TECHNICAL FIELD 0001 The present invention relates to a protective agent for pericytes present at the periphery of vascular endothelial cells in microvessels.

TECHNICAL BACKGROUND 0002 Pericytes are cells present surrounding the vascular endothelial cells in microvessels and they act not only in tension regulation in the microvessels but also in the maintenance of endothelial cell functions such as endothelial cell proliferation suppression and prostacyclin production, and they play an important role in the maintenance of microvascular homeostasis. 0003 If disorders of these pericytes occur, the functions of the endothelial cells constituting the blood vessels are impaired and there is thought to be a link with the onset and progress of various vascular disorders such as diabetic retinopathy, diabetic neuropathy, diabetic nephropathy and other such diabetic microvascular disorders, hypertension, arteriosclerosis, peripheral circulatory disturbance (intermittent claudication and the like), cerebrovascular disorders and ischaemic heart disease. 0004 Prostanoic acid derivatives are known to have various physiological activities and they can be divided into several groups according to the modification of the five-membered ring moiety comprising C-8 to C-12. Among these, the compounds where the carbon atoms at positions 6 and 9 are linked via an oxygen atom are referred to as PGI, and prostaglandin I2 (PGI2, prostacyclin) is regarded as typical. This PGI2 is known to be a substance with a powerful platelet aggregation suppressing action and a peripheral blood vessel dilating action (see Nature Vol.268, p.688, 1976). 0005 Furthermore PGI derivatives having a skeletal structure where the exoenol ether moiety structure, which is the characteristic structure of PGI2, has been converted to the inter-m-phenylene form have been described in JP-B-2-12226, JP-B-2-57548 and JP-B-1-53672 as compounds where the instability of PGI2 has been substantially improved. Again, in addition to PGI derivatives with the carbon atoms at positions 6 and 9 via an oxygen, there are known PGI derivatives where this oxygen atom has been replaced with a carbon atom or some other heteroatom. Examples include Ataprost, Iloprost, Clinprost, Ciprostene, Naxaprostene, Taprosten, Cicaprost, Pimilprost, CH-169 and CS570 (see Gendai Iryosha, Sosetsu Purosutaguranjin An Outline of Prostaglandins No. 1, p123, 1994, Asu no Shinyaku 15-IV-p185, 1996, and Asu no Shinyaku 15-III-p551, 1996). 0006 However, hitherto, drugs capable of maintaining pericyte function have been entirely unknown, and the fact that prostaglandins have a pericyte protecting action has also been completely unknown.

OBJECT OF THE INVENTION 0007 The present invention offers a pericyte protective agent which, by displaying a vascular pericyte protecting action, is effective in the prevention and treatment of various vascular disorders such as diabetic retinopathy, diabetic neuropathy, diabetic nephropathy and other such diabetic microvascular disorders, hypertension, arteriosclerosis, peripheral circulatory disturbance (intermittent claudication and the like), cerebrovascular disorders and ischaemic heart disease.

DISCLOSURE OF THE INVENTION 0008 As a result of painstaking research, the present inventors have discovered that prostanoic acid derivatives exhibit an outstanding pericyte protecting action, and the present invention has been perfected based thereon. 0009 Specifically, the present invention offers a pericyte protective agent in which a prostanoic acid derivative is the effective component.

BRIEF EXPLANATION OF THE DRAWINGS 0010 FIG. 1 shows the cell protection effect of Beraprost sodium on pericytes.

OPTIMUM FORM FOR PRACTISING THE INVENTION 0011 As aforementioned, the pericyte protective agent of the present invention has a prostanoic acid derivative as its effective component. Here a prostanoic acid derivative may be any type of prostaglandin having the prostanoic acid skeletal structure, or derivative thereof. Among these, preferred prostanoic acid derivatives are the prostaglandin I derivatives and, as such prostaglandin derivatives, there may be used prostaglandin I1 derivatives, prostaglandin I2 derivatives and prostaglandin I3 derivatives or pharmacologically acceptable salts thereof, preferably prostaglandin I2 derivatives or pharmacologically acceptable salts thereof. Still further preferred is the use of the 4,8-inter-m-phenylene prostaglandin I2 derivatives represented by the following general formula (I) 1

0012 where R1 is 0013 (A) COOR2 0014 (where R2 is 0015 1) hydrogen or a pharmacologically acceptable cation, 0016 2) a C1-12 straight chain alkyl or C3-14 branched alkyl, 0017 3) -Z-R3 0018 (where Z is a valence bond or a straight chain or branched alkylene represented by CtH2t, where t denotes an integer in the range 1-6, and R3 is a C3-12 cycloalkyl or C3-12 substituted cycloalkyl which is substituted with from one to three R4 groups, and R4 denotes hydrogen or a C1-5 alkyl), 0019 4) (CH2CH2O)nCH3 0020 (where n denotes an integer in the range 1 to 5), 0021 5) -Z-Ar1 0022 (where Z is defined as above and Ar1 denotes phenyl, -naphthyl, -naphthyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, -furyl, -furyl, -thienyl, -thienyl or substituted phenyl (where the substituent groups are at least one of chlorine, bromine, fluorine, iodine, trifluoromethyl, C1-4 alkyl, nitro, cyano, methoxy, phenyl, phenoxy, p-acetamidobenzamido, CHNNHC(O)NH2, NHC(O)-Ph, NHC(O)CH3 and NHC(O)NH2)), 0023 6) CtH2tCOOR4 0024 (where CtH2t and R4 are as defined above), 0025 7) CtH2tN(R4)2 0026 (where CtH2t and R4 are as defined above), 0027 8) CH(R5)C(O)R6 0028 (where R5 denotes hydrogen or benzoyl, and R6 denotes phenyl, p-bromophenyl, p-chlorophenyl, p-biphenyl, p-nitrophenyl, p-benzamidophenyl or 2-naphthyl), 0029 9) CpH2pWR7 0030 (where W is CHCH, CHCR7 or CC, and R7 is hydrogen or a C1-30 straight chain or branched alkyl or aralkyl), and p denotes an integer in the range 1-5), or 0031 10) CH(CH2OR8)2 0032 (where R8 denotes a C1-30 alkyl or acyl), 0033 (B) CH2OH 0034 (C) C(O)N(R9)2 0035 (where R9 represents hydrogen, a C1-12 straight chain alkyl, C3-12 branched alkyl, C3-12 cycloalkyl, C4-13 cycloalkylalkylene, phenyl, substituted phenyl (where the substituent groups are as defined in (A) 5) above), C7-12 aralkyl or SO2R10, and R10 represents a C1-10 alkyl, C3-12 cycloalkyl, phenyl, substituted phenyl (where the substituent groups are as defined in (A) 5) above) or a C7-12 aralkyl, and the two R9 groups may be the same or different, but where one represents SO2R10 then the other R9 is not SO2R10), or 0036 (D) CH2OTHP (where THP is a tetrahydropyranyl group), 0037 Y is hydrogen, a C1-4 alkyl, chlorine, bromine, fluorine, formyl, methoxy or nitro, 0038 B is XC(R11)(R12)OR13 0039 (where R11 is hydrogen or a C1-4 alkyl, R13 is hydrogen, C1-14 acyl, C6-15 aroyl, tetrahydropyranyl, tetrahydrofuranyl, 1-ethoxyethyl or t-butyl, 0040 X is 0041 1) CH2CH20042 2) CHCH or 0043 3) CC and 0044 R12 represents 0045 1) a C1-12 straight chain alkyl or C3-14 branched alkyl, 0046 2) -Z-Ar2 0047 (where Z is as defined above and Ar2 represents phenyl, -naphthyl, -naphthyl or phenyl substituted with at least one of chlorine, bromine, fluorine, iodine, trifluoromethyl, C1-4 alkyl, nitro, cyano, methoxy, phenyl or phenoxy), 0048 3) CtH2tOR14 0049 (where CtH2t is as defined above and R14 represents a C1-6 straight chain alkyl, C3-6 branched alkyl, phenyl, phenyl substituted with at least one of chlorine, bromine, fluorine, iodine, trifluoromethyl, C1-4 alkyl, nitro, cyano, methoxy, phenyl or phenoxy, cyclopentyl, cyclohexyl, or cyclopentyl or cyclohexyl substituted with from one to four C1-4 straight chain alkyls), 0050 4) -Z-R3 0051 (where Z and R3 are as defined above), 0052 5) CtH2tCHC(R15)R16 0053 (where CtH2t is as defined above, and R15 and R16 each independently represent hydrogen, methyl, ethyl, propyl or butyl), or 0054 6) CuH2uCCR17 0055 (where u is an integer in the range 1-7, CuH2u represents a straight chain or branched alkylene, and R17 represents a C1-6 straight chain alkyl, 0056 E represents hydrogen or OR18 (where R18 represents a C1-12 acyl, C7-15 aroyl or R2 (where R2 is as defined above)), 0057 and where the general formula represents the d-form, l-form or dl-form, 0058 or pharmacologically acceptable salts thereof. 0059 Among the PGI2 derivatives represented by the aforementioned general formula (I), Beraprost (the general name of ()-(1R*,2R*,3aS*,8bS*)-2,3,3a,8b-tetrahydro-2-hydroxy-1-(E)-(3S*)-3-hydroxy-4-methyl-1-octen-6-ynyl-1H-cyclopentabbenzofuran-5-butyric acid), Iloprost, Clinprost, Ataprost, Ciprostene, Naxaprostene, Taprosten, Cicaprost, Pimilprost, CH-169 and SM-10902, and their salts, can be cited as especially preferred but there is no particular restriction thereto. 0060 The prostanoic acid derivatives used in the present invention can be produced by known methods and, for example, the compounds represented by aforementioned general formula (I) can be produced by the method described in JP-B-1-53672. 0061 The pericyte protective agent of the present invention has an outstanding pericyte protecting action and is effective in the prevention and treatment of hypertension and arteriosclerosis where vascular damage is closely involved in the onset and worsening of the disease state, and also of peripheral circulatory disturbance (intermittent claudication and the like), cerebrovascular disorders, ischaemic heart disease and various other such vascular disorders. Furthermore, it is effective in the prevention and treatment of diabetic angiopathy such as diabetic retinopathy, diabetic neuropathy and diabetic nephropathy which are associated with the loss of pericytes due to advanced glycation endproducts (AGE), and also complications of diabetes like arteriosclerosis, ischaemic heart disease, cerebrovascular disorders and other such macrovascular disorders. 0062 The prostanoic acid derivatives of the present invention bring about a marked pericyte protecting effect as a result of oral or parenteral administration. 0063 In the present invention, for adults the prostanoic acid derivatives are administered 1-3 times a day at a dose of 0.1 g-500 mg/person. 0064 Regarding the pericyte protective agent of the present invention, one or several types of prostanoic acid derivative may be used directly as they are, but oral administration can also be carried out in the form of a solid material containing undermentioned additives. 0065 As examples of additives, there are starches, lactose, sucrose, glucose, mannitol, calcium carbonate, calcium sulphate and other such excipients; starches, dextrin, gum Arabic, tragacanth, methyl cellulose, gelatin, polyvinyl pyrrolidone, polyvinyl alcohol and other such binders; starches, polyvinyl pyrrolidone, crystalline cellulose and other such disintegrants; magnesium stearate, talc and other such lubricants; and colorants and fragrances. 0066 The pericyte protective agent of the present invention can be used in various dosage forms; specifically, as tablets, sugar-coated tablets, powders, granules, troches, capsules, pills, syrups and other such conventionally-used dosage forms. 0067 Furthermore, parenteral administration in the form of a sterilized solution may also be carried out and, moreover, other dissolved substances can also be employed, for example sufficient sodium chloride, glucose or the like, to make the solution isotonic. 0068 The pericyte protective agent of the present invention can be orally or parenterally administered. As parenteral administration routes, there are intravenous injection, subcutaneous injection, intramuscular injection, perintestinal administration, transdermal administration, eye instillation and transnasal administration. 0069 As a specific example of a formulation, there is the following. 1prostanoic acid derivative500gphysiological saline1ml

EXAMPLES 0070 Below, the present invention is explained in more specific terms by giving examples, but the present invention is not restricted to the examples provided.

Reference Example 1

Preparation of Materials 0071 (1) Preparation of AGE-BSA 0072 AGE-BSA was formed by incubating bovine serum albumin (BSA) (fraction V, fatty acid-free, endotoxin-free, produced by Boehringer Mannheim GmbH, Germany) with 0.5 M glucose at 37 C. for 6 weeks under sterilized conditions. Unbound glucose was removed by dialysing against phosphate-buffered saline (PBS), and the glucose-modified macromolecular substance was purified by heparin-Sepharose CL-4B column (produced by Pharmacia LKB, Uppsala, Sweden) chromatography and used as the AGE-BSA. Control non-glycated BSA was prepared by carrying out incubation under the same conditions as aforementioned, except for not including the glucose. The fact that non-glycated BSA had been separated from the AGE-BSA was confirmed by SDS-PAGE. The AGE-BSA concentration was measured by the Bradford method (Bradford, M.: Anal. Biochem. 72: 248-254, 1976).

Example 1

Pericyte Protecting Action 0073 Pericytes were isolated from bovine retina and maintained in Dulbecco's modified Eagle medium containing 20% foetal calf serum. 0074 The suppression of pericyte proliferation attributable to AGE was measured by adding 0.25 mg of AGE-BSA (prepared in Reference Example 1) to a culture plate into which 3.0104 cells had been plated, then culturing at 37 C. for 5 days and performing measurements using a Packard Cyto Life. Separately, Beraprost sodium (BPS) was added at a final concentration of 0.1-10 M prior to AGE addition. The results are shown in FIG. 1. As controls, measurements were carried out in the same way for the case when treatment was carried out with AGE-BSA only and for the case where no treatment was carried out. For each group, the tests were carried out 10 times respectively. 0075 As shown in FIG. 1, proliferation of pericyte was significantly suppressed by AGE. As a result of BPS addition, this suppression of proliferation was improved in a concentration-dependent manner and was significant at 1 M. Thus, based on these results, BPS exhibited a pericyte protecting action. 0076 Next, the quantity of the AGE receptor RAGE mRNA present in the pericytes was analyzed by means of a quantitative reverse transcription-polymerase chain reaction method, in accordance with the technique described in Biochemical and Biophysical Research Communications, Vol. 213, 682-683, 1995. By simultaneously carrying out 1 M BPS treatment, expression of RAGE was suppressed and it was concluded that suppression of the expression of RAGE, which is the AGE receptor, plays a role in the pericyte protecting action of BPS.

Industrial Application Potential 0077 In both oral and parenteral administration, the protective agent of the present invention has an outstanding protecting action of pericyte vascular and it is effective in the prevention and treatment of various types of vascular disorder such as diabetic retinopathy, diabetic neuropathy, diabetic nephropathy and other such diabetic angiopathy, hypertension, arteriosclerosis, peripheral circulatory disturbance (intermittent claudication and the like), cerebrovascular disorders and ischemic heart disease.