JP5263349B2 - Biologically active molecule-containing crosslinked heparin gel composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a photo-crosslinked heparin solvation hydrogel of a novel substance. <P>SOLUTION: The photo-crosslinked heparin solvation hydrogel is obtained by a method of preparing a solution containing components B-D shown hereinafter, to be frozen thereafter, by irradiating an obtained frozen matter with light, to be gelled, and by melting the frozen gel, where B is a photoreactive heparin wherein cinnamic acid amino propyl is coupled with a 6-desulfated heparin derivative obtained by removing partially or completely a sulfate group bonded to 6-hydroxyl group of hexosamine (N-sulfated glucosamine or N-acetylglucosamine) residue of a heparin skeleton; C is a polyethylene glycol or sodium edetate; and D is an aqueous solvent capable of dissolving the B and C components. <P>COPYRIGHT: (C)2012,JPO&amp;INPIT

Description

  The present invention relates to a crosslinked heparin gel composition containing a bioactive molecule.

  Fibroblast growth factor (hereinafter also referred to as FGF) is a kind of heparin-binding growth factor (hereinafter also referred to as HBGF) present in the body, and has an action such as angiogenesis and has an affinity for heparin. It is known to have. HBGF such as FGF is rapidly degraded by various proteolytic enzymes in the living body, and therefore generally has a short active half-life (hereinafter also referred to as stability), but HBGF forms a complex with heparin. As a result, resistance to proteolytic enzymes is obtained, and experimental results have been reported such as improved stability. However, in animal experiments and the like, if heparin and b-FGF were simply administered in a mixed manner, they disappeared easily from the administration site, and thus a sufficient effect could not be expected.

  Therefore, recently, in order to prevent disappearance from the administration site, a sustained release agent by incorporating a growth factor into a crosslinked product obtained by crosslinking heparin and one or more polysaccharides other than heparin has been proposed ( Patent Document 1). However, such a sustained-release agent is a xerogel obtained by imparting mechanical strength to a cross-linked product (base material) containing a growth factor. Therefore, the administration method thereof is a method of applying to a biological defect part or a sewing method. Yes, not an injection method.

  By the way, conventionally, photocrosslinked GAG obtained by introducing a photoreactive group into glycosaminoglycan (hereinafter also referred to as GAG) and crosslinking the photoreactive group by irradiation with light such as ultraviolet rays has been reported. In addition to the types of GAGs, various photocrosslinked GAGs have also been reported due to differences in properties such as gels and sponges (Patent Documents 2 to 5). However, conventionally, there is no known gel that contains HBGF in photocrosslinked GAG and that can be injected into a site where HBGF is to be administered by an injection tool.

JP 2001-233786 A JP-A-6-073102 JP-A-8-143604 JP-A-9-087236 WO02 / 060971 pamphlet

  The present invention has been made in view of the above circumstances, and its purpose is to achieve the stability of HBGF at the administration site and to exert its effect continuously, and is an optimal form for local administration. The object is to provide a crosslinked heparin gel composition containing a physiologically active molecule to which an injection method can be applied.

  As a result of intensive studies to solve the above problems, the present inventors have obtained the following knowledge. That is, if a solution containing a crosslinked heparin and a heparin-binding physiologically active molecule is crosslinked under a specific condition, particularly a method of photocrosslinking heparin having a photoreactive group bonded thereto, it has not been conventionally known. It was found that a crosslinked heparin gel composition having a viscosity to which the injection method can be applied was obtained, and that the heparin-binding growth factor was stabilized according to the crosslinked heparin gel composition.

  The present invention has been completed on the basis of the above findings, and is composed of a plurality of related inventions, the gist of which is as follows.

  The first gist of the present invention resides in a bioactive molecule-containing cross-linked heparin gel composition containing at least cross-linked heparin and a heparin-binding bioactive molecule and having a viscosity that can be extruded from an injection tool.

  The second gist of the present invention is obtained by a method in which a solution containing the following components A to D is prepared and then frozen, and the resulting frozen product is irradiated with light to gel, and then the frozen gel is melted. It exists in the crosslinked heparin gel composition containing the bioactive molecule | numerator which has the viscosity which can be extruded from the injection tool characterized by the above-mentioned.

A: Heparin-binding physiologically active molecule B: Heparin having a photoreactive group bound thereto (hereinafter also referred to as “photoreactive heparin”)
C: Component having miscibility with any aqueous solvent selected from the group consisting of alcohol, surfactant and chelating agent D: Aqueous solvent capable of dissolving components A, B and C

  The third gist of the present invention resides in a medical preparation characterized by including the above-mentioned bioactive molecule-containing crosslinked heparin gel composition as an active ingredient.

  The fourth gist of the present invention resides in a preparation for topical administration, characterized in that it contains the above-mentioned crosslinked heparin gel composition containing physiologically active molecules as an active ingredient.

  The fifth gist of the present invention resides in a preparation for sustained release of a heparin-binding bioactive molecule characterized in that it contains the above-mentioned cross-linked heparin gel composition containing a bioactive molecule as an active ingredient.

  The sixth gist of the present invention resides in an angiogenesis-promoting agent comprising the above-mentioned bioactive molecule-containing crosslinked heparin gel composition as an active ingredient.

  The seventh gist of the present invention resides in a therapeutic agent useful for peripheral ischemic disease characterized by containing each of the above-mentioned preparations or the above-mentioned angiogenesis promoting agent as an active ingredient.

  The eighth gist of the present invention resides in a method for stabilizing a heparin-binding bioactive molecule characterized in that a heparin-binding bioactive molecule is allowed to coexist with a crosslinked heparin gel.

  The ninth gist of the present invention resides in a kit characterized by filling the extrudable container with the bioactive molecule-containing crosslinked heparin gel composition.

  The tenth gist of the present invention is characterized in that a solution containing the following components A to D is prepared and then frozen, and the resulting frozen product is irradiated with light to gel, and then the frozen gel is melted. The present invention resides in a method for producing a crosslinked heparin gel composition containing a bioactive molecule having a viscosity that can be extruded from an injection tool.

A: Heparin-binding physiologically active molecule B: Heparin to which a photoreactive group is bound C: Any aqueous solvent miscible component selected from the group consisting of alcohol, surfactant and chelating agent D: A, B And an aqueous solvent capable of dissolving component C

  The present invention provides a crosslinked heparin gel composition containing a bioactive molecule that can be extruded from an injection device. The bioactive molecule-containing crosslinked heparin gel composition of the present invention can be sustained-released by improving the stable storage of heparin-binding bioactive molecules such as FGF at the administration site, thereby treating peripheral ischemic diseases. Useful for.

It is a graph which shows the effect (angiogenesis) of b-FGF containing photocrosslinking 6DSH in a rat, "6DS-Hep / b-FGF" is b-FGF containing photocrosslinking 6DSH gel, and "b-FGF" is b-FGF aqueous solution. "6DS-Hep" represents a photocrosslinked 6DSH gel.

  Hereinafter, the present invention will be described in detail. First, for convenience of explanation, a method for producing a bioactive molecule-containing crosslinked heparin gel composition according to the present invention will be described. In the following description, photocrosslinked heparin obtained by photocrosslinking reaction of heparin having a photoreactive group bonded thereto will be described as an example. However, the heparin-binding bioactive molecule has a viscosity that can be extruded from an injection tool. Any other cross-linked heparin is applicable to the present invention as long as it does not inhibit the above.

  In the present invention, the heparin-binding physiologically active molecule used as component A is known to have heparin-binding cytokines, and similarly heparin-binding, and has cell growth promoting and differentiation promoting effects. Refers to a group of heparin binding growth factors (HBGFs). Examples include fibroblast growth factor (FGF), hepatocyte growth factor (HGF), bone morphogenetic factor (BMP), vascular endothelial growth factor (ECGF), epidermal growth factor (EGF), transforming growth factor. (TGF), vascular endothelial growth factor (VEGF), midkine (MK), interleukin 8 (IL8), vitronectin (VN), heparin-binding brain cell mitogenic factor (HBBM), heparin-binding neurite outgrowth promotion Factor (HBNF), human androgen-induced growth factor (AIGF), insulin-like growth factor (IGF), ciliary nerve growth factor (CNTF), platelet-derived growth factor (PDGF), brain-derived growth factor (BNDF), Nerve cell growth factor (NGF), stem cell growth factor (CSF), interferon γ (IFN-γ), keratinocyte growth Factor (KGF), CXC chemokine and the like. Among these, fibroblast growth factor (FGF) is preferable, and basic fibroblast growth factor (b-FGF) is particularly preferable.

  In the present invention, heparin bonded with a photoreactive group used as component B is obtained by reacting heparin with a compound having a photoreactive crosslinking group (photoreactive substance).

  Heparin as a raw material for producing the above photocrosslinked heparin may be one having a heparin skeleton as a basic skeleton, which is a repetitive structure of a disaccharide unit composed of a uronic acid residue and a hexosamine residue. There is no particular limitation. For example, not only a compound usually called heparin but also a glycosaminoglycan having affinity with a heparin-binding physiologically active molecule including HBGF such as heparan sulfate and heparinoid is also included in the present invention. In addition, derivatives obtained by chemical modification etc. to these (for example, sulfated derivatives with sulfate groups bonded, desulfated derivatives with sulfate groups removed) and normal heparin extracted and purified from natural products It also includes heparin that has been degraded by enzymes, nitric acid, sulfuric acid, hydrochloric acid, etc. to reduce its molecular weight.

  However, as described later, considering the use of the physiologically active molecule-containing crosslinked heparin gel composition of the present invention for pharmaceutical purposes, heparin derivatives in which the bleeding tendency of heparin is attenuated or eliminated are preferred. Examples of such heparin derivatives include desulfated heparin derivatives in which sulfate groups have been partially or completely removed from heparin, redox heparin derivatives obtained by subjecting heparin to a redox reaction, and heparin subjected to a redox reaction and a desulfation reaction. And redox-desulfated heparin derivatives.

  Among the above, desulfated heparin derivatives and redox-desulfated heparin derivatives are preferable. Examples of the desulfated heparin derivative include a 6-position desulfated heparin derivative in which the sulfate group bonded to the 6-position hydroxyl group of the hexosamine (N-sulfated glucosamine or N-acetylglucosamine) residue of the heparin skeleton is partially or completely removed (for example, , WO00 / 06608 pamphlet), 2-position desulfated heparin derivatives (for example, those described in JP-A No. 2003-1113090), fully desulfated heparin derivatives, and the like. Redox-desulfated heparin Examples of the derivatives include periodate redox-desulfated heparin derivatives as described in JP-A No. 11-310602.

  As described above, the anticoagulant activity of these desulfated heparin derivatives and redox-desulfated heparin derivatives is attenuated. For example, an activated partial thromboplastin time (hereinafter also referred to as “APTT”) in a 6-position desulfated heparin derivative in which the sulfate group bonded to the 6-position hydroxyl group of the N-acetylglucosamine residue of the heparin skeleton is partially or completely removed. Is 50 seconds or less when measured by adding the heparin derivative to plasma at a final concentration of 30 μg / mL, and the thromboplastin time (hereinafter also referred to as “TT”) is 100 μg / mL. When it is measured by adding the heparin derivative, it does not exceed 50 seconds.

  APTT is an index indicating anticoagulant activity and can be obtained by the following measurement method. Blood was collected from the lower aorta of the rat at a ratio of 9 volumes to 1 volume of 3.2 wt% sodium citrate, and centrifuged at 1000 × g for 10 minutes to obtain 100 μL of plasma, and this was the measurement target 100 μL of a heparin derivative solution having a concentration is placed in a measuring cup, kept at 37 ° C. for 1 minute, and then 100 μL of actin (trade name: Mitsubishi Pharma Corporation) previously kept at 37 ° C. is added. Incubate for 2 minutes. Next, 100 μL of a 0.02 M calcium chloride solution kept at 37 ° C. is added, and the time from this time until coagulation occurs is measured with an automatic blood coagulation measuring device.

  TT is also an index showing anticoagulant activity and can be obtained by the following measuring method. 100 μL of plasma prepared in the same manner as in the above APTT measurement and 100 μL of an aqueous solution of heparin derivative having a known concentration are dispensed into a measuring cup and kept at 37 ° C. for 1 minute. Thereafter, 100 μL of thrombin (10 U / mL: manufactured by Yoshitomi Pharmaceutical Co., Ltd.) that has been kept warm at 37 ° C. for 5 minutes in advance is added to start coagulation, and the coagulation time is measured with a blood coagulation automatic measuring device.

  The heparin used in the present invention may be derived from a natural product, chemically synthesized, or produced by a microorganism such as yeast by genetic engineering techniques, and may be commercially available heparin, heparan sulfate, or heparinoid. Absent. In general, it can be prepared by extraction from a biological material (small intestine, lung, skin, etc.) or chemical modification after extraction. The weight average molecular weight of heparin is usually 1,500 to 30,000, preferably 3,500 to 25,000.

  The photoreactive group is not particularly limited as long as it is a residue of a compound that undergoes a photodimerization reaction or a polymerization reaction upon irradiation with light such as ultraviolet rays. Specific examples of the photoreactive substance having a photoreactive crosslinking group include cinnamic acid, substituted cinnamic acid, acrylic acid, maleic acid, fumaric acid, sorbic acid, coumarin, thymine, and derivatives thereof. Examples of the substituted cinnamic acid include amino cinnamic acid (preferably p-amino cinnamic acid), which is cinnamic acid in which any hydrogen of the benzene ring is substituted with an amino group, and examples of acrylic acid derivatives include Thiophene acrylic acid, furyl acrylic acid and the like. Among these, cinnamic acid or substituted cinnamic acid is preferable from the viewpoint of photoreactivity and safety, and aminocinnamic acid is preferable as the substituted cinnamic acid.

  The photoreactive substance may be bonded with a spacer that improves the photoreactivity and facilitates the photocrosslinking reaction or facilitates the reaction of introducing a photoreactive group into heparin. As the spacer, a compound having a divalent or polyvalent functional group having a linear or cyclic hydrocarbon residue having 2 to 18 carbon atoms is preferable, and examples thereof include diaminoalkanes having 2 to 18 carbon atoms, aminoalkyl alcohols and amino acids. It is done. Further, for example, when the photoreactive substance is cinnamic acid, an amino alcohol having 2 to 8 carbon atoms is preferable as the spacer. In this case, the amino alcohol is ester-linked or amide-bonded to the carboxyl group of cinnamic acid. Particularly preferred spacers are n-aminopropanol or n-aminobutanol.

  Photoreactive heparin can be prepared according to known methods such as JP-A-6-73102, JP-A-8-143604, JP-A-11-512778, and the like.

  Furthermore, in the present invention, as the component C, any aqueous solvent miscible component selected from the group consisting of alcohols, surfactants and chelating agents is used. Such components are substances conventionally known as antifreeze forming additives.

  In the present invention, as the component D, any component having miscibility with an aqueous solvent selected from the group consisting of the heparin-binding physiologically active molecule, photoreactive heparin and alcohol, surfactant and chelating agent is dissolved. An aqueous solvent that can be used is used. Such an aqueous solvent acts as a solvent for the photocrosslinking reaction, and the type thereof is not particularly limited as long as it contains water. For example, water, water for injection, physiological saline, Tris-HCl buffer, phosphate buffered physiological saline and the like can be mentioned.

  That is, in gelation of a polysaccharide into which a photoreactive group has been introduced, heparin having a low molecular weight is extremely difficult to gel by light irradiation (crosslinking reaction) as compared to hyaluronic acid, which is a typical glycosaminoglycan. However, surprisingly, the cross-linking reaction of photoreactive heparin can be advanced very efficiently by light irradiation in a frozen state. However, in this case, if an aqueous solvent miscible component such as the alcohol is not present in the reaction system, a heparin sponge is produced by the crosslinking reaction, and the desired photocrosslinked heparin gel cannot be obtained. However, if a component having an aqueous solvent miscibility such as the alcohol is present in the reaction system, sponging is suppressed and a gel is formed. The reason is not necessarily clear, but it is presumed that a part of the solute component has become antifreeze liquefaction and is related.

  As said alcohol, surfactant, and chelating agent, what is conventionally used as an additive for antifreeze formation can be selected suitably. As the alcohol having an aqueous solvent miscibility, alcohols represented by the following general formula (1), particularly polyethylene glycols, are preferred.

  Examples of the chain alkyl having 1 to 10 carbon atoms include methyl and ethyl, and examples of the alkyl having 3 to 10 carbon atoms include isopropyl and t-butyl.

  Examples of the alcohol as described above include lower alcohols, polyhydric alcohols, and polysaccharide alcohols.

  Examples of the lower alcohol include alcohols having 1 to 10 carbon atoms, more preferably 1 to 8 carbon atoms, and examples thereof include methanol, ethanol, isopropyl alcohol, and t-butyl alcohol. That is, the polyhydric alcohol is an alcohol having 2 or more hydroxyl groups present in the molecule, and more preferably an alcohol having 3 or more hydroxyl groups. Examples thereof include ethylene glycol and glycerin, and ethylene glycol (PEG) is preferable. As the sugar alcohol, a chain sugar alcohol or a cyclic sugar alcohol can be used, but a chain sugar alcohol is preferable. As the sugar alcohol, inositol, mannitol, xylitol, sorbitol and the like are preferable, but mannitol, xylitol and sorbitol are more preferable, and mannitol or sorbitol is more preferable.

  As the surfactant, a nonionic surfactant or an anionic surfactant is preferably mentioned. Among them, the nonionic surfactant is polyethylene glycol, the anionic surfactant is an alkyl sulfate, and especially dodecyl sulfate. Sodium is preferred.

  Examples of the chelating agent include oxycarboxylic acids such as citric acid or a salt thereof, and polyaminocarboxylic acids such as edetic acid or a salt thereof (for example, ethylenediaminetetraacetic acid or a salt thereof).

  In addition, when using commercially available “Fiblast” (manufactured by Kaken Pharmaceutical Co., Ltd.) as basic fibroblast growth factor (b-FGF), sodium edetate contained as an excipient is itself Since it is a chelating agent having miscibility with an aqueous solvent and an additive for forming an antifreeze, it is not necessary to separately use the aforementioned component C.

  First, in the present invention, a solution containing each of the components A to D is prepared. In this case, the usage ratio of each component is as follows. The concentration of component A is usually 1 ng / mL to 100 μg / mL, and the concentration of component B is appropriately selected according to the relationship of the photoreactive group introduction rate, but is usually 0.1 to 10% by weight, 0.5-9 weight% is more preferable. Moreover, the density | concentration of the component C is 0.1 to 10 weight% normally, and 0.8 to 9 weight% is more preferable. The order of dissolution of other components in the aqueous solvent (component D) can be selected as appropriate.

  The photoreactive group introduction rate is the number of moles of “introduced photoreactive group” relative to the number of moles of “functional group capable of introducing photoreactive group” present in the polysaccharide (= photoreactivity). This is the value expressed as a percentage of the number of substances introduced). In addition, the functional group of a polysaccharide into which a photoreactive group can be introduced differs depending on the type of the photoreactive group and spacer, but when a hydroxyl group contained in the photoreactive group or spacer is used for binding to the polysaccharide. , A carboxyl group in a polysaccharide is exemplified, and when a carboxyl group contained in a photoreactive group or spacer is used for binding to the polysaccharide, an amino group or a hydroxyl group in the polysaccharide is exemplified, and a photoreactive crosslinking group or When the amino group contained in the spacer is used for binding to the polysaccharide, the carboxyl group of the polysaccharide is exemplified. Examples of the functional group capable of introducing a photoreactive group in normal heparin include a carboxyl group and a hydroxyl group. For example, when a carboxyl group is selected as a functional group for introducing a photoreactive group in heparin, the introduction rate of the photoreactive group is based on the fact that heparin has one carboxyl group per constituent disaccharide unit. It is shown as a percentage value of the number of photoreactive groups introduced per constituent disaccharide unit. In the solution prepared by mixing the above components, the heparin-binding physiologically active molecule and the photoreactive heparin are not a mere physical mixture but constitute a complex based on biochemical affinity.

  The above solution is then frozen. The solution is stored in a suitable container for freezing. At this time, as a photoreactive substance, for example, when using a compound having an unsaturated double bond that absorbs ultraviolet rays and causes a crosslinking reaction, such as a cinnamoyl group (cinnamic acid residue), use a solvent for the crosslinking reaction. Since it has the property of absorbing ultraviolet light by certain water, it is preferable to select a form in which the optical path length of the ultraviolet light is 1 cm or less. The material of the container must be a material that does not absorb light having a wavelength necessary for the crosslinking reaction of the photoreactive group and transmits such light. As such a material, for example, when ultraviolet rays are used for the crosslinking reaction, polymer compounds such as polypropylene having a low ultraviolet absorption rate, glass (particularly quartz glass or hard glass), and the like can be mentioned. The freezing conditions are not particularly limited, and may be frozen rapidly using, for example, a cryogenic substance such as liquid nitrogen, a refrigerant such as alcohol cooled below the freezing temperature of the cross-linking reaction solution, It may be frozen relatively slowly using a freezer. The freezing temperature is usually −10 to −40 ° C., preferably −20 to −30 ° C.

  Next, the frozen product obtained above is irradiated with light to carry out a crosslinking reaction. The light to be irradiated is not particularly limited as long as it acts on the photoreactive substance and causes a reaction such as polymerization or dimerization. For example, visible light, ultraviolet rays, infrared rays, electron beams, radiation and the like are also included in the light in the present invention. Among these, visible light or ultraviolet light is preferable, and ultraviolet light is more preferable. And as a wavelength, 180-650 nm is preferable. For example, when cinnamic acid is used as the photoreactive substance, an ultraviolet ray of 260 to 350 nm is preferable.

  Next, in the present invention, the frozen gel obtained above is melted. Thereby, the bioactive molecule containing crosslinked heparin gel composition which has a viscosity which can be extruded from an injection tool is obtained. Here, the gel means a solvated gel, and when water is used as an aqueous solvent during the crosslinking reaction, it is obtained as a hydrogel by hydration.

  Next, the bioactive molecule-containing crosslinked heparin gel composition of the present invention (hereinafter sometimes simply referred to as a composition) will be described. The composition of the present invention can be obtained, for example, by the specific production method as described above. The composition (solvated gel) of the present invention contains at least a heparin-binding bioactive molecule and a photocrosslinked heparin, and in a preferred embodiment thereof, is selected from the group consisting of alcohols, surfactants and chelating agents. Ingredients containing any aqueous solvent miscibility.

  The photocrosslinked heparin constituting the composition of the present invention dimerizes the photoreactive groups by irradiating light such as ultraviolet rays to photoreactive heparin obtained by chemically binding a photoreactive group to heparin, It is a photocrosslinked heparin obtained by crosslinking the heparin. Such photocrosslinked heparin usually does not dissolve in an aqueous solvent, that is, is often insoluble.

The greatest characteristic of the gel of the present invention is that it has a viscosity that can be extruded from an injection tool. The specific viscosity is usually 100 to 10,000 mPa · s, preferably 500 to 8,000 mPa · s, as the viscosity measured at 20 ° C. using a rotational viscometer. An example of the rotational viscometer used for the viscosity measurement is an E-type rotational viscometer manufactured by Toki Sangyo Co., Ltd., and a standard cone (W1 °, 34 ′ × R24) is used as the rotating rotor. Is preferred. In addition, for example, when the gel of the present invention is filled in a 9 mm inner diameter syringe at room temperature and extruded from a 23 gauge injection needle, the pressing pressure (pressurization) is 200 to 8000 g / cm ² .

  The cross-linked heparin gel composition containing the heparin-binding bioactive molecule obtained according to the present invention produces a heparin-binding bioactive molecule by utilizing the action of the heparin-binding bioactive molecule and the action brought about by the cross-linking gel. It can be used as a medical preparation used for continuous release in the body.

  In accordance with the present invention, a photocrosslinked heparin containing a heparin-binding bioactive molecule is gelled to stabilize the heparin-binding bioactive molecule for a long period of time, and a heparin-binding bioactive molecule for a certain period in vivo. It becomes possible to release slowly so that the density | concentration of can be maintained. That is, the composition of the present invention can be used as a sustained-release preparation for heparin-binding bioactive molecules.

  In addition, the composition of the present invention, which is a gel that can be extruded from an injection device, can be administered locally, has little metabolic loss during the delivery to the treatment site, can maintain a therapeutically effective concentration at the treatment site, Effective effects can be obtained. For example, an angiogenesis-promoting agent containing the composition of the present invention containing FGF as a heparin-binding physiologically active molecule as an active ingredient is locally administered to an ischemic site to promote angiogenesis and develop peripheral ischemic intractable diseases Can be improved.

  Currently, heparin is commonly used as a pharmaceutical (anticoagulant), and especially as described above, a heparin derivative that attenuates or eliminates the bleeding activity of heparin has anticoagulant activity and bleeding activity compared to normal heparin. Since such a heparin derivative is used as a raw material for the composition of the present invention, the preparation containing the composition of the present invention can be used as a pharmaceutical preparation such as a pharmaceutical or a medical device (equipment). Even when administered to a living body, the anticoagulant activity or bleeding activity is low, which is preferable.

  The dosage form and administration method of the composition of the present invention can be appropriately selected depending on the nature and severity of the target disease, but parenteral administration is preferred, specifically, infusion And application. The dose should be appropriately determined individually according to the administration method, dosage form, patient's weight and metabolic function state, disease state, etc., and is not particularly limited.

  For example, the dose is basically selected based on the effective amount of the bioactive molecule contained in the prepared preparation, but is contained when the composition of the present invention is used as a preparation or therapeutic agent for angiogenesis. Examples of the physiologically active molecule to be used include about 5 μg to 60 μg per day. Basically, it is preferable to select appropriately depending on changes in the amount of bioactive molecule, pathological condition, and blood flow contained in the prepared preparation, for example, several times at a time for the shin and thigh of the ischemic foot. It can be divided into several hundreds and administered in small amounts. On the other hand, since the composition of the present invention is in a gel form and has high stability at the administration site, and it is also possible to release the physiologically active molecules contained therein once every 3 days to 4 weeks. Administration is also possible. The dosage form is not particularly limited, and various dosage forms are possible by a usual formulation method. Among them, an injection and an injection that take advantage of the characteristics of the composition of the present invention are particularly preferable.

  EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, this invention is not limited to a following example, unless the summary is exceeded. The definitions of technical terms and measurement methods used in the following examples are as follows.

(1) Measuring method of introduction rate of photoreactive group:
The rate of introduction of photoreactive groups in the photocrosslinked heparin gel composition means a value representing the number of photoreactive groups introduced per repeating disaccharide unit constituting heparin as a percentage. The amount of the constituent disaccharide unit of heparin necessary for calculating the introduction rate was measured by a carbazole measurement method using a calibration curve, and the amount of the photoreactive group was measured by an absorbance measurement method using a calibration curve. Incidentally, when cinnamic acid or aminocinnamic acid was used as the photoreactive group, the absorbance at a measurement wavelength of 269 nm was used.

(2) Method for measuring crosslinking rate:
The crosslinking rate was determined by hydrolyzing 1 g of a test substance (photocrosslinked heparin gel composition) with 1 ml of 1 mol / l sodium hydroxide for 1 hour, and then acidifying the resulting solution with ethyl acetate to obtain a photoreactive group-derived substance ( Photoreactive group monomer and photoreactive group dimer) are extracted and analyzed by high performance liquid chromatography (HPLC), and the amount of photoreactive group dimer is determined using a calibration curve. It was measured. And the number of moles of the photoreactive group which became a dimer with respect to the photoreactive group introduced into heparin measured by said (1) was computed by the percentage (%).

Example 1:
(1) 3 g of 6-O-desulfated heparin (derived from porcine small intestine, weight average molecular weight: about 9000, hereinafter also referred to as 6DSH) prepared according to the method described in WO00 / 06608 pamphlet is dissolved in 100 ml of distilled water. , 50 ml of 1,4-dioxane was added. Next, N-hydroxysuccinimide (hereinafter also referred to as HOSu) 205.8 mg, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (hereinafter also referred to as EDCI · HCl) 171.4 mg and cinnamon Acid aminopropyl hydrochloride 216.1 mg was sequentially added and allowed to react at room temperature for 2 hours. Thereafter, 3 g of sodium chloride (hereinafter also referred to as NaCl) was added, ethanol was poured, and the deposited precipitate was recovered. The collected precipitate was washed with ethanol and then dried under reduced pressure at 40 ° C. to obtain 2.7 g of photoreactive 6DSH. The introduction rate of the photoreactive group was 16.0%.

(2) A basic fibroblast growth factor (hereinafter also referred to as b-FGF) is added to a solution prepared by dissolving the photoreactive 6DSH obtained in (1) above in water for injection so that the weight concentration is 4%. ) Was added and dissolved to a concentration of 50 μg / mL to obtain a reaction solution containing a complex of photoreactive 6DSH and b-FGF. As the basic fibroblast growth factor (b-FGF), commercially available “Fiblast Spray 500” (manufactured by Kaken Pharmaceutical Co., Ltd., composition: b-FGF 500 μg, sodium edetate 460 mg) was used. The reaction solution was filtered through a 0.22 μm membrane filter (manufactured by Nihon Millipore), poured into a Pyrex (registered trademark) plate adjusted to have a gap of 1 mm, and frozen in a −20 ° C. atmosphere. While maintaining the frozen state, an 800 W high-pressure mercury lamp (manufactured by Oak Manufacturing Co., Ltd.) was used, and UV irradiation was performed so that the irradiation light amount was 5.0 J / cm ² to cause a photocrosslinking reaction. Then, it returned to room temperature, the frozen body was thawed, and the gel-like b-FGF containing photocrosslinking 6DSH gel composition was obtained. When the crosslinking rate of this gel substance was measured, it was 24.5%. Further, when the viscosity was measured using a rotational viscometer, the viscosity under 1 ° cone and 20 ° C. conditions was 3810 mPa · s.

Reference example 1:
In a solution obtained by dissolving the photoreactive 6DSH obtained in (1) of Example 1 in water for injection so as to have a weight concentration of 4%, polyethylene glycol 400 (hereinafter also referred to as PEG400) has a weight concentration of 4%. It was made to melt | dissolve and it was set as the solution. This solution was subjected to a photocrosslinking reaction in the same manner as (2) of Example 1, and then returned to room temperature to melt the frozen body, thereby obtaining a gel-like photocrosslinked 6DSH gel. It was 13.4% when the crosslinking rate of this gel-like substance was measured. Moreover, when the viscosity was measured using a rotational viscometer, the viscosity under 1 ° cone and 20 ° C. conditions was 3700 mPa · s.

Test Example 1 (Effect of ultraviolet rays on b-FGF):
Since the photocrosslinking reaction in Example 1 uses ultraviolet rays, the influence of ultraviolet rays on b-FGF was examined. About the b-FGF solution prepared so that it might become 40 mg / mL, the change of the peak area by HPLC before and behind irradiating a 10 J / cm < ² > ultraviolet-ray was measured. As a result, it was confirmed that the peak area decreased by 28% before and after the ultraviolet irradiation. Considering that the b-FGF concentration of Example 1 is about 1/1000 of Test Example 1 and the amount of irradiation light of Example 1 is 1/2 of Test Example 1, UV light in the photocrosslinking reaction of Example 1 Is considered to have little influence (decomposition) on b-FGF.

  In HPLC, “TOSOHODS-120T” (4.6 mmφ × 150 mm) is used for the column, 0.01 nHCl is used as eluent A, acetonitrile is used as eluent B, and the gradient conditions are B%: Measurement was performed at 25 (0 min) -31 (15 min) -55 (25 min), a flow rate of 1.0 mL / min, a measurement temperature of 40 ° C., and a detection wavelength of 214 nm. A sample prepared so that the b-FGF content was about 50 mg / mL was used.

Test Example 2 (Effect of b-FGF-containing photocrosslinking 6DSH in rats):
8 SD male rats (SPL, 14 weeks old, body weight about 480 g) were used, and the back was shaved under ketamine / xylazine anesthesia, and the b-FGF-containing photocrosslinked 6DSH gel obtained in Example 1, Reference 100 μL each of the photocrosslinked 6DSH gel containing no b-FGF obtained in Example 1 and a b-FGF aqueous solution having a b-FGF concentration of 50 μg / mL was subcutaneously injected into 6 sites in total at 2 sites. After 3, 6, 10 and 15 days after sacrifice by massive anesthetic injection, the tissue at the injection site was collected, and the specimen was prepared by staining with hematoxylin and eosin, and a peripheral image of the remaining heparin gel using a microscope. And the capillary blood vessels containing red blood cells per field at 100 times were measured with the naked eye. The results are shown in FIG. 1 as a blood vessel number graph. In the figure, “6DS-Hep / b-FGF” represents a b-FGF-containing photocrosslinked 6DSH gel, “b-FGF” represents a b-FGF aqueous solution, and “6DS-Hep” represents a photocrosslinked 6DSH gel. As shown in FIG. 1, angiogenesis was clearly observed in the administration group of the b-FGF-containing photocrosslinked 6DSH composition. Further, even after 15 days, the residual b-FGF-containing photocrosslinked 6DSH composition was observed.

Claims (1)

A photocrosslinking heparis obtained by preparing a solution containing the following components B to D, freezing the gel, irradiating the obtained frozen product with light and gelling, and then melting the frozen gel Solvated hydrogel.
B: Aminopropyl cinnamate bound to a 6-position desulfated heparin derivative in which the sulfate group bonded to the 6-position hydroxyl group of the hexosamine (N-sulfated glucosamine or N-acetylglucosamine) residue of the heparin skeleton was partially or completely removed. Photoreactive heparin C: polyethylene glycol or sodium edetate D: a solvent containing water that can dissolve the B and C components

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