JPH09286803A - Medium-molecular heparin, amino acid derivative, and medicine composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain medium-molecular heparin which gives an amino acid deriv. effectively usable for various diseases reduced in bleeding tendency by depolymerizing heparin to a specified average mol.wt. SOLUTION: This medium-molecular heparin has an average mol.wt. of 8,500-9,500 and, when converted into an amino acid deriv., gives a medium- molecular heparinylamino acid deriv., more specifically a medium-molecular heparinylamino acid or its lower alkyl ester. The deriv. is used as the active component for compsns. for coagulation inhibition, nephric mesangial cell proliferation inhibition, cancer metastasis inhibition, complement activation inhibition, pulmonary inhibition, nephric disease inhibition, radical scavenging, allergic disease prevention, and a remedial medicine.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an amino acid derivative of medium-molecule heparin. More specifically, it relates to medium molecular weight heparinyl-amino acids or -amino acid lower alkyl esters. The present invention also relates to a pharmaceutical composition containing an amino acid derivative of medium-molecule heparin as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Heparin is a heteropolysaccharide having a complicated structure which exists in the liver, small intestine, lung, skin and the like, and is an acidic mucopolysaccharide having a molecular weight of 4,000 to 20,000. That is, the sugar chain is D-glucosamine,
It contains D-glucuronic acid, and the amino group of glucosamine is partially sulfated. It has a strong blood anticoagulant activity, and it has a generalized intravascular blood coagulation syndrome (D
IC), various thromboembolisms (venous thrombosis, myocardial infarction, pulmonary embolism, cerebral embolism, limb arterial thromboembolism, intraoperative)
It is used for the treatment and prevention of postoperative thromboembolism, etc., as well as for the prevention of blood coagulation during the use of extracorporeal circulation devices such as hemodialysis and artificial heart-lung machines, insertion of vascular catheters, and blood transfusions and blood tests. .

However, since heparin has a strong blood anticoagulant activity based on its anti-Xa activity, it has a serious side effect of exacerbating the bleeding tendency associated with the prolongation of blood coagulation time.
It is also known that hypertriglyceridemia and increase in free fatty acids due to lipolytic action, hypoHDLemia, or development of thrombocytopenia due to long-term continuous use.

On the other hand, proteolytic enzyme inhibitors such as gabexate mesylate and nafamostat mesylate are also used for the prevention of coagulation of perfused blood and the treatment of DIC during extracorporeal blood circulation, but these drugs are metabolized. The product guanidine compound causes gastrointestinal disorders. Furthermore, since the molecular weight is as small as 539 dalton and it is dialyzed out from the dialyzer, coagulation occurs in the circuit, especially in the venous drip chamber after the dialyzer.

In recent years, low molecular weight heparin (dalteparin sodium), which is a low molecular weight fraction having a molecular weight of about 5,000 daltons or less obtained by depolymerization of heparin, has been added to the inhibitory activity against blood coagulation factor X and prolongation of activated partial thromboplastin time. An increased rate of action has been confirmed and is being used as a blood anticoagulant with a relatively low bleeding tendency. However, even this low-molecular-weight heparin preparation has not been able to completely improve the above-mentioned side effects, and it has been recognized that osteoporosis occurs at a higher frequency than heparin when used at a high dose.

Heparinylglycine, which is a compound of heparin and glycine, has been reported to be a blood anticoagulant having less bleeding tendency, but this also reduces blood anticoagulant activity. Due to the points, it has not been put to practical use.

As described above, the above blood anticoagulants such as heparin may cause serious side effects such as an increase in bleeding tendency, but in particular, extracorporeal circulation such as hemodialysis and artificial heart lung. It is a drug that is indispensable for preventing blood coagulation when using the device, and since there are no other suitable drugs to substitute, be aware of some risks,
It was the current situation that I had no choice but to use it.

On the other hand, heparin, in addition to the aforementioned blood anticoagulant activity, lipoprotein lipase activation activity, antiplatelet aggregation activity, blood pressure lowering activity, anticomplement activity, cancer metastasis inhibitory activity and degranulation inhibition from mast cells. It is known to have many physiological activities such as action. However, the bleeding tendency associated with blood anticoagulant activity is so strong that it cannot be used for purposes other than blood anticoagulation.

The present inventors have studied blood anticoagulants having few side effects such as bleeding tendency, and as a result, depolymerized heparin to collect a fraction of medium molecular weight heparin having an average molecular weight of 8,500 to 9,500. Furthermore, they have found that a compound obtained by derivatizing this medium-molecule heparin with an amino acid achieves this object, and completed the present invention. In addition, the medium-molecular-weight heparinyl amino acid derivative of the present invention can reduce the bleeding tendency without impairing various physiological activities originally possessed by heparin, and thus cannot be conventionally applied due to a serious side effect. It can be effectively used for various diseases.

[0010]

The first aspect of the present invention is a medium molecular heparin having an average molecular weight of 8,500 to 9,500.

The second aspect of the present invention is a medium molecular weight heparinyl amino acid derivative derived from the above medium molecular weight heparin, and more specifically, a medium molecular weight heparinyl amino acid or a medium molecular weight heparinyl amino acid lower alkyl ester.

More specifically, the medium molecular weight heparinyl amino acid derivative is represented by the general formula: NH ₂ CH (R ₁ ) COOR ₂ [wherein R ₁ is H; N and α of the α-amino group of the amino acid. Form a pyrrolidine ring with C at position; or
Phenyl optionally substituted with OH, OH, NH ₂ ,
SH, SCH ₃ , COOH, CONH ₂ , guanidino (N
_{H 2 C (= NH) NH-} ), optionally substituted with a group selected from the group consisting of indolyl and 1H- imidazolyl is also lower alkyl, R ₂ is a compound represented by H or lower alkyl] Is.

The amino acids of the present invention are preferably natural amino acids or more preferably essential amino acids.

The medium-molecular-weight heparinyl amino acid derivative of the present invention includes anticoagulant activity, renal mesangial cell proliferation inhibitory activity, cancer metastasis inhibitory activity, complement activation inhibitory activity, pulmonary edema inhibitory activity, renal disease inhibitory activity, radical scavenger. Since it has been confirmed that the compound has an activity and an inhibitory effect on degranulation from mast cells, the third aspect of the present invention is a pharmaceutical composition having these activities, which contains a medium molecule heparinyl amino acid derivative.

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 and the maximum molecular weight is 12,500, and the molecular weight of 5,000 to 10,000 is 70% or more of the medium molecular heparin.

The amino acid used in 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. Glycine, alanine, valine, leucine, isoleucine, phenylalanine and tyrosine are used. Α-amino acids such as serine, threonine, cysteine, methionine, aspartic acid, asparagine, glutamic acid, glutamine, lysine, arginine, histidine, tryptophan and proline, or derivatives thereof are preferably used. These amino acids are D
It may be a body, L body or DL body. The lower alkyl of the present invention means a saturated linear or branched carbon atom having 1 to 1 carbon atoms.
It refers to a hydrocarbon residue containing 6, preferably 1 to 5, and more preferably 1 to 4. For example, methyl, ethyl, propyl, isopropyl, butyl, t-butyl and the like are included. The compound of the present invention generally exhibits substantially the same physiological activity or pharmacological activity as the free form in vivo, for example, a derivative of the compound of the present invention and a pharmaceutically acceptable salt, addition salt or hydrate thereof. Objects 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 depolymerization of ordinary heparin by a conventional method such as enzymatic decomposition method, nitrous acid decomposition method, hydrogen peroxide decomposition method, for example, It is fractionated using a fractionation means such as external filtration, and is composed 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 filtered. Hydrogen peroxide solution is added to the filtrate and heated under pressure to depolymerize. After heating, sodium hydroxide aqueous 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-weight heparinyl amino acid derivative The above-mentioned medium-molecular-weight heparin is used, for example, in 1-ethyl-3-
After esterifying the carboxyl group with (3-dimethylaminopropyl) carbodiimide hydrochloride, the ester form of the above amino acid is added to give a medium molecular weight heparinyl amino acid ester, which is further hydrolyzed under alkaline conditions to give a medium molecular weight heparinyl. Get an amino acid. For example, medium molecular heparin is dissolved in water and adjusted to pH 4.75, and then the corresponding amino acid ester hydrochloride (molar ratio to medium molecular heparin, 1:
100) is added.

Further, an aqueous solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (molar ratio to medium-molecular heparin, 1:50) was gradually added,
After stirring and reacting for about 4 hours, water and an aqueous solution of hexadecyltrimethylammonium bromide are added until precipitation of ammonium salt does not occur. Then, after separating the precipitate, a sodium iodide solution in ethanol is added to the precipitate. After stirring, the mixture is filtered and the precipitate is recrystallized from ethanol to obtain a white powder of medium molecular weight heparinyl amino acid ester.

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

The thus-obtained medium-molecular-weight heparinyl amino acid derivative can be formulated by a usual method and administered as an injection or an oral preparation. For example, it is administered by the following administration method, but the dose or administration rate is usually determined by measuring the whole blood coagulation time or whole blood activated partial thromboplastin time after administration of this drug, and the age, case, or indication range. Or 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 a 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. An intravenous drip is administered at a rate of about 20 to 30 drops per minute. In addition, in the intravenous intermittent injection method, an amount corresponding to 5,000 to 50,000 heparin units of the medium molecule heparinyl amino acid derivative is intravenously injected every 4 to 8 hours. In the subcutaneous injection / intramuscular injection method, a medium-molecular-weight heparinyl amino acid derivative equivalent to 5,000 to 10,000 heparin units is subcutaneously or intramuscularly injected every 4 hours.

In the use during extracorporeal circulation (hemodialysis / artificial heart lung), the appropriate dose for each patient in the artificial kidney is calculated based on the result of heparin sensitivity test before dialysis, but in the case of whole body heparinization method, Usually, prior to the start of dialysis, a medium molecular weight heparinyl amino acid derivative corresponding to 1,000 to 3,000 heparin units is administered, and after the start of dialysis, an amount equivalent to 500 to 1,500 heparin units per hour is administered. 500 to 1.5 continuously or hourly
An amount corresponding to 00 heparin units is intermittently added. In the case of the local heparinization method, 1,500 to 2, per hour,
A continuous infusion of 500 heparin units is infused. At the time of artificial cardiopulmonary perfusion, an amount equivalent to 150 to 300 heparin units / kg is administered, which is different depending on the operation method / method, and further additional administration is appropriately performed depending on the extension of the extracorporeal circulation time.

In the case of oral administration, a medium-molecular-weight heparinyl amino acid derivative corresponding to 500 to 2,000 heparin units / g is taken once to several times a day. 1 for topical agents
It is used as an ointment of a medium-molecular-weight heparinyl amino acid derivative corresponding to 00 to 500 heparin units / g, and an appropriate amount is applied to the gauze or the like by rubbing it once or several times a day.

In the case of suppositories, the medium molecular weight heparinyl amino acid derivative corresponding to 1,000 to 4,000 heparin units / g is applied to the anus or vagina once or twice a day.

Pharmacological Experiments I. In Vitro APTT Measurement In order to examine the effect of reducing the bleeding tendency of the medium-molecule heparinylamino acid derivative of the present invention, the effect of each compound on the activated partial thromboplastin time (APTT) was measured. Normal human plasma (Coagulation Control Plasma)
(Normal) Level 1: Pacific Hemostasis) 1 volume of aqueous solution of heparin, medium molecular weight heparin and medium molecular weight heparinyl amino acid derivative prepared at various concentrations is added to 9 volume to prepare a test plasma. Separately, take 1 volume of PBS (-),
Operate in the same manner as the test plasma, and use it as the control plasma. After adding the activated partial thromboplastin reagent (APTT Test Wako: Wako Pure Chemical Industries) and calcium chloride solution to the test plasma and the control plasma, the plasma coagulation time automatic measuring device (A
The APTT of each plasma was measured using a melung-coagulometer). The results are shown in Table 1. AP in normal human plasma
The TT prolonging action was milder than that of heparin in all the compounds, and it was revealed that the bleeding tendency was reduced.

II. In vitro measurement of anti-Xa activity In order to investigate the blood anticoagulant activity of the medium-molecule heparinylamino acid derivative of the present invention, the anti-Xa activity of each compound was measured,
Comparison was made with heparin, low molecular weight heparin and low molecular weight heparinyl amino acid derivatives. The low-molecular-weight heparin is obtained by depolymerizing heparin by a conventional method, and the low-molecular-weight heparinyl amino acid derivative is produced from this low-molecular-weight heparin as a raw material.
It was produced according to the above-mentioned method for preparing a medium molecule heparinyl amino acid derivative. Normal human plasma (Coagulation Control Plas
ma (Normal) Level 1: Pacific Hemostasis) 2 volumes of heparin, medium molecular weight heparin and medium molecular weight heparinyl amino acid derivative aqueous solution prepared at various concentrations were added, and a buffer solution (50 mM 2-amino-2- Add 6 volumes of hydroxyl-1,3-propanediol, pH 8.4) to make the test plasma.

After adding factor Xa, a substrate solution and a reaction stop solution to the test plasma, the remaining factor Xa was measured from the absorbance at 405 nm. The results are shown in Table 1. From the results of APTT and anti-Xa activity, the anti-Xa / APTT activity ratio, which is an index of blood anticoagulant ability, was determined. The results are shown in Table 1. It was revealed that by depolymerizing heparin, selecting a medium molecular fraction, and using it as an amino acid derivative, the serious bleeding tendency can be reduced while maintaining blood anticoagulant activity.

[0030]

[Table 1] APTT, anti-Xa activity and anti-Xa / APTT activity ratio of medium molecular heparin and medium heparinyl amino acid APTT anti-Xa activity anti-Xa / APTT ratio compound dose heparin ratio IC50 1 / heparin ratio (μg / ml ) * (μg / ml) Heparin 4.79 1.00 0.69 1.00 1.00 Low molecular weight heparin 65.0 13.57 2.842
0.2442 3.31 Low molecular weight heparinyl arginine 2000.0 417.54
6000 0.0001 0.05 low molecular heparin Riniruasuha ° Raginsan 516.7 107.87 11 138 0.0001 0.01 low molecular heparin Li sulfonyl phenylalanine 303.6 63.38 609.36 0.0011 0.07 low molecular heparin Rinirufu Bu Lorin 650.0 135.70 106.24 0.0065 0.89 low molecular heparin Riniruserin 240.0 50.10 59.09 0.012 0.59 in molecular weight heparin 14.79 3.09 1.23 0.562 1.74 Medium molecular heparinylglycine 53.70 11.21 18.88 0.037 0.41 Medium molecular heparinylalanine 30.90 6.45 4.38 0.159 1.02 Medium molecular heparinyl alkyne 85.11 17.77 27.61 0.025 0.45 Medium molecular heparinyl asparachiral acid 31.56 0.07 Medium 63.21 0.06 0.07 63.21 Histidine 57.54 12.01 26.37 0.026 0.32 Medium molecule Heparinyl leucine 35.96 7.51 8.71 0.080 0.60 Medium molecule Heparinyl methionine 35.48 7.41 24.61 0.028 0.21 Medium molecule Heparinyl phenylalanine 40.27 8.41 30.98 0.022 0.19 Medium molecule Heparinylproline 28.18 5.88 1.91 0.363 2.14 Medium molecule Heparinylserine 26.86 5.61 10.96 0.063 0.36 Medium molecule Heparinyl Tyrosine 38.90 8.12 8.34 0.083 0.68 * Concentration that prolongs coagulation time to 100 seconds

Regarding APTT which is an index of side effects,
The concentrations of the medium-molecular-weight heparinyl amino acid derivatives of the present invention, which extended the blood coagulation time to 100 seconds, were 6 to 17 times that of heparin, and a clear reduction in side effects was recognized as compared with heparin. In addition, low molecular weight heparin (1
In comparison with 3.57), it was confirmed that side effects were reduced to the same extent or higher. Conventionally, the anti-Xa / APTT activity ratio has been used as an index of the blood anticoagulant action of low-molecular-weight heparin. The higher this ratio, the weaker the bleeding tendency and the stronger the blood anticoagulant ability (Shinji Naganuma et al. Medical Journal, 27, 55, 1991). As for the anti-Xa / APTT activity ratio when normal heparin was set to 1, medium-molecule heparinylproline showed high activity of 2.14, and also showed activity equal to or higher than that of low-molecular-weight heparinyl amino acid. It was

III. Growth inhibitory effect of medium-molecular-weight heparinyl amino acid on renal mesangial cells Since the inhibitory effect on proliferation of renal mesangial cells has been reported as one of the pharmacological actions of heparin in recent years (Cas
tellot, JJ et al., American Journal of
Pathology (Am. J. Pathol.), Volume 120, 427
(1989, 1985), heparin was examined for its inhibitory effect on renal mesangial cell proliferation, of medium molecular weight heparinyl amino acids, medium molecular weight heparinylphenylalanine was used as a test drug, and heparin was used as a control.

(1) Culture of glomerulus and renal mesangial cells A method of continuously using different types of sieves for the glomerulus (sievin
g method). Wistar weighing about 150g
Male male rats (Charles River Japan) were anesthetized with ether, the back was shaved, the carotid artery was cut, and the blood was exsanguinated. After the exsanguination, the rat was placed in a clean bench, the kidney was removed from the back side, and the rat was stored in a petri dish filled with PBS (-) (phosphate buffer solution containing no calcium and magnesium). The kidney capsule was peeled off and the renal cortex was minced. A metal sieve (sieve size 355 μm, 125
μm, 75 μm in this order) and passed through a sieve while crushing with a spatula, and the glomeruli remaining on the sieve having a sieve size of 75 μm were collected and suspended in PBS (−). This suspension is
The mixture was centrifuged at 000 rpm for 5 minutes, the supernatant was removed, and the obtained glomeruli were treated with 0.2% trypsin solution at 37 ° C for 20 minutes.
Furthermore, it was incubated at 37 ° C. for 40 minutes with 0.1% collagenase solution. Then, wash the glomerulus, 20%
The cells were suspended in RPMI 1640 culture solution (manufactured by Nissui Pharmaceutical Co., Ltd.) containing fetal bovine serum (manufactured by Biowhittaker) and 0.66 U / ml of insulin, and cultured at 37 ° C. in the presence of 5% carbon dioxide. The culture medium was exchanged on the 7th day of culture, and after that, 2-3
The culture medium was exchanged at a frequency of once / week. On day 21 of culture, renal mesangial cells that had grown around the glomerulus were detached from the culture flask with a trypsin-EDTA solution and subcultured. In this example, the 3rd to 10th passage cells were used.

(2) Effect of medium molecular weight heparinyl phenylalanine on the proliferation of renal mesangial cells Renal mesangial cells were added to a culture solution containing 20% fetal bovine serum, and a 24-well microplate (made by Corning).
For 24 cells under the condition of 5-8 × 10 ³ cells / well.
Cultured for hours. Further, the medium was replaced with 0.4% fetal calf serum-added culture medium and cultured for 3 days, and then the medium was replaced with 10% fetal bovine serum-added culture medium and a test drug or a control drug was added. Four
After a day, the cells were detached with a trypsin-EDTA solution, and Cou
The cell number was measured using an lter Counter (manufactured by Nikkaki). The amount of each drug added was 1/10 volume of the total volume. The inhibitory effect on renal mesangial cell proliferation was evaluated by the control rate shown in the following formula.

[Equation 1]

As a result of the experiment, the control heparin was 1-100.
At a dose of μg / ml, renal mesangial cell proliferation was suppressed by 40.5 to 83.5% in a dose-dependent manner. On the other hand, the inhibition rate of 100 μg / ml of medium-molecule heparinylphenylalanine was 95.0%, and the effect of inhibiting the proliferation of renal mesangial cells was confirmed.

IV. Cancer metastasis inhibitory effect of medium-molecule heparinyl amino acid Since heparin glycine, a glycine derivative of heparin, has been reported to have cancer metastasis inhibitory effect (Villa
nueva, GB et al., Anals New York Academy of Sciences (Ann.NY.Acad.Sc)
i.), 556, 496, 1989), the in vitro chemoinvasion assay method and the experimental tumor-lung metastasis system of mice were used to control the cancer metastasis of the medium-molecular-weight heparinylamino acid derivative with heparin as a control. It examined using.

(1) Cancer cells Fidler method (Fidler, IJ, Nature
e), Vol. 242, p. 148, 1973), and highly metastatic lung B16 mouse melanoma cells (B16).
-F7 or F10) was used. The cells cultured in F12 / DME (1: 1) culture medium containing 5% fetal bovine serum at 37 ° C. in the presence of 5% carbon dioxide were used for the experiment.

(2) Experimental Animal Male C57BL / 6NCrj mice (7 days old, Charles River Japan) were used.

(3) Examination of Cancer Metastasis Inhibitory Effect Manipulation of Chemoinvasion Inducing Factor 3T3 cells in a sub-confluent state were serum-free F1.
The culture supernatant after culturing in 2 / DME (1: 1) medium in a 37 ° C. carbon dioxide incubator for 24 hours was serum-free F12.
A 2-fold dilution with a / DME (1: 1) medium was used as a chemoinduction factor.

In Vitro Chemo-Invasion Method 700 μl of the chemo-invitation-inducing factor prepared above was placed in a 24-well plate well, and the inside of the chamber was adjusted to 0.
5 × 10 ⁴ B16-F7 cells (200 μl) suspended in F12 / DME (1: 1) medium containing 1% BSA were added. A medium-molecular-weight heparinyl amino acid derivative was added to the chamber at 500 μg / ml, the 24-well plate was covered, and then left standing in a carbon dioxide gas incubator for 24 hours. After that, the chamber was taken out, B16-F7 cells invading the lower surface of the membrane in the chamber were stained with Giemsa, and the membrane was cut out and mounted on a slide glass. After completion of mounting, B16-F7 cells were examined under a microscope (×
200), using a Miller ocular disc, 0.202.
The number of infiltrating cells per 5 mm ² was counted.

Results The results are shown in Table 2. As can be seen from the table, medium heparin and medium heparinyl amino acids reduced the number of invasive cancer cells except for methionine and phenylalanine derivatives. Especially for medium molecular heparin, aspartic acid derivatives, serine derivatives and arginine derivatives,
It was confirmed that the effect of suppressing the number of invasive cancer cells was higher than that of heparin.

[0042]

[Table 2] Compounds inhibiting invasion of B16-F7 cells Number of infiltrating cells (%) Mean ± SE (N) No addition 100 Heparin 76 ± 11 (6) Medium molecular heparin 65 ± 11 (6) Medium molecular heparinyl aspartic acid 61 ± 16 (6) Medium molecule heparinyl serine 67 ± 16 (3) Medium molecule heparinyl arginine 72 ± 6 (6) Medium molecule heparinyl histidine 81 ± 26 (3) Medium molecule heparinyl tyrosine 85 ± 24 (3) Medium molecule Heparinyl proline 90 ± 22 (6) Medium molecule heparinyl leucine 93 ± 16 (3) Medium molecule heparinyl glycine 97 ± 5 (3) Medium molecule heparinyl alanine 97 ± 17 (3) Medium molecule heparinyl methionine 104 ± 8 (3) Medium molecule heparinyl phenylalanine 133 ± 7 (3)

(4) Examination of Cancer Metastasis Inhibitory Effect of Medium Molecular Weight Heparinyl Aspartic Acid and Medium Molecular Weight Heparinyl Serine Further investigation was conducted on medium molecular weight heparinyl aspartic acid and medium molecular weight heparinyl serine. B16-F10
Cancer cells (1 × 10 ⁵ cells / mouse) were used as control drugs (10, 3
0, 100 μg / mouse) or test drug (100, 3
(00, 1000 μg / mouse) simultaneously with administration into the tail vein of the mouse, and 20 days later, the lung was excised and the number of lung nodules due to cancer cell engraftment was measured. A control group and an untreated normal group that were administered with the same amount of cancer cells and the same volume of solvent alone were set and compared. The number of cases in one group was 6 to 7. In addition, the same amount of control drug or test drug was administered into the tail vein of the mouse, and 1 hour later, blood was collected from the abdominal aorta under Nembutal anesthesia (3.8% sodium citrate solution: blood = 1: 9).
Then, a plasma sample was obtained after centrifugation. A reagent is added to this plasma sample, and an Amelung-Coagulometer KC1 is added.
APTT was measured using 0A (made by Amelung). As a control, a group was prepared in which the same volume (0.1 ml) of PBS (−) (solvent) was administered. The number of cases in one group was 3 or 5.

Table 3 shows the measurement results of the number of lung melanoma nodules and the APTT values at the doses. Medium molecule heparinyl aspartic acid and medium molecule heparinyl serine are
A statistically significant (p <0.01) inhibitory effect on nodule formation was shown. Judging from the APTT value, heparin cannot be applied as a cancer metastasis suppressor due to the side effect of promoting bleeding, whereas the medium molecular weight heparinyl amino acid reduces the bleeding tendency. Clinical application as an inhibitor can be expected.

[0045]

[Table 3] APTT value of medium molecular weight heparinyl amino acid and compound for suppressing increase in pulmonary melanoma nodule compound APTT (sec) Pulmonary melanoma nodule number (% decrease relative to control) Mean ± SD (N) Mean ± SD (N) No treatment Normal group 0 ± 0 (6) ** Control 23.7 ± 1.9 (3) 271 ± 77 (7) 10 μg / mouse 22.9 ± 1.2 205 ± 49 (7) [24] Heparin 30 μg / mouse 180 <111 ± 36 (7) ** [59] 100 μg / mouse 180 <49 ± 20 (7) ** [82] Medium molecule 100 μg / mouse 24.3 ± 2.3 (3) 164 ± 62 (7) [39] Heparinil 300 μg / mouse 27.9 ± 0.7 (3 ) 170 ± 20 (7) [37] Asparagine 1000 μg / mouse 32.4 ± 2.4 (3) 118 ± 30 (7) ** [56] Medium molecule 100 μg / mouse 21.9 ± 1.2 (3) 202 ± 98 (7) [25 ] Heparinil 300 μg / mouse 29.2 ± 0.9 (3) 158 ± 81 (7) [42] Serine 1000 μg / mouse 61.4 ± 11.3 (5) 111 ± 44 (7) ** [59] ** Hazard 1 against control There is a significant difference below% (by the Tukey method)

V. Inhibitory effect of medium molecular weight heparinyl amino acid on complement activation.
Nafamostat mesylate was used as a positive control with the hemolysis reaction of sensitized sheep erythrocytes based on activation of the sical pathway as an index. The test drug was dissolved in gelatin veronal buffer (GVB) in the in vitro test and in physiological saline in the in vivo test.

A 6-week-old male Hartley guinea pig (Charles River Japan) was purchased, and a 12-hour light-dark cycle (light period: 7:00 to 19:00) at a temperature of 23 ± 2 ° C. and a humidity of 50 ± 5%. After preliminarily breeding for 1 week under the conditions, it was subjected to an experiment. During the preliminary breeding period until the day of the experiment, you can eat and drink freely.
Watered.

(2) Examination of inhibitory effect on complement activation In vitro guinea pig complement classical pathway
Effects on the hemolytic response of sensitized sheep red blood cells by activation

Procedure: Sensitized sheep red blood cells (Denka biopsy) are suspended in GVB so that the concentration becomes 5 × 10 ⁸ cells / ml. This liquid 200
Complement (dry complement, Denka biopsy) dissolved and diluted with GVB was added to μl, and GVB was further added to make a total volume of 1.5 ml. After reacting for 1 hour in a constant temperature bath at 37 ° C, 300
Centrifugation was performed at 0 rpm for 10 minutes, and the absorbance value of the supernatant hemoglobin amount at a measurement wavelength of 542 nm was measured (UVID
EC-77Σ). At the same time, the absorbance values of a sample to which GVB was added instead of complement as physical hemolysis and a sample to which pure water was added instead of complement and GVB as 100% hemolysis were measured. The action of each test drug was examined under the condition that the hemolysis rate due to activation of complement was about 50%. The amount of the test drug added was 1/100 of the total volume.

Results Heparin was 9.6-8 at a dose of 2-50 μg / ml.
It has a hemolytic inhibitory effect of 3.4% and an IC50 value of 20.
It was 1 μg / ml. Similarly for medium-molecular heparin,
At the dose of 400 μg / ml, it showed 10 to 97.8% hemolytic inhibitory effect, but its IC50 value was 66.8 μg / ml.
It was about 3.3 times that of heparin and a decrease in the action was observed. However, when phenylalanine was introduced into the medium molecular heparin, the IC50 value was 0.0 of that of the medium molecular heparin.
It was 9 times as much as 6.1 μg / ml, and when tyrosine was introduced, the IC50 value was 0.34 times as much as that of medium molecular heparin, 21.4 μg / ml, and the hemolytic inhibitory effect was enhanced compared with medium molecular heparin. (Table 4).

Guinea pig complement classic in vivo
Effect on the hemolytic reaction of sensitized sheep erythrocytes by activation of al pathway Operation 1 ml of sensitized sheep erythrocyte suspension of 10 ¹⁰ cells / ml was intravenously administered to the guinea pig's back leg vein, and 3 minutes later, to 0.5 ml from the external jugular vein. Blood was collected. Immediately, add 4.5 ml of 0.01% EDTA-Saline to the blood, and add 20
Centrifugation was performed at 00 rpm for 10 minutes, and the absorbance value of the supernatant hemoglobin amount at a measurement wavelength of 542 nm was measured. The test drug was intravenously administered through the dorsal leg vein 1 minute before the administration of sensitized sheep red blood cells.

[0052]

[Table 4] Inhibitory effect of medium-molecule heparinyl amino acid on sensitized sheep erythrocyte hemolysis by activation of complement classical pathway in vitro Inhibitory effect of complement activation on complement activation Concentration to extend APTT to 100 seconds IC50 (μg / ml) (μg / Ml ) Amino acid Heparin Heparin Heparin Heparin Heparin Heparin Medium Small molecule Small medium Small molecule No small molecule 47.9 147.9 650.0 20.1 66.8 208.0 Glycine 104.7 537.0-35.2 157.6-Aspartic acid 144.5 315.6-25.9 79.2-Phenylalanine 239.9 402.7 3036.0 2.9 6.1 55.8 Alanine 129.9 − 45.8 150.9 − Methionine 153.1 354.8 − 26.4 71.9 − Arginine 245.5 851.1 − 56.1 166.4 − Tyrosine 323.6 389.0 − 9.2 21.4 − Histidine 288.4 575.4 − 64.9 191.5 − Proline 117.5 281.8 − 46.5 80.5 − Serine − 268.6 − − 119.4 − Leucine − 359.6 − − 78.6 −

Results The positive control nafamostat mesylate was 0.1 to
Intravenous administration of 1.0 mg / kg showed a suppressive effect of 8.5 to 76.6%. Medium molecular weight heparinyl phenylalanine is also 23.8 to 91. by intravenous administration of 3 to 30 mg / kg.
It exhibited a hemolytic suppression effect of 1%. The medium molecular weight heparinyl amino acid clearly showed stronger hemolytic inhibitory effect and complement activation inhibitory effect than the low molecular weight heparinyl amino acid.

VI. The inhibitory effect of medium molecular weight heparinyl amino acid on pulmonary edema was investigated for the inhibitory effect of medium molecular weight heparinyl amino acid on scorpion venom-induced pulmonary edema. (1) Experimental animal 6-week-old male Wistar rats (Charles, Japan
Purchased a river and a 12-hour light-dark cycle (light period: 7
After 1 to 1 week of preliminary breeding under the conditions of temperature 23 ± 2 ° C., humidity 50 ± 5%, and ventilation all fresh 15 times / hour, the sample was subjected to an experiment.

(2) Examination of Pulmonary Edema Inhibitory Effect Operation: Operation according to the method of Lineu, FM and Ione, MDM (Toxicon, Vol. 31, p. 1207, 1993). did. That is, heparin, medium molecular heparin or medium molecular heparinyl amino acid or Saline of the same volume was intravenously administered, and 30 minutes later, scorpion toxin (Tityus serrulatus venom, manufactured by Sigma) was intravenously administered. One hour later, the rat was killed, and after exsanguination, the lung was removed and the wet weight was measured. LBI (L
ung / Body Index: Lung weight (g) × 100 / B
ody weight (g)) and flame photometer (MF-303
Type, manufactured by JASCO Corporation) and the Na ⁺ / K ⁺ ratio in lung tissue was determined, and the pulmonary edema inhibitory effect of each test compound was calculated. The number of cases in one group was 4 to 14, and the significance between groups was tested by the Duncan method after confirming the uniformity of dispersion by the Bartlett method.

Results The inhibitory effects of heparin, medium molecular heparin and medium molecular heparinyl amino acid on scorpion venom-induced pulmonary edema are shown in Table 5. As a result, heparin was not found to have an inhibitory effect on pulmonary edema induced by scorpion venom, but both of the medium-molecule heparin and the medium-molecule heparinylamino acid were compared with the control group in LBI.
It was confirmed that histidine, methionine, arginine and tyrosine derivatives significantly reduced pulmonary edema.
In addition, the phenylalanine, histidine, arginine and tyrosine derivatives were also found to have an improving effect on the Na ⁺ / K ⁺ ratio value.

[0057]

[Table 5] Effect of heparin, medium molecular heparin and medium molecular heparinyl amino acid on scorpion venom-induced pulmonary edema Compound LBI Na ⁺ / K ⁺ (average value ± SE) (average value ± SE) Normal group 0.47 ± 0.01 ** 0.81 ± 0.02 ** Control group 0.73 ± 0.02 1.54 ± 0.13 Heparin 0.81 ± 0.02 1.87 ± 0.26 Medium molecular heparin 0.69 ± 0.05 1.77 ± 0.18 Medium molecular heparinylalanine 0.69 ± 0.07 1.54 ± 0.20 Medium molecular heparinyl asparagine Acid 0.72 ± 0.01 1.70 ± 0.03 Medium heparinyl phenylalanine 0.66 ± 0.08 1.39 ± 0.29 Medium heparinyl glycine 0.70 ± 0.05 1.61 ± 0.18 Medium heparinyl histidine 0.64 ± 0.03 * 1.36 ± 0.15 Medium heparinyl leucine 0.75 ± 0.06 1.94 ± 0.19 Medium Heparinylmethionine 0.65 ± 0.03 * 1.59 ± 0.13 Medium Heparinyl Proline 0.69 ± 0.06 1.79 ± 0.25 Medium Heparinyl Arginine 0.52 ± 0.02 ** 1.00 ± 0.07 ** Medium Heparinylserine 0.66 ± 0.03 1.64 ± 0.15 Medium molecule Paris sulfonyl tyrosine 0.59 ± 0.04 ** 1.18 ± 0.11 doses: 5000 [mu] g / kg * and **: significant difference, respectively a critical rate less than 5% and 1% relative to control

VII. Medium molecule heparinyl amino acid therapeutic effect on renal disease Using the puromycin aminonucleoside (PAN) renal disease model known to cause pathological conditions similar to minimal change nephrotic syndrome, medium molecule heparinylphenylalanine was selected as the test drug. Then, the therapeutic effect of the medium-molecular-weight heparinyl amino acid derivative on renal diseases was examined.

(1) Experimental Animal Six-week-old male Crj: CD rats (Charles River Japan) were used.

(2) Examination of inhibitory effect on renal disease An anesthesia rat was anesthetized with ether, and an Alza osmotic pump (model 2002, Alza) equipped with a silicon tube (inner diameter 0.7 mm, outer diameter 1.3 mm, made by Imma) at the tip Was manufactured under the skin of the neck. The silicone tube was inserted and fixed in the right jugular vein. 100 mg / kg PA after awakening in rats
A single dose of N was intraperitoneally administered to induce renal disease. On the 1st, 4th, 7th, 10th and 14th day after the induction of renal disease, the rat was placed in a metabolic cage and urine was collected to measure the urine volume and urinary protein concentration (micro TP-Test Wako, Wako Pure Chemical Industries). Then, the amount of excreted protein in urine / day was calculated. Medium molecular weight heparinyl phenylalanine doses of 0.3, 1 and 3 mg / kg / d
ay and the administration period was 2 weeks. The number of animals in each group was 6. Table 6 shows the results. As a result, in the medium molecular weight heparinyl phenylalanine, 3 mg / kg / day administration group, 4
A significant urinary protein excretion inhibitory effect was observed after the first day.
This significantly exceeded the urinary protein excretion suppressive effect of heparin in the literature.

[0061]

[Table 6] Inhibitory effect of heparinyl phenylalanine on protein excretion in urine (inhibition rate (%)) 1 day 4 days 7 days 10 days 14 days Heparin 3.45 mg / kg / day 44.7 ¹⁾ Medium molecule heparinyl phenylalanine 0.3 mg / kg / day -25.0 52.8 8.3 11.5 37.1 1mg / kg / day 25.0 67.1 30.1 -5.4 43.7 3mg / kg / day 33.3 93.7 ** 65.6 * 63.7 * 61.2 1) Diamond, JR, Karnovsky, MJ, Rinal Physiology ( Renal Physio l.), Vol. 9, p. 366 (1986) * and **: Significantly different from controls at risk rates of 5% and less than 1% (Dunkan method).

VIII. Radical scavenger effect of medium-molecular-weight heparinyl amino acids Heparin has been reported to act as a radical scavenger (Hiebert, LM, Liu, Ji-mi
n, Atherosclerosis, No. 83
Vol., P. 47, 1990) using xanthine (X) and xanthine oxidase (XO) to inhibit injury of medium molecular heparinyl amino acid derivatives as radical scavengers against injury of cultured cells by free radicals generated from these compounds. The effect was examined.

(1) Cultured cells Human umbilical vein endothelial cells (HUV-EC: Dainippon Pharmaceutical Co., Ltd.) were used as cultured cells, and 20% fetal bovine serum, bovine brain extract, Epidermal Growth Factor (EGF), hydro The cells were cultured using M-199 medium (manufactured by Dainippon Pharmaceutical) containing a heparin-free additive factor set for vascular endothelial cell culture containing cortisone, gentamicin and amphotericin (EGM-MV additive factor set: manufactured by Sanko Junyaku).

(2) Effect of medium molecular weight heparinyl phenylalanine, medium molecular weight heparinyl tyrosine and medium molecular weight heparinyl leucine on HUV-EC damage caused by free radicals. HUV-EC in a subconfluent state in a flask.
Was treated with a mixed solution of 0.25% trypsin: 0.25% EDTA = 1: 1, and then collected, and then added to a 6-well plate at 1.5 × 1.
After dispensing at a cell density of 0 ⁵ cells / well, the cells were cultured for 24 hours using M-199 medium containing only 1% fetal bovine serum. Then X (0.01 μM / ml) and XO
Free radicals were generated by allowing (0.2 U / ml) (both manufactured by Wako Pure Chemical Industries, Ltd.) to act in the medium for 24 hours. HUV in the plate 24 hours after X and XO action
-EC was treated again with the above trypsin: EDTA = 1: 1 mixed solution and then recovered. The same amount of 0.2% trypan blue solution was added to the recovered cell suspension to distinguish between blue-stained cells (dead cells) and unstained cells (viable cells) in the mixed solution, Count the number of each cell using a hemocytometer, and check cell viability (number of viable cells /
The total cell number) was determined. The heparin and the medium-molecular-weight heparinyl amino acid derivative were dissolved in PBS (-) and then 5
X and XO were allowed to act at a dose of 0 μg / ml and simultaneously added to the medium. In addition, a normal group in which X and XO were not actuated and a control group in which only a solvent for heparin or a medium molecular weight heparinyl amino acid derivative was added were also provided.

The results of the experiment are shown in Table 7. Although the cell viability was significantly decreased in the control group due to the injury caused by X and XO, the addition of medium molecular weight heparinyl phenylalanine, medium molecular weight heparinyl tyrosine or medium molecular weight heparinyl leucine statistically The decrease in cell viability could be suppressed significantly. Moreover, this inhibitory effect was superior to that of heparin.

[0066]

[Table 7] Inhibitory effect of medium molecular weight heparinyl amino acid derivative on xanthine and xanthine oxidase-induced cytotoxicity Cell viability (%) Compound mean ± standard error (number of cases) Normal 100.0 ± 0.0 (3 ) ** Control 51.6 ± 3.3 (3) Heparin 84.2 ± 9.6 (3) Medium molecule heparinyl phenylalanine 94.8 ± 5.4 (3) * Medium molecule heparinyl tyrosine 93.1 ± 6.3 (3) * Medium molecular weight heparinyl leucine 92.6 ± 8.2 (3) ** Significant difference at a risk rate of less than 5% with respect to the control ** Risk rate of less than 1% with respect to the control Significant difference (by Tukey method)

XI. Allergic suppressive effect of medium-molecular-weight heparinyl amino acids In recent years, allergic diseases such as bronchial asthma, allergic rhinitis, and chronic urticaria tend to increase, but these diseases are one of the main causes of degranulation from mast cells. And On the other hand, heparin is known to have an action of inhibiting degranulation from mast cells. (T.Ahmed et.a
l., American Review of Respiratory
Decembers (Am. Rev. Respir. Dis.), Volume 145,
566, 1992). Therefore, regarding the allergy suppressive effect of the medium molecule heparinyl amino acid derivative of the present invention,
Rat 48-hour allogeneic passive cutaneous anaphylaxis (P
The effect on the (CA) reaction was used as an index and examined.

(1) Experimental Animal A male Wistar rat of 8 weeks old (Charles River Japan) and a female Wistar rat of 5 weeks old (Charles River Japan) were subjected to a 12-hour light-dark cycle (light period: 7 o'clock). ˜19: 00), room temperature 23 ± 2 ° C., humidity 50 ± 5%, and ventilation all-fresh 15 times / hour, and preliminarily breeded for 1 week, and then subjected to the experiment.

(2) Action of medium-molecule heparinylphenylalanine on rat homologous PCA reaction for 48 hours Operation Stotland et al.'S method (Canadian Journal of
Physiology and Pharmacology (Can. J.
Physiol. Pharmacol.), 52, 1119, 19
1974), anti-ovalbumin (OVA) rat serum was prepared. That is, the spleen of a female rat was removed and 4
After a day, 1.0 ml of an equal volume mixture of physiological saline containing 2.0 mg / ml of OVA (manufactured by Seikagaku Kogyo) and 2% aluminum hydroxide gel (manufactured by Wako Pure Chemical Industries) was divided into four subcutaneously on the extremities. , And then pertussis killed cells (manufactured by Wako Pure Chemical Industries) 2 × 10
Sensitize by intraperitoneally administering ¹⁰ cells / 1.0 ml. Five days after the administration, 1.0 ml of an equal volume mixture of a physiological saline solution containing 2.0 mg / ml of OVA and a 2% aluminum hydroxide gel was administered for additional sensitization. Two weeks after the final sensitization, blood was collected from the abdominal aorta under anesthesia, the blood was centrifuged, and anti-OVA rat serum was collected. The rat 48-hour PCA antibody titer was 128.

The anti-OVA rat serum thus obtained was diluted with physiological saline to an antibody titer of 16, and 100 μl / site thereof was administered to 6 sites within the shaved back skin of male rats. I made it passively sensitized. 48 hours after the sensitization, 0.3, 3.0 and 30.0 mg of medium-molecular-weight heparinylphenylalanine per 1 kg of body weight was administered into the tail vein so that the liquid volume was 10 ml / kg. As a control, the same volume of physiological saline was used. Five minutes after the administration, 1 ml of Evans blue physiological saline solution containing 1 mg of OVA was administered into the tail vein. This OVA induces a PCA response, the intensity of which is proportional to the extent to which Evans blue (pigment) diffuses into the tissues surrounding the sensitized site. 30 minutes after the administration of OVA-containing Evans Blue saline solution, the animals were killed by decapitation and killed by the method of Katayama et al. (Microbiology and Immunology (Mi
crobiol.Immunol.), Vol. 22, p. 89, 1978).
The amount of dye leakage in the tissue (μg / site) and the area of dye leakage site (mm ² ) at the sensitized site of the rat dorsal skin were measured by. The number of cases in one group was 4 to 5. The significance between groups was confirmed by confirming the homogeneity of dispersion by the Bartlett method and then by using the Spjotvoll & Stoline method (corrected Tukey method).

Results Rat 48-hour PC of medium molecular weight heparinyl amino acid derivative
Table 8 shows the inhibitory effect on the A reaction.

TABLE 8 Medium molecular f suppressive effect dosage tissue dye leakage amount dye leakage site area against the rat 48 hr PCA reaction in Paris sulfonyl phenylalanine (mg / kg) (μg / site) (mm 2) ( mean ± standard error) (Mean ± standard error) Saline (control) 40.02 ± 4.46 130.50 ± 11.39 Medium molecular weight heparinyl phenylalanine 0.3 31.80 ± 2.89 191.14 ± 79.08 3.0 29.40 ± 4.13 90.84 ± 10.10 30.0 13.08 ± 0.43 ** 82.02 ± 3.74 **: There is a significant difference with a risk rate of less than 1% compared to the control (Spjotvoll & Stoline method)

In the medium-molecular-weight heparinylphenylalanine-administered group, there was a tendency to suppress the amount of dye leakage in the tissue and the area of dye leakage site in a dose-dependent manner.
In the g / kg administration group, the amount of dye leakage in the tissue was about 30%, which was significant (P <0.0) compared with the physiological saline (control) administration group.
1), and the effect of suppressing allergic reaction was remarkable. A drug used for an allergic disease is required to have not only an improvement in symptoms but also an effect of preventing the onset. From the above results, the medium-molecular-weight heparinyl amino acid derivative of the present invention can be used as a preventive or therapeutic agent for allergic diseases.

Example 1 Preparation of medium-molecular heparin 3 g of heparin (manufactured by Scientific Protein Laboratories) was dissolved in water to 52.5 ml, and a cation exchange resin (Dowex HCR-W2 20-50mesh) was used. ) Is added to adjust the pH to 3.30 to 3.40 and then filtered. 3 in the filtrate
Add 3.5 ml of 0% hydrogen peroxide solution (Mitsubishi Gas Chemical Co., Ltd.),
123 ° C, 1.4 kgf / cm ² , 1 in autoclave
Depolymerization is performed by heating under pressure for 0 minutes. After heating,
The reaction solution was adjusted to pH 6.8 by adding an aqueous sodium hydroxide solution, and subjected to ultrafiltration (Ultrafree CL: Millipore, Ultracent-10: Tosoh, Molcut II: Millipore, Molcut L: Millipore) to repeat the molecular weight. Fractionation was performed. Recrystallization with ethanol gave a white powder of medium molecular weight heparin (2.1 g, yield 70.10%).

Example 2 Preparation of medium molecular weight heparinyl amino acid (alanine, arginine, aspartic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, tyrosine) 0.64 g of medium molecular weight heparin was added to make 15 ml. ,
After adjusting the pH to 4.75, the corresponding amino acid ester hydrochloride (100 M to 1 M of medium-molecule heparin) was added (L-alanine methyl ester hydrochloride: manufactured by Sigma, L-
Arginine methyl ester dihydrochloride: Sigma, L-aspartic acid dimethyl ester hydrochloride: Sigma, L-
Glycine methyl ester hydrochloride: manufactured by Hanui Tesque, L-histidine methyl ester dihydrochloride: manufactured by Hanai Tesque, L-
Leucine methyl ester hydrochloride: manufactured by Tokyo Kasei, L-methionine methyl ester hydrochloride: manufactured by Sigma, L-phenylalanine methyl ester hydrochloride: manufactured by Aldrich, L-
Proline methyl ester hydrochloride: manufactured by Tokyo Kasei, L-serine methyl ester hydrochloride: manufactured by Hanai Tesque, L-tyrosine methyl ester hydrochloride: manufactured by Wako Pure Chemical Industries, Ltd.).

Furthermore, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Hanui Tesque)
After gradually adding 5 ml of 0.98 g of an aqueous solution and stirring for 4 hours, a small amount of water and a 5% aqueous solution of hexadecyltrimethylammonium bromide (manufactured by Hanai Tesque) are added until no ammonium salt precipitates. Then, the precipitate is completely separated by centrifugation, and 50 ml of a 5% ethanol solution of sodium iodide (manufactured by Hanai Tesque) is added to the precipitate. After stirring for 1 hour, it is filtered and the precipitate is recrystallized from ethanol to obtain a white powder of medium molecular weight heparinyl amino acid ester. To this medium-molecular-weight heparinyl amino acid ester, 10 ml of a 0.1N sodium hydroxide aqueous solution was added, and the mixture was stirred at 0-5 ° C for 2 days under a nitrogen stream. P with acetic acid
After adjusting to H5, the mixture was filtered, and ethanol was added to the filtrate to obtain white powder of medium molecular weight heparinyl amino acid. The respective yields are shown in Table 9.

[0076]

[Table 9] Yield of medium molecular weight heparinyl amino acid (Yield obtained from medium molecular weight heparin) Amino acid yield (%) Amino acid yield (%) Glycine 44.1 Methionine 63.2 Alanine 39.4 Phenylalanine 68. 6 Arginine 36.6 Proline 38.3 Aspartic acid 70.1 Serine 60.3 Histidine 65.0 Tyrosine 57.5 Leucine 49.7

Instrumental analysis of medium molecular weight heparinyl amino acid (1) Measurement of molecular weight by HPLC analysis Heparin, medium molecular weight heparin, and each amino acid derivative of medium molecular weight heparin were treated with 0.1 M sodium sulfate aqueous solution for 5 m.
Adjust to g / ml and use this as the sample solution. On the other hand, polyethylene glycol (molecular weight 10,000: made by Aldrich,
A molecular weight of 8,500 and an average molecular weight of 3,000: manufactured by Tokyo Chemical Industry Co., Ltd., an average molecular weight of 1,450: manufactured by Kanto Kagaku Co., Ltd.) are used as standard substances, and the standard solution is prepared in the same manner as the sample solution. For the sample solution and standard solution, perform HPLC analysis under the following measuring instruments and operating conditions, and use the calibration curve method to determine the amount of heparin,
The molecular weights of medium molecular weight heparin and medium molecular weight heparinyl amino acid were measured. The results are shown in Table 10. Equipment used Liquid feed pump: LC-10AD (Shimadzu) Differential refractometer: RI-8020 (Tosoh) Autosampler: KMT-60A-11 (Kyowa Seimitsu) Data processor: C-R3A ( Shimadzu Corporation) Column: TSK-Gel G3000PWXL (7.8 mm x 30 cm: made by Tosoh) are used by connecting two operating conditions Flow rate: 1.0 ml / min Pressure: 43 kg / cm ² Sensitivity: RI 512 x 10 ^-6 RIVFS Column temperature: 40 ° C. Detection sensitivity: 8-10 Injection volume: 200 μl

[0078]

[Table 10] Molecular weight compound of heparin, medium molecular weight heparin and medium molecular weight heparinyl amino acid Maximum molecular weight Minimum molecular weight Average molecular weight Heparin 18,000 7,000 13,000-14,000 Medium molecular heparin 12,500 4,500 8,500-9,500 Medium molecular heparin glycine 12,500-13,500 3,500- 4,500 8,500-9,500 Medium molecular weight heparinylalanine 12,500-13,500 3,500-4,500 8,500-9,500 Medium molecular weight heparinyl alkyne 12,500-13,500 3,500-4,500 8,500-9,500 Medium molecular weight Heparinyl asparagine acid 12,500-13,500 3,500-4,500 9,000-10,000 Medium molecular weight heparin Nylhistidine 12,500-13,500 3,500-4,500 8,500-9,500 Medium molecular weight heparinyl leucine 12,500-13,500 3,500-4,500 8,500-9,500 Medium molecular weight heparinylmethionine 12,500-13,500 3,500-4,500 8,500-9,500 Medium molecular weight heparinylphenylalanine 12,500-13,500 3,500-4,500 8,500-9,500 Medium molecular weight Heparinylproline 12,500-13,500 3,500-4,500 8,5 00-9,500 Medium molecule Heparinyl Serine 12,500-13,500 3,500-4,500 8,500-9,500 Medium molecule Heparinyl Tyrosine 12,500-13,500 3,500-4,500 8,500-9,500

(2) Measurement of infrared absorption spectrum About 3 mg of each of the medium molecular weight heparin and each of the amino acid derivatives of medium molecular weight heparin were weighed and mixed with about 100 mg of potassium bromide to prepare pellets by a handy press. About this thing, by potassium bromide tablet method,
An infrared absorption spectrum was measured with the following measuring equipment and operating conditions. The results are shown in Table 11. Equipment used Fourier Transform Infrared Spectrometer: FT-200 (manufactured by Horiba Ltd.) Operating conditions Number of scans: 50 Measurement resolution: 4 cm ^-1 Gain: AUTO Measurement range: 4,000-400 cm ^-1 Device function: Happganzel ( H-G) Spectrum:% T Scan speed: 5 mm / sec

[0080]

[Table 11] Infrared absorption spectrum of medium molecular weight heparinyl amino acid Compound frequency (cm ^-1 ) Medium molecular weight heparin 3,316-3,645 (OH, COOH), 1,727 (COO), 1,622 (CO) Medium molecular weight heparinylglycine 3,273-3,623 (OH, COOH), 1,727 (COO), 1,668 (CO), 1,550 (NH) Medium molecular weight heparinyl arginine 3,200-3,600 (OH, COOH), 1,739 (COO), 1,667 (CO), 1,541 (NH ) Medium molecule heparinyl 3,300-3,600 (OH, COOH), 1,739 (COO), 1,676 (CO), 1,543 (NH) Asparabic acid Medium molecule heparinyl 3,150-3,600 (OH, COOH), 1,730 (COO), 1,667 (CO) , 1,541 (NH) Phenylalanine Medium molecule Heparinylproline 3,140-3,630 (OH, COOH), 1,748 (COO), 1,657 (CO), 1,531 (NH) Medium molecule Heparinylserine 3,280-3,610 (OH, COOH), 1,729 (COO) , 1,667 (CO), 1,531 (NH)

(3) Amino Acid Analysis Medium molecular weight heparin and about 100 μg of each amino acid derivative of medium molecular weight heparin were weighed in a sample test tube, and the test tube was placed in a reaction vial and dried under reduced pressure. Then, at the bottom of the reaction vial, 6N hydrochloric acid for hydrolysis (containing 1% phenol)
After adding 100 μl and replacing the air in the reaction vial with nitrogen under reduced pressure, the mixture was heated at 105 ° C. for 24 hours for hydrolysis. After cooling, take out the test tube for the sample, 0.02N
100 μl of hydrochloric acid was added and mixed well, and the mixture was filtered through a filter, and the filtrate was used as a sample solution. Using the sample solution and the raw material before hydrolysis, the amino acid content was measured with the following measuring instruments and operating conditions. As a result, in all of the medium-molecular-weight heparinyl amino acids, substitution was made with amino acids at a ratio of about 90% or more of carboxylic acid to medium-molecular-weight heparin. Equipment used Amino acid automatic analyzer: L-8500 (manufactured by Hitachi, Ltd.) Operating conditions Eluent: Citrate buffer (pH 3.5, 4.0, 5.5) Flow rate: Eluent 0.15 ml / min Ninhydrin solution 0. 2 ml / min Column: Ion exchange resin (Hitachi Custom Ion # 2620, 4.6 mm × 80 mm) Column temperature: 50 to 65 ° C. Injection volume: Standard solution 10 μl Sample solution 30 μl

(4) Measurement of ¹ H and ¹³ C-NMR spectra Medium heparin and amino acid derivatives of medium heparin were dissolved in D ₂ O, and ¹ H and ¹³ C-NMR spectra were measured (XL- 300: Varian, 300MH
z, 75.4 MHz). The results are shown in FIGS.

(5) Elemental analysis CHN CORDER (manufactured by Yanagimoto) for each amino acid derivative of heparin, medium molecular heparin and medium molecular heparin
Was measured by the combustion method. Table 12 shows the results.

[Table 12] Elemental analysis compounds of heparin, medium molecular heparin and medium molecular heparinyl amino acid Carbon (%) Hydrogen (%) Nitrogen (%) Oxygen / sulfur (%) Heparin 22.25 3.82 2.11 71.83 Medium molecular heparin 22.23 3.95 2.04 71.79 Medium molecular weight heparinyl alkynine 26.73 4.79 7.70 60.79 Medium molecular weight heparinyl asparagine acid 26.53 4.71 4.00 64.77 Medium molecular weight heparinyl phenylalanine 30.33 4.63 3.63 61.42 Medium molecular weight heparinyl fluorin 30.33 4.63 3.63 61.42 Medium molecular weight 60.03.

(6) Determination of uronic acid Carbazole-sulfuric acid method (improved method of Bitter-Muir: Analytical Biochemistry (Anal. Biochem.), Volume 4,
Page 330, 1962) to determine the uronic acid content. Transfer 0.5 ml of an aqueous solution of heparin, medium molecular heparin, and each amino acid derivative of medium molecular heparin to a test tube,
While cooling with ice water, a 3.0 ml sulfuric acid solution (sodium tetraborate decahydrate (manufactured by Kishida Chemical Co., Ltd., 0.95 g dissolved in concentrated sulfuric acid 100 ml)) was added dropwise, and 0.1 ml of 0.1 ml was added.
A 125% carbazole solution (12.5 mg of carbazole (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in 10 ml of ethanol) was added and mixed thoroughly, and then capped with a glass ball and heated in a boiling water bath for 20 minutes. After cooling to room temperature, 530n
The absorbance at m was measured (UVIDEC-77Σ: manufactured by JASCO Corporation).

(7) Quantification of Sulfate Group The method of TT Terho et al. (Anal. Biochem., Vol. 41, p. 471, 19).
71), and the sulfate group was measured by the rhodizonic acid method. That is, 0.5 ml of a 1N hydrochloric acid solution containing about 0.2 mg of each amino acid derivative of heparin, medium molecular heparin and medium molecular heparin is placed in a glass stoppered test tube and sealed. After heating for 1 hour in a boiling water bath for hydrolysis, the mixture was dried under reduced pressure to remove hydrochloric acid, and this was dissolved in 1.0 ml of water. Take 0.5 ml of this solution in a centrifuge tube with a glass stopper, and add 2.0 ml of ethanol. Further, 1.0 ml of barium chloride solution (10 ml of 2.0 M acetic acid, 5 ml of 5 mM barium chloride and 8 ml of 20 mM sodium hydrogen carbonate were mixed, and ethanol was added to make 100 ml),
And 1.5 ml of sodium rhodizonate solution (5 mg of sodium rhodizonate (manufactured by Wako Pure Chemical Industries, Ltd.) in 20 ml of water.
100 mg of L-ascorbic acid was added to dissolve, and ethanol was added to make 100 ml), and the mixture was stirred well. After leaving it in a dark place at room temperature for 10 minutes, the absorbance at 520 nm was measured (UVIDEC-77
Σ: manufactured by JASCO Corporation). Table 13 shows the results.

[0086]

[Table 13] Sulfate and hexosamine content compounds of heparin, medium molecular heparin and medium molecular heparinyl amino acid derivatives Nitrogen (%) Sulfur (%) Nitrogen / sulfur ratio Hexosamine heparin 2.11 12.06 5.73 30.34 Medium molecular heparin 2.04 11.01 5.41 27.14 Medium molecular weight heparinyl arginine 7.70 13.91 1.81 25.13 Medium molecular weight heparinyl asparagine acid 4.00 11.97 2.99 22.36 Medium molecular weight heparinyl phenylalanine 3.63 12.19 3.36 35.22 Medium molecular weight heparinyl porolin 3.63 10.96 3.02 29.47 Medium molecular weight heparinyl serine 25.21 10.08 2.80

Example 3 Injectable Medium Heparinyl Amino Acid Derivative 250 μg-2.5 mg
After taking 10 ml of a physiological saline solution to dissolve it, it is filled in an ampoule, sealed and sterilized to obtain an injection.

Example 4 Tablets 2.5 to 10 mg of a medium-molecular-weight heparinyl amino acid derivative was added, 300 mg of lactose as an excipient was added, and the mixture was evenly mixed to give tablets by direct compression molding.

Example 5 Capsule A medium-capacity heparinyl amino acid derivative (2.5-10 mg) was taken, and lactose (300 mg) was added as an excipient and the mixture was evenly mixed to prepare a capsule.

Example 6 Suppository A suppository is prepared by taking 5.0 to 20 mg of a heparinyl amino acid derivative of a medium molecule, adding 2 g of macrogol as a base, and mixing the mixture evenly, and shaping into a certain shape.

[Brief description of drawings]

FIG. 1 shows a ¹³ C NMR spectrum of medium molecular heparin.

FIG. 2 shows a ¹ H NMR spectrum of medium molecular heparin.

FIG. 3: ¹³ C of medium-molecule heparinylphenylalanine
3 shows an NMR spectrum.

FIG. 4 ¹ H N of medium-molecule heparinylphenylalanine
An MR spectrum is shown.

FIG. 5 ¹³ C NMR of medium molecule heparinyl arginine
The spectrum is shown.

FIG. 6 shows a ¹ H NMR spectrum of medium molecular weight heparinyl arginine.

FIG. 7 shows a ¹³ C NMR spectrum of medium-molecule heparinylserine.

FIG. 8 shows a ¹ H NMR spectrum of medium molecular weight heparinyl serine.

FIG. 9 shows a ¹³ C NMR spectrum of medium-molecule heparinylproline.

FIG. 10 ¹ H NMR of medium molecule heparinylproline
The spectrum is shown.

FIG. 11: ¹³ C of medium molecule heparinyl aspartic acid
3 shows an NMR spectrum.

FIG. 12: ¹ H N of medium molecule heparinyl aspartic acid
An MR spectrum is shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location A61K 31/725 ACV A61K 31/725 ACV ADU ADU (72) Inventor Kazuki Nakamura Jojo, Osaka City, Osaka Prefecture 2-3-30 Morinomiya, Ward Fuso Kuwa Pharmaceutical Co., Ltd. Research and Development Center (72) Inventor Yuya Otani 2-3-3 Morinomiya Morinomiya, Joto-ku, Osaka City, Osaka (72) Invention Yuko Nishimura 2-3-30 Morinomiya, Joto-ku, Osaka-shi, Osaka Prefecture Fuso Pharmaceutical Co., Ltd. Research and Development Center (72) Inventor Chiaki Shimada 2-3-30 Morinomiya, Joto-ku, Osaka-shi, Osaka Osaka Fuso Pharmaceutical Co., Ltd. Company Research & Development Center (72) Inventor Seiichi Takeda 2-3-30 Morinomiya Morinomiya, Joto-ku, Osaka-shi, Osaka Fuso Pharmaceutical Co., Ltd. Research & Development Center (72) Inventor Kenzo Kawai 2-3-30 Morinomiya, Joto-ku, Osaka-shi, Osaka Prefecture Research & Development Center, Fuso Pharmaceutical Co., Ltd. (72) Inventor Chizuko Kitagawa 2-Morinomiya, Joto-ku, Osaka-shi, Osaka Prefecture 3-30 Fuso Pharmaceutical Co., Ltd. Research and Development Center (72) Inventor Masaaki Kuwahara 3-7-16 Kashiwagi, Aoba-ku, Sendai City, Miyagi Prefecture (72) Tomoyuki Abe 2-3-3 Morinomiya, Joto-ku, Osaka City, Osaka Prefecture 30 Fuso Pharmaceutical Co., Ltd. Research and Development Center

Claims (14)

[Claims] 1. A medium molecular weight heparin having an average molecular weight of 8,500 to 9,500. 2. A medium-molecule heparinyl amino acid derivative derived from the medium-molecule heparin according to claim 1. 3. The medium molecule heparinyl amino acid derivative according to claim 2, wherein the amino acid derivative is an amino acid or an amino acid lower alkyl ester. 4. The above amino acid derivative has the general formula: NH ₂ CH (R ₁ ) COOR ₂ [wherein R ₁ is: H; Or phenyl, optionally substituted with OH, OH, N
_{H 2, SH, SCH 3,} COOH, CONH 2, guanidino, indolyl and lower alkyl which may be substituted with a group selected from the group consisting of 1H- imidazolyl, R ₂ is H or lower alkyl] The medium molecular weight heparinyl amino acid derivative according to claim 2, which is represented by 5. The medium molecule heparinyl amino acid derivative according to claim 4, wherein the amino acid is a naturally occurring amino acid. 6. The medium molecule heparinyl amino acid derivative according to claim 5, wherein the amino acid is an essential amino acid. 7. An anticoagulant active pharmaceutical composition comprising as an active ingredient at least one kind of the middle molecule heparinyl amino acid derivative according to claim 2. 8. A pharmaceutical composition for inhibiting renal mesangial cell proliferation, which comprises at least one kind of the medium-molecular-weight heparinyl amino acid derivative according to claim 2 as an active ingredient. 9. A pharmaceutical composition for suppressing cancer metastasis, which comprises at least one kind of the middle molecule heparinyl amino acid derivative according to claim 2 as an active ingredient. 10. A pharmaceutical composition for inhibiting complement activation, which comprises at least one kind of the medium-molecular-weight heparinyl amino acid derivative according to claim 2 as an active ingredient. 11. A pharmaceutical composition for suppressing pulmonary edema, which comprises at least one kind of the medium-molecular-weight heparinyl amino acid derivative according to claim 2 as an active ingredient. 12. A pharmaceutical composition for treating renal diseases, which comprises at least one kind of the medium-molecular-weight heparinyl amino acid derivative according to claim 2 as an active ingredient. 13. A radical scavenger active pharmaceutical composition comprising as an active ingredient at least one kind of the medium molecule heparinyl amino acid derivative according to claim 2. 14. A pharmaceutical composition for preventing and treating allergic diseases, which comprises at least one kind of the medium-molecular-weight heparinyl amino acid derivative according to claim 2 as an active ingredient.