WO2018159604A1

WO2018159604A1 - Heparin conjugate

Info

Publication number: WO2018159604A1
Application number: PCT/JP2018/007240
Authority: WO
Inventors: 恭平東
Original assignee: 国立大学法人千葉大学
Priority date: 2017-03-02
Filing date: 2018-02-27
Publication date: 2018-09-07
Also published as: JP2020066638A

Abstract

The present invention relates to a novel conjugate of heparin and polyamine, an anticoagulant comprising the conjugate, a pharmaceutical composition containing the conjugate, a kit for producing the conjugate, a sustained release agent, a method for producing the conjugate, etc. (1) A conjugate of heparin and a linear or branched polyamine that has at least 3 amino groups; (2) an anticoagulant comprising the conjugate described in (1); (3) a pharmaceutical composition containing the conjugate described in (1); (4) a kit for producing the conjugate, which contains heparin or a salt thereof and a linear or branched polyamine having at least 3 amino groups or a salt thereof; (5) a sustained release agent for providing a sustained release behavior for heparin, comprising a linear or branched polyamine having at least 3 amino groups or a salt thereof; and (6) a method for producing the conjugate, the method comprising mixing an aqueous solution of heparin or a salt thereof with an aqueous solution of a linear or branched polyamine having at least 3 amino groups or a salt thereof.

Description

Heparin aggregate

The present invention relates to an aggregate of heparin and polyamine, an anticoagulant comprising the aggregate, a pharmaceutical composition containing the aggregate, a kit for producing the aggregate, a sustained-release imparting agent, and a method for producing the aggregate Etc.

Unfractionated heparin cocoon (UFH) cocoon is a linear acidic polysaccharide in which uronic acid and glucosamine are alternately bonded and a sulfate group is added to a part of each sugar hydroxyl group, and is widely used as an anticoagulant. Indications: 1) Treatment of generalized intravascular blood coagulation syndrome, 2) Prevention of blood coagulation when using hemodialysis, cardiopulmonary bypass or other extracorporeal circulation devices, 3) Prevention of blood coagulation when inserting a vascular catheter, 4 ) Prevention of blood coagulation during blood transfusion and blood tests, 5) Thromboembolism (venous thrombosis, myocardial infarction, pulmonary embolism, cerebral embolism, limb arterial thromboembolism, thromboembolism during and after surgery And the like).

In recent years, low-molecular-weight heparin modified from unfractionated heparin (for example, trade name: Arikstra (registered trademark)) and a new oral anticoagulant drug to replace warfarin (for example, trade name: plazaxa (registered trademark), trade name: XXARElt ( The development of the registered trademark)) is expected to increase year by year and continue to increase to 164.7 billion yen in 2022. However, UFH is still in high demand because it is withdrawn and replaced with heparin at the time of surgery, and 10 pharmaceutical companies are currently producing only in Japan. During surgery, UFH is administered by intravenous infusion, intravenous intermittent injection, or subcutaneous injection.

In Patent Document 1, by gradually releasing heparin or low molecular weight heparin locally over a long period of time, the production of hepatocyte growth factor (HGF) is efficiently promoted in a lesion while reducing bleeding tendency as a side effect. In order to provide a composition capable of suppressing fibrosis in the kidney, a sustained release gelatin hydrogel composition containing cationized gelatin and heparin has been disclosed (Patent Document 1).

JP 2013-75835 JP

Since UFH has a high negative charge and a high number average molecular weight of about 13,000, oral absorption efficiency is extremely low, and administration is limited to intravenous injection or subcutaneous administration. Furthermore, it binds nonspecifically to blood and blood vessel proteins. Therefore, intravenous administration of UFH has a rapid elimination phase due to binding with vascular endothelial cells and macrophages and a slow elimination phase due to excretion from the kidney. In addition, UFH has a short half-life (about 40-60 minutes) and a narrow therapeutic window.
Low molecular weight heparin has been developed to extend half-life, but its indication is limited compared to UFH. Therefore, development of new heparin is desired.
Accordingly, the present invention provides a novel association product of heparin and polyamine, an anticoagulant comprising the association product, a pharmaceutical composition containing the association product, a kit for producing the association product, a sustained release agent, and an association product It relates to the manufacturing method of
Further, in one aspect, the present invention relates to an aggregate having a long half-life of heparin after administration and a long therapeutic range, an anticoagulant comprising the aggregate, a pharmaceutical composition containing the aggregate, and production of the aggregate The present invention relates to a kit, a sustained-release imparting agent, and a method for producing an aggregate.

The present invention relates to the following [1] to [16].
[1] An association product of heparin and a linear or branched polyamine having 3 or more amino groups.
[2] The aggregate according to [1], wherein the polyamine is a branched polyamine.
[3] The polyamine has the formula (1):

[Wherein R ¹ , R ² , R ³ and R ⁴ are each independently a divalent aliphatic hydrocarbon group having 3 or 4 carbon atoms. ] The association according to [1] or [2], which is a compound represented by
[4] The aggregate according to any one of [1] to [3], wherein the heparin is unfractionated heparin or low molecular weight heparin.
[5] The polyamine includes at least one selected from the group consisting of spermidine, spermine, theremin, cardopentamine, tris (3-aminopropyl) amine, and tetrakis (3-aminopropyl) ammonium. To [4].
[6] The aggregate according to any one of [1] to [5], wherein the polyamine contains tetrakis (3-aminopropyl) ammonium.
[7] An anticoagulant comprising the aggregate according to any one of [1] to [6].
[8] A pharmaceutical composition comprising the aggregate according to any one of [1] to [6].
[9] A kit for producing an aggregate comprising heparin or a salt thereof and a linear or branched polyamine having 3 or more amino groups, or a salt thereof.
[10] A sustained release agent for imparting sustained release of heparin, comprising a linear or branched polyamine having 3 or more amino groups, or a salt thereof.
[11] A method for producing an aggregate, comprising mixing an aqueous solution of heparin or a salt thereof and an aqueous solution of a linear or branched polyamine having 3 or more amino groups or a salt thereof.
[12] The aggregate according to any one of [1] to [6] for anticoagulation.
[13] Use of the aggregate according to any one of [1] to [6] for anticoagulation.
[14] Use of the aggregate according to any one of [1] to [6] for the production of an anticoagulant.
[15] Use of a linear or branched polyamine having 3 or more amino groups or a salt thereof for imparting sustained release to an anticoagulant containing heparin.
[16] A method for preventing blood coagulation, comprising administering the association according to any one of [1] to [6] in vivo.

According to the present invention, a novel aggregate of heparin and polyamine, an anticoagulant comprising the aggregate, a pharmaceutical composition containing the aggregate, a kit for producing the aggregate, a sustained-release imparting agent, and an aggregate And the like.
Further, in one aspect, according to the present invention, the heparin has a long half-life after administration and a long-lasting treatment area, an anticoagulant comprising the aggregate, a pharmaceutical composition containing the aggregate, and an aggregate And the like, a sustained-release imparting agent, and a method for producing an aggregate.

SPM and UFH contained in the aggregate prepared under each pH condition were dissociated, and the SPM in the aggregate was quantified by the method shown in [SPM quantification]. The results shown in FIG. 1A and UFH in the aggregate [quantification of heparin] The results quantified by the method shown in Fig. 1B are shown. FIG. 2 is a graph showing the relationship between the amount of UFH and Taa in the supernatant and the ratio between the negative charge of UFH and the positive charge of Taa (negative / positive ratio, N / P ratio). FIG. 3 shows changes over time in plasma Anti-Factor Xa activity after subcutaneous administration of UFH and UFH-Taa aggregates to mice. FIG. 3A shows the results at a UFH dose of 4 mg / kg (body weight), FIG.3B shows the results at a UFH dose of 30 mg / kg weight (body weight), and FIG. The results at a dose of 30 mg / kg (body weight) of Taa aggregates are shown. FIG. 4 shows the time course of plasma Anti-Factor IIa activity after subcutaneous administration of UFH and UFH-Taa aggregates to mice. FIG. 4A shows the results at a UFH dose of 4 mg / kg (body weight), FIG.4B shows the results at a UFH dose of 30 mg / kg weight (body weight), and FIG. The results at a dose of 30 mg / kg (body weight) of Taa aggregates are shown. FIG. 5 shows changes over time in plasma Anti-Factor Xa activity after subcutaneous administration of LMWH and LMWH-Taa aggregates to mice.

In this specification, the following abbreviations may be used for various terms.
Unfractionated heparin or its salt may be abbreviated as “UFH”.
Low molecular weight heparin or a salt thereof may be abbreviated as “LMWH”.
Spermine may be abbreviated as “SPM”.
Spermidine is sometimes abbreviated as “SPD”.
Putrescine may be abbreviated as “PUT”.
Tetrakis (3-aminopropyl) ammonium may be abbreviated as “Taa”.

[Meeting]
The aggregate of the present invention is an aggregate of heparin and a linear or branched polyamine having 3 or more amino groups. By forming an association between heparin and the polyamine, a gel-like association or a solid association is obtained in water. The aggregate is considered to form a polyion complex. By forming the aggregate, heparin is gradually released in the blood after administration, and the heparin half-life is increased and the therapeutic area is prolonged.
Heparin is an acidic polysaccharide and has a high negative charge. The surface charge is neutralized with polyamine to form an aggregate. The aggregate is bound by electrostatic interaction due to, for example, a negative charge based on an acidic group present in the heparin molecule and a positive charge based on a basic group present in the polyamine molecule.

<Heparin>
Heparin is a linear acidic polysaccharide in which uronic acid and glucosamine are alternately bonded, and a sulfate group is added to a part of each sugar hydroxyl group.
Examples of heparin include unfractionated heparin (UFH), photolytic heparin (for example, HP7000 described later), and low molecular weight heparin (LMWH).

As a raw material of the aggregate, heparin or a salt thereof is used.
Examples of heparin salts include sodium salts and calcium salts.
Heparin sodium is obtained, for example, from the liver, lungs, and intestinal mucosa of healthy food animals.
Heparin calcium is obtained, for example, from the intestinal mucosa of healthy pigs.
For the preparation of heparin, for example, a method disclosed in the Japanese Pharmacopoeia can be used. Sodium salt is available as a Japanese pharmacopoeia product, and calcium salt is available as a British pharmacopoeia product or, for example, CarboMer Inc.
In addition, when a salt is used as a raw material, a part of the salt may remain.

The number average molecular weight of unfractionated heparin is preferably 5,000 or more, more preferably 8,000 or more, further preferably 10,000 or more, more preferably 12,000 or more, and preferably 15,000 or less, more preferably 14,000 or less, and still more preferably Is less than 13,000. The number average molecular weight is measured by the method described in “Heparin molecular weight measurement” in the Examples.
Examples of commercially available unfractionated heparin used as a raw material include heparin sodium salt (manufactured by New Zealand Pharmaceutical) and “heparin calcium subcutaneous injection 5,000 units / 0.2 mL syringe” (manufactured by Mochida Pharmaceutical Co., Ltd.). .

Photolytic heparin is heparin obtained by reducing the molecular weight of unfractionated heparin by a photolytic reaction using titanium dioxide. Photolytic heparin is not particularly limited, and can be obtained, for example, by the method described in International Publication No. WO2008 / 059869.
The number average molecular weight of the photolytic heparin is preferably 3,000 or more, preferably 5,000 or more, more preferably 6,000 or more, and preferably 10,000 or less, more preferably 9,000 or less, and further preferably 8,000 or less. The number average molecular weight is measured by the method described in “Heparin number average molecular weight measurement” in the Examples.

Low molecular weight heparin is, for example, depolymerized heparin obtained by decomposing heparin derived from bovine or porcine intestinal mucosa with hydrogen peroxide and cupric sulfate.
The number average molecular weight of the low molecular weight heparin is preferably 2,000 or more, more preferably 2,500 or more, still more preferably 3,000 or more, and preferably 7,000 or less, more preferably 6,000 or less, and even more preferably 5,000 or less. The number average molecular weight is measured by the method described in “Heparin number average molecular weight measurement” in the Examples.
Examples of the low molecular weight heparin include parnaparin, dalteparin (number average molecular weight of about 5,500), danaparoid, leviparin, and enoxaparin (number average molecular weight of about 3,500 to 4,500).
Examples of commercially available low molecular weight heparin used as a raw material include enoxaparin sodium injection “Clexane Subcutaneous Injection Kit 2000 IU” (manufactured by Sanofi Corporation).

Among these heparins, unfractionated heparin or low molecular weight heparin is preferred, and unfractionated heparin is more preferred from the viewpoint of sustained release of heparin in blood, a long heparin half-life, and a long therapeutic range.

The titer of heparin is preferably 80 IU / mg or more, more preferably 90 IU / mg or more, and preferably 220 IU / mg or less, more preferably 210 IU / mg or less, and even more preferably 200 IU / mg. mg or less. The unit of heparin titer is expressed in international units (IU).
The average negative charge per disaccharide of heparin is preferably -3.00 or less, more preferably -3.20 or less, further preferably -3.30 or less, and preferably -4.00 or more, more preferably -3.80 or more. Is -3.60 or more, more preferably -3.40 or more.
The average negative charge per disaccharide of heparin can be measured by the method described later.

A heparin preparation may be used as a raw material. Examples of heparin preparations include pharmaceutically acceptable heparin-containing solutions and heparin-containing solid preparations (powder preparations, freeze-dried preparations, etc.) that are used after dissolution. Among these, a solution is preferable.

<Polyamine>
An association is formed by a combination of heparin and a linear or branched polyamine having 3 or more amino groups.
The polyamine is preferably a branched polyamine, more preferably a cross-linked polyamine from the viewpoint of enhancing the formation of aggregates, and from the viewpoint of sustained release of heparin in the blood, a long heparin half-life, and a long therapeutic area. A polyamine.
A cruciform polyamine means an amine having four or more substituents bonded to one nitrogen atom.
In the polyamine, each nitrogen atom may be connected to each other by a divalent aliphatic hydrocarbon group having 3 or 4 carbon atoms.
The number of amino groups of the polyamine is 3 or more, preferably 4 or more, and preferably 6 or less, more preferably 5 or less, from the viewpoint of forming an aggregate.
The number of carbon atoms of the polyamine is preferably 5 or more, more preferably 6 or more, still more preferably 8 or more, still more preferably 10 or more, still more preferably 11 or more, from the viewpoint of enhancing the formability of the aggregate. Is 20 or less, more preferably 18 or less, still more preferably 16 or less, and still more preferably 14 or less.
The polyamine preferably has a quaternary ammonium moiety in the molecule from the viewpoint of enhancing the formability of the aggregate and from the viewpoint of sustained release of heparin in the blood, a long heparin half-life, and a long therapeutic range.

The polyamine preferably has the formula (1): from the viewpoint of sustained release of heparin in the blood, long heparin half-life, and long treatment area.

[Wherein R ¹ , R ² , R ³ and R ⁴ are each independently a divalent aliphatic hydrocarbon group having 3 or 4 carbon atoms. It is a compound represented by this.
Examples of the divalent aliphatic hydrocarbon group include a 1,3-propanediyl group and a 1,4-butanediyl group. Among these, 1,3-propanediyl is preferable.
The polyamine may contain a counter anion X ⁻ . Examples of X ⁻ include halide ions such as Cl ⁻ , Br ⁻ and I ⁻ .

Examples of the polyamine include spermidine, spermine, theremin, cardopentamine, tris (3-aminopropyl) amine (hereinafter also referred to as “mitubicin”), and tetrakis (3-aminopropyl) ammonium.
Among these, tetrakis (3-aminopropyl) ammonium is preferable from the viewpoint of enhancing the formation of aggregates and from the viewpoint of sustained release of heparin in the blood, long heparin half-life, and long treatment area.
The tetrakis (3-aminopropyl) ammonium described above is, for example, (dentification, chemical synthesis, and biological functions of unusual polyamines produced by extreme thermophiles By: Oshima, Tairo; Moriya, Toshiyuki; Terui, Yusuke Methods in Molecular Biology (New York , NY, United States) Vol. 720, Issue Polyamines, Pages 81-111. General Review, 2011].

In the aggregate, the molar ratio of polyamine per 1 g of heparin is, for example, preferably 0.5 mmol / g or more, more preferably 0.7 / mmol / g or more, still more preferably 0.8 mmol / g or more, and preferably It is 8 mmol / g or less, more preferably 7 mmol / g or less, still more preferably 6 mmol / g or less.

In the aggregate, the ratio of the negative charge of heparin to the positive charge of polyamine (negative / positive ratio, N / P ratio) is preferably 5 or less, more preferably 3 or less, still more preferably 2 or less, and still more preferably. 1.5 or less, and preferably 0.2 or more, more preferably 0.5 or more, and still more preferably 0.8 or more.
In addition, the positive charge of a polyamine calculates the whole positive charge from the molar amount of a polyamine by making the number of amino groups and ammonium groups into the number of positive charges per molecule from molecular structure. For example, in the case of Taa, the charge per molecule is +5.
The negative charge of heparin is calculated from the average molecular weight per disaccharide and the average charge per disaccharide. For example, in the case of UFH, the calculation is performed using an average charge per disaccharide of −3.38. The charge ratio is an absolute value ratio. The average molecular weight per disaccharide and the average charge per disaccharide are measured by the following methods.

(Measurement of disaccharide composition of heparin, average molecular weight per disaccharide, and average charge per disaccharide)
After heparin is cleaved with heparinase I, II and III, unsaturated disaccharides are separated by reversed-phase ion pair chromatography, and the disaccharide composition is identified by a fluorescence detector.
Detected disaccharide composition (ΔUA-GlcNAc (carboxy group number 1, sulfate group number 0, molecular weight 379.3), ΔUA-GlcNS (carboxy group number 1, sulfate group number 1, molecular weight 417.3), ΔUA-GlcNAc6S (carboxy group number 1, sulfate group number 1, molecular weight 459.3), ΔUA-GlcNS6S (carboxy group number 1, sulfate group number 2, molecular weight 497.4), ΔUA2S-GlcNS (carboxy group number 1, sulfate group number 2, molecular weight 497.4), ΔUA2S-GlcNS6S (carboxy group number 1, sulfate group number 3, The average molecular weight per disaccharide is calculated from the molecular weight 577.4)). The average charge per disaccharide is calculated from the average value of carboxy group and sulfate group per disaccharide.

[Method for producing aggregate]
The aggregate includes, for example, an aqueous solution of heparin or a salt thereof (hereinafter also simply referred to as “heparin aqueous solution”), a linear or branched polyamine having three or more amino groups, or an aqueous solution of a salt thereof (hereinafter simply referred to as an “aqueous solution”). And "polyamine aqueous solution").

The mixing ratio and concentration of the heparin aqueous solution and the polyamine aqueous solution are adjusted so that the concentration of each component after mixing is preferably as follows.
The heparin concentration after mixing the heparin aqueous solution is preferably 0.5 mg / mL or more, more preferably 1 mg / mL or more, still more preferably 3 mg / mL or more, and further preferably 5 from the viewpoint of enhancing the formation of aggregates. mg / mL or more, more preferably 8 mg / mL or more, and preferably 20 mg / mL or less, more preferably 15 mg / mL or less, and further preferably 13 mg / mL or less.
The heparin aqueous solution may be, for example, a heparin preparation as described above.

The polyamine concentration after mixing with the polyamine aqueous solution is preferably 5 μmM or more, more preferably 10 μmM or more, further preferably 30 μmM or more, and preferably 80 μmM or less, from the viewpoint of enhancing the formability of the aggregate. More preferably, it is 60 μm or less, and still more preferably 50 μm or less.

The conditions at the time of mixing the heparin aqueous solution and the polyamine aqueous solution are not particularly limited, and the aggregate can be easily formed by mixing these aqueous solutions at room temperature.
The pH of the mixed solution containing the aggregate is preferably 1 or more, more preferably 3 or more, still more preferably 4 or more, and preferably 6 or less from the viewpoint of the formability of the aggregate.

[Application of the aggregate]
The aggregate is preferably used as an anticoagulant. By using the aggregate as an anti-coagulant and administering the aggregate in vivo, heparin is gradually released in vivo, so that the half-life of heparin after administration is long and the treatment area is long. That is, it is used as a blood coagulation preventing method in which an aggregate is administered in vivo.

From the viewpoint of use as an anticoagulant or the like, the aggregate is preferably an aggregate of unfractionated heparin and the compound represented by the above formula (1), more preferably unfractionated. It is an association of heparin and Taa. By using the aggregate, the maximum blood concentration reaching time and half-life of heparin are extended, and the maximum blood concentration can be kept low.

In another aspect, from the viewpoint of use as an anticoagulant or the like, the aggregate is preferably an aggregate of low molecular weight heparin and a compound represented by the above formula (1), and more Preferably, it is an aggregate of low molecular weight heparin and Taa. By using the aggregate, the time for reaching the maximum blood concentration of heparin is extended, and the maximum blood concentration can be kept low.

The administration method includes, for example, oral administration, subcutaneous administration, and intravenous administration, preferably subcutaneous administration or intravenous administration, more preferably subcutaneous administration.

The aggregate is used as a drug composition containing the aggregate.
From the viewpoint of oral administration, for example, a mixture of the above-mentioned heparin aqueous solution and a polyamine aqueous solution may be used as it is as the drug composition.
From the viewpoint of use in subcutaneous administration, the pharmaceutical composition may be used by mixing a mixture of the above-mentioned heparin aqueous solution and the polyamine aqueous solution by centrifugation, and mixing the separated aggregate with a vegetable oil such as corn oil.
The pharmaceutical composition may be suspended in physiological saline or the like from the viewpoint of intravenous administration.

The dose of the aggregate is, for example, from 1 to 60 mg / kg (body weight), more preferably from 10 to 50 mg / kg (body weight), more preferably from 20 to 20 in terms of the mass of heparin contained in the aggregate. 40 mg / kg (body weight).

[kit]
The aggregate of the present invention can be easily obtained by mixing two aqueous solutions. Therefore, it can also be implemented as a kit containing heparin or a salt thereof and polyamine or a salt thereof.
A kit for producing the aggregate (hereinafter also simply referred to as “kit”) includes heparin or a salt thereof, and a linear or branched polyamine having three or more amino groups, or a salt thereof.
In the kit, heparin or a salt thereof is preferably a heparin aqueous solution.
In the kit, the polyamine or a salt thereof is preferably a polyamine aqueous solution.
The concentration of the aqueous heparin solution and the aqueous polyamine solution is preferably in the range of the concentration after the above-mentioned mixing.
The kit may contain heparin or a salt thereof and polyamine or a salt thereof as separate packaging units.
The kit may include instructions for mixing heparin or a salt thereof and polyamine or a salt thereof. In addition, the kit may include instructions for centrifuging the mixture, mixing the separated aggregate with a vegetable oil such as corn oil, and administering subcutaneously.
Any of the compounds and drugs contained in the kit can contain one or more pharmaceutically acceptable carriers (pharmaceutical carriers) as described above, if necessary.

[Slow release agent]
Since the aggregate of the present invention can be easily obtained by mixing two kinds of aqueous solutions, a linear or branched polyamine having three or more amino groups or a salt thereof is imparted with sustained release of heparin. It may be used as a sustained release imparting agent.
The sustained-release imparting agent can be used, for example, by mixing with a commercially available heparin preparation or the like to form an aggregate. That is, the use of the above-mentioned polyamine or a salt thereof for imparting sustained release of heparin, or the use of the above-mentioned polyamine or a salt thereof for forming an association with heparin is contemplated.

As described above, according to the aggregate of the present invention, a novel aggregate, an anticoagulant comprising the aggregate, a pharmaceutical composition containing the aggregate, a kit for producing the aggregate, a sustained release agent, and the aggregate In addition, the heparin has a long half-life after administration and has a long therapeutic window, an association comprising the association, a pharmaceutical composition containing the association, a kit for producing the association, and imparting sustained release The agent and the method for producing the aggregate are obtained.

Hereinafter, although the Example of this invention is shown concretely, it is not limited to the said aspect.

[sample]
Heparin sodium salt (Unfractionated Heparin, used as “UFH”) (from porcine small intestine, titer 100 IU / mg, number average molecular weight 13,000, average molecular weight per disaccharide 534, average negative charge per disaccharide -3.38) was purchased from New Zealand Pharmaceutical. Clexane Subcutaneous Injection Kit 2000 IU (enoxaparin sodium injection solution) was purchased from Sanofi Corporation. Heparin hexasaccharide (MW: 1800), 10 sugar (MW: 3000) and 20 sugar (MW: 5750) standard products were purchased from iduron. 1,3-diaminopropane, thermine, caldopentamine, mitsubishine and Tetrakis (3-aminopropyl) ammonium (Taa, chloride salt) were donated by Dr. Yusuke Terui of Chiba University of Science. All other reagents used were commercially available special grades and HPLC grade, and water was purified with a Milli-Q ultrapure water system.

[apparatus]
1-1. Determination of polyamine The eluent pump is Chromaster 5110 Pump manufactured by Hitachi High-Tech Science Co., Ltd., the autosampler is ELITE LaChrom L-7200 manufactured by Hitachi High-Tech Science Co., Ltd., and the column thermostat is ELITE LaChrom L- manufactured by Hitachi High-Tech Science Co., Ltd. 7300, JASCO Corporation Intelligent Fluorescence Detector FP-1520S was used as the fluorescence detector, and Tosoh Corporation TSKgel Polyaminepak (4.6 mm x 50 mm) was used as the column.

1-2. Heparin determination The eluent pump is DP8020 manufactured by Tosoh Corporation, the degasser is DGU-12A manufactured by Shimadzu Corporation, the post pump is MINICHEMI PUMP manufactured by Japan Precision Science Co., Ltd., and the autosampler is manufactured by Tosoh Corporation AS-8020. , Dry reactor is Reactor 522 manufactured by FROM Co., Ltd., Fluorescence detector is Intelligent Fluorescence Detector FP-1520S manufactured by JASCO Corporation, Asahipak NH2P-50 4E (4.6 mm x 250 mm) manufactured by Showa Denko KK, guard column Asahipak NH2P-50G 4A (4.6 mm x 10 mm) manufactured by Showa Denko KK was used.

1-3. Measurement of heparin molecular weight 1-2. The same apparatus as that used for heparin determination was used. Asahipak GF-510 HQ (7.6 mm x 300 mm) manufactured by Showa Denko KK was used as the column.

1-4. Data analysis Tosoh Corporation LC data analysis application was used for heparin quantification, and Runto Instruments Inc. chromatoPRO-GPC and chromatoPRO were used for other data analysis.

2. Photolytic reaction The light source is Sen Special Light Source Co., Ltd. high pressure mercury lamp HL100CH-4, the power source is Sen Special Light Source Co., Ltd. HB100A-1, the lamp jacket is Sen Special Light Source Co., Ltd. water-cooled lamp jacket JW-1G, the reaction tank Used a reaction vessel VG500 manufactured by Sen Special Light Source Co., Ltd.

[Production example]
LMWH Purification Clexane Subcutaneous Injection Kit 2000 IU was dialyzed using Spectra Pore Dialysis Membrane 1K, and then freeze-dried to obtain LMWH (Low Molecular Weight Heparin, titer 120 IU / mg, average per disaccharide The molecular weight was 535, and the average negative charge per disaccharide was -3.43).

Preparation of photolytic heparin (HP 7,000) The molecular weight of UFH was reduced using a photolysis reactor (K. Higashi, S. Hosoyama, A. Ohno, S. Masuko, B. Yang, E. Sterner, Z. Wang , RJ Linhardt, T. Toida, Photochemical Preparation of a Novel Low Molecular Weight Heparin, Carbohydr. Polym., 67 (2012) 1737-43.). The apparatus consists of a high-pressure mercury lamp, a reaction vessel, and a water-cooled lamp jacket. It is possible to cool the heat generated during the reaction by allowing the water-cooled lamp jacket to react while flowing cooling water. 130 mg of UFH was weighed into a glass tube and dissolved in 13 mL of double distilled water (DDW), and then 13 mg of titanium dioxide was added and stirred well. A total of three samples were prepared, and the sample was prepared by irradiating light for a predetermined time while stirring in the apparatus. The reaction time was 0, 0.5, 1, 2, 3, 3.5, 3.75, 4, 4.5 hours, and each sample was collected when it reached about MW: 6,700. Thereafter, centrifugation is performed, and the supernatant is filtered through a syringe filter to remove titanium dioxide, followed by dialysis, lyophilization, photolysis heparin (hereinafter also referred to as “HP 7,000”, titer 100 IU / mg, An average molecular weight of 523 per disaccharide and an average negative charge per disaccharide of 3.20) were obtained.

[Measuring method]
[Quantification of heparin]
UFH obtained from pH 1 to pH 3 aggregates was dissolved in 5 mL of purified water, and UFH obtained from pH 4 to pH 9 aggregates was dissolved in 500 μL of purified water. Heparin quantification was performed using fluorescence post-column detection (Y. Huang, Y. Washio, M. Hara, H. Toyoda, I. Koshiishi, T. Toida, T. Imanari., Simultaneous determination of dermatan sulfate and oversulfated dermatan sulfate. in plasma by high-performance liquid chromatography with postcolumn fluorescence derivatization, Anal. Biochem., 240 (1996) 227-34.). Asahipak NH ₂ column was used, the flow rate was 0.5 mL / min, and the column temperature was room temperature. Because of the two-liquid gradient, the eluent was (A) 0.1 M sodium carbonate buffer (pH 10.0) containing 0.1 M NaCl and (B) 0.1 M sodium carbonate buffer (pH 10.0) containing 1.0 M NaCl. Gradient elution under the conditions of (0-100 v / v% B), 20-25 min (100 v / v% B), 25-27 min (100-0 v / v% B), 0 v / v Equilibrated with% eluent B for 8 minutes. 50 mM guanidine and 1.0 M NaOH used as post-column reagents were both sent by a double plunger pump at a flow rate of 0.25 mL / min. While passing through the reaction coil (0.5 mm id × 10 m), the mixed solution was heated and reacted at 120 ° C. using a dry reaction tank, and cooled with a cooling coil (0.25 mm id × 5 m). After the reaction, fluorescence was detected at an excitation wavelength of 320 nm and a fluorescence wavelength of 425 nm. UFH was quantified by drawing a calibration curve using UFH 25 ppm, 50 ppm, 75 ppm, and 100 ppm as standard products.

[SPM quantification]
Quantification of SPM was performed using a fluorescent post-column method (K. Igarashi, K. Kashiwagi, H. Hamasaki, A. Miura, T. Kakegawa, S. Hirose, S. Matsuzaki, Formation of a compensatory polyamine by Escherichia coli polyamine-requiring mutants during growth in the absence of polyamines, J. Bacteriol., 166 (1986) 128-34.). Using TSKgel polyaminepak (4.6 mm x 50 mm), Buffer II (0.08 w / v% Briji-35, 20 v / v% MeOH, 0.64 mM hexanoic acid, 2 M NaCl, 0.35 M sodium citrate) as the eluent flow rate The solution was fed at 0.42 mL / min. Post column reagent orthophthalaldehyde solution (0.63 v / v% MeOH, 0.1 w / v% Briji-35, 0.4 M boric acid, 0.35 M NaOH, 4 mM orthophthalaldehyde, 28 mM 2-mercaptoethanol) The solution was fed at 0.42 mL / min. After the reaction, detection was performed under conditions of an excitation wavelength of 336 nm and a fluorescence wavelength of 470 nm. The column temperature was 50 ° C. As a standard, 20 μL of 5 μM SPM solution was used.

[Taa quantitative]
Taa was quantified by modifying the above SPM quantification. A buffer II adjusted to pH 3.2 with 3 M hydrochloric acid was used as an eluent, and the solution was fed at a flow rate of 0.7 mL / min. The column temperature was 70 ° C. As a standard, 20 μL of Taa 20 μM solution was used. Other conditions followed the SPM quantification method.

[Heparin number average molecular weight measurement]
The number average molecular weight of heparin was measured using a fluorescent post column method. Asahipak GF-510 HQ column was used, the flow rate was 0.3 mL / min, and the column temperature was room temperature. The eluent was 10 mM ammonium hydrogen carbonate and analyzed under isocratic conditions. Detection was performed with UV at 204 nm. The number average molecular weight of heparin was measured by drawing a calibration curve using dp 6 (MW 1,800), dp 10 (MW 3,000) and dp 20 (MW 5,750) standards. For analysis of data, chromatoPRO-GPC manufactured by Instruments Inc. was used.

[Transmissivity]
The transmittance was measured with SPECTRONIC 20D + (Thermo).

[Manufacture of aggregates]
Effect of Concentration Ratio on Aggregate Formation The concentrations of 100 μL of UFH aqueous solution and 100 μL of polyamine aqueous solution were adjusted with purified water so that the concentration was twice the target aggregate solution (Table 1). A solution of the desired concentration was prepared by mixing the polyamine aqueous solution with the same volume of UFH aqueous solution and mixed well. The degree of cloudiness was evaluated by transmittance (% T). Table 1 shows the results of mixing the UFH aqueous solution and each polyamine aqueous solution. The concentration is shown as the final concentration after mixing.
Among the three polyamines, SPM showed high formation efficiency, and the aggregate formation efficiency decreased as the number of SPD, PUT and amino groups decreased. This result is thought to be due to the fact that PUT has a shorter molecular chain than other polyamines and has a small charge per molecule, so the interaction with UFH was weak. It was suggested that the number was affected. The higher the UFH and SPM concentrations in the solution, the higher the formation efficiency. However, when either the UFH or SPM concentration was extremely high, the formation efficiency tended to decrease. These results suggest that the aggregate formation efficiency is affected by the ratio of UFH to polyamine in the solution.

Effect of heparin molecular weight on aggregate formation The concentrations of the heparin aqueous solution and the SPM aqueous solution were adjusted with purified water so that the concentration of the target aggregate solution was doubled (Table 2). A solution of the desired concentration was prepared by mixing the polyamine aqueous solution with the same volume of heparin aqueous solution and mixed well. The degree of cloudiness was evaluated by transmittance (% T). Table 2 shows changes in aggregate formation efficiency when the molecular weight of heparin is changed. Here, as the polyamine, SPM having the highest aggregate formation efficiency among SPM, SPD and PUT was used. The formation of aggregates was also confirmed when HP 7,000 and LMWH were used, but the formation efficiency tended to decrease as the molecular weight decreased.

Effect of pH on aggregate formation After the formation of aggregates by mixing equal volumes of 20 mg / mL UFH aqueous solution and 20 mmol / L SPM aqueous solution, 1 mol / L HCl and 0.2 mol / L NaOH were used. The pH was adjusted from 1 to 8. 2 mol / L NaOH was used to make the pH 9 aggregate solution. The solution was centrifuged at 6,000 × g for 5 minutes to precipitate the aggregate layer, remove the aqueous layer, and then washed the aggregate twice with water adjusted to the same pH. The dissociation of the aggregate was performed by using 2.0 mol / L NaOH to adjust the pH of the aggregate solution to 9 or more, and UFH and SPM were separated by centrifuging at 6,000 x g and 4 ° C for 30 minutes using Amikon Ultra 10K. The residue on Amikon Ultra 10K was made into a UFH solution and freeze-dried to obtain UFH. On the other hand, the passing fraction was used as the SPM fraction, and SPM was obtained by lyophilization.
The SPM and UFH contained in the aggregate prepared under each pH condition were dissociated, and the SPM in the aggregate was quantified by the method shown in the above [SPM quantification]. The results quantified by the method shown in [Heparin quantification] are shown in FIG. 1B. On the acidic side, both the amount of UFH and SPM forming aggregates increased. Aggregates prepared from UFH and SPM are negatively charged, and when the zeta potential of the aggregate solution with varying pH was measured, the surface charge approached 0 as it proceeded to the acidic side. We thought that tilting the pH to the acidic side neutralized the negative charge and increased the formation of aggregates (Table 3).
Table 4 shows the ratio of the number of SPM molecules to one UFH molecule. In the range where the pH was less than 6, 30 to 40 SPM molecules were bound per UFH molecule.

Formation of aggregates using polyamine The concentrations of 20 μL of the UFH aqueous solution and 20 μL of the polyamine aqueous solution were adjusted with purified water so that the concentration of the target aggregate solution was doubled (Table 5). A solution of the desired concentration was prepared by mixing the polyamine aqueous solution with the same volume of UFH aqueous solution and mixed well. The degree of cloudiness was judged visually. Table 5 shows the results of mixing UFH and each polyamine. The displayed density is the final density after mixing. Further, the aggregate formed using Taa was in a solid state, and its form was greatly different from the liquid aggregate formed using other polyamines. From these results, it was suggested that the number of amino groups contained in the polyamine molecule and the branched chain structure are important. Of these, the interaction between UFH and Taa was found to be particularly strong. The aggregate of UFH and Taa was glued. Therefore, since the transmittance could not be measured, it was shown by visual evaluation.

Effect of charge ratio on aggregate formation PIC solution prepared by mixing 20 μL of 20 mg / mL UFH aqueous solution and 20 μL of 80, 40, 20, 10, 5 mM Taa aqueous solution was centrifuged at 6,000 xg for 5 minutes. The aggregate and supernatant were separated. Taa was quantified by adding 80 μL of 5 w / w% TCA (trichloroacetic acid) to 20 μL of the supernatant. Further, 20 μL of the supernatant was diluted by adding 80 μL of water, and UFH was quantified. The negative charge of UFH was calculated using an average molecular weight per disaccharide of 534, an average charge per disaccharide of -3.38. The positive charge possessed by Taa was calculated using the charge +5 per molecule. Using the calculated charge, the ratio of the negative charge of UFH to the positive charge of Taa (negative / positive ratio, N / P ratio) was determined.
As described above, after the aggregate was prepared, the UFH and Taa remaining in the supernatant were quantified to examine the aggregate formation. FIG. 2 is a graph showing the relationship between the amount of UFH and Taa in the supernatant and the ratio of the negative charge of UFH to the positive charge of Taa (negative / positive ratio, N / P ratio). In the figure, the concentration of Taa is 80 mM for a, 40 mM for b, 20 mM for c, 10 mM for d, and 5 mM for e. As the Taa concentration was increased to 5 mM (e), 10 mM (d), and 20 mM (c), the ratio of UFH and Taa in the supernatant decreased, confirming an increase in PIC formation efficiency. . In particular, when 20 mM (c) Taa is mixed, almost all UFH and Taa form PIC, and the ratio of the negative charge of the mixed UFH to the positive charge of Taa (negative / positive ratio, N / P ratio) ) Was 1.3. When the concentrations of 40 mM (b), 80 mM (a), and Taa were further increased, free Taa increased in the supernatant, which revealed that Taa was excessive under these conditions. These results suggest that aggregates composed of UFH and Taa have the highest aggregate formation efficiency at a concentration ratio where the N / P ratio is 1.
In addition, approximately 20 molecules of Taa were associated with each UFH molecule, suggesting that approximately 1 molecule of Taa was associated with each UFH disaccharide unit.

[In vivo experiment]
1. Mouse Female ddY mice (hereinafter also referred to as “ddY mice”) were purchased from Japan SLC. The animal was preliminarily raised for more than a week in a gauge with a chip maintained at 25 ° C. and 55% relative humidity in the animal department of the Faculty of Pharmaceutical Sciences, Chiba University, and then used for the experiment. Food (MF, oriental yeast) and drinking water were all ad libitum. In the case of an oral administration experiment, fasting was performed for 24 hours in consideration of the effect of heparin contained in the diet.

2. Sample
2.1. Standard Protein, Synthetic Substrate Factor Xa, Normal Human Plasma, Antithrombin III and Chromogenic Synthetic Substrate S-2222 were purchased from Sekisui Medical Co., Ltd. using Heparin Kit Test Team (registered trademark) Heparin S. Thrombin and chromozyme TH were purchased from Roche Diagnostics GmbH.
2.2. Reagents Heparin sodium salt (20 kDa, derived from porcine small intestine, titer 100 IU / mg, number average molecular weight 13,000, average molecular weight per disaccharide 534, average negative charge per disaccharide −3.38, the heparin “UFH” Was purchased from New Zealand Pharmaceutical. Clexane subcutaneous injection 2000 IU (enoxaparin sodium injection solution) purchased from Sanofi Co., Ltd. was dialyzed using Spectra Pore Dialysis Membrane 1K, and then freeze-dried to use the heparin obtained from enoxaparin as LMWH (titer) 120 IU / mg, average molecular weight 535 per disaccharide, average negative charge per disaccharide -3.43). H3393-50KU heparin sodium salt, grade IA: from porcine imtestinal mucosa (212 USP units / mg) was purchased from Sigma Aldrich.

2.3. Others All other reagents used were commercially available special grades and HPLC grade, and water was purified using a Milli-Q ultrapure water system.

3． apparatus
3.1. Absorbance measurement TECAN Austria GmbH Austrian Sunrise Thermo was used as a detector.

Anti-Factor Xa activety assay
・ Preparation of standard solution
Clexane subcutaneous injection kit (2000 IU / 0.2 mL) was used as a standard product. The standard product was diluted to 10 IU / mL with 0.9 w / w% sodium chloride aqueous solution, and further diluted to 0.2 IU / mL with 50 mM Tris buffer (pH 8.4). Dissolve

dilutions

0, 6, 12, 18, 24 μL in 50 mM Tris buffer (pH 8.4) 48, 42, 36, 30, 24 μL, respectively, and add 1 U / mL ATIII 6 μL and human normal plasma 6 μL. In addition, standard solutions of 0, 0.2, 0.4, 0.6, 0.8 U / mL plasma were prepared.
・ Measurement 6 μL of the measurement sample was dissolved in 48 μL of 50 mM Tris buffer (pH 8.4). To this, 6 μL of 1 U / mL ATIII was added to prepare a sample solution. After 60 μL of the standard solution or measurement sample was heated at 37 ° C. for 5 minutes, 30 μL of Factor Xa solution adjusted to 7.1 nkat / mL was added, and heated exactly at 37 ° C. for 30 seconds. To this was added 60 μL of S-2222 solution adjusted to 0.75 mg / mL, and the mixture was heated at 37 ° C. for exactly 3 minutes, and then 90 μL of 50 w / w% acetic acid aqueous solution was added to stop the reaction. After stopping the reaction, measurement was performed at 405 nm. A standard curve was used to plot the anti-factor Xa activity (IU / mL plasma) and absorbance to create a calibration curve, and the anti-factor Xa activity of each sample was calculated. At this time, Anti-Factor Xa activity in the specimen after 0 hour was treated as a blank of the mouse. When Anti-Factor Xa activity in the measurement sample was larger than 0.8 U / mL, the measurement sample was diluted with human normal plasma to prepare a measurement sample solution, and measurement was performed again.
The therapeutic range for Anti-Factor Xa activity is 0.3 to 0.8 IU / mL.

Anti-Factor IIa activity assay
-Preparation of standard solution As a standard product, heparin sodium salt (212 USP units / mg, sigma Aldrich) was used. Standards were diluted to 0.2 USP / mL with purified water.

Diluted solutions

0, 10, 20, 30, 40 μL are dissolved in buffers (pH 8.4) 270, 260, 250, 240, 230 μL each containing 50 mM Tris-HCl and 227 mM NaCl, and 3 U / mL FIIa solution 6 μL and 9 μL of human normal plasma were added to prepare standard solutions of 0, 0.2, 0.4, 0.6, 0.8 USP / mL plasma, respectively.
・ Measurement 9 μL of the measurement sample was dissolved in 270 μL of buffer. To this was added 6 μL of 3 U / mL FIIa solution to prepare a sample solution. After heating 285 μL of the standard solution or measurement sample at 25 ° C. for 5 minutes, 15 μL of Chromozyme TH solution adjusted to 1.9 mmol / L was added, and the mixture was heated accurately at 25 ° C. for 5 minutes. The reaction was stopped by adding 100 μL of a 10 w / w% aqueous citric acid solution. After stopping the reaction, measurement was performed at 405 nm. A standard curve was used to plot the anti-factor IIa activity (USP / mL plasma) and absorbance to create a calibration curve, and the anti-factor IIa activity of each sample was calculated. At this time, Anti-Factor IIa activity in the specimen after 0 hour was treated as a blank of the mouse. When Anti-Factor IIa activity in the measurement sample was larger than 0.8 U / mL, the measurement sample was diluted with human normal plasma to prepare a measurement sample solution, and measurement was performed again.

Subcutaneous administration of an aggregate consisting of UFH and Taa An aggregate consisting of UFH and Taa was subcutaneously administered to ddY mice. As for the aggregate, 20 mg / mL UFH and 80 mM Taa were mixed in the same volume, and then centrifuged at 800 × g for 10 minutes to remove the supernatant. Cone oil (1.7 mL / kg (body weight)) was added to the resulting precipitate, the aggregate was suspended in the oil, and then subcutaneously administered to the back of the mouse. The amount of UFH was 4 mg / kg (body weight) administered UFH (heparin calcium subcutaneous injection 5,000 units / 0.2 mL syringe “Mochida” package insert, Mochida Pharmaceutical Co., Ltd., revised in January 2016, 5th edition) The volume of UFH and Taa to be mixed was calculated and adjusted. That is, to 30 g of mice, 120 μg of UFH was subcutaneously administered by preparing an aggregate in which 6 μL of UFH and Taa were mixed. Thereafter, blood was collected from the tail vein after 2, 4, 8, 12, 16, 20, and 24 hours. Before subcutaneous administration, blood was collected from the tail vein and used as a sample after 0 hour. Plasma was obtained by centrifuging the collected blood at 4 ° C. and 800 × g for 15 minutes. Moreover, the aggregate which mixed UFH and Taa was prepared so that the administered UFH might be set to 30 mg / kg (body weight), and it administered subcutaneously to the mouse | mouth after performing the same process. That is, for 30 g of mice, 45 μL each of UFH and Taa were mixed and 900 μg UFH was subcutaneously administered. As a control group, mice with UFH 4 mg / kg (body weight) and UFH 30 mg / kg (body weight) suspended in cone oil and administered subcutaneously were also administered subcutaneously 0, 0.5, 1, 2, 4, 6 8 and 12 hours later, blood was collected from the tail vein, and plasma was prepared in the same manner as in the aggregate group. Specifically, 20 mg / kg UFH 6 μL and 100 mg / kg UFH 9 μL were suspended in cone oil and administered subcutaneously to 30 g mice.

As described above, aggregates prepared from UFH and Taa were subcutaneously administered to 12 ddY mice, and changes in plasma Anti-Factor Xa activity over time were examined (FIG. 3).
FIG. 3 shows changes over time in plasma Anti-Factor Xa activity after subcutaneous administration of UFH and UFH-Taa aggregates to mice. FIG. 3A shows the results at a UFH dose of 4 mg / kg (body weight), FIG. 3B shows the results at a UFH dose of 30 mg / kg (body weight), and FIG. The results at a dose of 30 mg / kg (body weight) of Taa aggregates are shown. In the 4 mg / kg (body weight) control group (FIG. 3A), the time to reach the maximum blood concentration was 0.5 hours after administration, and the half-life of Anti-Factor Xa activity was about 1 hour. It disappeared from the blood in 4 hours. On the other hand, in mice administered 30 mg / kg (body weight) as UFH, the time to reach the maximum blood concentration was extended 4 hours after administration, and Anti-Factor Xa activity could be detected until 20 hours later. (FIG. 3C). The half-life of Anti-Factor Xa activity was extended to 6 hours. When the same volume of UFH (20 mg / kg body weight) as the aggregate was administered subcutaneously to the mouse, the maximum blood concentration time was 1 hour, and a rapid increase in Anti-FXa activity was confirmed immediately after administration. (Figure 3B).
At a UFH dose of 4 mg / kg (body weight), the treatment duration of Anti-FXa activity is 0.86 hours, and at a UFH dose of 30 mg / kg (body weight), the treatment range of Anti-FXa activity is sustained. The time was 1.3 hours, and at a dose of 30 mg / kg (body weight) of the UFH-Taa aggregate, the therapeutic range duration of Anti-FXa activity was 4.5 hours. The duration of these treatment areas was calculated by calculating the duration of each treatment area from the blood concentration transition of each mouse.
From the above results, it was suggested that the aggregate prepared from UFH and Taa has utility as a sustained-release preparation of UFH.

The time course of plasma Anti-Factor IIa activity in this group of mice was examined.
FIG. 4 shows changes over time in plasma Anti-Factor IIa activity after subcutaneous administration of UFH and UFH-Taa aggregates to mice. FIG.4A shows the results at a UFH dose of 4 mg / kg (body weight), FIG.4B shows the results at a UFH dose of 30 mg / kg (body weight), and FIG.4C shows UFH- The results at a dose of 30 mg / kg (body weight) of Taa aggregates are shown. Anti-Factor IIa activity in plasma was also detected in the group of mice that received the aggregate.

Subcutaneous administration of an aggregate consisting of LMWH and Taa For an aggregate consisting of LMWH and Taa, an aggregate in which 20 mg / mL LMWH and 80 mM Taa were mixed in the same volume was prepared in the same manner as UFH and subcutaneously administered to mice. The amount of LMWH should be 2 mg / kg (body weight) of the administered LMWH (Pharmaceutical Interview Form Clexane (Registered Trademark) Subcutaneous Injection Kit 2000 IU, Kaken Pharmaceutical Co., Ltd., Revised March 2015, 8th Edition) The volume of LMWH and Taa to be mixed was calculated and prepared. That is, to 30 g of mice, 60 μg of LMWH was subcutaneously administered by preparing an aggregate in which 3 μL each of LMWH and Taa were mixed. Thereafter, blood was collected from the tail vein 0.5, 1, 2, 4, 6, and 8 hours later, and the blood was centrifuged at 4 ° C. and 800 × g for 15 minutes to obtain plasma. Moreover, the aggregate which mixed LMWH and Taa was prepared so that the administered LMWH might be 10 mg / kg, and it administered subcutaneously to the mouse | mouth after performing the same process. That is, for 30 g of mice, 15 μL each of LMWH and Taa were mixed and 300 μg LMWH was subcutaneously administered.
As a control group, LMWH 2 mg / kg (body weight) and LMWH 10 mg / kg (body weight) were suspended in cone oil and administered subcutaneously, blood was collected over time in the same manner as in the association group. Got. That is, for 30 g mice, 20 mg / kg LMWH 3 μL or 15 μL was suspended in cone oil and administered subcutaneously.
Three ddY mice were administered subcutaneously with an aggregate prepared from LMWH and Taa, and the time course of plasma Anti-Factor Xa activity was examined.
FIG. 5 shows changes over time in plasma Anti-Factor Xa activity after subcutaneous administration of LMWH and LMWH-Taa aggregates to mice.
In the 2 mg / kg (body weight) control group and the 10 mg / kg (body weight) control group, the time to reach the maximum blood concentration was 0.5 hours after administration. On the other hand, in mice administered with 2 mg / kg (body weight) and 10 mg / kg (body weight) LMWH as an aggregate, the time to reach the maximum blood concentration was prolonged 2 hours after administration.

The aggregate of the present invention can be used, for example, as an anticoagulant having sustained release properties.

Claims

An association of heparin and a linear or branched polyamine having 3 or more amino groups.
The aggregate according to claim 1, wherein the polyamine is a branched polyamine.
The polyamine has the formula (1):

[Wherein R 1 , R 2 , R 3 and R 4 are each independently a divalent aliphatic hydrocarbon group having 3 or 4 carbon atoms. The association according to claim 1 or 2, which is a compound represented by the formula:
The aggregate according to any one of claims 1 to 3, wherein the heparin is unfractionated heparin or low molecular weight heparin.
The polyamine includes at least one selected from the group consisting of spermidine, spermine, theremin, cardopentamine, tris (3-aminopropyl) amine, and tetrakis (3-aminopropyl) ammonium. The association according to any one of the above.
The aggregate according to any one of claims 1 to 5, wherein the polyamine contains tetrakis (3-aminopropyl) ammonium.
An anticoagulant comprising the aggregate according to any one of claims 1 to 6.
A pharmaceutical composition comprising the aggregate according to any one of claims 1 to 7.
A kit for producing an aggregate comprising heparin or a salt thereof and a linear or branched polyamine having 3 or more amino groups, or a salt thereof.
A sustained release imparting agent for imparting sustained release of heparin, comprising a linear or branched polyamine having 3 or more amino groups, or a salt thereof.
A method for producing an aggregate, comprising mixing an aqueous solution of heparin or a salt thereof and an aqueous solution of a linear or branched polyamine having 3 or more amino groups or a salt thereof.