Use of oxazopyrroloquinolines and pyrroloquinolinequinones for the manufacture of nerve growth factor production accelerators

Abstract: Nerve growth factor production accelerating agents containing oxazopyrroloquinolines, pyrroquinolinequinones and/or their esters as active ingredient are provided. As the oxazopyrroloquinolines and their esters exhibit such production accelerating activity, they are suitably utilized for preventing and treating functional disorders of central nervous system, particularly, Alzheimer's dementia, cerebral ischemia and spinal trauma, as well as for functional disorders of peripheral nervous system, particularly, peripheral nervous system trauma and diabetic neurosis. As the pyrroquinolinequinones and their esters exhibit strong nerve growth factor production accelerating activity, they are suitably utilized for preventing and treating functional disorders of peripheral nervous system, particularly, peripheral nervous system trauma, diabetic neurosis, etc.

Description: This invention relates to production accelerators for nerve growth factor (hereinafter referred to as NGF) which works for the recovery of neural function in central and peripheral nervous system diseases. Furthermore, it relates to compositions for preventing or treating neuronal degeneration. NGF is a nutrition and growth factor necessary for the growth and maintenance of neuronal tissues, which is considered to be essential for maturation and differentiation of sensory and sympathetic nerves in the peripheral nervous system, and magnocellular cholinergic neurons in the central nervous system, as well as for life maintenance. However, NGF is a protein with the molecular weight of 13,000 as monomer and 26,000 as dimer, so that it can not pass through the blood-brain barrier. Accordingly, it has been thought preferable that an agent accelerating the production of NGF in the living body, rather than NGF itself, is administered to promote biosynthesis of NGF. Thus, investigations to seek NGF production accelerators have been attempted. Cathecholamines, such as epinephrine, norpinephrine and dopamine, have been found as agents having NGF production accelerating activity. However, because these compounds are a kind of hormones, their administration to accelerate the NGF synthesis accompanies some side effects due to quantitatively ill-balanced hormones in the living body. Therefore, satisfactory drugs have not yet been discovered from the practical point of view. The present inventors have extensively studied on NGF production accelerating agents on account of the reasons as mentionned above, and accomplished the present invention, based upon the findings that oxazopyrroloquinolines, pyrroloquinolinequinones, and their esters exhibit the NGF production accelerating activity. Accordingly, the present invention is to provide an NGF production accelerating agent containing an oxazopyrroloquinoline or pyrroloquinolinequinone and/or their esters as active ingredient. Compositions comprising a pyrroloquinolinequinone or its salt as an active ingredient are known form JP-A-2262581 and JP-A-2196720 for being useful in the treatment of dementia due to brain senescence or of Alzheimer type and other diseases of the nervous system. The term, oxazopyrroloquinolines (hereinafter referred to as OPQS), used herein means 2,8,10-tricarboxy-1H-oxazo[5,4-h]-pyrrolo[2,3-f]quinolines (OPQ) and 5-substituted compounds thereof. The OPQS and their esters are represented by the following formula : [wherein R represents a hydrogen atom or an alkyl group having 1-4 carbon atoms, which may be substituted with a hydroxyl, carboxyl, mercapto, carbamoyl, hydroxyphenyl, guanidyl, imidazolyl, or methylmercapto group, and R 1 , R 2 and R 3 represent a hydrogen atom or an alkyl, alkenyl or benzyl group which may be same or different.] OPQs employed in the present invention can readily be prepared by a process wherein a pyrroloquinolinequinone compound or its salt (hereinafter referred to as PQQ, which will be explained more minutely below) is allowed to react with an α-amino acid, methylamine or the like in the presence of oxygen. The reaction is usually undertaken in a aqueous medium like a microbial culture. The pH of the reaction mixture is usually in the range from 2 to 10, and the reaction temperature is practically in the range from 20 to 100°C. And the reaction time is preferably within 24 hours. OPQs in the present invention include OPQ (R=H) obtained from a PQQ and any of glycine, threonine, tryptophan, proline, tyrosine, serine, and monomethylamine [Japanese Patent Publication (Laid-Open) No. 294281/1991]; hydroxymethyl-OPQ obtained from a PQQ and serine [Japanese Patent Publication (Laid-Open) No. 123782/1991]; 1-methylethyl-OPQ obtained from a PQQ and valine [Japanese Patent Publication (Laid-Open) No. 170484/1991]; 1-methylpropyl-OPQ obtained from a PQQ and isoleucine [Japanese Patent Publication (Laid-Open) No. 170485/1991]; 2-methylpropyl-OPQ obtained from a PQQ and leucine [Japanese Patent Publication (Laid-Open) No. 170486/1991; methyl-OPQ obtained from a PQQ and alanine [Japanese Patent Publication (Laid-Open) No. 188081/1991]; 2-carboxyethyl-OPQ obtained from a PQQ and glutamic acid [Japanese Patent Publication (Laid-Open) No. 190882/1991]; 2-carbamoylethyl-OPQ obtained from a PQQ and glutamine [Japanese Patent Publication (Laid-Open) No. 188082/1991]; 2-methylthioethyl-OPQ obtained from a PQQ and methionine [Japanese Patent Publication (Laid-Open) No. 19088/1991]; benzyl-OPQ obtained from a PQQ and phenylalanine [Japanese Patent Publication (Laid-Open) No. 190881/1991]; 4-hydroxyphenylmethyl-OPQ obtained from a PQQ and tyrosine [Japanese Patent publication (Laid-Open) No. 9387/1992]; carboxymethyl-OPQ obtained from a PQQ and aspartic acid; carbamoylmethyl-OPQ obtained from a PQQ and asparagine; 4-imidazolylmethyl-OPQ obtained from a PQQ and histidine; 4-aminobutyl-OPQ obtained from a PQQ and lysine; 3-guanidinopropyl-OPQ obtained from a PQQ and arginine; and mercaptomethyl-OPQ obtained from a PQQ and cysteine. As shown above, R group of each OPQs basically corresponds to the R group of α-amino acid [R-CH(NH 2 )COOH] which is used for substrate to produce one of OPQs. When the tyrosine is used as α-amino acid, OPQ (R=H) is main product in the case that the pH value of the reaction mixture is low and 4-hydroxyphenylmethyl OPQ (R=CH 2 C 4 H 4 OH) is main product in the case that the value is high. Salts of these OPQs include alkali metal salts, alkali earth metal salts, ammonium salts and substituted ammonium salts, which are also effective as the NGF production accelerating agents. Typical examples are the salts of sodium, potassium, magnesium, calcium, ammonium, trimethylammonium, triethylammonium, and triethanolammonium. Esters of these OPQs include those in which R 1 , R 2 and R 3 in the formula mentioned above represent a hydrogen atom, or an alkyl, alkenyl, or benzyl group, which may be same or different, to form mono-, di- or triesters. These OPQs esters can be prepared by a conventional process wherein an OPQs or its salt is allowed to react with an alcohol. The alkyl group may be a methyl or ethyl group, and the alkenyl group may be an allyl group. These objective OPQs esters can also be obtained by a conventional process wherein a PQQ or its salt is allowed to react with an alcohol to give the corresponding PQQ ester, and then the ester is allowed to react with an amino acid or methylamine. PQQs have been found as a coenzyme for methanol dehydrogenase in methanol-metabolizing bacteria. The term, PQQs, used herein means 4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid (PQQ) and its salts. The PQQs and their esters are represented by the following formula: [wherein in R 1 , R 2 and R 3 represent a hydrogen or an alkyl, alkenyl, benzyl, propargyl or alkoxycarbonylalkyl group, which may be same or different.] PQQs and their esters actively accelerate NGF production, but they can not increase the NGF level in cerebral cortex, though they exhibit the production accelerating activity to sciatic nerve in animal experiments. Therefore, they are suitably used as preventing and therapeutic agents for degenerations of peripheral nervous system, such as peripheral nervous trauma and diabetic neurosis. PQQs employed in this invention can be prepared by any of organic chemical syntheses [for example, that mentioned in J.A.C.S. Vol. 103, pages 5599-5600 (1981)] and fermentation methods [for example, that mentioned in Japanese Patent Publication (Laid-Open) No. 218597/1989]. PQQs referred to in this invention means PQQ and its salts, such as sodium and potassium salts of PQQ. In the above formula for PQQ esters, R 1 , R 2 and R 3 represent a hydrogen atom, or alkyl, alkenyl, benzyl, propargyl or alkoxycarbomethyl group, which may be same or different, to form mono-, di- or triesters. The alkyl group may be a methyl or ethyl group, and the alkenyl group may be an allyl group. PQQ triesters are readily synthesized by reaction of a PQQ with an alcohol [see, for example, Japanese Patent Publications (Laid-Open) Nos. 123781/1991 and 145492/1991]. PQQ monoesters or diesters can be obtained by partial hydrolysis of a PQQ triester under basic condition. PQQ diesters can also be obtained by reaction of a PQQ monoester with an alcohol under suitably selected reaction conditions of the temperature and the period of time. OPQs and PQQs and their esters in this invention may be administered orally or non-orally. In case of oral administration, they may be administered in the form of conventional formulations such as capsules, tablets, and powders. In case of non-oral administration, the formulations may be those for injections and parenteral fluids. Sustained release formulations are also effective. Dosage and dosing time vary depending on symptoms, ages, body weights, dosing formulations, and others, but they may be administered ordinarily in an amount of 1-500 mg a day for adults in case of oral administration, or 0.1-100 mg at once or in several times in case of non-oral administration. In preparing formulations of the active ingredients of this invention, any additives, such as surface active agents, excipients, coloring agents, preservatives, coating auxiliaries, and the like may be suitably used. They may also be used in combination with other pharmaceuticals. Hereinafter, examples are illustrated in order to show the NGF production accelerating activity of PQQs, OPQs and their esters, according to the present invention. These examples are, however, never construed to limit the invention. Example 1 L-M cells of fibroblast cell line originated from a mouse connective tissue were suspended in a 199 culturing medium (manufactured by Flow Laboratories) containing 0.5% peptone (manufactured by Difco Laboratories), and the suspension was placed in a microplate with 96 flat bottom holes to make the cell numbers of 2 x 10 4 /hole, which was then incubated in a CO 2 incubator (at 37°C, in an atmosphere of 5% CO 2 and 95% air) for 3 days. Each incubated liquid was replaced by a 199 medium containing OPQ of each given concentration and 0.5% bovine serum albumin (manufactured by Armour Pharmaceutical) or the same medium containing no OPQ, and incubated in a CO 2 incubator. After 24 hour incubation, the amount of NGF contained in the supernatant fluid was estimated by enzyme immunoassay [see Korsching and Thoenen, Proc. Natl. Acad. Sci., U.S.A., 80 , 3513-3516, (1983)]. The results are shown in Table 1. Amount of OPQ added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 170 100 3.1 203 119 6.3 278 164 12.5 371 218 25 367 216 50 422 248 100 288 169 NGF assay A solution of anti-mouse β-NGF antibody (made by using β-NGF prepared from mouse submaxillary gland as antigen) was dispensed to each hole on a 96 hole microplate made of polystyrene (MS-3496F, manufactured by Sumitomo Bakelite Co. Ltd.) in an amount of 50µl/hole, and stood for 4 hours at 37°C. The antibody not adsorbed to each hole of the microplate was removed, and each hole was rinsed 3 times with a cleansing liquor. A solution of standard β-NGF ( manufactured by Toyobo Co., Ltd.) or a sample solution was dispensed to each hole in an amount of 40 µl/hole, and stood for 18 hours at 4°C. Then, the standard β-NGF or sample solution (incubated supernatant as mentioned above) was removed, and each hole was rinsed 3 times. A solution of anti-β-NGF monoclonal antibody labeled with β-glactosidase (manufactured by Boehringer Mannheim) (40 mU/ml, pH 7.6) was dispensed to each hole in an amount of 50 µl/hole, and stood for 4 hours at 37°C. Then, the enzyme-labeled antibody was removed, and each hole was rinsed 3 times. A solution of 4-methylumbelliferyl-β-D-galactoside (manufactured by Sigma) was dispensed to each hole in an amount of 100 µg/hole, and allowed to react for 1.5 hours at room temperature, and then a 0.2 M glycine-sodium hydroxide buffer (pH 10.3) was dispensed to each hole in an amount of 100 µl/hole to stop the enzyme reaction. Fluorescent intensity of 4-methyllumbelliferone produced was estimated using a plate reader, and NGF amount was calculated from .the standard curve. The results are shown in Table 1. NGF production accelerating activity of the tested compound was shown as a relative value (%) of the NGF amount produced by cells treated with the testing compound against the NGF amount produced by untreated cells without testing compound. Example 2 Procedure of Example 1 was repeated using hydroxymethyl-OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 2. Amount of hydroxymethyl-OPQ added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 384 100 0.8 525 137 1.6 554 144 3.1 565 147 6.3 582 152 12.5 593 154 25 670 174 50 708 184 100 786 205 200 982 258 400 1,453 378 Example 3 Procedure of Example 1 was repeated using methyl-OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 3. Amount of methyl-OPQ added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 384 100 0.8 525 137 1.6 604 157 3.1 599 156 6.3 665 173 12.5 665 173 25 632 165 50 676 176 100 681 177 200 873 227 400 1,075 280 Example 4 Procedure of Example 1 was repeated using 2-carboxyethyl -OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 4. Amount of 2-carboxyethyl-OPQ added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 264 100 1.6 330 125 3.1 387 147 6.3 330 125 12.5 349 132 25 356 135 50 368 139 100 381 144 200 381 144 400 454 172 Example 5 Procedure of Example 1 was repeated using benzyl-OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 5. Amount of benzyl-OPQ added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 264 100 1.6 375 142 3.1 368 139 6.3 375 142 12.5 349 132 25 375 142 50 356 135 100 317 120 200 298 113 400 271 103 Example 6 Procedure of Example 1 was repeated using 1-methylpropyl -OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 6. Amount of 1-methylpropyl-OPQ added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 264 100 1.6 442 167 3.1 446 177 6.3 430 163 12.5 418 158 25 484 183 50 430 163 100 381 144 200 337 128 400 245 93 Example 7 Procedure of Example 1 was repeated using 2-methylpropyl -OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 7. Amount of 2-methylpropyl-OPQ added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 264 100 1.6 460 174 3.1 400 152 6.3 460 174 12.5 424 161 25 436 165 50 478 181 100 418 158 200 393 149 400 324 123 Example 8 Procedure of Example 1 was repeated using 2-methylthioethyl -OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 8. Amount of 2-methylthioethyl-OPQ added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 264 100 1.6 490 186 3.1 442 167 6.3 484 183 12.5 430 163 25 466 177 50 480 186 100 424 161 200 356 135 400 349 132 Example 9 Procedure of Example 1 was repeated using 2-carbamoylethyl -OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 9. Amount of 2-carbamoylethyl-OPQ added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 382 100 0.8 443 116 1.6 512 134 3.1 505 132 6.3 546 143 12.5 632 165 25 639 167 50 652 171 100 566 148 200 512 134 400 408 107 Example 10 Procedure of Example 1 was repeated using 1-methylethyl -OPQ, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 10. Amount of 1-methylethyl-OPQ added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 382 100 0.8 386 101 1.6 478 125 3.1 492 129 6.3 526 138 12.5 546 143 25 553 145 50 606 159 100 573 150 200 539 141 400 485 127 Example 11 Procedure of Example 1 was repeated using 4-hydroxyphenylmethyl -OPQ, in place of OPQ, to estimate its NGF product ion accelerating activity. The results are shown in Table 11. Amount of 4-hydroxyphenylmethyl-OPQ added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 382 100 0.8 560 147 1.6 580 152 3.1 553 145 6.3 632 165 12.5 613 160 25 613 160 50 652 171 100 429 112 200 335 88 Example 12 Procedure of Example 1 was repeated using OPQ methyl ester at 2-position (OPQ-2-ME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 12. Amount of OPQ-2-ME added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 259 100 0.8 290 112 1.6 299 115 3.1 299 115 6.3 344 133 12.5 308 119 25 234 90 Example 13 Procedure of Example 1 was repeated using OPQ trimethyl ester (OPQ-TME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 13. Amount of OPQ-TME added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 336 100 0.8 508 151 1.6 677 201 3.1 636 189 6.3 628 187 12.5 653 194 25 563 168 50 524 156 100 516 154 200 587 175 400 508 151 Example 14 Procedure of Example 1 was repeated using OPQ 2-methyl-8,10-diethyl ester (OPQ-2-ME-8,10-DEE), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 14. Amount of OPQ-2-ME-8,10-DEE added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 381 100 0.8 476 125 1.6 558 146 3.1 542 142 6.3 576 151 12.5 716 188 25 625 164 50 608 160 100 421 110 Example 15 Procedure of Example 1 was repeated using PQQ·Na 2 , in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 15. Amount of PQQ·Na 2 added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 204 100 0.8 319 156 1.6 302 148 3.1 398 195 6.3 677 332 12.5 1,076 527 25 2,515 1,233 50 5,915 2,900 100 8,034 3,938 200 5,428 2,661 400 2,487 1,219 Example 16 Procedure of Example 1 was repeated using PQQ dipotassium salt (PQQ·K 2 ), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 16. Amount of PQQ·K 2 added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 384 100 0.8 560 146 1.6 554 144 3.1 729 190 6.3 812 211 12.5 1,453 378 25 3,288 856 50 7,047 1,835 100 7,281 1,896 200 4,425 1,152 400 1,237 322 Example 17 Procedure of Example 1 was repeated using PQQ methyl ester at 2-position (PQQ-2-ME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 17. Amount of PQQ-2-ME added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 727 100 0.8 1,434 197 1.6 1,663 229 3.1 2,501 344 6.3 3,126 430 12.5 4,510 620 25 5,611 772 50 5,188 714 100 2,397 330 Example 18 Procedure of Example 1 was repeated using PQQ methyl ester at 7-position (PQQ-7-ME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 18. Amount of PQQ-7-ME added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 219 100 1.6 258 118 3.1 282 129 6.3 493 225 12.5 1,238 565 25 1,448 661 50 928 424 100 335 153 200 266 121 Example 19 Procedure of Example 1 was repeated using PQQ dimethyl ester at 2- and 9-positions (PQQ-2,9-DME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 19. Amount of PQQ-2,9-DME added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 727 100 0.8 1,165 160 1.6 1,777 244 3.1 3,126 430 6.3 3,790 521 12.5 4,790 659 25 6,829 939 50 5,499 756 100 4,584 631 200 1,852 255 Example 20 Procedure of Example 1 was repeated using PQQ trimethyl ester (PQQ-TME), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 20. Amount of PQQ-TME added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 341 100 0.8 429 126 1.6 464 136 3.1 600 176 6.3 954 280 12.5 1,448 425 25 2,852 836 50 3,015 884 100 1,678 492 Example 21 Procedure of Example 1 was repeated using PQQ triethyl ester (PQQ-TEE), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 21. Amount of PQQ-TEE added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 381 100 0.8 2,190 575 1.6 2,160 567 3.1 1,889 496 6.3 1,882 494 12.5 1,249 328 25 550 144 Example 22 Procedure of Example 1 was repeated using PQQ triallyl ester (PQQ-TAE), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 22. Amount of PQQ-TAE added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 204 100 0.8 403 198 1.6 414 203 3.1 611 300 6.3 776 380 12.5 1,063 521 25 707 347 Example 23 Procedure of Example 1 was repeated using PQQ triethoxycarbonylmethyl ester (PQQ-TECE), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 23. Amount of PQQ-TECE added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 381 100 3.1 428 112 6.3 455 119 12.5 558 146 25 669 176 50 1,908 508 100 3,092 812 200 304 80 Example 24 Procedure of Example 1 was repeated using PQQ tripropargyl ester (PQQ-TPGE), in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 24. Amount of PQQ-TPGE added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 381 100 1.6 389 102 3.1 599 157 6.3 764 201 12.5 2,453 644 25 6,022 1,581 50 3,926 1,030 100 505 133 Example 25 L-M cells were incubated in similar way as in Example 1 using epinephrine which has been known as an NGF production accelerator, in place of OPQ, to estimate its NGF production accelerating activity. The results are shown in Table 25. Amount of epinephrine added (µg/ml) Amount of NGF produced (pg/ml) Relative activity (%) 0 179 100 0.8 180 101 1.6 168 94 3.1 180 101 6.3 187 104 12.5 224 125 25 319 178 50 431 241 100 872 487 200 259 144 Epinephrine showed NGF production accelerating activity at an amount of not less than 12.5 µg/ml, and exhibited the maximum value (about 500%) at an amount of 100 µg/ml. On the other hand, OPQs and OPQ esters showed almost the same degree of NGF production accelerating activity as shown in Examples 1-17. Also, PQQs and PQQ esters gave high activity values even at a lower concentration, having markedly higher NGF production accelerating activity, when compared with epinephrine. Example 26 L-M cells were incubated in similar way as in Example 1. In three groups (A, B and C) of L-M cell incubation systems, Group A contained no tested compound, Group B contained 100 µg/ml of PQQ·Na 2 , and Group C contained 275 µg/ml of epinephrine. Amounts of NGF produced during the course of times (3, 6, 9, 12, 24, 30, 36 and 48 hours from the beginning of incubation) were estimated. The results are shown in Table 26. As obvious from the table, addition of PQQ·Na 2 definitely increases NGF production, as compared with the cases of no addition and adding epinephrine. Incubating period of time (hr) Amount of NGF produced (pg/ml) (A) No addition (B) PQQ·Na 2 100 µg/ml added (C) Epinephrine 275 µg/ml added 0 63 81 66 3 124 180 92 6 219 667 202 9 254 2,657 288 12 271 4,947 347 24 378 9,924 556 30 420 8,635 689 36 458 8,334 873 48 465 7,769 1,960 Example 27 SD female rats (7 weeks age, 160-190 g) were put under anesthesia by intramuscularly administering 25 mg of ketamine hydrochloride and 0.25 mg of doloperidol. Left femor sciatic nerve was exposed and cut off, and the cut ends were connected with a gap of about 2 mm using a silicone tube (1 mm inner diameter, 6 mm length). The gap between the cut ends was filled with an isotonic sodium chloride solution. After reduction of the operational cut, each 0.5 ml of an aqueous 2% gum arabic solution containing a given concentration of OPQ was administered intraperitoneally. As control, 0.5 ml of an aqueous 2% gum arabic solution containing no OPQ was administered intraperitoneally. As for positive control, the gap of the cut ends of sciatic nerve was filled with an isotonic sodium chloride solution containing 1 mg/ml of NGF, and 0.5 ml of an aqueous 2% gum arabic solution containing no OPQ was administered intraperitoneally. After 4 weeks from the operation, animals were sacrificed by cervical bertebral luxation, and the reproduced sciatic nerves were collected. Cross sectional slices of the reproduced sciatic nerves were prepared and dyed with hematoxylin-eosine, and number of the reproduced nerve fibers was counted. The results are shown in Table 27. As obvious from the table, number of the reproduced sciatic nerves was much increased by administering OPQ, which was comparable to direct injection of NGF. Amount of OPQ administered (µg/kg rat) Number of reproduced sciatic nerves Relative activity (%) 0 12,590 100 5 12,600 100 10 12,848 102 50 13,912 111 100 19,839 158 500 17,870 142 Positive control (NGF gap injection) 20,932 166 Example 28 Procedure of Example 27 was repeated using OPQ trimethyl ester (OPQ-TME), in place of OPQ, to estimate the reproduction accelerating activity of OPQ-TME for sciatic nerve. The results are shown in Table 28. As obvious from the table, number of the reproduced sciatic nerves was much increased by administering OPQ·TME, which was comparable to NGF. Amount of OPQ·TME administered (µg/kg rat) Number of reproduced sciatic nerves Relative activity (%) 0 12,590 100 5 11,860 94 10 11,193 89 50 12,780 102 100 22,966 182 500 26,313 209 Positive control (NGF gap injection) 20,932 166 Example 29 Procedure of Example 27 was repeated using PQQ·Na 2 , in place of OPQ, to estimate the reproduction accelerating activity for sciatic nerve. The results are shown in Table 29. As obvious from the table, number of the reproduced sciatic nerves was much increased by administering OPQ·Na 2 , which was comparable to NGF. Amount of PQQ·Na 2 administered (µg/kg rat) Number of reproduced sciatic nerves Relative activity (%) 0 12,590 100 5 16,984 135 10 21,822 173 50 26,142 208 100 18,247 145 500 14,031 111 Positive control (NGF gap injection) 20,932 166 Example 30 Procedure of Example 27 was repeated using PQQ trimethyl ester (PQQ-TME), in place of OPQ, to estimate its reproduction accelerating activity for sciatic nerve. The results are shown in Table 30. As obvious from the table, number of the reproduced sciatic nerves was much increased by administering PQQ-TME, which was comparable to NGF. Amount of PQQ-TME administered (µg/kg rat) Number of reproduced sciatic nerves Relative activity (%) 0 12,590 100 5 19,047 151 10 21,054 167 50 24,940 198 100 17,882 142 500 17,902 142 Positive control (NGF gap injection) 20,932 166 Example 31 OPQ trimethyl ester (OPQ-TME) was suspended in 0.5 ml of an aqueous 2% gum arabica in a given concentration, and the suspension was administered to Wistar male rats (8-10 weeks age, 200-250 g) intraperitoneally. Administrations were conducted every other days once a day, 4 times in total. After 2 days from the last administration, rats were dissected under anesthesia, and the neocortex, submaxillary grand and hippocampus were collected. The following procedures were conducted under ice-cooling. Each of these tissues was weighed, and mixed with a 20 time-volume of a phosphate buffer (8 g/l of NaCl, 0.2 g/l of KCl, 1.15 g/l of Na 2 HPO 4 and 0.2 g/l of KH 2 PO 4 ), and the mixture was homogenized by an ultrasonic crusher, followed by centrifugation at 10,000 x G for 30 minutes to separate the supernatant. Amount of NGF contained in the supernatant was estimated by enzyme immunoassay. The results are shown in Table 31. NGF amount (ng) per 1 mg (wet weight) of neocortex, submaxillary gland or hippocampus was set forth as the average value ± standard error from 3 heads tested simultaneously. As for former two tissues, relative activities were also shown against the case of no OPQ-TME administration. As obvious from the table, NGF contents in neocortex and submaxillary gland were increased by administering OPT-TMF. Particularly, the degree of increase was high in neocortex. Amount of OPQ-TME administered (µg/kg rat) Neocortex Submaxillary gland Hippocampus NGF (ng/mg tissue wet weight) Relative activity (%) NGF (ng/mg tissue wet weight) Relative activity (%) NGF (ng/mg tissue wet weight) 0 1.83± 0.05 100 0.63± 0.04 100 2.44± 0.23 0.1 2.64± 0.17 144 0.70± 0.05 111 2.31± 0.20 0.5 2.60± 0.40 142 0.85± 0.06 135 2.20± 0.46 1.0 3.17± 0.44 173 0.67± 0.09 106 - Example 32 Procedure of Example 31 was repeated using PQQ trimethyl ester (PQQ-TME), in place of OPQ-TME, to estimate the accelerating activities of PQQ-TME for NGF contents of neocortex, submaxillary gland and hippocampus. The results are shown in Table 32. Administration of PQQ-TME did not increase the NGF contents in neocortex, submaxillary gland and hippocanpus. Supposedly, PQQ-TME have no NGF production accelerating activity to central nervous system. Amount of PQQ-TME administered (mg/kg rat) NGF production amount (ng/mg tissue wet weight) Neocortex Submaxillary gland Hippocampus 0 2.51± 0.23 1.42± 0.07 2.53± 0.23 0.1 2.25± 0.10 1.67± 0.15 2.44± 0.04 0.5 2.19± 0.33 1.58± 0.07 2.10± 0.15 1.0 2.71±0.30 1.30±0.08 - Thus, as OPQs and their esters exhibit NGF production accelerating activity, and, in animal experiments, they increase the NGF content in neocortex, and accelerate reproduction of sciatic nerve,

Claims: The use of an oxazopyrroloquinoline or its ester represented by the following formula: [wherein R represents a hydrogen atom or an alkyl group having 1-4 carbon atoms, which may be substituted with a hydroxyl, carboxyl, mercapto, carbamoyl, hydroxyphenyl, guanidyl, imidazolyl or methylmercapto group, and R 1 , R 2 and R 3 represent a hydrogen atom or an alkyl, alkenyl or benzyl group, which may be same or different] for the manufacture of a nerve growth factor production accelerating composition. The use of pyrroloquinolinequinone or its ester represented by the following formula: [wherein R 1 , R 2 and R 3 represent a hydrogen atom or an alkyl, alkenyl, benzyl, propargyl or alkoxycarbonylalkyl group, which may be same or different.] for the manufacture of a nerve growth factor production accelerating composition.

Inventors: KONDO KIYOSI, TSUJI TOMOKO, URAKAMI TEIZI, YAMAGUCHI KOHJI, KONDO, KIYOSI, TSUJI, TOMOKO, URAKAMI, TEIZI, YAMAGUCHI, KOHJI, Urakami, Teizi, c/o MITSUBISHI GAS CHEMICAL CO.INC
Applicants: MITSUBISHI GAS CHEMICAL CO, SAGAMI CHEM RES, MITSUBISHI GAS CHEMICAL COMPANY, INC., SAGAMI CHEMICAL RESEARCH CENTER
Application year: 1993
Main classification: A61K31
IPC codes: A61K31