JP2021155334A - Pharmaceutical composition containing middle-molecular-weight heparin or amino acid derivatives of middle-molecular-weight heparin - Google Patents

Abstract

To provide a pain therapeutic drug that prevents or treats pain, and further, has a very low risk of hemorrhage.SOLUTION: Provided is a pharmaceutical composition which is for preventing or treating pain and the anticoagulation action of which is suppressed, the composition containing, as an active ingredient, middle-molecular-weight heparin having an average molecular weight of 8500-9500, or amino acid derivatives of the middle-molecular-weight heparin.SELECTED DRAWING: None

Description

The present invention relates to a pharmaceutical composition for the prevention or treatment of pain, which comprises medium-molecular-weight heparin or an amino acid derivative of medium-molecular-weight heparin as an active ingredient.

Numbness and pain due to peripheral neuropathy are frequently seen as side effects of chemotherapy for cancer. The pain seen during this chemotherapy is classified as neuropathic pain and is intractable. Currently, pregabalin (trade name: Lyrica) has been approved as a treatment for neuropathic pain including peripheral neuropathy caused by anticancer drugs, but the response rate is not sufficient and side effects such as severe wobbling are common. In some cases, the onset of neuropathic pain may force the patient to discontinue chemotherapy or change the type of anticancer drug. For these reasons, the development of highly effective preventive and therapeutic agents for neuropathic pain is desired.

On the other hand, heparin has strong anticoagulant activity and is used for treatment of disseminated intravascular coagulation (DIC) and various thromboembolisms (venous thrombosis, myocardial infarction, pulmonary embolism, cerebral embolism). In addition to the treatment and prevention of (disease, limb artery thromboembolism, intraoperative / postoperative thrombosis, etc.), when using an extracorporeal circulation device such as hemodialysis / artificial cardiopulmonary, when inserting a vascular catheter, or during blood transfusion and blood test, etc. It is used to prevent blood coagulation in.

However, since heparin has a strong blood anticoagulant effect based on anti-Xa and antithrombin (IIa) activities, it has a serious side effect of exacerbating the bleeding tendency with the prolongation of blood coagulation time. It is also known that hypertriglyceridemia, increased free fatty acids, hypoHDLemia due to lipolytic action, and thrombocytopenia due to long-term continuous use.

As described above, heparin and other blood anticoagulants may cause serious side effects such as increased bleeding tendency, but in particular, blood when using an extracorporeal circulation device such as dialysis or heart-lung machine. Since it is an indispensable drug for preventing coagulation and there is no other suitable drug to be replaced, the current situation is that it must be used with some risk.

On the other hand, in addition to the blood anticoagulant activity described above, heparin has a lipoprotein lipase activating effect, an antiplatelet aggregation effect, a blood pressure lowering effect, an anticomplement effect, a cancer metastasis inhibitory effect, and a degranulation inhibitory effect from mast cells. It is known to have many physiological activities of. However, the bleeding tendency associated with blood anticoagulant activity was so strong that it could not be used for purposes other than blood anticoagulant purposes.

As a result of investigating a blood anticoagulant having few side effects such as bleeding tendency, the present inventors depolymerized heparin and collected a fraction of medium-molecular-weight heparin having an average molecular weight of 8,500 to 9,500. We have found that compounds obtained by derivatizing medium-molecular-weight heparin with amino acids achieve the purpose of reducing this bleeding tendency, and have already obtained patent rights for these substances (Patent Document 1).

In addition, the present inventors have added blood anticoagulant activity, renal mesangial cell growth inhibitory activity, cancer metastasis inhibitory activity, complement activation inhibitory activity, pulmonary edema inhibitory activity, and renal disease treatment to medium molecule heparin amino acid derivatives. It has been found to have an effect, a radical scavenger activity and an allergy-suppressing effect, and a patent right has already been obtained for a pharmaceutical composition containing these substances (Patent Document 1).

Patent No. 40477945

Here, we present that such medium-molecular-weight heparin and amino acid derivatives of medium-molecular-weight heparin have an effect of preventing or treating pain in addition to the above-mentioned effects, particularly neuropathic pain, and particularly an anticancer agent. We have found that it has an effect of preventing or treating neuropathic pain derived from the above, and have completed the present invention.

That is, the present invention uses medium-molecular-weight heparin having an average molecular weight of 8,500 to 9,500, or an amino acid derivative of the medium-molecular-weight heparin (hereinafter, may be referred to as "medium-molecular-weight heparinyl amino acid derivative") as an active ingredient. Provided is a pharmaceutical composition for the prevention or treatment of pain in which the blood anticoagulant effect is suppressed.

This is because the medium-molecular-weight heparinyl amino acid derivative of the present invention can prevent and treat pain, suppress blood anticoagulant action and reduce bleeding tendency without impairing various physiological activities originally possessed by heparin. , Myelosuppression reduces platelets and can be effectively used for numbness and pain caused by peripheral neuropathy, which is seen during cancer chemotherapy, which tends to cause bleeding tendency.

The graph which shows the inhibitory effect of the medium molecule heparin and the medium molecule parinylphenylalanine on the anticancer drug oxaliplatin-induced hyperalgesia is shown. The graph which shows the anti-Xa activity of the medium molecule heparin and the medium molecule heparinyl phenylalanine is shown. The graph which shows the anti-IIa activity of the medium molecule heparin and the medium molecule heparinyl phenylalanine is shown.

The present invention is a pharmaceutical composition for preventing or treating pain with suppressed blood anticoagulant action, which comprises a medium molecular weight heparin or a medium molecular weight heparinyl amino acid derivative having an average molecular weight of 8,500 to 9,500 as an active ingredient. Is to provide.

The medium-molecular-weight heparinyl amino acid derivative contained in the pharmaceutical composition of the present invention is a medium-molecular-weight heparin having an average molecular weight of 8,500 to 9,500, and a general formula:
NH ₂ CH (R ₁ ) COOR ₂
[In the formula, R ₁ is
・ H;
• Form a pyrrolidine ring with the N and α-position C of the α-amino group of the amino acid; or
_{-Selected from the group consisting of phenyl, OH, NH 2} , SH, SCH ₃ , COOH, CONH ₂ , guanidino (NH ₂ C (= NH) NH-), indrill and 1H-imidazolyl which may be substituted with OH. It is a lower alkyl that may be substituted with a group;
R ₂ is an H or lower alkyl] is a compound in which the amino acid derivative represented by] is amide-bonded between the amino group or carboxyl group of the medium-molecular-weight heparin and the carboxyl group or amino group of the amino acid derivative.

The amino acids of the medium molecular weight heparinyl amino acid derivatives of the present invention are preferably natural amino acids, or more preferably amino acids having an aromatic ring, particularly preferably phenylalanine and tyrosine.

The medium-molecular-weight heparinyl amino acid derivative of the present invention includes anticoagulant activity, renal mesangial cell growth inhibitory activity, cancer metastasis inhibitory activity, complement activation inhibitory activity, pulmonary edema inhibitory activity, renal disease inhibitory activity, and radical scavenger activity. It has been confirmed that it has an inhibitory effect on degranulation from mast cells.

The medium molecular weight heparin used in the present invention refers to a fraction of depolymerized heparin having an average molecular weight of 8,500 to 9,500. The minimum molecular weight is 4,500, the maximum molecular weight is 12,500, and the molecular weight is 5,000 to 10,000, which is 70% or more of the medium molecular weight heparin.

The amino acid used in the medium-molecular-weight heparinyl amino acid derivative of the present invention is not particularly limited as long as it is a compound having an amino group and a carboxyl group regardless of whether it is a synthetic product or a natural product, but glycine, alanine, valine, leucine and isoleucine are not particularly limited. , Phenylalanine, tyrosine, serine, threonine, cysteine, methionine, aspartic acid, aspartic acid, glutamic acid, glutamine, lysine, arginine, histidine, tryptophan, proline and other α-amino acids or derivatives thereof are preferably used. These amino acids may be D-form, L-form or DL-form.

The lower alkyl of the medium molecular weight heparinyl amino acid derivative of the present invention is a saturated linear or branched hydrocarbon residue containing 1 to 6, preferably 1 to 5, and more preferably 1 to 4 carbon atoms. Refers to the group. For example, methyl, ethyl, propyl, isopropyl, butyl, t-butyl and the like are included.
The compounds of the present invention generally exhibit physiological or pharmacological activity substantially similar to that of the free form in vivo, for example, derivatives of the compounds of the present invention and pharmaceutically acceptable salts, addition salts, hydrates. Things and the like are included in the technical scope of the present invention.

Production of medium-molecular-weight heparinyl amino acid derivative (1) Preparation of medium-molecular-weight heparin After depolymerizing ordinary heparin by conventional methods such as enzymatic decomposition method, nitrite decomposition method, and hydrogen peroxide decomposition method, for example, ultrafiltration and the like. It is fractionated using the fractionation means of the above, and consists of fractions having an average molecular weight of 8,500 to 9,500.

For example, heparin is dissolved in water, a cation exchange resin is added to adjust the pH to 3.30 to 3.40, and then filtration is performed. Add hydrogen peroxide solution to the filtrate and heat under pressure to depolymerize. After completion of heating, an aqueous sodium hydroxide solution is added to the reaction solution, and ultrafiltration is repeated to carry out molecular weight fractionation. Recrystallization with ethanol is performed to obtain medium-molecular-weight heparin.

(2) Preparation of Medium-Molecular Heparinyl Amino Acid Derivative After esterifying the carboxyl group of the above-mentioned medium-molecular-weight heparin with, for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, the above amino acid An ester is added to obtain a medium-molecular-weight heparinyl amino acid ester, which is further hydrolyzed under alkaline conditions to obtain a medium-molecular-weight heparinyl amino acid.
For example, medium-molecular-weight heparin is dissolved in water, adjusted to pH 4.75, and then the corresponding amino acid ester hydrochloride (molar ratio to medium-molecular-weight heparin, 1: 100) is added.

Further, an aqueous solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (molar ratio to medium-molecular-weight heparin, 1:50) was gradually added, and the mixture was stirred and reacted for about 4 hours, and then water and bromide were added. Add an aqueous hexadecyltrimethylammonium solution until no ammonium salt precipitates form.
Then, after separating this precipitate, an ethanol solution of sodium iodide is added to the precipitate. After stirring, the mixture is filtered and the precipitate is recrystallized from ethanol to obtain a white powder medium-molecular-weight heparinyl amino acid ester.

An aqueous sodium hydroxide solution is added to this ester, and the mixture is stirred at 0 to 5 ° C. for a long time under a nitrogen stream. The reaction mixture is adjusted to pH 5 with acetic acid, filtered, and ethanol is added to the filtrate to obtain a white powder of medium-molecular-weight heparinyl amino acid.

The pharmaceutical composition of the present invention contains a medium-molecular-weight heparin or a medium-molecular-weight heparinyl amino acid derivative as an active ingredient, is formulated by a usual method, and is administered as an injection, an oral preparation, an external preparation, a suppository, a transdermal absorbent, or the like. can do.
For example, it is administered by the following administration method, and the dose or administration rate is usually determined by age, case, and indication range while measuring whole blood coagulation time or whole blood activated partial thromboplastin time after administration of this drug. Alternatively, it is determined by the purpose.

For example, in the intravenous drip administration method, an amount corresponding to 5,000 to 50,000 heparin units of medium-molecular-weight heparin or medium-molecular-weight heparinyl amino acid derivative is diluted with 1,000 ml of 5% glucose injection solution, physiological saline solution or Ringer's solution. Then, it is intravenously administered at a rate of about 20 to 30 drops per minute.

In the intravenous intermittent injection method, an amount corresponding to 5,000 to 50,000 heparin units of medium-molecular-weight heparin or medium-molecular-weight heparinyl amino acid derivative is injected intravenously every 4 to 8 hours. In the subcutaneous injection / intramuscular injection method, a medium-molecular-weight heparinyl amino acid derivative in an amount corresponding to 5,000 to 10,000 heparin units is injected subcutaneously or intramuscularly every 4 hours.

For use during extracorporeal circulation (hemodialysis / artificial cardiopulmonary), the appropriate amount for each patient is calculated based on the results of the heparin susceptibility test before dialysis, but in the case of the systemic heparinization method, normal dialysis is started. Prior to this, a medium-molecular-weight heparinyl amino acid derivative corresponding to 1,000 to 3,000 heparin units was administered, and after the start of dialysis, an amount corresponding to 500 to 1,500 heparin units was continuously administered per hour. , Or an amount corresponding to 500 to 1,500 heparin units is added intermittently every hour. In the case of the local heparinization method, an amount corresponding to 1,500 to 2,500 heparin units is continuously injected per hour. In addition, at the time of artificial heart-lung machine perfusion, although it depends on the surgical method and method, an amount corresponding to 150 to 300 heparin units / kg is administered, and further administration is appropriately added according to the extension of the extracorporeal circulation time.

In the case of a pharmaceutical composition for oral administration, the amount of medium-molecular-weight heparin or medium-molecular-weight heparinyl amino acid derivative corresponding to 500 to 2,000 heparin units is taken one to several times a day.
In the case of a pharmaceutical composition for an external preparation, it is formulated as an ointment containing a medium-molecular-weight heparin or a medium-molecular-weight heparinyl amino acid derivative in an amount corresponding to 100 to 500 heparin units, and an appropriate amount is rubbed or rubbed once or several times a day. Spread it on gauze and attach it.
In the case of suppositories, an amount of medium-molecular-weight heparin or medium-molecular-weight heparinyl amino acid derivative corresponding to 1,000 to 4,000 heparin units is applied to the anus or vagina once or twice daily.
For transdermal absorbents, apply medium-molecular-weight heparin or medium-molecular-weight heparinyl amino acid derivative in an amount equivalent to 1,000 to 4,000 heparin units once or twice daily in the chest, upper abdomen, back, upper arm or large arm. Apply to the thigh.

Example 1
Inhibitory effect of medium-molecular-weight heparin and medium-molecular-weight heparinylphenylalanine on the anticancer drug oxaliplatin-induced allodynia (neuropathic pain) Experimental method The mechanical pain threshold was measured by the von Frey test using the up-down method. Eight kinds of filaments with different strengths of 0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6 and 1.0g are used, and the filament is light on the sole of the mouse. The mice were pressed for 6 seconds to bend and stimulated, and it was observed whether or not the mice showed an escape reaction. Stimulation with the filament was started with the one with the lowest intensity, and if there was no response to the stimulus, the stimulation with one stronger filament was used again. If a reaction was observed, it was stimulated with one weak intensity at intervals of 30 seconds. The measurement was performed 5 times from the place where the reaction occurred for the first time, and the 50% threshold value was calculated.
Medium-molecular-weight heparin (MMWH) and medium-molecular-weight heparinylphenylalanine (MMWH-F) were intraperitoneally administered at a dose of 2.5 mg / kg, and oxaliplatin (OHP) was intraperitoneally administered 1 hour later. The pain threshold was measured from 2 hours after OHP administration to 6 hours every hour, and then on days 1, 3, 5 and 7.
The results are shown in FIG.

As shown in FIG. 1, intraperitoneal administration of OHP (− ● −) lowered the pain threshold from 2 hours after administration and maintained a low value until 7 days after administration. Premedication of 2.5 mg / kg of MMWH (-□-) suppressed this decrease in pain threshold. On the other hand, the pre-administration of 2.5 mg / kg of MMWH-F (-■-) strongly suppressed the decrease in the pain threshold, and the action was maintained until 7 days after the intraperitoneal administration of OHP.

Example 2
Inhibitory action of blood coagulation factors Xa and IIa (thrombin) of unfractionated heparin, medium-molecular-weight heparin and medium-molecular-weight heparinylphenylalanine (anti-Xa and anti-IIa activity)
Experimental method The anti-Xa and anti-IIa activities of unfractionated heparin, medium-molecular-weight heparin, and medium-molecular-weight heparinylphenylalanine were measured by the synthetic substrate method. The results are shown in FIGS. 2 and 3, respectively.
As shown in FIG. 2, the anti-Xa activity of MMWH (− □ −) tended to be lower than that of unfractionated heparin (HE, − ○ −). In addition, MMWH-F (-■-) showed much lower anti-Xa activity as compared with HE and MMWH.
Further, as shown in FIG. 3, MMWH (-□-) tends to have lower anti-IIa activity as compared with HE (-○-), and MMWH-F (-■-) has anti-IIa activity. Was not shown at all.

Blood coagulation is an important mechanism for stopping bleeding by producing fibrin by a chain reaction of many coagulation factors. Inhibition of any blood coagulation factor can suppress blood coagulation, but it is known that direct inhibition of IIa increases the risk of bleeding. The main target molecules for the anticoagulant effect of heparin are Xa and IIa, but the anti-Xa / anti-IIa activity ratio is used as an index showing the relationship between the anticoagulant effect and the risk of bleeding. In general, the anti-Xa / anti-IIa activity ratio of unfractionated heparin is 1/1, whereas MMWH-F has anti-Xa activity but almost no anti-IIa activity (10, in this experiment). 000 μg / mL or more). From this, it is considered that MMWH-F has a blood anticoagulant effect but has a low risk of bleeding.
From these experimental results, MMWH and MMWH-F suppress the decrease in pain threshold (allodynia) induced by administration of the anticancer drug oxaliplatin to mice, and the anti-IIa activity is significantly attenuated. Therefore, it was shown that it is a therapeutic drug for neuropathic pain with an extremely low risk of bleeding.

The pharmaceutical composition of the present invention has industrial applicability as a pain therapeutic agent having an extremely low risk of bleeding.

Claims (6)

A pharmaceutical composition for preventing or treating pain with suppressed anticoagulant action, which comprises medium-molecular-weight heparin having an average molecular weight of 8,500 to 9,500 or an amino acid derivative of the medium-molecular-weight heparin as an active ingredient. Amino acid derivatives of medium-molecular-weight heparin are medium-molecular-weight heparin having an average molecular weight of 8,500 to 9,500, and the general formula: NH ₂ CH (R ₁ ) COOR _2.
[In the formula, R ₁ is
・ H;
• Form a pyrrolidine ring with the N and α-position C of the α-amino group of the amino acid; or
• With a lower alkyl optionally substituted with a group selected from the group consisting of _{phenyl, OH, NH 2} , SH, SCH ₃ , COOH, CONH ₂ , guanidino, indolyl and 1H-imidazolyl which may be substituted with OH. The pharmaceutical composition for the prevention or treatment of pain according to claim 1, wherein R ₂ is a compound formed by amide-bonding an amino acid derivative represented by H or a lower alkyl to a carboxyl group. The pharmaceutical composition for the prevention or treatment of pain according to claim 2, wherein the amino acid derivative of the medium molecule heparin is parinylphenylalanine to the medium molecule or parinylphenylalanine alkyl ester to the medium molecule. The pharmaceutical composition for preventing or treating pain according to any one of claims 1 to 3, wherein the pain is neuropathic pain. The pharmaceutical composition for the prevention or treatment of pain according to claim 4, wherein the pain is pain derived from an anticancer drug. The pharmaceutical composition for the prevention or treatment of pain according to claim 5, wherein the pain is oxaliplatin-derived pain.

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