JP2010013411A - Anti-inflammatory agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an anti-inflammatory agent which can efficiently suppress inflammation and is highly safe with little side effect. <P>SOLUTION: The anti-inflammatory agent contains 4-amino-6-hydroxypyrazolo(3,4-d)pyrimidine, its pharmaceutically acceptable salt, its derivative, or a micelle containing 4-amino-6-hydroxypyrazolo(3,4-d)pyrimidine. Combining 4-amino-6-hydroxypyrazolo(3,4-d)pyrimidine with a polymer such as a styrene-maleic acid copolymer, PEG, polyacrylic acid, and polyvinylacetic acid improves water solubility and retention in blood and allows the resulting anti-inflammatory agent to be more efficiently accumulated in a focus to thereby reduce side effect. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a water-soluble pharmaceutical preparation of 4-amino-6-hydroxypyrazolo (3,4-d) pyrimidine, and particularly relates to an anti-inflammatory agent.

When the tissue is damaged, prostaglandins and inflammatory cytokines are produced in the vascular endothelial cells at the damaged site. Prostaglandins and pro-inflammatory cytokines include fever, vasodilation, increased vascular permeability, leukocyte chemotaxis, leukocyte and vascular endothelial cell activation, activated leukocyte adhesion and invasion to vascular endothelial cells, Shows various physiological effects. Furthermore, prostaglandins and leukotrienes are produced from activated leukocytes, and the inflammatory reaction is accelerated. Leukocytes with enhanced chemotaxis infiltrate inflammatory sites outside the blood vessels from the junctions of vascular endothelial cells, where they phagocytose microorganisms, pathogens and unwanted substances to protect against infection. As a result, harmful unnecessary substances such as pathogens and antigens are reduced and tissue cells proliferate, leading to healing. However, if the infiltration of leukocytes continues, on the contrary, further tissue damage occurs and inflammation becomes chronic.
As such anti-inflammatory agents that suppress inflammatory reactions, steroidal anti-inflammatory agents and non-steroidal anti-inflammatory agents such as cyclooxygenase (hereinafter sometimes referred to as COX) inhibitors have been used. . Non-steroidal anti-inflammatory agents include aspirin, salicylic acid and indomethacin. These anti-inflammatory agents exhibit anti-inflammatory activity by inhibiting the binding of COX and arachidonic acid in the arachidonic acid cascade leading to prostaglandin biosynthesis. Thus, steroidal anti-inflammatory agents and non-steroidal anti-inflammatory agents can suppress inflammation and pain in the affected area by inhibiting cyclooxygenase activity. On the other hand, it has been pointed out that inhibition of COX activity reduces blood flow in the stomach and liver, which adversely affects gastric ulcers and renal function.

In addition, regarding the activity of 4-amino-6-hydroxypyrazolo (3,4-d) pyrimidine (hereinafter sometimes referred to as AHPP), the inventors have a blood pressure lowering action. Is disclosed. However, since AHPP is insoluble in water, it is necessary to make it water-soluble. So far, no blood pressure-lowering preparation in which AHPP is water-soluble has been obtained.
JP-A-9-118622 T. T. Oda et al, Science, 244, 974-976 (1989) T. T. Akaike et al, J.A. Clin. Invest. , 85, 739-745 (1990) T. T. Akaike et al, PNAS. , 93, 2448-2453 (1996) Y. Matsumura, and H.M. Maeda, Cancer Res. , 46, 6387-6392, (1986)

An object of the present invention is to provide a highly safe anti-inflammatory agent that can efficiently suppress inflammation and that exhibits no side effects.
Furthermore, another object of the present invention is to provide an AHPP preparation having the ability to inhibit water-soluble xanthine oxidase (hereinafter sometimes referred to as XO). The water-soluble AHPP preparation suppresses the generation of superoxide (hereinafter also referred to as O ₂ ⁻ ), which is an active oxygen, and therefore may be described as nitric oxide (hereinafter referred to as NO) due to the O ₂ ^−. ), And conversely, by increasing the concentration of NO that is not consumed, antihypertensive preparations and, furthermore, proinflammatory peroxynitrite (ONOO ⁻ ) produced from O ₂ ⁻ and NO are suppressed, and thus anti- Can be used as an inflammatory agent.

The present inventors have clarified that O ₂ ⁻ and NO are deeply involved in the mechanism of influenza exacerbation so far (Non-Patent Documents 1 to 3).
Furthermore, it is also clarified that O ₂ ⁻ is produced by XO. O ₂ ⁻ reacts with NO, that is, vascular endothelium-derived relaxing factor (hypertensive substance EDRF) to become ONOO ⁻ , and consumes NO. Therefore, NO is deficient in blood pressure, resulting in normal blood pressure. It becomes difficult to cause hypertension. Therefore, the present inventors have found that the suppression of O ₂ ⁻ by AHPP has the effect of suppressing the increase in blood pressure of rats, and have filed a patent application (Patent Document 1).

In addition, the present inventors have clarified that reactive oxygen molecular species such as O ₂ ⁻ and ONOO ⁻ are deeply involved in the cause of inflammation caused by viral infection, drugs, and the like. Therefore, it is thought that by administering an inhibitor against XO, which is one of the main enzymes that catalyze the production of O ₂ ⁻ , it is possible to suppress inflammation involving active oxygen molecular species produced from XO. The present invention has been completed.

The present invention relates to 4-amino-6-hydroxypyrazolo (3,4-d) pyrimidine, pharmaceutically acceptable salts thereof, derivatives thereof, or 4-amino-6-hydroxypyrazolo (3,4-d). ) An anti-inflammatory agent whose active body is a micelle containing pyrimidine.
The present invention also includes a derivative of 4-amino-6-hydroxypyrazolo (3,4-d) pyrimidine, or a micelle containing 4-amino-6-hydroxypyrazolo (3,4-d) pyrimidine. It is a pharmaceutical preparation. The pharmaceutical preparation of the present invention can be used as an anti-inflammatory agent or antihypertensive agent with the aim of achieving a medicinal effect obtained by suppressing the enzyme activity of XO.
4-Amino-6-hydroxypyrazolo (3,4-d) pyrimidine is a compound represented by Formula 1. The derivative is a compound represented by Formula 2. In Formula 2, X is any one substituent represented by Formula 3 to Formula 6.

Here, k represents a natural number of 3 to 200. The upper limit of the degree of polymerization was set to 200. When the micelle formation was composed of three strands of SMA, the SMA was tripled with respect to one drug active site (AHPP), and the AHPP was relatively high. The concentration (content) of is diluted. Therefore, in order to maintain a high specific activity, the amount of SMA was kept relatively low.

Here, l in the formula represents a natural number of 1 to 1000. In the case of 1000 or more, the solubility in an aqueous system is lowered, so that practicality is lacking.

Here, m and n each represent a natural number of 1 to 1000. Note that m> n is preferable. In the case of 1000 or more, the solubility in an aqueous system is lowered, so that practicality is lacking.

  A micelle containing 4-amino-6-hydroxypyrazolo (3,4-d) pyrimidine is a styrene maleic acid copolymer of 4-amino-6-hydroxypyrazolo (3,4-d) pyrimidine ( Hereinafter, it is a micellized preparation formed by non-covalent bonding using SMA). Specifically, it is expressed by Equation 7. It can be said that this is characterized in that AHPP and SMA are non-covalently bound (complex).

Here, p represents a natural number of 3 to 200. The range of the degree of polymerization is limited for the same reason as k above.

  According to the present invention, it is possible to provide a highly safe anti-inflammatory agent that can efficiently suppress inflammation and has few side effects. The anti-inflammatory agent of the present invention is a water-soluble derivative of AHPP. Since these derivatives (dosage forms) behave as a polymer in solution, blood, and body, the retention in blood at the time of administration in blood and the directivity to the lesion, i.e., the inflamed area, are further improved. It is getting stronger. This can be said to be an improvement in accumulation property due to the Enhanced Permeability and Retention effect (EPR) effect (Non-patent Document 4) in the tumor part and the inflammatory part, which is a characteristic of the property. In other words, these techniques have made it possible to make pharmaceuticals even better.

AHPP is a known compound and can be synthesized by the reaction of 3-amino-4-cyanopyrazole and urea. Further, the pharmaceutically acceptable derivatives of AHPP are not particularly limited to those exemplified here, and can be appropriately selected depending on the purpose. For example, amino acids (aspartic acid, glutamic acid, succinic acid, etc.) Acid), organic acid derivatives such as citrate, gluconate, and ascorbate, glucose, maltose, fructose, sorbitol, xylose, mannose, and ε-caprolactam.
The AHPP derivative is a water-soluble low to high polymer residue such as AHPP, such as SMA, polyethylene glycol (hereinafter sometimes referred to as PEG), acrylic acid or polyvinyl acetic acid (hereinafter sometimes referred to as PV). A compound in which an amide bond is formed with a group. In addition, AHPP using SMA, PEG, acrylic acid or PV, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hydroxypropyl methacrylate (HPMA) copolymer, divinylmaleic acid (anhydrous) copolymer (pyran copolymer), etc. It is a micellized formulation formed by covalent bonding.
AHPP is a water-insoluble substance, but not only becomes water-soluble but also becomes a more excellent pharmaceutical product by being polymerized by binding or micellizing with a water-soluble polymer or PEG having a low polymerization degree.

The administration form of the anti-inflammatory agent of the present invention may be an oral administration agent, an injection, or a suppository. The oral dose may be adjusted according to the age, weight, and degree of disease of the patient. For example, 100 to 9000 mg per adult is preferably administered once to several times a day. The injection is preferably administered intravenously, and may be administered intravenously by infusion or the like. In the case of injection, the AHPP content is preferably in the range of 3 to 500 mg / kg.
The present invention will be described below based on examples.

SMA-AHPP was synthesized according to the scheme shown in FIG.
To 36.3 mg of AHPP (molecular weight 151.1, fine powder, Wako Pure Chemicals, Osaka), 5.0 ml of 0.1 M NaOH or 0.1 M KOH was added and dissolved to obtain 7.3 mg / ml (48 mM). An alkaline solution was prepared. To the resulting AHPP solution, about 50-fold molar amount (assuming the molecular weight is assumed to be 1200) of 576 mg (96 mM) of SMA anhydride (fine powder form) was slowly added over about several hours over several hours with stirring. . After the SMA anhydride was completely added, the mixture was further reacted for 6 hours with stirring. The reaction solution was adjusted to pH 9.0 with 0.1M HCl and further reacted in the dark for 24 hours. After 24 hours, 0.1M HCl was added, and the mixture was precipitated at pH 6.0 or lower and obtained separately. Next, a cooled 0.01 M HCl and 0.1 M acetic acid weakly acidic aqueous solution was added to form a suspension, which was washed by centrifugal precipitation. The resulting precipitate was then redissolved with 5 ml of 0.1 M Na ₂ CO ₃ , diluted to 100 ml to make a solution around pH 9.5, and using a molecular sieve membrane with a molecular weight of 3000, a Laboscale TFF system (Millipore, Millipore, Billerica, MA) was concentrated and washed under pressure while removing low molecules. Further, 0.01 M Na ₂ CO ₃ / NaHCO ₃ (pH 9.0) was added to the concentrated solution, diluted, and concentration and washing were repeated again using the above system. The concentrated polymer fraction solution was lyophilized. The lyophilizate was dissolved in 0.1 M Na ₂ CO ₃ and purified by Sephadex G-100 gel (GE Healthcare, Uppsala, Sweden) column chromatography [size: 35 cm (h) × 2.5 cm (d)]. AHPP was detected by absorption at 260 nm.

_{O 2} by XO-derived milk ^- the inhibitory activity of SMA-AHPP on the production of, 10 [mu] M lucigenin, xanthine 10 [mu] M, 10 mM phosphate buffer containing SMA-AHPP of AHPP or 10~5000μM of 10, 100 (pH 7 X.4) was added to 20 mU / ml and chemiluminescence was measured (multiplate reader MTP-700CL, CoronaCo. Ltd., Ibaraki, Japan). In addition, the inhibitory activity of SMA-AHPP on the enzyme activity of XO is 10 mM phosphate buffer solution (pH 7) containing SMA-AHPP at 22, 44 and 132 μM with respect to the concentration of substrate xanthine of 2, 5, 10, 20 μM. 4) The reaction was carried out in Enzyme XO was added to 3.33 mU / ml and stirred, and the uric acid produced was measured by absorption at 290 nm (visible ultraviolet spectrophotometer model UV / Vis-550, JASCO Corp, Japan). (Ki) was determined.

As shown in FIG. 2, the FT-IR spectrum of the SMA-AHPP conjugate is 1375 and 1397 cm ⁻¹ (derived from C—O—H of carboxylic acid), 1477 and 1570 cm ⁻¹ (N—H), 1638 cm ⁻¹ ( Absorption peaks around 3350-3500 cm ⁻¹ (O—H stretch) were observed.
From the results of elemental analysis, SMA-AHPP contained 2.86% of N (derived from AHPP) that was not found in SMA. From this, it was considered that one AHPP was bonded to one SMA chain (degree of polymerization 6).
FIG. 3 shows the inhibitory activity of the AOPP and SMA-AHPP XO enzyme activities. The Ki of SMA-AHPP was 0.25 μM, and AHPP showed a strong inhibitory activity almost the same as 0.17 μM. FIG. 4 shows the activity of inhibiting O ₂ ⁻ production by XO of AHPP and SMA-AHPP. As shown in FIG. 4, the inhibition of O ₂ ⁻ production by XO was also suppressed in a concentration-dependent manner. The measurement was performed according to the method described above.

The protective effect of SMA-AHPP against liver damage caused by ischemia-reperfusion was examined.
Six-week-old male Wistar rats (average weight of about 250 g, about 5 mice per group, Kudo, Kumamoto) were subjected to the experiment after preliminary breeding for 1 week. Rats were fasted 9 hours before the test, and SMA-AHPP was dissolved in physiological saline at 0.5, 10 and 20 mg / kg per rat and administered from the tail vein. Two hours later, the rat was laparotomized under anesthesia, the portal vein was ligated with a clip for 30 minutes for ischemia, the clip was removed from the portal vein, reperfusion (Ischemia reperfusion, I / R) was performed, and the laparotomy was sutured with a thread. . Three hours later, the rats were sacrificed under ether anesthesia, blood was collected, and lactate dehydrogenase (LDH), alanine aminotransferase (AST) and aspartic acid in plasma were used as indicators of liver damage. Aspartate aminotransferase (ALT) was measured (N. Ikebe et al, J. Pharm. Exp. Ther., 295 904-911 (2000)). In addition, in order to observe histopathologically, the liver was partially fixed with 10% formalin, and the paraffin block was prepared, and the 5-10 μm section was stained with hematoxylin eosin, followed by microscopic observation. Was observed. Furthermore, the degree of liver peroxidation was determined by preparing liver homogenate and generating peroxide in the liver by the thiobarbitur (TBA) method (J. Fung et al, Cancer Res., 62 3138-3143 (2002)). Was also measured.

As shown in FIG. 5, the enzyme LDH, which is an indicator of hepatic damage in blood in I / R-treated rats, was significantly increased in a concentration-dependent manner by administration of SMA-AHPP compared to control (no SMA-AHPP administration). The increase of serum LDH was suppressed.
FIG. 6 is a graph showing changes in ALT and AST by SMA-AHPP. Similar to LDH, SMA-AHPP significantly suppressed ALT and AST increases in a concentration-dependent manner. FIG. 7 shows the amount of peroxide produced in the I / R-treated rat liver. Production of peroxide in the liver of I / R-treated rats is also significantly suppressed by administration of SMA-AHPP. As described above, it is clear that SMA-AHPP suppresses the production of O ₂ ⁻ due to the activation of XO caused by I / R and suppresses liver damage.

The therapeutic effect on colitis caused by dextran sulfate sodium salt of SMA-AHPP (DSS, MP Biomedicals, LLC, CA, USA) was investigated. DSS was dissolved as 2% in distilled water and given as drinking water for a week.
5 weeks old male ICR (average weight about 25 g) mice were preliminarily raised for 1 week, and the body weight was measured.
(A) Normal group (no DSS administration),
(B) Control colitis group (DSS administered and no SMA-AHPP administered from 1 day later),
(C) Treatment group (100 mg / kg SMA-AHPP oral administration group from 1 day after DSS administration), and (d) Treatment group (30 mg / kg SMA-AHPP intravenous administration group 3 days after DSS administration) Four groups were established every day.
The administration of 2% DSS for 7 days to 3 groups (b), (c) and (d) other than the normal group was started. One day after the start of DSS administration, SMA-AHPP was orally administered at 100 mg / kg with a sonde in (c). In (d), SMA-AHPP was administered at 30 mg / kg for 3 consecutive days from the tail vein.
Seven days later, all mice were weighed and then sacrificed by ether anesthesia, and blood, liver and large intestine were removed. Liver was measured for weight and large intestine for length. In addition, interleukin-12 (IL-12) and tumor necrosis factor (TNF-α), which are inflammatory cytokines in serum, were used as Mouse TNF-α ELISA kit and Total IL-12 Mouse ELISA kit (Pierce Biotechnology, Rockford, IL). And measured by ELISA.

FIG. 8 shows the body weight, liver weight, and length of the large intestine of each group on the 7th day after administration of 2% DSS drinking water. Data were expressed as mean ± SD (n = 5). Here, the symbols *: P <0.05 and **: P <0.01 indicate the normal group or the SMA-AHPP administration group vs. the control colitis group.
As shown in FIG. 8, (b) the control colitis group (no treatment) further showed a decrease in body weight compared to (a) the normal group (DSS untreated). On the other hand, as for (c) group and (d) group of the treatment group of SMA-AHP administration, the weight loss was not seen like (a). In addition, liver weights (c) and (d) did not change as compared with (a), and no side effects were observed.
In addition, (b) the control colitis group (no treatment) showed a significant decrease in the length of the large intestine compared with (a) the normal group (DSS untreated), and was atrophyed. At that time, the occurrence of diarrhea was significantly increased in the (b) control colitis group (no treatment). On the other hand, (c) and (d) of the treatment group by administration of SMA-AHPP suppressed significant decrease in the length of the large intestine as compared with (b). (C) and (d) were not significantly different from (a), and there was no atrophy of the length of the large intestine.
Furthermore, SMA-AHPP administration (c) and (d) suppressed the occurrence of diarrhea.
Regarding the suppression of inflammatory cytokines, as shown in FIG. 9 and FIG. 10, in the (c) and (d) of the SMA-AHPP administration group, the IL-12 in serum was compared with that in the non-treatment group (b) And production of TNF-α was significantly suppressed.

PEG (2000) -AHPP was synthesized according to the scheme shown in FIG.
A solution prepared by dissolving 100 mg (0.7 mmol) of fine powder of AHPP in 15 ml of 0.1 M NaOH and 1.3 g of activated PEG (molecular weight 2000, Sunbright MEC-20AS manufactured by NOF) in 20 ml of chloroform. Was added in a suspended state for 40 minutes while stirring dropwise on ice with stirring. After complete addition of PEG, the reaction was further continued at room temperature for 2 hours. The reaction solution consisting of two components was adjusted to pH 1.0 by adding 0.1 M HCl. The chloroform phase of the reaction solution was separated with a separatory funnel, dried over anhydrous sodium sulfate. The solution was filtered with a filter paper, and chloroform was removed under reduced pressure using a rotary evaporator to obtain a dried sample. Subsequently, this sample was dissolved in 5 to 8 ml of distilled water and subjected to gel chromatography using the Sephadex G-100. The eluted fraction was detected by absorption at 260 nm, and the peak was collected and lyophilized to obtain a purified product. The sample was subjected to dynamic light scattering analysis. In addition, the dry sample was measured in the same manner as in Example 1 for elemental analysis, FT-IR, UV absorption spectrum, measurement of particle size distribution by dynamic scattering, and inhibition of O ₂ ⁻ production by XO.

Next, PEG (300) and AHPP were synthesized. An example is shown.
The synthesis of the activated ester for the condensation reaction begins with 5.0 g of PEG (molecular weight 300, Wako Pure Chemicals, 164-09055, Lot No. WKG6642, Osaka) and 0.6 g of p-nitrophenyl chloroform. The mate was dissolved in 50 ml of tetrahydrofuran (THF), 0.29 g of triethylamine was further added, and the mixture was allowed to react with stirring at room temperature for 24-30 hours. Gradually, triethylamine hydrochloride precipitated and was removed by filtration. To this solution, 300 ml of ethyl ether was added to cause precipitation to obtain a precipitate. This was further washed with cold ethyl ether. About 50 ml of THF was again added to dissolve this precipitate, ethyl ether was added to this THF solution, and a crystallized product of activated PEG (p-nitrophenyl chloroformate) was obtained from a mixture of both solvents. The yield was ˜80 w / w% based on PEG. 1.0 g of this activated PEG was taken and dissolved in 20 ml of THF, and an AHPP solution (0.5 g / 25 ml of NaOH) dissolved in 0.1 M NaOH was added dropwise thereto with stirring for 30 to 40 hours at room temperature. Reacted. At this time, all are in a solubilized state. The reaction solution was concentrated to dryness using a rotary evaporator to obtain a dried product. This was further dissolved in THF, precipitated with ethyl ether, Whatman no. The precipitate was separated by filtration through one filter. To this precipitate, 10 ml of distilled water was added, dissolved, and freeze-dried to obtain a PEG (300) -AHPP preparation. The particle size distribution was measured by UV absorption spectrum and photodynamic scattering method.

  The absorption spectrum of AHPP is shown in FIG. 12, and the absorption spectra of PEG (2000) -AHPP and PEG (300) -AHPP are shown in FIGS. 13A and 13B, respectively. As shown in FIG. 12, AHPP showed maximum absorption at 222 and 270 nm. Further, as shown in FIG. 13A, PEG (2000) -AHPP showed the maximum absorption at 270 nm, which is characteristic of 230 and AHPP. As shown in FIG. 13B, PEG (300) -AHPP showed maximum absorption at 270 nm, which is characteristic of 222 and AHPP.

FIG. 14 shows the FT-IR spectrum of AHPP, and FIG. 15 shows the FT-IR spectrum of PEG (2000) -AHPP. As shown in FIG. 14, in PEG (2000) -AHPP, characteristic absorption peaks of 1638 cm ⁻¹ (C═O stretching) and around 2850 cm ⁻¹ (—OCH ₃ ) were observed.
FIGS. 16A and 16B show the results of measurement of particle size distribution by dynamic scattering in a solution of PEG (2000) -AHPP and PEG (300) -AHPP. As a result, the average particle sizes of PEG (2000) -AHPP and PEG (300) -AHPP were about 287 nm and 235.5 nm, respectively. This data demonstrates that the PEG-AHPP molecule forms an aggregate in solution.
Moreover, in FIG. 17, the inhibitory activity with respect to the enzyme activity of XO of AHPP and PEG (2000) -AHPP was shown. As shown in FIG. 17, the inhibition of PEG (2000) -AHPP on the enzyme activity of XO was suppressed to almost the same extent as AHPP.

An AHPP-acrylate monomer-binding compound was synthesized according to the scheme shown in FIG.
A solution of 500 mg (3.3 mmol) of AHPP fine powder dissolved in 15 ml of 0.075 M NaOH and a solution of 299 mg (3.3 mmol) of acryloyl chloride (acryloyl chloride) in 10 ml of chloroform were stirred on ice. It was added and reacted for 30 minutes while dropping. After complete addition of acryloyl chloride, the reaction was continued for 30 minutes at room temperature with further stirring. The chloroform phase of the aqueous phase reaction solution is removed by a separatory funnel, and this aqueous solution is adjusted to about pH 3.0 using 0.1 M HCl to form a precipitate. The precipitate was collected by filtration, washed twice with 50 ml 0.1 M HCl, twice with 50 ml acetone and dried. The yield of AHPP-acrylate monomer was 0.6 g (98%).

  The absorption spectrum of the AHPP-acrylate monomer is shown in FIG. The sample solution was measured as a 1.5 mg / ml 0.1 M NaOH solution. As shown in FIG. 19, the AHPP-acrylate monomer showed maximum absorption at 224 and 270 nm.

The synthesis of AHPP-oligoethylene glycol (OEG) methacrylate copolymer was reacted as in the scheme of FIG.
1.0 g (4.0 mmol) of oligoethylene glycol ethyl ester methacrylate (3-4 ethylene glycol was bonded, so that it was not poly but oligoethylene glycol. Average molecular weight 246, OEG-MA, Cat No. 409545, Batch No-02824AH (Aldrich) was dissolved in 20 ml of dimethyl sulfoxide (hereinafter sometimes referred to as DMSO), and 0.2 g of AHPP-acrylate monomer-binding compound was dissolved in 15 ml of DMSO. 2.0 mg of 2,2′-azobis [2-methylpropanenitrile] as an initiator was added and reacted at 65 ° C. for 24 hours. The reaction solution was concentrated to dryness by a rotary evaporator, and the dried product was washed with diethyl ether three times and purified. This sample was dissolved in distilled water in the same manner as described above and subjected to gel chromatography using Sephadex G-100. The eluted fraction was detected by absorption at 260 nm, and the peak was collected and lyophilized to obtain a purified product. The yield of AHPP-OEG methacrylate copolymer was 0.95 g (79%).

FIG. 21 shows an absorption spectrum of the AHPP-OEG methacrylate polymer.
As shown in FIG. 21, the AHPP-OEG methacrylate polymer showed maximum absorption at 230 and 270 nm. Further, the FT-IR spectrum of the AHPP-OEG methacrylate copolymer is shown in FIG. As shown in FIG. 22, in the AHPP-OEG methacrylate polymer, a peak (C═O stretching of amide) was observed in the vicinity of 1650 cm ⁻¹ .

The molecular weight of the AHPP-OEG methacrylate polymer was determined by separating human immunoglobulin IgG (155 kDa), bovine serum albumin (67 kDa) by Sephadex G-100 gel (GE Healthcare) chromatography [size: 35 cm (h) × 2.5 cm (d)]. , Ovalbumin (43 kDa), lysozyme (14 kDa), and phenolred (0.3 kDa) were used as molecular weight markers, and the molecular weight in the apparent solution state was estimated. The results are shown in FIG. The AHPP-OEG methacrylate polymer apparently showed a molecular weight of 99 kDa.
When the inhibition of XO enzyme activity was measured in the same manner as in Example 1, the obtained AHPP-OEG methacrylate copolymer inhibited XO enzyme activity to the same extent as AHPP. The results are shown in FIG.

A micelle of AHPP by SMA (also referred to as SMA-AHPP micelle inclusion) was prepared.
SMA anhydride (200 mg) was added with 0.1 M NaOH or 0.1 M KOH, and stirred with a stirrer while maintaining a temperature of 50 to 60 ° C., and the hydrolysis reaction of SMA anhydride was allowed to proceed over about 12 hours, This SMA hydrolysis was performed. To the reaction solution, 0.1M HCl was added dropwise with stirring to precipitate the SMA hydrolyzate. The resulting precipitate was collected through a glass filtration filter (2 to 4 μm), further washed with cold 0.001 M HCl and lyophilized to prepare an SMA hydrolyzate.
Separately from SMA, 378 mg of AHPP (fine powder) was added to 50 ml of 0.1 M NaOH and allowed to stand overnight at 37-40 ° C. to prepare a saturated solution of AHPP. The saturated solution of AHPP was filtered through a PTFE filter (0.45 μm, Toyo Roshi Kaisha, Ltd, Tokyo, Japan), and 576 mg of SMA hydrolyzate was added to 10 ml of 0.01 M Na ₂ CO ₃ / NaHCO ₃ (pH 9.0). The solution dissolved in was slowly added dropwise with stirring with a stirrer at room temperature. After complete addition of the SMA hydrolysis solution, the reaction was allowed to proceed for 5 hours with further stirring. Under stirring, 0.01 M HCl was added to the resulting water-soluble micelle to adjust the pH to 4.0, and the resulting precipitate was centrifuged and washed once more with cold 0.001 M HCl. Next, 0.1 M NaHCO ₃ was added and redissolved, and then concentrated according to Example 1 using a molecular sieve membrane having a molecular weight of 1000 to 10,000 to obtain a lyophilized product. This lyophilizate was dissolved in 0.1 M Na ₂ CO ₃ and purified as a single peak by Sephadex G-100 gel (GE Healthcare) chromatography [size: 35 cm (h) × 2.5 cm (d)]. AHPP was detected by absorption at 260 nm. Estimated micellar association / inclusion is shown in FIG.

  SMA-AHPP was dissolved in physiological saline so as to be 15 and 30 mg / kg (converted to AHPP) in 0.5 ml per rat, and spontaneously hypertensive rats (spontaneous hypertensive rat, SHR, average body weight of about 300 g, 1 group 3- 4 animals) were administered via the tail vein. In another group, 100 mg / kg rat (AHPP equivalent) of SMA-AHPP was orally administered to the SHR with a sonde. Three hours after administration, blood pressure fluctuations were measured using an unheated non-invasive blood pressure monitor (Model MK-2000MT, Muromachi Kikai).

FIG. 26 shows the results of tail vein administration of SMA-AHPP. When SMA-AHPP 15 and 30 mg / kg were observed over 24 hours after the tail vein administration, the SMA-AHPP administration group showed a blood pressure lowering effect (antihypertensive effect) compared to the control group. Intravenous administration of SMA-AHPP at 30 mg / kg significantly suppressed the blood pressure in the untreated control group of SHR hypertensive rats to about 75% (−25% reduction). This action sustained the antihypertensive effect for 24 hours or more by a single administration. In the figure, the ◆ plot indicates the control, the ■ plot indicates 30 mg / kg, and the ▲ plot indicates 30 mg / kg.
More importantly, as shown in FIG. 27, oral administration of 100 mg / kg SMA-AHPP significantly reduced the blood pressure by 20 mmHg lower than the original value of blood pressure 24 hours after administration, and further increased for 48 hours. Significantly lower blood pressure was maintained until later. However, after that, while showing a gradual increase, the second treatment after 72 hours showed a significant antihypertensive effect as compared with the control group until 216 hours (P <0.01). In the figure, the ▲ plot represents the control, and the ● plot represents the SMA-AHPP dose of 100 mg / kg.

The synthesis scheme of SMA-AHPP concerning this invention is shown. The FT-IR spectrum of SMA and SMA-AHPP is shown. The inhibitory activity of AHPP and SMA-AHPP on the activity of XO enzyme is shown. According to XO of AHPP and SMA-AHPP _{O 2} ^- it shows the inhibitory activity of production. It is a graph which shows the LDH change in a rat liver. 2 shows changes in ALT and AST in rat liver. The change of the amount of peroxide in a rat liver is shown. The change of the body weight of a mouse | mouth, a liver weight, and the length of a large intestine by DSS drinking water administration is shown. The change of IL-12 in mouse serum is shown. The change of TNF-α in mouse serum is shown. The synthesis scheme of PEG-AHPP concerning this invention is shown. The absorption spectrum of AHPP is shown. (A) shows the absorption spectrum of PEG (2000) -AHPP, and (B) shows the absorption spectrum of PEG (300) -AHPP. The FT-IR spectrum of AHPP is shown. The FT-IR spectrum of PEG-AHPP is shown. (A) shows the particle distribution in the solution by light dynamic scattering of PEG (2000) -AHPP, and (B) shows PEG (300) -AHPP. _{O 2} by XO of PEG and PEG-AHPP ^- shows the inhibitory activity of production. The synthetic scheme of an AHPP-acrylate monomer coupling | bonding compound is shown. The absorption spectrum of an AHPP-acrylate monomer is shown. 1 shows a synthesis scheme of AHPP-oligoethylene glycol (OEG) methacrylate copolymer. The absorption spectrum of an AHPP-OEG methacrylate polymer is shown. 2 shows an FT-IR spectrum of an AHPP-OEG methacrylate copolymer. The molecular weight of AHPP-OEG methacrylate polymer is shown. According to XO of AHPP-OEG methacrylate polymers _{O 2} ^- shows the inhibitory activity of production. It is a structure conceptual diagram of the aggregate / inclusion of the SMA micelle of AHPP. The blood pressure-lowering effect by the injection administration of SMA-AHPP of AHPP is shown. The blood pressure-lowering effect by oral administration of SMA-AHPP of AHPP is shown.

Claims (6)

  Contains 4-amino-6-hydroxypyrazolo (3,4-d) pyrimidine, pharmaceutically acceptable salts, derivatives thereof, or 4-amino-6-hydroxypyrazolo (3,4-d) pyrimidine An anti-inflammatory agent containing a micelle body as an active ingredient. The anti-inflammatory agent according to claim 1, wherein the 4-amino-6-hydroxypyrazolo (3,4-d) pyrimidine is a compound represented by Formula 1, and a derivative thereof is a compound represented by Formula 2. [In Formula 2, X is one substituent represented by Formula 3 to Formula 6. ]



Note that k in the formula represents a natural number of 2 to 300.


In the formula, l represents a natural number of 1 to 1000.


In the formula, m and n represent natural numbers of 1 to 1000.
The 4-amino-6-hydroxypyrazolo (3,4-d) pyrimidine derivative is a micellar product of 4-amino-6-hydroxypyrazolo (3,4-d) pyrimidine and a styrene maleic acid copolymer (formula 7 The anti-inflammatory agent according to claim 1, wherein In addition,

Here, p represents a natural number of 3 to 200.
  The anti-inflammatory agent according to any one of claims 1 to 3, wherein the inflammatory reaction is inflammation caused by active oxygen.   The anti-inflammatory agent according to any one of claims 1 to 3, wherein the inflammation is inflammation derived from ischemia reperfusion.   The anti-inflammatory according to any one of claims 1 to 3, wherein the inflammation is one of various inflammatory diseases such as pneumonia, hepatitis, gastritis, colitis, dermatitis, stomatitis, pharyngitis, bronchitis, pancreatitis and the like. Agent.