to 200 g. I did it. Animals were treated with bentoparbital sodium (40 mg/kg i, p). The animal was anesthetized and its ventral side was disinfected with alcohol. Starting from the ribs and along the center of the abdomen A 4cm incision was made. Isolate the vena cava from the aorta to the normal iliac artery; A cotton thread was tied just below the junction with the left renal vein. Constant and continuous due to double occlusion pressure was applied for 5 seconds. The abdominal wall was then closed. 2 hours later it opens again. Formation of abdominal thrombus was confirmed. 0 vena cava and collateral circulation corresponding to the normal iliac arteries After closing with hemostatic forceps, the portion of the tube below the occlusion was cut lengthwise with a needle. Blood 5 Remove the clot removed, soaked in distilled water, blotted dry on paper, then placed in a drying box for 24 hours. The dry residue was measured and expressed in mg.

Results Blood clot severity 1 after 2 hours of venous p blood! (mg) (Table 3) and incidence of thrombus (%) The data on (Table 4) indicate that PE has antithrombotic efficacy. Ru. Its protective effect (before treatment) is significant at doses of 1, 5 mg/kgiV .

・The efficacy of PE is the efficacy of unfractionated heparin UFH (0, 5 mg/kg i, v .

(which already has a noticeable effect).

Table 3 In vivo antithrombotic efficacy of PE compared to BiFl in a venous impact model 20-animals of intravenous 11f [1 (performed 15 minutes after 1.v administration of test product)] number of Table 4 vein! In vivo antithrombotic effect of PE compared to UFH in blood model Intravenous blood transfusion (1v of test product, performed 15 minutes after administration) in 26 rats. Effect on bleeding time The experiment described below shows that intravenous administration of the test product 1 The effect of PE on bleeding time after 5 minutes was evaluated. Sterile saline test product and administered i, v at concentrations ranging from 1 to 3 mg/kg.

Exam description Experiments were conducted using male cD-GOBS rats (175-200 g), and test items The substance was administered acutely by intravenous route. After 15 minutes, by standard molding method of cutting off the tail. Bleeding time (seconds) was evaluated [Dejana et al.

“Bleeding time in the vert: Comparison of various experimental conditions”, Thromb, H. The obtained data (Table 5) show the following: P for "bleeding time" The effect of E increases significantly at doses of 2.0 mg/kg i.v.

・The effect of PE is similar to that obtained with unfractionated heparin UFH (bleeding value is 1 mg/ (already markedly increases at doses of kg i, v), but , comparable to the effect of CY216 (data not reported).

To obtain the same value for bleeding time, use at least 3 times the dose of UFH. It is interesting to note that PE needs to be administered.

Table 5 Test product i, v, bleeding time (seconds) 15 minutes after administration of PEn-animals number 7. In vivo tolerance after i, v and sc administration in mice This experiment Maximum tolerance of compound PE after intravenous and subcutaneous route administration in mice Dosage was evaluated.

The test product was dissolved in sterile saline, 125-2000 mg/kgi, v , and a 1 degree range between s, c.

Exam description Using male and female CD-1v mice (C, RiverX 25-35 g), the following An experiment consisting of the following two stages was conducted.

1) Screening Groups of Kawanishi male mice and one female mouse were administered the following: Treated with one of the amounts: 125.250.500, l 000 and 200 0 tag/kg, the number of deaths within 17B was monitored.

2) Groups of 5 male mice and 5 female mice were screened. Treatment was performed at the highest tolerated dose (for each compound and each route of administration).

Animals were observed for 14 days, during which time the number of deaths and altered behavior and general toxicity were determined. The presence of symptoms was observed.

In mortality experiments, lower doses are used until the maximum tolerated dose is found. was used. When possible, necropsies were performed on dead animals.

Results As shown in Tables 6 and 7, the maximum tolerated dose of the test compound is: It turned out that ” PE: 1000mg/kg i, v unfractionated heparin UFH: 125mg/kg i, v.

・CY216: 1000mg/kg i, v.

Therefore, PE is characterized as having a greater tolerated dose than unfractionated heparin UFH. I have to write it down. to unfractionated, (500 and 250 m of phosphorus UFH Necropsy of animals that died after treatment with g/kg did not reveal any gross findings. However, in some animals there was evidence of what appeared to be the remains of internal bleeding in the intestines. found.

2. Subcutaneous tolerance dose The results obtained, as shown in Tables 8 and 9, indicate that the maximum tolerated dose of the test product is shown to be as follows...P E: 2000 mg/kg s, c .

・Paris'I to non-fractionation: FH: 125mg/kg s, c CY216 : 2000 mg/kg s, c Therefore, by intravenous route, the tolerated dose of PE is much larger than the tolerated dose of unfractionated heparin (UFH) (for CY216). They are similar. However, it is important to emphasize the following...CY All animals (10/10) treated with 216 (2000 mg/kg s, c,) , the round area of skin affected by necrosis corresponding to the injection site is located on the back. Its size varies depending on the animal, with a maximum diameter of approximately 1 cI11).

An autopsy revealed that the necrosis was confined to the skin: the underlying muscles were therefore unaffected. It wasn't making any impact.

・Animals treated with PE (2000 mg/kg s, c,) had no abnormalities observed. There wasn't.

・Animals that died after treatment with UFH (250 mg/kg) should not be treated. There was an expansible scar at the site of subcutaneous administration.

Therefore, the subcutaneous tolerance data shows the differences between compounds PE and CY216. difference Furthermore, although the maximum tolerated dose was the same, CY216 showed no significant increase in skin necrosis. A cut site exists at the injection site, which allows CY216 to be Local resistance was shown to be lower than that of

Takashi-1 Compound PE, CY216 and unfractionated PE 125 0/ after one intravenous administration 1 0/I CY216 500 0/1 0/1 0 Table 7 Intravenous administration 1 and 0 compounds PE・CY2i6i, h non-fractionated (mg/k g i, V, ) Male Female Number of days after treatment Immediately *1234 PE 1000 015 015 - - - - CY216 1000 0 15 015 - - - - Table 8 Lethality of compounds PE, CY216 and unfractionated heparin UPH after one subcutaneous administration Sex (Screening) CY216 2000 0/1 0/ICY216 10 00 0/1 0/ICY216 500 0/1 0/1 Heparin IJFH20001/1 1/1 Hepawan UFH10001/1 0/ 1 Heparin UFH5001/1 0/1 Heparin [JFH2500/1 0/1 Table 9 Compound PE, CY216 and unfractionated (mg/kg i, v ,) Male Female Number of days after treatment Immediately 1234 CY2162000015 015 − − − Conclusion Based on the in vitro and invivo results described above, unfractionated heparin The pharmacological activity of heparin derivative PE is different from that of heparin derivative (UHF). Its interesting pharmacological properties that make it valuable are explained.

The activity of PE is one of the best low molecular weight heparins currently commercially available in France. The activity of CY216 is slightly different, especially after subcutaneous administration to mice. Shows good local tolerance. In fact, the maximum tolerated dose is the same, but CY216 In all animals treated with (2000 mg/kg s, c,), the skin at the injection site While skin necrosis is evident, the skin of animals treated with PE remains unaffected. It was.

For in Vitr□ and inv:vo pharmacological activity, PE is It is interesting to note that it has the properties of... far inferior anticoagulant than UFH. Solid activity.

-Much more effective than UFH, which can require less daily dosing to achieve the same pharmacological effect Great bioavailability after subcutaneous administration.

・Less side effects] For the same FH, reduction of bleeding time and injection site skin demonstrated by the absence of necrosis, CY216 (the best commercially available in France) (low molecular weight heparin), local non-tolerance was observed in all treated animals. phenomenon (necrosis)].

・Both a model of venous blood in horses and a model of arterial thrombosis in rabbits. In vivo antithrombotic activity shown in Therefore, this product has 15 and It is effective in preventing these lesions at each dose of 6 mg/kg i, v. , does not cause any significant changes in side effects. In particular, venous antithrombotic activity Therefore, unlike UFH, the derivative PE can be used at dosages of 1, 5 mg/kg i, v. It does not cause any unpleasant changes in bleeding time depending on the dose.

Therefore, the novel low molecular weight heparin derivatives of the present invention are suitable for all indications. , can be used in place of unfractionated heparin as an anticoagulant and clot aggregation inhibitor . Dosage will be adapted to each particular case, but usually about It is 1 to 7 mg/kg.

The heparin used in the described method of the invention can be of any type and of various origins. Commonly extracted from, for example, pig, cow, and sheep fried and bull hearts Heparin, especially any of the products commercially available or described in the literature There may be. Such products have a wide range of molecular weights, e.g. ．． 000 and 30,000 daltons, especially unfractionated heparins UFH1 and Furthermore, low molecular weight heparin obtained by fractionation of standard heparin (2,00~ 10,000 daltons) and standard heparin by chemical or enzymatic methods. Molecule 11500A-10,000 daltons obtained by partial depolymerization of There are heparin fragments.

The quaternary ammonium salt used as starting material for the present method can be prepared in a known manner. Can be manufactured. For example, as a sodium or potassium salt in an aqueous solution. Resins based on quaternary ammonium salts (e.g. quaternary ammonium salts) It is produced by ion exchange using a sulfone resin (chlorinated with a rubber base). Ru. Quaternary ammonium salt can be obtained by freeze-drying the eluate .

As a quaternary ammonium salt, tetraal-4-ammonium obtained from lower alkyl um salts, especially tetraalkyl ammonium salts with alkyl groups having up to 6 carbon atoms. It would be convenient to use a nium salt.

Optionally, alkyl-aryl-ammonium salts, e.g. It is also possible to use salts with Tetrabu among tetraalkylammonium salts It is preferable to use a tylammonium salt.

The quaternary ammonium salt used as a starting material for the method of the present invention is a commercially available product. alkaline salts (e.g., sodium or potassium salts) of the type of heparin that is A salt that can be obtained by ion exchange in the above method, i.e., a neutral salt. be. These salts are reacted with excess quaternary ammonium ions to form the corresponding neutral salts. get.

The quaternary ammonium salt can be added to the above-mentioned heterocyclic solvent and an aprotic organic solvent ( soluble in both dimethyl sulfoxide and dimethyl formamide) . The reaction with the alkylating agent in the first step of this method is carried out in the above-mentioned heterocyclic solvent. or in a solution (preferably concentrated) of the substance in any of the above aprotic organic solvents. (in solution). Particularly preferred among the above heterocyclic solvents are: A heterocyclic ring whose ring structure is unsubstituted (i.e., N-alkyl-0-aryl-2- pyrrolidone), or its derivatives containing heterocyclic atoms in the cyclic structure (e.g. for example imidacilline, piperazine or the corresponding derivatives of morpholine).

The N-alkyl group of the heterocyclic solvent is from lower alkyl having up to 6 carbon atoms. (preferably, the aryl group is optionally 1 to 3 lower alkyl groups) (In particular, a phenyl group substituted with a methyl group). N-methyl-2-pyro Preferably, lydones are used.

Hydrocarbons having 6 to 30 carbon atoms (preferably 8 to 18 carbon atoms) Alkylating agents derived from compounds, such as alcohols with a corresponding number of carbon atoms (preferably the most Aliphatic or arylaliphatic alcohols having up to 18 carbon atoms, e.g. reagent (which may be alcohol, heptyl, octyl or/and acid) It is an ester group. The reagent ester can be hydrogen acid, sulfuric acid or sulfite, or Alkyl or aryl sulfonic acids (e.g. methanesulfonic acid or p4 can be derived from inorganic or organic acids such as enesulfonic acid). hydrogen acid s Specifically, the compounds are chloride, bromide and iodide. 4 of the above alkylating agents The reaction with grade ammonium salts is carried out at room temperature (20°C) or slightly higher temperature ( For example, at a temperature of 30-35°C and not exceeding 60°C) for an extended period of time, about 5 to 20 hours (usually about 16 hours).

The reaction product is isolated and then subjected to the second step of the method (i.e. alkaline treatment) can also be added to the solution of the reaction product of the first step (concentrated if desired). It can also be directly converted into the final product by direct alkali treatment. Ma In addition, the solvent is evaporated under mild conditions after the first step of the method. It is also possible to carry out alkali treatment on this residue.

Therefore, the method of the invention can be practiced as a "one pot" method.

Base treatment of the reaction product between the quaternary ammonium salt of heparin and the alkylating agent Temperatures between 5 and 120°C (temperatures between 50 and 120°C are suitable, and temperatures around 70°C are preferred) (for about 2 hours). Alkaline hydrates, such as N a OH or KOH or other inorganic and/or organic bases at concentrations between 01 and IM? preferable. This base is used in an aqueous or alcoholic solution; - other solvents that are miscible with the alcohol mixture (e.g. in the case of the "one container" method described above) residual solvent used in the first step of the method) may also be present.

If it is desired to isolate the reaction product of the first step of the process, it can be Organic polar solvents (aliphatic alcohols such as methyl or ethyl alcohol are preferred) and a basic buffer, especially a basic salt of a carboxylic acid, such as acetic acid or It is preferable to add sodium or potassium salts of propionic acid). It can be precipitated by addition. Alkali bicarbonate or alkaline phosphate Other basic buffers can also be used, such as. This precipitated product is It is preferable to wash with the polar solvent used for the reaction (for example, methanol). This precipitation By reprecipitating the resulting product one more time or several times, i.e. , the product by dissolving it in water and precipitating it with alcohol. Further purification is recommended.

The reaction product, which contains a low molecular weight heparin derivative and has the properties described above, is It can be released in free form from metal salts or organic bases in a conventional manner. Book The alkali salt corresponding to the alkali hydrate used in the second step of the method is treated with an acid (e.g. Neutralize the solution using 2M hydrochloric acid) and purify the solution to remove the water-immiscible organic solvent. extraction medium (e.g. methylene chloride) and repeating this operation several times. It can be obtained by This salt is then dialyzed against distilled water and sodium chloride. and lyophilize.

The products of the invention are primarily used in the form of metal salts or salts with organic bases. the above Gold other than the alkali salt corresponding to the ion of the alkali hydrate used in the alkali treatment of If it is desired to obtain a genus salt, ion exchange methods according to known methods are used. be able to. Heparin products can also be isolated in acid form. Obtained A calculated amount of diluted strong acid (such as hydrochloric acid or sulfuric acid) is added to the alkali salt obtained to form the product. is extracted with a suitable organic solvent to isolate the heparin compound in free form, which is then extracted with a suitable organic solvent. can be converted into the desired salt.

Of particular interest among the salts with metals or organic bases that can be used according to the invention are Salts include therapeutically acceptable salts, such as alkali and alkaline earth metal salts ( sodium, potassium, ammonium, calcium and magnesium salts, etc.) , or salts of heavy metals (such as copper and iron salts). Salts of organic bases are primary, secondary or tertiary aliphatic, aromatic or heterocyclic amines, such as methylamine, Thylamine, propylamine, piperidine, morpholine, ephedrine, furf Derived from rylamine choline, ethylenediamine and amine ethanol etc. Can be done.

Salts that are part of the invention and cannot be used directly in therapy are e.g. Salts that can be used to purify new heparin derivatives (e.g. heavy metal salts) part of).

The present invention also provides novel compounds obtainable by the method described herein as active substances. containing standard low molecular weight heparin derivatives, particularly in the form of therapeutically acceptable salts. Includes pharmaceutical preparations. This preparation is suitable for parenteral, e.g. subcutaneous or intravenous administration. for administration, or for topical use, e.g. in the form of a cream or ointment, or as a bulk, or or in the form of a nasal spray. Therefore, they can be made compatible with physiological fluids. one or more pharmaceutically acceptable excipients convenient for the above uses, having an osmotic pressure of or as a lyophilized powder of the active compound mixed with excipients or diluents; It can be formulated as a solution of the compound. The present invention provides novel low molecular weight heparin In addition to the above-described methods for producing these, it is intended to include the above-mentioned methods for producing them. Ma In addition, a single step of the present manufacturing method, i.e., the above-defined alkaline in any of the above-mentioned solvents, The reaction of a quilating agent with a quaternary ammonium salt of heparin and the alkali Reprocessing is also encompassed by the present invention.

The present invention will be explained below by giving examples, but these are not intended to limit the present invention. There isn't.

1) Preparations for injection I PE (mg) IQ 20 30 40 Water for injection (ml) 0.1 0.2 0.3 0.4 with a ready-to-use syringe containing sterile solution for subcutaneous administration /f , 7 cages.

1.2 P E (mg) 150 300 Other ingredients/ Water for injection (ml) 4 8 Place in a small cage with a vial containing sterile solution for intravenous administration.

PE (mg) 10 20 30 40 Other ingredients.

Sodium chloride (mg) 0.3 0.6 0.9 1.2 Water for injection (ml ) 0.1 0.2 0.3 0.4 Ready to use with sterile solution for subcutaneous administration One trick is to cage it with a syringe.

I P E (mg) l 50 300 other ingredients Sodium chloride (mg) 35 70 Water for injection (ml) 5 10 Packaged with a vial containing sterile solution for intravenous administration.

P E (mg) 150 300 Other ingredients: Sodium chloride (mg) 35 70 Sodium metabisulfite (mg) 5 10 Chlorobutanol (*) (mg) 25 50 Water for injection (IIl) 5 10 Package with vials containing sterile solution for multiple intravenous administration.

(*) The preservative chlorobutanol in Formulation Example 1.5 can be replaced with the following. Ru Benzyl alcohol (mg) 50 100 or Chlorocresol (mg) 5 102) Preparation for oral administration PE (mg) 30 60 120 Other ingredients/ phosphatidyl coria +Ho xyyf Jilsen’) O) (tag) 80 160 320 Lactose (IIlg) 50 100 200 Microcrystalline cellulose (agn 10 20 40 talc (tag) 3 6 12 Colloidal phosphoric acid (IIlgn 3 6 12 (*) The ratio of sphatidylcholine to phosphatidylserine can be varied.

22 (pills or capsules) PE (mg) 30 60 120 Other ingredients: phosphatidyl coria + Phosphatidyl serif 0) (mg) 80 160 320 Cholesterol (II+g) 2 4 8 Lactose (sag) 50 100 200 Slight Crystal cell o-se (mg) 10 20 40 Talc (mg) 3 6 12 Colloidal/Rica (mg) 3 6 12 (*) Phosphatide present in the preparation The ratio of zirconia to phosphatidylserine can be varied.

23 (powder) PE (mg) 60 120 180 Other ingredients phosphatidyl coria + phosphatidylserine (*) (mg) 250 500 750 cholesterol Le (mg) 5 10 15 Lactose (mg) l OO200300 required Flavoring and sweetening agents required Sucrose (**) (up to this amount) (mg) 2000 4000 6000 The ratio of phosphatidylcholine to phosphatidylserine present in the formulation may vary. can be done.

(**) Sucrose can be replaced with fructose.

(delayed action tablets) PE (mg) 30 60 120 Other ingredients: phosphatidylcholine Hon phosphatidyl serif (*) Cmg> 80 160 320 Lactose (mg) 30 60 120 Microcrystalline cellulose (ag) 5 10 20 h Droxypropyl- Methylcellulose Cmg) 10 20 40 Talc (sag) 3 6 12 Colloidal phosphoric acid (mg) 3 6 12 (*) Phosphatide present in el The ratio of zircholine to phosphatidylserine can be varied.

2.5 (stomach-resistant tablets) PE (mg) 30 60 120 other ingredients.

phosphatidyl coria - Phosphatidylserine (*) (mg) 80 160 320 Lactose (mg) 50 100 200 Microcrystalline cellulose (mg) 10 20 4 0 talc (ng) 30 60 120 colloidal licorice (mg) 3 6 12 Methacrylic acid copolymer (mg) 15 30 60 (*) Present in the formulation The ratio of phosphatidylcholine to phosphatidylserine present can be varied. Ru.

1 亙 (stomach-resistant delayed action tablet) PE (mg) 30 60 120 Other ingredients: phosphatidylcholine - Phosphatidylserine (*) (mg) 80 160 320 Lactose (mg) 50 100 200 Microcrystalline cellulose (mg) 10 20 4 0 hydroxypropyl- Methylcellulose (mg) 5 10 20 Talc (mg) 30 60 120 Colloidal/Rica (drunken) 3 6 12 Methacrylic acid copolymer (m g) 15 30 60 (*) Phosphatidylcholine and phosphatide present in the formulation The ratio of atidylserine can vary.

Liquid formulation PE administered orally as 2L<m liquid (mg) 30 60 120 other ingredients.

Citric acid (mg) 7.5 15 30 Purified water (container) (+nl) 0.25 0.5 1Z Yuken (granules *) PE (mg) 30 60 120 Other ingredients: Citric acid (II + g) 7.5 15 30 sucrose (tag) 30 6 0 60 Corn Starch (mg) 12.5 25 25 Talc (mg) 5 10 10 Polyvinylpyrrolidone (tag) 4 8 8 (*) 3 types of granules mixed with hard gelatin Administer in capsules.

2niji (stomach-resistant granules*) PE (mg) 30 60 120 Other ingredients: Citric acid (mg) 7.5 15 30 Sucrose (sag) 30 60 60 Corn Starch (ag) 12.5 25 25 Talc (11g) 5 10 10 Polyvinylpyrrolidone (tag 4 8 8 methacrylic acid fupolymer (m g) 10 15 15 (*) Administer three types of granules in a hard gelatin capsule. Ru.

3) Nasal or pulmonary formulations PE fine white powder (mg) 20 40 active ingredients as fine white powder It is placed in a gelatin capsule, which is opened at the time of use and placed in a suitable device for inhalation.

32 (powder for inhalation) PE fine white powder (mg) 20 40 phosphatidylcholine + Phosphatidylserine (*) (mg) 30 60 active ingredients in fine white powder Place it in a gelatin capsule, open it at the time of use, and place it in a suitable inhalation device. It will be done.

(*) The ratio of phosphatidylcholine and phosphatidylserine present in the drug may vary. can be converted into

Semi-synthetic glyceride I+ng) l 940 1880 phosphatidylserine + Phos 7y Tidylcoli 7 (mg) 100 200 Semi-synthetic glyceride (mg ) 1840 1680 In summary, the low molecular weight heparin derivatives and and its salts are N-alkylpyroCJ di/~2-one, N-alkylbiveridine -2-one (substituted or unsubstituted with a lower alkyl group) and -〇-1-8- and The heterocycle is interrupted by another heteroatom or group selected from -NH- and -NH-. Dissolved in a heterocyclic organic solvent selected from the group consisting of derivatives thereof Concentration of the quaternary ammonium salt of heparin or the compound in an aprotic solvent having 6 to 30 carbon atoms (preferably 8 to 18 carbon atoms) in solution. using an alkylating (etherifying) agent derived from a hydrocarbon that ) to 60'C for an extended period of time (e.g., about 5 to 20 hours). You can get it. Next, the obtained reaction product is dissolved in an inorganic or organic solution in an aqueous solution. Treat with a machine base at a temperature of 5 to 120'C (preferably about 70"C). Then The product obtained by this alkaline treatment can be used in free form or as a It is isolated as alkali metal or alkaline earth metal salt. From one salt to another Salts of other metals or organic bases may also be obtained from the above products by conversion. I'll come. In the second step of the method, the reaction product is converted into alkali hydroxide in an aqueous solution. Therefore, it is preferable to treat it. Obtained low molecular weight heparin derivative and its salt are therapeutically useful, eg, as antithrombotic drugs.

Fl(,URε　1 Full; URE '1 Summary book Quaternary ammonium heparin dissolved in a heterocyclic organic solvent or an aprotic solvent aluminum salt having 6 to 30 carbon atoms for an extended period of time at a temperature of about 20 to 60'C. The resulting reaction product is heated in an aqueous solution at about 5 to 120°C. treatment with an inorganic or organic base (such as alkali hydroxide) at By isolating the parin derivative in free form or as its alkali salt. to produce low molecular weight heparin derivatives and salts thereof. By the method of the invention The resulting heparin derivatives are largely depolymerized with a narrow range of molecular weights. It consists of a mixture of products. These products are useful as antithrombotic drugs.

International search report orT/GD Q1/n747Q

Claims (1)

[Claims] 1. (1) N substituted or unsubstituted by a lower alkyl group -alkylpyrrolidin-2-one, N-alkylpiperidin-2-one or the heterocycle is another heteroatom selected from -O-, -S- or -NH- or is dissolved in a heterocyclic organic solvent selected from derivatives blocked by a hetero group. the quaternary ammonium salt of heparin, or the compound in an aprotic solvent. The concentrated solution is mixed with 6 to 30 carbon atoms at room temperature or slightly above. (2) the resulting reaction product; is treated with an inorganic or organic base in aqueous solution at a temperature of 5 to 120°C; and (3) The obtained heparin derivative in free form or its alkali metal A low molecular weight heparin, characterized in that it is isolated as a salt of an alkaline earth metal; A method for producing a carbon derivative and/or a salt thereof. 2. Converting the obtained heparin derivative or salt into other metal salts or organic base salts. 2. The method of claim 1, further comprising: 3. converting heparin derivatives obtained in free form into pharmaceutically acceptable salts; , or converting the salt into the free form of the heparin derivative. The method described in Section 1. 4. In step (2), the reaction product is treated with alkali hydroxide in an aqueous solution. 2. The method according to claim 1. 5. A product obtained by the reaction of a quaternary ammonium salt of heparin with an alkylating agent. Claim 1 or Claim 4, wherein the product is isolated before being treated with an inorganic or organic base. How to put it on. 6.4 of heparin with molecular weight varying from 2,000 to 30,000 daltons 5. The method according to claim 1 or claim 4, wherein a grade ammonium salt is used as a starting material. . 7. Claim 1 or 2, wherein a quaternary ammonium salt of unfractionated heparin is used as a starting material. The method according to claim 4. 8. fraction obtained by partial chemical or enzymatic depolymerization of heparin. Heparin fragments between 500 and 10,000 daltons and molecular weight 2.0 Low molecular weight heparin obtained by fractionation of heparin between 00 and 10,000 daltons Claim 1 or Claim 4, wherein a quaternary ammonium salt of N is used as a starting material. the method of. 9. The quaternary ammonium salt is a tetra-ammonium salt in which the alkyl group has 1 to 6 carbon atoms. The method according to claim 1 or claim 4, which is a rukylammonium salt. 10. Claim 1 or 2, wherein the quaternary ammonium salt is a tetrabutylammonium salt. The method according to claim 4. 11. A lower alkyl group in which the N-alkyl group of the heterocyclic organic solvent has 1 to 6 carbon atoms 5. The method according to claim 1 or 4, wherein the method is Rukyl. 12. Claim 1 or Claim 4 wherein the heterocyclic organic solvent is N-methylpyrrolidone. The method described in. 13. Aliphatic or arylaliphatic alcohols having 6 to 30 carbon atoms; Process according to claim 1 or claim 4, in which an ester of an acid is used as the alkylating agent. . 14. 14. The method of claim 13, wherein the alkylating agent has 8 to 18 carbon atoms. 15. If the alkylating agent is an alkyl iodide, alkyl bromide or alkyl chloride, or an ester of an alkyl or aryl sulfonic acid. Method. 16. The alkylating agent was added to quaternary ammonium heparin dissolved in a heterocyclic organic solvent. 5. The method according to claim 1 or claim 4, wherein the reaction is carried out at a temperature of about 20 to 60°C. . 17. The reaction obtained by the reaction of an alkylating agent with a quaternary ammonium salt of heparin. 1 or 2, wherein the reaction product is isolated by precipitation by addition of a polar organic solvent. The method described in claim 4. 18. 18. The method according to claim 17, wherein the polar organic solvent is an aliphatic alcohol. 19. 19. The method of claim 18, wherein a basic buffer is added to the aliphatic alcohol. 20. 20. The method of claim 19, wherein the basic buffer is a basic salt of a carboxylic acid. 21. 18. The precipitated product is reprecipitated several times using the same organic solvent. How to put it on. 22. In step (2), the reaction product is dissolved in an aqueous solution or an alcohol-aqueous solution. or by another inorganic and/or 2. A process according to claim 1, characterized in that it is treated with an organic base at a temperature of 5 to 120<0>C. 23. The alkaline treatment step was carried out using a 0.1-1M sodium hydroxide aqueous solution for about 7 hours. At a temperature of 0°C, sufficient Claim 22, wherein the reaction product obtained in step (1) is How to put it on. 24. Neutralize the reaction product obtained in step (2) with an aqueous alkaline solution and do not mix with water. Isolated by extraction with organic solvents and dialysis against distilled water and sodium chloride. A method according to any of the preceding claims. 25. (1) Hydrogen with a molecular weight of 15,000 Daltons dissolved in N-methylpyrrolidone The tetrabutylammonium salt of parin was added to the 1-iodoocta Processed with (2) By pouring the obtained solution into methanol added with sodium acetate, (3) To the obtained product, sodium acetate was added. Purified by several reprecipitations from methanol; (4) dried; treating the product with a 0.5N aqueous sodium hydroxide solution at about 70° C. for about 2 hours; (5) Neutralize the resulting solution with 2M hydrochloric acid; (6) Neutralize this solution several times with methylene chloride. (7) Dialyze the solution against distilled water and 0.1M sodium chloride; and hand (8) Lyophilize the obtained solution; 1. A method for producing a low molecular weight heparin derivative and/or a salt thereof. 26. A low molecular weight hepamine produced by the method according to any one of claims 1 to 25. Phosphorus derivatives and their salts. 27. Low molecular weight heparin produced by the method according to any one of claims 1 to 25. alkali metal or alkaline earth metal salts of derivatives. 28. A heparin derivative produced according to any one of claims 1 to 25. Lium, potassium or calcium salts. 29. Treatment with heparin derivatives produced according to any of claims 1 to 25. Scientifically acceptable salts of bases. 30. Sodium and potassium heparin derivatives produced as described in claim 29 um or calcium salts. 31. A pharmaceutically acceptable low molecular weight heparin derivative according to claim 26 as an active ingredient. Pharmaceutical compositions containing together with acceptable carriers, diluents or excipients. 32. Alkali gold of the low molecular weight heparin derivative according to claim 26 as an active ingredient pharmaceutically acceptable carriers, diluents or excipients for salts of genus or alkaline earth metals A pharmaceutical composition containing the same. 33. Use of the low molecular weight heparin derivative according to claim 26 as a therapeutic drug. 34. Alkali metal or alkali of the low molecular weight heparin derivative according to claim 27 Use of salts of lithium earth metals as therapeutic drugs. 35. The low molecular weight heparin derivative or its salt according to claim 26 for thrombosis treatment. Use for. 36. The medicament according to claim 31 and claim 32 for use in the treatment of thrombosis. Composition.