TW200528095A - Therapeutic combinations - Google Patents

Abstract

The use of a combination of (A) a vasopressin receptor family antagonist or a pharmaceutically acceptable derivative thereof, and (B) a COX inhibitor or a pharmaceutically acceptable derivative thereof, is described for treatment or prophylaxis of dysmenorrhoea.

Description

200528095 IX. Description of the invention: [Technical field to which the invention belongs] The present invention relates to a synergistic combination of an antagonist of a vasopressin receptor system and a cyclooxygenase inhibitor. The use of such a combination in the treatment of dysmenorrhea, using this Method for treating dysmenorrhea by combination and medicine containing such combination. [Prior art] There is a highly unmet need for menstrual disorders, and it is estimated that 90% of women who menstruate are affected to some extent. Up to 42% of women lose work or other activities due to menstrual pain, and it is estimated that approximately 600 million working hours are lost each year in the United States {Coco, AS (1999). Primary dysmenorrhoea. [Review] [30 refs]. American Family Physician, 60 , 489-96} ° Dysmenorrhea can be divided into two types: primary dysmenorrhea and secondary dysmenorrhea. Primary dysmenorrhea is generally defined as cramping pain in the lower abdomen during menstruation, and there is no verifiable pelvic disease. This accounts for approximately 50% of the women's population {Coco, AS (1999) · Primary dysmenorrhoea. [Review] [30 refs]. American Family Physician, 60, 489-96; Schroeder, B. & Sanfilippo, JS (1999). Dysmenorrhoea and pelvic pain in Adolescents. [Review] [78 refs]. Pediatric Clinics of North America, 46, 555-71}. Secondary dysmenorrhea is dysmenorrhea related to specific pathological conditions, such as endometriosis, pelvic inflammatory diseases, fibroids, intrauterine contraception, etc. The cause of dysmenorrhea is unknown, although the pain of this woman seems to be closely related to excessive uterine muscle activity and reduced uterine blood flow. Only about 25% of women with dysmenorrhea can be diagnosed with secondary dysmenorrhea. Dysmenorrhea can occur at the same time as menstruation. 96335.doc 200528095 In healthy women, uterine contraction changes during the menstrual cycle {Akerlund, M. (1997), Contractility in the non-pregnant uterus. [Review] Annals of the New York Academy of Science, 828, 213 -twenty two}. This change is not consistent with fluctuations in the plasma concentration of ovarian hormones, but may be related to the amount of tissue because there is a time difference between the two. In the first few days of the menstrual cycle, uterine contraction is coordinated throughout the uterus, its contraction is regular, its intensity is greater, and there is a clear line between secondary contractions. During the follicular phase, especially before and after ovulation, uncoordinated uterine contractions occur, with a higher contraction frequency, lower intensity, and higher basal tone. This situation continues in the luteal phase (丨 plus 丨 phase) until the first 2-3 days of menstruation, at which time the uterine activity becomes more coordinated. At this time, the contraction has a cyclical activity, and it contracts from the cervix to the uterine floor. This orientation can change within minutes for a patient. Diffusion to the cervix may be important for blood excretion in the endometrium and during menstruation. In contrast, women with menstrual pain have stronger uterine activity. Its contraction is irregular. + Palace blood flow is also reduced, and ischemia on the base is in the non-pregnant {Akerlund, Μ. (1997), Contractility uterus. [Review] Annals 〇f the New Y〇rk Academy 〇f SCeinCeS, 828,213 · 22 }. The decrease in blood flow can be due to two factors: the compression of blood vessels caused by Zi Lu Zengnan. I believe this is related to the effect of vasoactive agents on the smooth muscle of arterial crests caused by abdominal colic experienced by such women. Decrease-This may be the cause of the persistent pain experienced by such women. In the current treatment of dysmenorrhea, fertility also requires fertility control. Non-steroidal anti-inflammatory drugs (NSAID ’S) should be the first choice. Oral contraceptives are used when fertility control is needed. During menstruation, primary dysmenorrhea is associated with an increase in endometrial prostaglandin F2a (PGF2a) {Pickles, V ·, Hall, W. & Best, F · (1965) · Prostaglandins in

endometrium and menstrual fluid from normal and dysmenorrheic subjects. BJOG: an International Journal of Obstetrics & Gynaecology, 72, 1 85-}, but not related to perimenstruation {Lundstrom, V. & Green, K. (1978). Endogenous level of Prostaglandin F2alpha and its main metabolites in plasma and endometrium of normal and dysmenorrheic women

American Journal of Obstetrics & Gynecology, 130, 640-6}. PGF2a is now known to promote uterine contraction and cause dysmenorrhea-like pain {Roth-Brandel, U.5 Bygdeman, M. & Wiqvist, N. (1970). Effect of introvenous administration of prostaglandin El, and F2 on the contractility of the non -pregnant human uterus in vivo. Acta Obstetricia et Gynecologica Scandinavica-Supplement, 5, 19-25}. It is also known that prostaglandins directly have pain-generating properties by sensitizing pain receptors, and this pain receptor can also be involved in menstrual pain sensations {Ferreira, S. (1976) · Pin nd Fever. In Prostoglandin and Thromboxanes: NATO advanced study institute on advances on prostagland-ins.pp. 433-442. New York: Plenum Press.}. Clinically, NS AID'S has shown that it relieves pain in some dysmenorrhea patients and restores uterine activity {Pulkkinen, MO & Csapo, Α · Ι (1978) · The effect of ibuprofen on the intruterine pressure and menstrual 96335.doc 200528095 pain of dysmenorrheic patients. Prostaglandins, 15, 105 5-62}. But it is not effective for all dysmenorrhea patients, especially those with severe dysmenorrhea. Moreover, it has side effects including upper gastrointestinal symptoms, sleepiness and tinnitus. The advantages of these agents over oral contraceptives are that they can be given in only 2-3 days per month and reduce the side effects associated with dysmenorrhea (vertigo, nausea and vomiting). U.S. Patent No. 5,466,823 discloses pyrazolyl cyclooxygenase-2 inhibitors for the treatment of inflammation and inflammation-related diseases, including menstrual cramps. U.S. Patent No. 5,932,598 discloses prodrugs of cyclooxygenase-2 inhibitors for the treatment of inflammation and inflammation-related diseases. Morrison et al. Described a study using rofecoxib, a cyclooxygenase-2 inhibitor, to treat primary dysmenorrhea (01: 61: Gynecol, 94 (4), 504-508 (1999)). U.S. Patent Nos. 5,521,207 and 5,633,272 also show that compounds that selectively inhibit cyclooxygenase-2 are useful in treating menstrual cramps. J. Talley in Prog. Med. Chem., 36, 201-234 (1999) has reviewed various selective inhibitors of cyclooxygenase-2. The following publications have also described compounds that selectively inhibit cyclooxygenase-2: US Patent Nos. 5,380,738, 5,344,991, 5.393,790, 5,434,178, 5,474,995 and 5,5 10,368; International Publication WO 96/06840, WO 96/03388, WO 96/03387, WO 96/19469, WO 96/25405, WO 95/15316, WO 94/15932, WO 94/27980, WO 95 / 00501, WO 94/13635, WO 94/20480 and WO 94/26731. When NSAIDs oral contraceptives are not effective alone in treating primary dysmenorrhea, 'a combination of both was used (Coco, A., American Family Physician, 60 (2), 489-496 (1999)) 〇96335.doc 200528095 U.S. Patent No. 5,811,416 discloses endothelin antagonists and / or endothelin synthase inhibitors and at least one progesterone, an estrogen, a combination of progesterone and estrogen, a cyclooxygenase inhibitor, nitrogen oxide A combination of donor or nitric oxide matrix treats menstrual disorders, including dysmenorrhea. U.S. Patent No. 5,912,006 discloses that a combination of omega fatty acids and cyclooxygenase inhibitors is used to reduce or alleviate uterine or vaginal pain associated with the onset of menstruation. W0 02/062391 discloses a combined treatment method for treating and preventing dysmenorrhea, which includes a COX-2 inhibitor and a sex steroid. Unless fertility control is also required, oral contraceptives are the second-line treatment for most women. Oral contraceptives must be taken continuously during the menstrual period, and may require 3 consecutive menstrual periods to treat menstrual pain for significant relief. Compared with NS AID'S, oral contraceptives reduce menstrual fluid volume to prevent menstrual pain {Nakano, R. & Takemura, Η. (1971). Treatment of functional dysmenrrhoea; a doube-blind study. Acta Obstetrica et Gynaecologica Japonica , 18, 41-4}, inhibit ovulation and reduce endometrial volume, which leads to a reduction in prostaglandin production {Chan, WY & Hill, JC (1978). Determination of menstrual prostaglandin level in non-dysmenorrheic and dysmenorrheic subjects. Prostaglandins, 15, 365-75} ° But a well-known problem is that NSAID's and oral contraceptives have a permanent failure rate (10-15%), especially in patients with severe dysmenorrhea {Coco, AS ( 1999). Primary dysmenorrhoea. [Review]. American Family Physician, 60, 489-96; Schroeder, B. & Sanfilippo, JS (1999). Dysmenorrhoea and pelvic pain in 96335.doc -10- 200528095 adolescents. [Review] . Pediatric Clinics of North America, 46, 5 5 5-71}. Newer, less specialized treatments are currently in the research phase and have not yet become modern treatment options. The physiological response of the uterus to vasopressin changes throughout the menstrual cycle, with the greatest sensitivity before menstruation {Bossmar, T ·, Akerlund, M ·, Szamatowicz, J ·, Laudanski, T ·, Fantoni, G. & amp Maggi, M. (1995). Re cep tor-mediated uterine effects of vassopressin and oxytocin in non-pregnant women. British Journal of Obstetrics & Gynaecology, 102, 907-12}. In the control group, there was no significant change in gold vasopressin content during the menstrual cycle {Forsling, ML, Akerlund, M. & Stromberg, P. (1981). Variations in plasma concentrations of vasopressin during the menstrual cycle · Journal of Endocrinology, 89, 263-6}. However, in women with dysmenorrhea, vasopressin content increases before or during menstruation {Hauksson, A ·, Akerlund, M ·, Forsling, ML & Kindahl, Η · (1987). Plasma concentrations of vasopressin and a prostaglandin F2 alpha metabolite in women with primary dysmenorrhoea before and during tretment with a combined oral contraceptive. Journal of Endocrinology, 115, 355-61}. This appears with increased pain, increased uterine muscle activity, and decreased uterine blood flow {Ekstrom, P., Akerlund, M., Forsling, M., Kindahl, H., Laudanski, T. & Mrugacz, G. (1992) . Stimulation of vasopressin release in women with primary dysmenorrhoea and after oral contraceptive treatment-effect on uterine contractility. British Jurnal of Obstetrics & 96335.doc 200528095

Gynaecology, 99, 680-4}. Details on the role of vasopressin in menstrual and dysmenorrhea mechanisms are unknown. But peptide activity on myometrial smooth muscle {Bossmar, T., Brouard, R., Doberl, A. & Akerlund, M. (1997). Effects of SR 49059, an orally active Via vasopressin receptor antagonist, on vasopressin- induced uterine contractions. British Journal of Obstetrics & Gynaecology, 104, 471-7} and uterine artery smooth muscle activity {Kpostrzewska, A., Laudanski, T., Steinwall, M., Bossmar, T., Serradeil-Le Gal, C & Akerlund, M. (1998). Effects of the vasopressin Via receptor antagonist, SR 49059, on the response of human uterine arteries to vasopressin and other vasoactive substances. Acta Obstetricia et Gynecologica Scandinavica, 77, 3-7} Vasopressin V1A receptors have significant contractile effects, and vasopressin V1A is different from V2 type that regulates renal function. There is clinical evidence that oral active V1A antagonists have a clear therapeutic benefit in preventing dysmenorrhea {Akerlund, M. (1987). Can primary dysmenorrhoea be allleviated by a vasopressin antgonist? Results of a pilot study. Acta Obstetricia et Bynecologica Scandinavica, 66, 45 9-61}. In a double-blind, randomized, placebo-controlled crossover trial, Relcovaptan (SR49059) did reduce pain intensity in women with dysmenorrhea without affecting the mechanisms that regulate menstruation {Brouard, R., Bossmar, T ., Fournie-Lloret, D., Chassard, D. & Akerlund, M. (2000). Effect of SR49059, an orally active via vasopressin receptor antagonist, in the prevention of 96335.doc -12- 200528095 dysmenorrhoea. BJOG: an International Journal of Obstetrics & Gynaecology, 107,614-9}. This compound also exhibits activity at the oxytocin receptor. Peptide vasopressin V1A antagonist / Oxytocin receptor antagonist 丨 _Amine_ 2-D-Tyr (Oet) -4-Thr-8-Orn-Oxytocin, also administered when given intravenously The effects of dysmenorrhea {Akerlund, M. (1987) Can primary dysmenorrhoea be alleviated by a vasopressin antagonist?

Results of a pilot study. Acta Obstetricia et Gynecologica Scandinavica, 66, 459-61} °

[Summary of the Invention] In order to explain the need to continue to seek safe and effective agents for the prevention and treatment of dysmenorrhea, the combination treatment of therapeutic agents is described here. It has now been surprisingly discovered that combination therapy with vasopressin receptor antagonists and cox inhibitors has led to unexpected and synergistic advances in the treatment of dysmenorrhea. There is an advantage when vasopressin antagonists and COX inhibitors are administered simultaneously, sequentially or separately. This is because the two active agents will produce a synergistic interaction and are more effective. To reduce the progress of the disease, thereby reducing the need for surgery, the two will have a wider range of activities, 1 more definitive,: less effect 'more selective (especially more beneficial for the treatment of dysmenorrhea): than this just technique The compounds or combinations used have more useful properties. The combination of the present invention not only provides treatment for excessive activity of uterine muscles, uterine arterial blood sacrifice, and the cramps caused by it, but also provides a treatment to reduce :! muscle: and the basal tension of the uterine arteries to maintain it more The state of relaxation is 'although dysmenorrhea is periodic'. If the uterine muscles and uterine arteries are maintained in a relaxed state every month, after the period of fork and fork, the inventors believe that 96335.doc -13- 200528095 will reduce the soothing Symptoms needed for treatment.疋 According to the present invention, a combination of (A) angiotensin receptor antagonist or a pharmaceutically acceptable derivative thereof and a WCOX inhibitor or a pharmaceutically acceptable and acceptable derivative thereof is provided in the manufacture of a medicament for the treatment or Uses for preventing dysmenorrhea. (B) is preferably a Cox_2 inhibitor. (B) More preferably, a cox_2 selective inhibitor. In another aspect, use of a combination of the aforementioned (A) and (B) for treating or preventing dysmenorrhea is provided. φ In another aspect, a combination of the aforementioned (A) and (B) is provided for use in the manufacture of a medicament by simultaneously and sequentially or separately administering ⑷ and (B) for the treatment or prevention of dysmenorrhea. Or 'provide a method for treating or preventing dysmenorrhea, which comprises administering to the subject in need thereof a therapeutic amount of the aforementioned ⑷ and (B)' The two are effective together, and provide a therapeutic composition comprising the aforementioned ( A) and (B), for use: to treat or prevent dysmenorrhea. In another aspect, a medicinal product is provided, which contains the aforementioned (A) and (B), and a pharmaceutically acceptable carrier as a combined preparation for simultaneous, separate or sequential administration for the treatment or prevention of dysmenorrhea. Indigenous medicine includes painful uterine cramps and preventive management of dysmenorrhea. This treatment can be given at the beginning of pain, before or after the pain. A key advancement over the prior art is that you do not experience moderate or severe pain before starting treatment, but this pain is preventable and has more satisfactory results. Another advantage is that lower doses of analgesics can be used when using this regimen. 96335.doc -14-200528095, oral or blood pressure vasopressin party system antagonist and cox inhibitor combination for the treatment or prevention of dysmenorrhea is better than either of these agents alone. It is believed that the effect provided by the combination of a vasopressin receptor antagonist and a COX inhibitor is superior to the effect obtained when the blood & vasopressin antagonist or cox inhibitor is administered separately. Combination therapy including vasopressin receptor antagonists or COX inhibitors can be used not only to improve the symptoms of dysmenorrhea, but also to reduce the amount of compound administered 1. Low doses reduce compound-related side effects. Dysmenorrhea can be primary or secondary. Secondary dysmenorrhea can be a consequence of increased uterine tension, such as caused by uterine fibroids or an intrauterine contraceptive device. The "treatment" ("treating," or "t0 treat,") means temporarily or permanently alleviating the symptoms or eliminating the cause, or preventing or slowing the appearance of symptoms. However, the term "treatment" means to alleviate or eliminate the causes associated with dysmenorrhea or prevent the symptoms and diseases associated with dysmenorrhea. The vasopressin receptor system includes Vla, Vlb, V2, and oxytocin receptors. Body {Thibonnier M., Exp. Opin. Invest. Drugs (1998) 7 (5), 729-740}. The vasopressin receptor antagonist used in the present invention is preferably selective for the Vla receptor and is related to oxytocin Receptors are closely related. It may be more beneficial for oxytocin receptors to be active. More preferably, the vasopressin receptor inhibitor used in the present invention is selective for V1 a receptors. Applicable Examples of vasopressin receptor antagonists in the present invention are disclosed in US Pat. No. 6,090,818; EP 0873309; WO 98/25901; WO 02/083685; JP 2000-63363; and WO 02/32864. ¥ 1 & receptor antagonists used in the present invention are: 81149049 (1 ^ 100 ¥ 301 & 11), 96335.doc • 15-200528095 atosiban (Tractocile®), Connie Conivaptan (Y ^ 4-087) and 0? 021268. In addition, ¥ 1 & receptor antagonism disclosed in 1 \ ¥ 0 01/58 880 Are also useful in the present invention. Other VI a suitable for the present invention are antagonists are disclosed in WO 2004/037809, i.e. the compound of formula (I),

Or a pharmaceutically acceptable salt or solvate thereof, wherein R1 represents (VC6 alkyl,-(CH2) e- [C3-C8 cycloalkyl]-, _ (CH2) eW, or-(CH2) cZ- ( CH2) dW; w represents CVC6 alkyl, 〇 ((6alkoxy, group), -CONR4R5, phenyl, nr4r5, het2 or het3, phenyl is optionally substituted with one or more groups, this The isogroup is independently selected from halogen, CF3,

R3 'OR3, C〇2r3' Conr4r5, CN, s〇2Nr4r5 and 3s〇2Me; Z represents o or s (0) g; g represents 0, 1 or 2; R2 represents phenyl 'which is fused as required An aryl or heterocyclic group at 5 ^ 6_M, which may contain-or more heteroatoms selected from N, 0 or 8; benzyl and optionally thick. The group is optionally substituted with one or more groups independently selected from the following: —, 疋,-or 6-membered at least one saturated heterocyclyl ring B represents phenyl Or hetl 'per-group is substituted with—or at most 96235.doc -16-200528095 selected from groups listed in the table defined below; R7 independently represents fluorene, C, C6 alkyl, OR3, -(CH2) e-R3 or-(CH2) f_〇- (CH2) e-R3; v r3 independently represents H at each occurrence, Cl-C6 alkyl substituted with Y if necessary, and CH2) g- [C3_C8 ring alkyl], phenyl, benzyl, pyridyl or pyrimidine; R and R5 independently represent Η at each occurrence, C! -C6 alkyl (substituted with CVC6 alkyloxy if necessary), (CH2) gC (V [Ci_C6 alkyl],-(CH2) g_ [C3_C8 cycloalkyl], so2Me, phenyl, benzyl, pyridyl or pentyl; or R4 and R5 are jointly connected with it The N atom represents a radical of 3 to 8 atoms; Y independently represents a phenyl group, NR4R5 or het4. This phenyl group is optionally selected from one or more of hydrogen, CF3, CF3, R4, OR4, C02R4. , CONR4R5, CN 'S02NR4R5, NR4s〇2MeA-NR4R, group substituted

Het represents a 4-, 5- or 6- or 7-membered saturated or unsaturated heterocyclic group, which contains at least one but it may also contain one or more 0 or 8 atoms); 7-shell saturated or unsaturated heterocyclic group containing at least one but it may also contain one or more 0 or § atoms Substituted by a group; ί et 3 4 七 db λ 5 or 6 or 7-shell saturated or unsaturated heterocyclic group, which contains at least -0 (but it can also contain one or more atoms ), If necessary, replaced by "groups independently selected from the groups listed in the table defined below;

Het4 represents 4-, 5- 痞 6, 7 ^^, or 7-shell saturated or unsaturated heterocyclic groups of eight or ^ N (but it may also contain one or more 0 or S atoms), as required乂 "Even independently selected from the group substituted by the group defined in the table defined below; 96335.doc-17 · 200528095 R2, ring B, het2, het2, het3 and het4 are independently selected from the following table: halogen, CF3, 〇CF3, R3,-(CH2) e-S02Me,-(CH2) e-OR3,-(CH2) e-C02R3 ^-(CH2) e-CONR4R5 ^-(CH2) e-CN ^-(CH2) e -S02NR4R5,-(CH2) e-NR3S02Me ^-(CH2) e-COR3 ^-(CH2) e-OCOR3,-(CH2) e-NHCOR3,-(CH2) e-NR3COR6 and-(CH2) eNR4R5; R6 Independently represents H at each occurrence, optionally substituted by Y — —'CH2) g_ [C3_C8 cycloalkyl], phenyl, benzyl, t! Than σ or σ definite; a and b are independent Represents 0 or 1; c, d, e, and g independently represent 0 ,! , 2, 3 or 4; f independently represents 1, 2, 3 or 4; the prerequisites are: (i) a + b cannot be equal to 0; and (Π) the prerequisite is that R1 represents _ (CH2) crZ_ (CH2) When d_W and w represent NR4R5 or any heterocyclic group having N, then d must not be 0 or 1; and (ui) a prerequisite is that R2 is represented by the formula-(CH2) e〇R3, _ (CH2) e-C02R3 or-(CH2) e〇COR3 group substituted phenyl; or het1 and / or het2 with the formula-(CH2) eOR3,-(CH2) e-C02R3 or _ (CH2) e〇c〇R3 group Group substitution; or when R7 represents _OR3 or-(CH2) r0- (CH2) e-R3 and e is 0; or when W represents a benzyl group substituted with _0R3 or _c〇2R3; When Y is substituted alkyl, and Y represents NR4R5 or ^ 3 with ^^; 96335.doc -18- 200528095, then R3 must represent C2-C6 alkyl substituted with γ. Other Vla antagonists using the present invention are disclosed in WO2004 / 074291, i.e. compounds of formula (I),

Or a medically acceptable derivative thereof, wherein V represents-(CH2) d (〇) e-, -CO-, or -CHCCrCs alkyl; W is -〇-, -S (〇) a-, or -R1 represents H, C! -C6 alkyl, (CH2) bCOR2, CO (CH2) bNR2R3, S02R2, (CH2) coor2, (Ch2) cNR2R3, or (CHJbhet1; het1 represents saturated or unsaturated 3 A heterocyclic ring of 8 to 8 atoms containing one or more heteroatoms selected from 0, N or S, optionally substituted with a C1-6 alkyl group; X and Y independently represent H, Cu alkyl, halogen, oh, CF3, 〇CF3, OR4; Z represents-(CH2) f (〇) g-, -CO- or -CHCCu alkyl)-; ring A represents a 4-7 membered saturated N-containing heterocyclic ring, which is apparent It needs to be substituted with 0H, and at least one ring N is optionally substituted with 0; ring B represents a phenyl group or a 4- to 7-membered unsaturated N-containing heterocyclic ring, which is optionally substituted with 3, OCF3 And at least one ring N substituted with OH, halogen, CN, CONH2, CF is 0; _6 alkyl [if necessary, 0H, halogen, N (Ci6 generation), Ck oxy, n (Ci_6 alkyl, R2 and R3 independently represent H, Ci_6 alkyl [alkyl] 2 or Cle6 alkyloxy substituted], or [C3-8 cycloalkyl]; 96335 .doc -19- 200528095 or the nitrogen to which R2 and R3 are jointly associated represents a heterocyclic ring of 3 to 8 atoms, which is optionally substituted with Cw alkyl; ”R4 represents a straight or branched CN6 alkyl A and c independently represent 0, 1 or 2; b, e and g independently represent 0 or 1; d and f independently represent 1 or 2. Specifically, 'preferably 8-gas-5-methyltetrahydro- Twisted, ^ bispyridin-4-yl), 5,6-dihydro-4H-2,3,5,10b_tetrazole-benzo [e] camomile ring and 8-qi-1- (1- Pyrimidin-2-yl-hexahydropyridin-4-yl) _5,6_dihydro_4H_2,3,1〇b_ triazine-benzo [e] guan Juhuan, or its pharmaceutically acceptable salt (especially dibesylate Salt) or solvates. The vasopressin receptor antagonists used in the present invention can be identified as follows: 1-0 鉴定 Identification by membrane binding 1.1 Preparation of membrane

Receptor binding identification is performed on the cell membrane, which is prepared from CHO cells that stably express the human v1A receptor (CHO-hV1A). CHO-hViJ ^, obtained according to a contract with Thibonnier, Dept, of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio. CHO-hV1A was routinely maintained in a DMEM / Hams F12 nutrient mixture at 37 ° C in a humid and 5% C02 atmosphere, supplemented with 10% fetal calf serum, 2 mM L-glutamine, 15 mM HEPES and 400 μg / ml G418. When producing cell pellets in batches, the adhered CHO-hV1A cells were grown to 90-100% confluence in a 85 0 cm cm roller bottle. This bottle contains DMEM / Hams F12 Nutrient Mix medium, plus 10% fetal bovine serum. , 2 mM 96335.doc -20- 200528095 L-glutamine and 15 mM HEPES. Confluent CHO-hV1A cells were washed with phosphate-buffered saline (PBS), harvested into ice-cold PBS, and centrifuged at 1,000 rpm. Cell pellets were stored at -80 ° C until use. Cell pellets were thawed on ice and homogenized in a membrane preparation buffer. The buffer consisted of 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, and a protease inhibitor cocktail (Roche) was added. The cell homogenate was centrifuged at 1000 rpm for 10 minutes at 4 ° C, and the supernatant was removed and stored on ice. The remaining pellets were homogenized and centrifuged as previously described. The supernatant was collected and centrifuged at 25,000 xg for 30 minutes at 4 ° C. The pellets were resuspended in a frozen buffer consisting of 50 mM Tris-HCl, ρΗ7.4, 5 mMMgCl2 and 20% glycerol, divided into small aliquots and stored at -80 ° C until use. The protein concentration was determined using Bradford reagent, with BSA as the standard. 1.2 V1A filter binding Protein linearity was completed, followed by saturation binding studies on each new batch of membranes. The selected cell membrane concentration can produce specific binding in the linear part of the curve. Then [3H] -arginine vasopressin was used for saturation binding studies to determine [3H] -AVP (0.05 nM-100 nM) & Kd and Bmax. Binding of [3H] -AVP to the compound (3110-11 ¥ 1 effect on eight membranes to test its effect (3H-AVP; specific activity 65.5 Ci / mmole; MEN Life Sciences). Compounds were dissolved in dimethylsulfine (DMSO) to identify the buffer The solution was diluted to 10% DMSO working concentration. The buffer contained 50 mM Tris-HCl pH 7.4, 5 mM MgCl2 and 0.05% BSA. 25 microliters of compound and 25 microliters of [3H] -AVP (at or below Kd Concentration measurement membrane batch, generally 96335.doc -21-200528095 (0.5 ηM-0.6 nM) is added to a 96-concave round-bottom polypropylene dish. Add 200 microliters of membrane to start the binding reaction, and shake the dish gently at room temperature for 60 Minutes. Filtermate Cell Harvester (Packard Instilments) was used to quickly stop the reaction by 96-concave GF / B UniHlter Plate. This GF / B Unifilter Plate was pre-soaked in 0.5% polyethyleneimine to prevent peptide adhesion. Use 1 Wash the filter three times with ml of ice-cold buffer containing 50 mM Tris-HCl pH 7.4 and 5 mM MgCl2. Dry the dish and add 50 to each well. Microliter Microscint-0 (Packard Instruments). Plates were sealed and counted on a TopCount Microplate Scintillation Counter (Packard Instruments). 1 μM unlabeled d (CH2) 5Tyr (Me) AVP ([p-hydrothio-β , Β-cyclopentamethylene propionyl, O-Me-Tyr2, Arg8] • Vasopressin (pMCPVP) (Sigma) Determines non-specific binding (NSB). Using a four-parameter logarithmic equation, the minimum value is set at 0%, analysis of radioligand binding data. Freely determine the slope, and use the effective curve between -0.75 and -1.25. Calculate specific binding from the average total minute count minus the average NSB minute count. Binding of the test compound to The amount of ligand on the receptor is expressed as% binding = (sample counts per minute-average NSB counts per minute) / specific binding counts per minute X 10 0. Draw the% binding based on the concentration of the test compound to make an S-shaped curve Calculate the inhibition dissociation constant by Cheng-Prusoff equation: 1 = 1 (: 50 / (1+ [L] / Kd), where [L] is the concentration of concave ligands, and Kd is the radio coordination obtained from the Scatchard plot analysis Volume dissociation constant. 2.0 V1A% can identify: AVP inhibition / FLIPR (Fluorescent Imaging Plate Reade 0 (Molecular Devices) caused Ca2 + movement caused by V1A-R Use FLIPR to measure intracellular calcium release in CHO-hV1A cells, this method 96335.doc- 22- 200528095 Calculate calcium quickly after receptor activation. This CHO-hVi ^ cell line is supplied by Marc Thibonnier, Dept, of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio according to contract. CHO- hV1A cells were stored in a DMEM / HamsF12 nutrient mixture in a humid atmosphere of 5% C02 at 37 ° C. The nutrient mixture was supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 15 mM HEPES, and 400 μg. / Ml G418. The cells were placed in a black sterilized 96-concave dish at a density of 20,000 cells / concave in the afternoon before the identification. This dish had a transparent bottom to observe the cells and fluorescence measurements in each cavity. A fresh wash buffer containing Dulbeccofs phosphate-buffered saline (DPBS), 2.5 mM probenecid, and loading dye is made of cell culture medium, containing 4 μM Fluo-3-AM (dissolved in DMSO and pluronic acid, (Molcular Probes) and 2.5 mM propionate. The compound was dissolved in DMSO and diluted in an identification buffer composed of DPBS. Contains 1% DMSO, 0.1% BSA, and 2.5 mM Prostone Huangshu. The cells were cultured at 37 ° C in a humid atmosphere with 5% C02 at 100 microliters per dye for 1 hour. After the dye was loaded Cells were washed three times with 100 microliters of buffer in a Denley dishwasher. 100 microliters of wash buffer was left in each well. FLIPR was used to measure intracellular decidue. 30 microseconds after the addition of 50 microliters of test compound, every 30 seconds Take a fluorescence reading every 2 seconds. Measure 155 times every 2 seconds to determine the compound anti-activity. Then add 50 microliters of arginine vasopressin (AVP) to make the final identification volume of 200 microliters Take fluorescence readings every 1 second for a total of 120 seconds. Measure the response with the peak fluorescence intensity (FI). For pharmacological characterization, subtract the substrate F1 from each decant photoreaction. Make an AVP dose response curve 96335. doc -23- 200528095, each response is expressed in% of the maximum AVP concentration among different AVP concentrations. When IC 5 was measured, each reaction was expressed as a% response to AV P. The Cheng-Prusoff equation is used to convert the IC5G value to a modified Kb value. This equation considers and counteracts the concentration, [A], counteracts EC5G, and slope: Kb = IC5G / (2 + [A] / A50) n) 1 / nl , Where [A] is the AVP concentration, A50 is the EC50 of AVP in the dose response curve, and n = AVP dose response curve slope. The terms "π cyclooxygenase-2 inhibitor" or "C0X-2 inhibitor" are used interchangeably and include compounds that inhibit COX enzymes and pharmaceutically acceptable salts of these compounds, regardless of whether they inhibit cyclooxygenase- 1 degree of enzyme. Therefore, for the purpose of the present invention, although the inhibition of the COX2 enzyme by this compound may be equal to, greater or less than the inhibition of the COX1 enzyme, this compound is still considered a COX2 inhibitor. Examples of COX2 inhibitor compounds are non-steroidal anti-inflammatory drugs (NSAIDs). Therefore, compounds that can be used as COX2 inhibitors in the present invention include non-steroidal anti-inflammatory drug compounds, pharmaceutically acceptable salts thereof, or pure hydrazone or (+) optical isomers thereof. Non-limiting examples of NS AID compounds include ibuprofen, naproxen, benoxprofen, flurbiprofen, fenoprofen, fenoprofen, fen Fenbufen, ketoprofen, indoprofen, pirprofen, carprofen, oxaprozin, pula Prapoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, tiaprofenic acid, fluprofen ( fiUprofen), bucloxic acid, indomethacin, 96335.doc -24- 200528095

(sulindac), tolmetin, zomepirac, diclofenac, fenclofenec, alclofenac, ibufenac, Isoxepac, furofenac, tiopinac, zidometacin, acetyl salicylic acid, σ introduced Duomeixin ( indometacin), piroxicam, tenoxicam 'nabumetone', S with ketorolac, azapropazone, mefenamic acid ( mefenamic acid), tolfenamic acid, diflunisal, podophyllotoxin derivatives, acemetacin, droxicam 'flofenine ( floctafenine, oxyphenbutazone, phenylbutazone, proglumetacin, acemetain, fentiazac, clinac , Oxipinac, mefenamic acid, mefenamic acid Acid (meclofenamic acid), flufenamic acid (flufenamicacid), diflunisal (niflumicacid), flufenisal (flufenisal) ,. Sudoxicam, etodolac, piprofen, salicylic acid, choline magnesium trisalicylate, salicylate, benolate ), Fentiazac, clopinac, feprazone, isoxicam and 2-fluoro-a-methyl [1,1,] biphenyl] 4-acetic acid, 4- (nitrooxy) butyl | impart (lWenk, etal., Europ. J. Pharmacol. 453: 319-324 (2002)). The compounds in the above examples include ibuprofen, naproxen, sulindac 96335.doc -25- 200528095 (sulindac), keto ibuprofen, fenoprofen calcium, thiamine Ibuprofen, suprofen, etodolac, carprofen, ketorolac, piprofen, indoprofen, Salicylic acid and flurbiprofen. The COX2 inhibitor is preferably a COX2 selective inhibitor. As used herein, the cyclooxygenase-2 selection inhibitor π or " COX2 selection inhibitor " is used interchangeably and includes compounds that selectively inhibit COX2 over COX1, and pharmaceutically acceptable salts of these compounds. Therefore, for the purpose of the present invention, the term "Cox" inhibitor "includes" COX2 selective inhibitor, and all other compounds that inhibit cox2 enzyme. In fact, the selectivity of COX2 inhibitors depends on The completion of the test and the inhibitor used will vary. However, for the purposes of this description, the selectivity of COX2 inhibitors can be expressed as the ratio of the icm value that inhibits coxi in vitro and in vivo divided by the ic50 value that inhibits COX2 (C0X1 IC5〇 / COX2 iC5〇). 00 The magic selection inhibitor is an inhibitor whose ratio of COX1 1 (^ () to (: 0 \ 2 IC5G is greater than 1. In a preferred embodiment, this ratio is greater than 2 More preferably, it is greater than 5, particularly preferably greater than 10, particularly preferably greater than 50, and most preferably greater than 100. The term iCw here refers to the concentration of the compound required to produce 50% of the cyclooxygenase activity. It is found that the COXIC5G of the preferred COX2 selection inhibitor is less than about 丨 μM, and more preferably At about 0.5 μM, particularly preferably less than about 0.2 μM. The cyclooxygenase-1 ICw of the preferred cyclooxygenase selection inhibitor is greater than about 1 μM, and more preferably greater than 20 μM. This is preferred Selectivity can indicate the ability to reduce the occurrence of general side effects caused by Ns Am. The following identification test of COX inhibitors used in the present invention 96335.doc 200528095 3.0 Evaluation of COX activity in vitro 3.1 Preparation of recombinant COX baculovirus such as Gierse et al. (J. Biochem., 305, 479-484 (1995)) to prepare recombinant COX1 and COX2. In a manner similar to that described by DR · O'Reilly et al. (Baculovirus Expression Vectors: A Laboratory Manual (1992)), A 2.0 kb fragment containing human or murine COX1 or human or murine COX2 coding region was cloned into a baculovirus transfer vector. \ ^ 1393 (111 ¥ 1: 1: 1: (^ 611) at positions 8 & 111 ^ 11 to generate COX1 and COX2 baculovirus transfer vector. Calls for the transfer of 4 micrograms of baculovirus transfer vector DNA and 200 nanograms of baculovirus-only plasmid DNA into SF9 insect cells (2x10 e8) using the calcium phosphate method. See MD Simmers and GE Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell C ulture Procedures,

Texas Agric. Exp. Station Bull. 1555 (1987). Recombinant virus was purified by three rounds of speckle purification, and a high titer (107-108 pfu / mL) stock solution of the virus was prepared. For mass production, SF9 insect cells were infected with recombinant baculovirus in a 10-liter fermentation tank (0.5x106 / ml), resulting in an infection rate of Oj. After 72 hours, the cells were centrifuged, and the cell pellets were obtained in Tr * is / sucrose (50%) containing 1% 3-[(3-chloroamidopropyl) diamidoamino] -1-propane sulfate (CHAPS) (50 mM; 25%, pH 8.0). The homogenate was centrifuged at 10,000 × G for 30 minutes, and the resulting supernatant was stored at -80 ° C for identification of COX activity. 3.2 Identification of COX1 and COX2 activity 0 ug 2 / mg protein / time using £ 1 ^ 18 eight identification (: 0 and activity to determine the prostaglandins released. CHAPS-lysed insect cell membrane containing appropriate COX enzymes in potassium phosphate buffer (50 mM, pH 8.0) culture, slow 96335.doc -27- 200528095 The eluate contains adrenaline, phenol, and heme 'and is added with arachidonic acid (10 μM). The compound is first cultured with enzyme before adding arachidonic acid 10 -20 minutes. Stop the reaction between arachidonic acid and enzyme by moving 40 μl of the reaction mixture to 160 μl of ELISA buffer and 25 μM indomethacin at 37 ° C for ten minutes at room temperature. ELISA technology (Cayman Chemical) was used to measure the generated PGE2. 3.3 Rapid identification of COX1 and COX2 activity. PGE2 / mg protein / time generated. ELISA was used to identify COX activity and the released prostaglandins were measured. CHAPS-containing appropriate COX enzyme was dissolved. The insect cell wall membrane was cultured in potassium dishate buffer (50 mM potassium phosphate, pH 7.5, 300 μM epinephrine, 2 μM phenol, 1 μM heme), and 20 μl of 100 μM arachidene was added to the buffer. Acid before the arachidonic acid is added The enzyme was incubated for 10 minutes at 37 ° C. After 2 minutes at 37 ° C at room temperature, 40 microliters of the reaction mixture was moved to 160 microliters of ELISA buffer and 25 μM indomethacin to stop the arachidonic acid and enzyme The reaction is determined by standard ELISA technology (Cayman Chemical). The scope of the present invention also includes compounds that act as prodrugs of COX2 selection inhibitors. As described in the previous COX2 selection inhibitors, the term "pidrug precursors" Refers to chemical compounds that are converted to COX2 selective inhibitors by simple chemical processes within the subject or the disease. An example of a prodrug of COX2 selective inhibitors is parecoxib, which is a tricyclic COX2 selective inhibitor paldecoxib. (valdecoxib) is a therapeutically effective prodrug. A preferred example of a COX2 selective inhibitor prodrug is parecoxib sodium. A class of COX2 inhibitor prodrugs is described in US Patent No. 5,932,598. 96335. doc • 28-200528095 Non-limiting COX2 selective inhibitors suitable for use in the present invention are described, for example, in (i)-(liv) below: B Formula B-1 'Meloxicam, (CAS No. 71125-3 8-7; see US Patent 4 for description No. 233,299), or a pharmaceutically acceptable salt or prodrug thereof; (ϋ)

-1H-pyrrole-2-yl] methyl] 3- (2H) -pyridazinone, (CAS Registry Number 1 793 82-91-3; EP 714895), or a pharmaceutically acceptable salt or prodrug thereof ;

(iii)

Substituted benzopyran derivatives are described in U.S. Patent 6,271,253. Additional benzopyran derivatives are shown in U.S. Patent Nos. 6,034,256 and 6,077,850 and International Publications Nos. Jia 98/47890 and WO 00/23433; (iv) The following Table 1 illustrates chromene COX2 selective inhibitors: 96335.doc -29- 200528095 Table 1: Examples of chromene COX2 selection inhibitors Compound Number Reference Structure B-3 US Patent No. 6,077,850 (Example 37)

0 cf3 6-Shissokyl-2-digasmethyl-2H-1-phenylhydrazone 0 Bran-3-unconic acid B-4 US Patent No. 6,077,850 (Example 38).

6-chloro-8-methyl-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid B-5 US Patent No. 6,077,850 (Example 68)

((S) -6-chloro-7-(, 1-dimethylethyl) _2- (trifluoromethyl-2H-1-benzopyran-3-carboxylic acid B-6 U.S. Patent No. 6,034,256 ( Example 64)

2-trifluoromethyl-2h-naphtho [2,3-b] pyran-3-carboxylic acid B-7 US Patent No. 6,077,850 (Example 203)

〇 V 0 6-Ran-7- (4-stonesynylphenyllactyl) -2- (difluoromethyl) -2H-1 -benzopyran-3-carboxylic acid B-8 US Patent No. 6,034,256 (Example 175 )

((S) -6,8-dichloro-2- (trifluoromethyl) -2Η-1 · benzo11pyran-3-acid acid 96335.doc -30- 200528095 compound number reference structure formula B- 9 U.S. Patent No. 6,077,850 (Example 143) 6-chloro-2- (trifluoromethyl) -4-phenyl-2H-1-benzoxan-3-carboxylic acid B-10 U.S. Patent No. 6,077,850 (Example 98 ) 6- (4-Hydroxybenzyl) -2- (trifluoromethyl) -2H-1 benzophenone ° Bran-3-unconic acid B-11 US Patent No. 6,077,850 (Example 155), 3 < 5γγχΛ〇Η Into 2- (trifluoromethyl) -6-[(trifluoromethyl) thio] -2pyridine-1-benzopyran-3-carboxylic acid B-12 US Patent No. 6,077,850 (Example 156) CI 6,8-digas-2-trifluoromethyl-2H-1-benzopyran-3-carboxylic acid B-13 US Patent No. 6,077,850 (Example 147) 6- (1,1-Dimethyl Ethyl) -2- (trifluoromethyl) -2H-1

96335.doc -31-200528095 Compound No. Reference Structure B-14 U.S. Patent No. 6,077,850 (Example 159) 0 CF3 6,7-di-l-1,2-dihydro-2- (trifluoromethyl) -3 -Sullenic acid B-15 US Patent No. 6,034,256 (Example 165) 0 human f3 ch3 6-gas-1,2-dihydro-1 -methyl-2- (trifluoromethyl) -3-quinoline carboxyl Acid B-16 U.S. Patent No. 6,077,850 (Example 174) clrrY ^-, N-human CF3 HJ 6-chloro-2- (trifluoromethyl) -1,2 · dihydro [1,8] diazanaphthalene- 3-carboxylic acid B-17 US Patent No. 6,034,256 (Example 172) ((S) -6-chloro-1,2-dihydro-2_ (trifluoromethyl) _3_quinolinecarboxylic acid B-18 US Patent 6,077,850 US Patent No. 6,034,256 (2S) -6,8-dimethyl-2- (trifluoromethyl) -2H-chromene_3-carboxylic acid B-19 US Patent No. 6,077,850 US Patent No. 6,034,256 (c). YAYAYA ^ OH human cf3 (2S) -8-ethyl-6- (trifluoromethoxy) -2- (trifluoromethyl) -2H-trypene-3-carboxylic acid 96335.doc -32- 200528095 Compound No. Cangco Literature Structural Formula B-20 US Patent 6,077,850 US Patent 6,034,256 aiicCrH F (2S) -6-chloro-5,7 · dimethyl-2- (trifluoromethyl) -2H-chromene -3-carboxylic acid (V) such as The following specific compounds: 1-methylsulfonyl-4- [1,1-dimethyl-4- (4-fluorophenyl) cyclopent-2-2,4-dien-3-yl] benzene (disclosed at WO 95/30656, WO 95/30652, WO 96/38418 and WO 96/38442); and 2- (4-methoxyphenyl) -4-methyl-1- (4-sulfamidinophenyl) ) -La π (disclosed in European Patent Application Publication No. 7 9 9 8 2 3); or a pharmaceutically acceptable salt or prodrug thereof; (vi) the compounds listed in Table 2 include celecoxib (B-18), valdecoxib (B-19), deracoxib (B-20), rofecoxib (B-21), itolecoxib (etoricoxib) (MK-663; B-22), JTE-522 (B-23), or a pharmaceutically acceptable salt or prodrug thereof; Table 2: Examples of tricyclic COX-2 selective inhibitors Compound No. Reference Structure B-18 US Patent No. 5,466,823 cf3 96335.doc -33- 200528095

(celecoxib), rofecoxib and etoricoxib; (vii) Parecoxib with a structure shown in B-24 (disclosed in US Patent No. 5,932,598) ) Select inhibitor Dixi 96335.doc -34- 200528095 for tricyclic COX-2, a therapeutically effective prodrug of Valdecoxib (B-19) (disclosed in US Patent No. 5,633,272);

N! 〇 Β · 24 parecoxib is preferably in the form of sodium parecoxib; (viii) ABT-963, a compound of formula B-25, disclosed in International Publication WO 00/24719;

OH

B-2S 〇 ^ γΛ > Λ = / Λ ^ · ρ

F (ix) Phenylacetic acid derivative COX-2 selective inhibitor disclosed in US Patent 2003/0013739, represented by the formula of Formula VI:

Where: R27 is methyl, ethyl, or propyl; 96335.doc -35- 200528095 R28 is chlorine or fluorine; R29 is hydrogen, fluorine, or methyl; R is hydrogen, fluorine, gas, methyl, ethyl , Methoxy, ethoxy or mesogen; R31 is hydrogen, fluorine, or methyl; and R32 is gas, fluorine, trifluoromethyl, methyl, or ethyl, prerequisites are R28, R29, and ethyl The base and ruler 30 are not fluorine when they are fluorene; the phenylacetic acid derivative COX2 selection inhibitor used in the present invention is disclosed

No less than WO 99/1 1605, which has a cox189 (CAS RN 346670-74_4) design, corresponding to a compound of formula ¥ 1, where: R27 is ethyl; R28 and R3G are gas; R29 and R31 are hydrogen; and R32 is methyl; Another suitable phenylacetic acid derivative COX2 selection inhibitor suitable for the present invention is a compound disclosed in US Patent 2003/0013739, which has formula VI, wherein: R27 is propyl; R28 and R3G are R29 and R31 are fluorenyl groups; and R32 is ethyl; 96335.doc -36- 200528095 Another suitable phenylacetic acid derivative COX2 selective inhibitor for use in the present invention is disclosed in WO 02/20090 and is called COX-189 (Also known as lumiracoxib) 'CAS Registry No. 220991-20-8; Other compounds having structures similar to Formula VI' are also suitable for use in the present invention as COX2 selective inhibitors and are disclosed in US Patent Nos. 6,310,099, 6,291 Nos. 523 and 5,958,978; (X) Compounds having the structure of formula VIII, wherein J is a carbocyclic or heterocyclic ring. The preferred embodiment has the following structure:

Nimesunde (disclosed in US Patent No. 3,840,597 to J. Carter, Exp. Opin. Ther. Patents, 8 (1), 21-29 (1997))-where: x is 0; J is 1 -Phenyl; R33 is 2-NHS02CH3; R34 is 4-N02; no R35 group; Hosulide (disclosed in J. Carter, Exp_ Opin. Ther. Patents, 8 (1), 21-29 ( 1997))-where X is 0; J is 1-oxo-inden-5-yl; R33 is 2-F; R34 is 4-F; and R35 is 6-NHS02CH3; NS-398 (CAS RN 123653-1 1 -2; disclosed in U.S. Patent No. 96335.doc -37- 200528095 4,885,367 and J. Carter, Exp. Opin. Ther. Patents, 8 (1), 21-29 (1997), Yoshimi, N. et al. Japanese J. Cancer Res., 90 (4): 406-412 (1999); Falgueyret, J.-P. et al. 3 Science Spectra; Iwata, K. et al, Jpn. J. Pharmacol., 75 (2): 191 -194 (1997))-where X is 0; J is cyclohexyl; R33 is 2-NHS02CH3; R34 is 5-N02; and there is no R35 group; L-745337 (disclosed in U.S. Patent 5,604.0,260 No. and Kirchner et al., J. Pharmacol · Exp Ther 282, 1094-1101 (1997))-where X is S; J is 1-oxo-indene-5-yl; r33 is eight F; rW Is 4-F; and R35 is 6_N-S02CH3.Na +; 11 \\ ^ 63 5 5 6 (disclosed in U.S. Patent No. 5,783,597)-wherein X is S; J is thiobenzene_2-yl; ruler 33 is 44; No R34 group; and R35 is 5-NHS02CH3; L-784512 (disclosed in US Patent Nos. 6,002,343; 5,981,576; 6,140,515)-wherein X is 0; J is 2-oxo- 5 (R > methyl-5- (2,2,2-trifluoroethyl) arafuran- (5H) -3-yl; R33 is 3-F; R34 is 4-F; and R35 is 4- ( p-S02CH3) C6H4; 96335.doc -38-200528095 (xi) diarylmethylene furan derivatives, described in US Patent No. 6,180,651; specific derivatives belonging to this class of compounds can be used in the present invention as COX2 selective inhibitors include N- (2-cyclohexyloxynitrophenyl) methanesulfonamide, and (E) -4-[(4-methylphenyl) (tetrahydro-2-oxo- 3-furanyl) methyl] benzenesulfonamide; (xii)

Darbufelone (Pfizer, described in US Patent No. 5,143,928), CS-502 (Sankyo), LAS 34475 (Almirall Profesfarma), LAS 34555 (Almirall Profesfarma), S-335 16 (Servier), SD 8381 (Pharmacia, described in US Patent No. 6,034,258), BMS-347070 (Bristol Myers Squibb, described in US Patent No. 6,180,651), MK-966 (Merck, described in US Patent No. 5,968,974), L-783 003 (Merck), T-614 (Toyama, in German Patent Publication

3 8/3 42〇4), 0-13 67 (〇111 * 〇8 as 61 ^ 0), 1 > -748731 (Merck, described in U.S. Patent No. 5,968,974), CT3 (Atlantic Pharmaceutical, in U.S. Patent No. 533,783), CGP-28238 (Novrtis, mentioned in EP88282 and EP56956), BF-389 (Biofor / Scherer, U.S. Patent Nos. 4,892,870 and 5 12,3 3 0), GR_253035 (Glaxo Wellcome ), 2,6-dioxo-9H-purin-8-yl-cinnamic acid (Glaxo Wellcome), and 8-2474 (811 丨 〇11 (^ 丨, mentioned in European Patent Publication No. 595 546)); Credits for S-335 16 can be found in Current Drugs Headline News at http://www.current-drugs.com/ 96335.doc -39- 200528095 (xiii) (xiv) (xv) (xvi) (xvii) (xviii) (xix) (xx) (xxi) (xxii) (xxiii) (xxiv) NEWS / Inflaml.htm, 10/04/20001, the information report 8-33516 is tetrahydroissond (eg 1 ^) 1? (^ 0 丨 50 丨 11 (^) derivatives with IC50 values against COX1 and COX2 of 0.1 and o.ooi mM, respectively; multi-binding compounds containing 2 to 10 ligands covalently Connected to one or more linkers, as described in US Patent 6,395,724; Oleic acid, described in U.S. Patent No. 6,077,868; heterocyclic aromatic oxazole compounds, described in U.S. Patent Nos. 5,994,381 and 6,362,209; U.S. Patent Nos. 6,080,876 and 6,133,292 and pharmaceutically acceptable Derivatives; U.S. Patent Nos. 6,369,275, 6,127,545, 6,130,334, 6,204,387 '6,071,936, 6,001,843, and 6,040,450 and International Patent Publications WO 96/03392 and WO 96/24585; Diaryl; Benzofuran derivatives, disclosed in U.S. Patent No. 6,340,694; 1- (4-Aminosulfonylaryl) -3-substituted-5-aryl-2 ^ bizoline derivatives, U.S. Patent No. 6,376,519 References; Heterocycles described in US Patent No. 6,153,787; 2,3,5-Trisubstituted. Bipyridine, U.S. Patent No. 6,040,217; Diaryl bicyclic heterocyclic ring, U.S. Patent No. 6,329,421; 5-amino or substituted amine salts of 1,2,3-triazole compounds, U.S. Patent No. 6,239,137 mentioned; ° Ratio ° derivative, US Patent 6,13 6,83 1 mentioned; 96335.doc -40- 200528095 (XXV) substituted benzosulfonamide derivatives, US patent No. 6,297,282; (xxvi) 3-phenyl-4- (4- (methylsulfonyl) phenyl) -2- (5H) -furanone, described in US Patent No. 6,239,173; (xxvii) Bicyclic carbonyl indole compounds, described in US Patent No. 6,303,628; (xxviii) benzimidazole compounds, described in US Patent No. 6,310,079; (xxix) indole compounds, described in US Patent No. 6,30,363; (XXXviii) ) Arylphenylhydrazine, described in US Patent No. 6,077,869; (xxxi) 2-aryloxy, 4-arylfuran-2-one, described in US Patent No. 6,140,515; (xxxii) bisaryl compounds , U.S. Patent No. 5,994,379; (xxxiii) 1,5-diarylpyrazole, U.S. Patent No. 6,028,202; (xxxiv) 2-Substituted imidazole, U.S. Patent No. 6,040,320 Refers to; (xxxv) 1,3- and 2,3-diarylcycloalkanes and cyclodipyrazoles, described in US Patent No. 6,083,969; (xxxvi) Derived from the purpose of 1-bitalol and derived from Introducing g-pyramidamine of pyridoxanthinamine, described in US Patent No. 6,306,890; (xxxvii) pyridazinone compounds, described in US Patent No. 6,307,047; (xxxviii) benzosulfonamide derivatives, US Patent No. 6 No. 04,948; (乂 乂 乂 parent) US Pat. No. 6,169,188, 6,020,343, 5,981,576 ((methylsulfonyl) phenylfuranone); US patent No. 6,222,048 (diaryl-2 (5H) -furanone); US Patent No. 6,0 5 7,3 19 5 Tiger (3,4 · —square group_ 2 _ Jingji-2,5 -dirat sigma (Num), U.S. Patent No. 6,046,236 (carbosulfanilamide); U.S. Patent Nos. 96335.doc -41-200528095 6,002,014 and 5,945,53 9 (oxazole derivatives); and U.S. Patent No. 6,359,182 (C-Asian) (Nitro compound); (xl) 1,5-diphenyl-3-substituted pyrazole, described in International Patent Application Publication No. WO97 / 13755; (xli) radicicol and its Pharmaceutically acceptable derivatives are described in International Patent Application Publication No. Wo 96/25928 and Kwon et al., Cancer Res. (1992), 52, 6296); (xlii) Compound TP-72 and its pharmacologically acceptable Accepted derivatives are discussed in Cancer Res. (1998), 58, 4, 717-723; (xliii) indomethacin derivatives derived from indomethacin, as described in International Patent Application Publication No. WO 96/374679; (xliv ) ° ratio σ seat type, as described in International Patent Application Publication Nos. WO 95/1 53 16, WO 95/153 No. 15, and WO 96/03385; 95/00501 and WO 94/15932; (xlvi) oxazoles, according to International Patent Application Publications WO 95/00501 and WO 94/27980; (xlvii) isoxazoles, as described in International Patent Application Publication WO No. 96/25405; (xlviii) Flavor sigma, according to International Patent Application Publications WO 96/03388 and WO 96/03387; (xlix) Cyclopentene derivatives, as described in US Patent No. 5,344,991 and International Patent Application Announcement WO 95/00501; (I) Triphenyl compound, according to International Patent Application Publication WO 96/16934 96335.doc -42- 20052809 No. 5; (Ii) Thiazole derivatives, as described in International Patent Application Publication No. WO 96/03392; (Ιϋ) Benzopyranopyrazolyl compounds, disclosed in International Patent Application Publication No. WO 96/09304; (Iiii) Benzopyran derivatives according to International Patent Application Publication No. WO 98/47890; and (Iiv) Arylpyridazinone, disclosed in International Patent Application Publication No. WO 00/24719. Preferred COX2 selection inhibitor compounds are selected from the group consisting of celecoxib, parecoxib, deracoxib, valdecoxib, and etoricoxib. ), Meloxiczm, rofecoxib, lumiracoxib, RS 57067, T-614, BMS-347070, JTE-522, S-2474, SVT-2016, CT -3, ABT-963, SC-58125, nimesulide, flosulide, NS_398, L-745337, RWJ-63556, L-784512, darbufelone, CS- 502, LAS-34475, LAS-34555, S-335 16, SD-8381 and other compounds and prodrugs and mixtures thereof. The COX2 selection inhibitor is preferably selected from the group consisting of celecoxib, parecoxib, deracoxib, valdecoxib, lumiracoxib, A group of etoricoxib, rofecoxib, and prodrugs and mixtures thereof. 96335.doc -43-200528095 Particularly preferred is a COX2 selection inhibitor containing celecoxib. The COX2 inhibitor used in the present invention can be supplied from any source as long as it is a pharmaceutically acceptable COX2 inhibitor. Similarly, coX2 inhibitors useful in the present invention are also synthesized, for example, as described in Examples. Several COX2 inhibitors suitable for the compositions and methods of the present invention are available. For example, synthetic COX2 inhibitors described in U.S. Patent No. 5,466,823 issued to TaUey et al. Can also be isolated and purified from natural sources. For the purposes of the present invention, the quality and purity of cox2 inhibitors should be commercially customary for pharmaceutical products. Any combination of the above-mentioned Vla antagonists and COX2 inhibitors can be used in the novel compositions, pharmaceutical compositions and kits of the present invention. For example, a cox2 inhibitor, such as celecoxib, can be combined with any of the aforementioned Vla antagonists, including 8_chloro_5_methyl_ 1- (3,4,5,6 · tetrahydro-2 ^ 1- [1,2,] bipyridyl_4_yl) _5,6_dihydro_4'-2,3,5,10-tetraaza-benzo [e] camomile ring and ^ chloro- 丨 pyrimidinyl-hexa Hydrogen ° pyridine_4_yl) _5,6-dihydro-4H_2,3,10b-triazine-benzo [e] chamomile ring. According to the present invention, the compounds (A) and / or (B) derivatives include salts, solvates, complexes, polymorphs, prodrugs, stereoisomers of compounds (A) and / or (B) , Geometric isomers, tautomeric forms, and isotopic vanation. Preferably, the pharmaceutically acceptable derivatives of the compound (A) and / or amidine include salts, solvates, esters, and amidines of the compounds (A) and / or (B) as salts and solvates. More preferably, the pharmaceutically acceptable derivatives of the compounds (A) and / or (B) are salts and solvates. The compounds used in the present invention may exist in a form other than a solvate and a solvate formula, including a hydrate form. In general, solvate forms include hydrate forms, which can be isotopically substituted (eg D20, d6- 96335.doc -44-200528095 acetone, d6-DMSO), are equivalent to non-solvate forms, and It is included in the scope of the present invention. The pharmaceutically acceptable salts of the compounds used in the present invention include the acid addition salts and test salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, argon sulfate, side acid salts, camsylate, citrate, edisylate, ethionate, formic acid Salt, acid salt, fumarate, gluceptate, gluconate, gluconate, hexafluoroscale, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodate / Iodide, isethionate, D- and L-lactate, malate, maleate, malonate, methanesulfonate, methyl sulfate, naphthate, 2-naphthalenesulfonate Acid salt, nicotinic acid salt, nitrate, orotate, oxalate, palmitate, paraben, phosphate, dihydrogen phosphate, sugar salt, stearate, succinate Salts, sulfates, D- and L-tartrate, tosylate, and trifluoroacetate. Particularly suitable salts are besylate derivatives of the compounds of the invention. A suitable salt test is a test that produces non-toxic salts. Examples include Ironium salt, arginine salt, benzathine salt, # 5 salt, bile salt, diethylamine salt, diolamine salt, glycine salt, lysine acid Salts, magnesium salts, olamine salts, potassium salts, sodium salts, tromethamine salts, and 0. For an overview of suitable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Wiley -VCH, Weiheim, Germany (2002) o 96335.doc -45- 200528095 used in the medical invention of this combined invention ☆ /, the salt can be mixed with the compound and the required acid or test solution simply borrow the door Prepare early. Salt will precipitate from the solution and can be recovered by transition or evaporation to remove the solvent. The degree of ionization of the salt can range from fully ionized to almost non-ionized. The compounds used in the present invention may have-or multiple palm centers, and each center may exist in the R⑼ or S (L) configuration. The invention includes all enantiomers and epi-structures / formulas and suitable mixtures thereof. Separation of non-mirror stereoisomers or cis and trans isomers can be accomplished by conventional techniques, such as fractional crystallization, chromatographic analysis or HPLC. The private day η # 人 μ & _ The isomer mixture or its suitable salt or derivative is separated. Also included within the scope of the compounds of this invention are their polymorphs. In a further embodiment, a pharmaceutical composition is provided, which contains an effective amount of a mixture of a compound of formula (I) as defined above and a compound of formula (B) as previously defined, optionally with a 4 pharmaceutically acceptable loading. The agent is for preventive administration or when pain is prescribed. In the W pharmaceutical composition of the present invention, the content of tincture is 1 mg to 1000 mg per dose, and the content of (B) is 1 mg to 1000 mmol per dose. The doctor can determine the dose that is most suitable for an individual patient based on the patient's age, weight 'and response. Above, the dose is an average amount. Naturally, there may be individual cases where higher or lower agents are used, and this is also included in the scope of the invention. The pharmaceutical composition of the present invention may be administered alone, but is generally administered as a formulation with one or more pharmaceutically acceptable excipients. As used herein, "excipients" are used to describe any ingredient other than the compounds of the present invention. The choice of excipients depends largely on the particular mode of administration. 96335.doc -46- 200528095 The compounds used in the present invention may be Oral administration. Oral administration includes iris, which causes the compound to enter the gastrointestinal tract, or it can be administered bucally or sublingually, whereby the compound can enter the bloodstream directly from the oral cavity. Formulations suitable for oral administration include solid-state formulations Substances, such as tablets, capsules containing microparticles, liquid or powder, sugar-coated tablets (including those containing liquids), and chews, polysaccharides, gels, films (including mucosal patches), ovoid bodies (ovules), sprays, and liquid formulations. Liquid formulations include suspensions, solutions, syrups, and tinctures. Such formulations can be used as fillers in soft or hard capsules, and generally contain carriers such as water, ethanol, and propylene Secondary fermentation, methyl cellulose, or suitable oils, and one or more emulsifiers and / or suspending agents. Liquid formulations can also be prepared by solid reconstruction, such as reconstruction with sachet. The compounds used in the present invention are also useful. Yu Su Fast disintegrating dosage forms, as described in Expert Opinion in Therapeutic pat (11) (6), 981-986 (2001), edited by Liang and Chen. Typical ingots are prepared by standard methods known to chemists, For example by direct extrusion, granulation (dry, wet or thawing), thawing freezing or extrusion. Ingot formulations may include one or more layers and are coated or uncoated. Suitable for oral administration Examples of excipients include carriers such as cellulose, calcium carbonate, dibasic calcium phosphate, mannitol and sodium citrate, particulate binders such as polyvinylpyrrolidine, hydroxypropylcellulose (Hpc ), Hydroxypropyl methylcellulose (HPMC) and gelatin, disintegrating agents, such as sodium starch glycolate and silicate 'lubricants' such as sodium lauryl filler, preservatives, antioxidants, flavoring agents and color enhancers 96335.doc -47- 200528095

Orally administered solid formulations are formulated for immediate and / or modified release. Modified release formulations include delayed-, sustained-, intermittent_, dual-control, targeted and programmed release, suitable modified release techniques such as high-energy dispersion, osmotic pressure, and coating Coated particles can be found in Verma et U

Pharmaceutical Technology On-line, 25 (2), 1-14 (2001). Other modified release formulations are found in U.S. Patent No. 6,106,864. The compounds used in the present invention can also be administered directly to the bloodstream, intramuscularly, or internal organs. Suitable parenteral administration methods include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, and subcutaneous administration. Devices suitable for parenteral administration include needle (including microneedle) injectors' needleless syringes and infusion technology. Parenteral formulations are generally aqueous and may contain excipients such as salts, carbohydrates and buffers (preferably at pH 3 to 9), but can sometimes be suitably formulated into a sterile, non-aqueous solution or dry form Use with a suitable sterilization carrier such as sterilized non-pyrogenic hydration. Preparation of parenteral formulations under sterilization conditions, such as lyophilization, can be accomplished by standard medical techniques known to those skilled in the art. The solubility of the compounds used in the present invention when made into solutions for parenteral administration can be improved by suitable processing, such as by using high-energy spray-drying dispersion (see WO 01/47495) and / or by using suitable formulation techniques, such as by using solubility Enhancer. Parenteral formulations can be formulated for immediate and / or modified release. Modified released formulations include delayed-, sustained-, intermittent-, controlled dual-, targeted and programmed release. 96335.doc • 48-200528095 The compounds used in the present invention can also be administered topically to the skin or mucosa, either transdermally or transdermally. Typical formulations used for this purpose include gels, hydrogels, lotions, solutions, creams, creams, dusting powders, dressings, foams, films, skin barriers, and wafers , Implants, sponges, fibers, bandages, and microemulsions. Liposomes are also available. Typical carriers include alcohol, water, mineral oil, liquid paraffin, white paraffin, glycerol and propylene glycol. May contain penetration enhancers—see, for example, Plant Pharm. Sci. 5 88 (10), 955-958 ^ Finnin and Morgan ^ (1999 ^ October). Other means of local administration include iontophoresis, electroporation, phonophoresis, sonophresis, and needleless microneedle injection. Topical administration formulations can be formulated for immediate and / or modified release. Modified released formulations include delayed-, continuous_, intermittent_, dual-control, targeted and sequential (pr-) released. The compound used in the present invention can be formulated into a more solid form as A antagonist, which can be implanted in storage to provide long-term release of the active compound. / x The compound used in the invention can also be administered intranasally or by inhalation, in the form of 妒 浐 (alone or in a mixture, for example, with fine particles of ingredients mixed with lactose, such as with phospholipids) by dry powder Inhaled or as a spray from a pressurized container, pump, sprayer or atomizer (preferably: hydrodynamic atomization produces fine mist) or a sprayer with or without the use of a suitable dosing agent such as digas methane, give. Or sprayer containing active compound pressurized container, pump, sprayer, nebulizer solution or suspension of 96335.doc -49- 200528095, such as ethanol (aqueous ethanol solution if necessary) or another suitable agent for dispersion and dissolution , Or prolonged release of the active compound, as a solvent for the propellant and a surfactant as needed, such as sorbitol: oleate or oligolactic acid. Prior to use in dry or suspension formulations, the drug product is micronized to a size suitable for inhalation (typically less than 5 microns). This can be achieved by a suitable comminuting method, such as a spiral jet mill, a liquid bed jet mill, supercritical liquid processing to produce nanometer particles, or spray drying. The solution formulation for a nebulizer which generates fine mists electrohydrodynamically can contain from 1 microgram to 10 milligrams of a compound of the present invention per launch, and the volume discharged can be from 1 microliter to 1001 microliters. A typical formulation may contain the compound used in the present invention, propylene glycol, sterilized water, ethanol, and sodium chloride. Instead of propylene glycol, another solvent 'such as glycerin and polyethylene glycol may be used. Capsules' foaming agents, and cartridges (for example, made of gelatin or HPMC) for users of inhalers or insufflators, formulated to contain the compound of the present invention, a suitable bulk base such as lactose or powder, and performance modification Powders such as leucine, mannitol or stearic acid. Formulations for inhalation / intranasal administration can be formulated for immediate and / or modified release. Modified release formulations include delayed-, sustained-, intermittent-, controlled dual-targeted and programmed releases. The compounds for use in the present invention may be formulated as a suppository, vaginal suppository, or enemas for rectal, vaginal or intrauterine administration. Coconut oil is a conventional suppository base, but other bases can also be used. Formulations for transrectal / vaginal administration can be formulated for immediate and / or modified release 96335.doc -50- 200528095. Modified release formulations include delayed-, continuous-, intermittent-, controlled dual-targeted and sequential releases. The compounds for use in the present invention can also be administered directly to the eyes or ears, generally in the form of pharmaceuticals in micronized suspensions or solutions in isotonic, sterilized saline. Other formulations for ocular and ear administration include ointments, biodegradable (e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g. polysiloxane) implants, seals, lenses , And particulate or vesicular systems, such as niSomes or liposomes. Polymers such as cross-linked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, and cellulose polymers such as hydroxypropylmethyl cellulose, hydroxyethyl cellulose, or methyl cellulose, or heteropolysaccharide polymers can also be added. , Such as gelan gum, and at the same time add preservatives, such as benzacet gas. Such formulations can also be administered by iontophoresis. Formulations for ocular / andial administration can be formulated for immediate and / or modified release. Modified release formulations include deferred, continuous_, intermittent_, double-control, targeted and pr0grammed releases. The compound used in the present invention may be combined with a soluble A molecule such as cyclodextrin or a polyethylene glycol-containing polymer to improve its solubility, dissolution rate, deodorization 'bioavailability and / or stability. For example, it has been discovered that drug-cyclodextrin complexes are generally useful in most dosage forms and routes of administration. Inclusion complexes and non-inclusion complexes can be used. In addition to being directly compounded with drugs, cyclodextrin can also be used as a co-additive, that is, as a carrier, diluent, or co-solvent. Α-, β_, and r-cyclodextrin are used for the most purposes and deductions. Examples can be found in the International Patent Application Agency Bading Xi ~ Jiaming Announcement WO91 / 11172, WO 94/02518, and WO 98/55 148. 96335.doc 51 200528095 This agent is preferred. 'Preferably, the composition of the present invention can be administered by injection. When used in this way, it is administered orally, so that the agent is preferably administered topically to the pharmaceutical composition of the present invention, which may contain 0.01% to 95% of the compound of the present invention, 1% to 70%. , Is the amount of ⑷ and (B) will induce the organism to be found or: response. The daily dose of ㈧ and (B) used in this treatment is similar to that of the pharmaceutical composition described above. In the treatment method of the present invention, (A) and (B) can be given in fresh-dose form or separately, mainly at the same time = each is given in its own dosage form, but as the same treatment plan Part of 'is expected and (B) can be given at different times and in different ways. [Embodiment] Examples: Example 1: Preparation of COX-2 inhibitor celecoxib Step 1: 1- (4-methylphenyl) -4,4,4-trifluorobutane Preparation of ketone. According to the disclosure of US Patent No. 5,760,068, 4, _methylacetamidobenzene (5.26 g '39.2¾ mole) was dissolved in 25 ml of methanol under argon, and 12 ml of methanol was added ( 52.5 mmol) sodium methoxylate (25%). Stir the mixture for 5 to add 5 · 5 liters (46 · 2 mol) of difluoro ^ ethyl acetate. After refluxing for 24 hours, The mixture was cooled to room temperature and concentrated. 100 ml of 10% HC1 was added, and the mixture was extracted with 4 x 75 ml of ethyl acetate. The extract was dried over MgS04, filtered and concentrated to give 8.47 g ( 94%) color-sweeping oil body, directly used without purification. Step 2: 4- [5- (4-methylphenyl) -3- (trifluoromethyl) bisalan-1-yl] 96335.doc- 52- 200528095 Preparation of this s & amine Preparation of dione (414 g, 180 mmol) obtained in step 1 in 75 ml of pure ethanol, 4.26 g (19.0 mmol) of 4-sulfamethoxamine hydrochloride Phenylhydrazine. The reaction was refluxed under argon for 24 hours. After cooling to room temperature and filtering, the reaction mixture was concentrated to give 6.13 g of an orange solid. This solid was recrystallized from methane / hexane to give 3.11 g (8.2 mmol, 46%) of the product as a grayish yellow Solid, melting point 157 ... _159 C; calculated composition C17H14N302SF3; C, 53_54; H, 3.70; N, 11.02. This composition was measured and analyzed as: C, 5317; η, 3.81; N, 10.90. Example 2: Clinical The study investigates the synergistic effects in the myocardium and uterine arteries. Women with primary dysmenorrhea are treated as oral treatments in clinical trials with two individual components and combination therapy. In a randomized double-blind placebo-controlled study, Twelve women with primary dysmenorrhea studied the effects of cox inhibitors or Via antagonists (such as SR49059) alone or in combination. Experiments were performed on the first three days of the three opportunities, usually a continuous menstrual cycle. Utilize a 3-D Doppler speed meter or a 2-D color Doppler (measure powder 0〇1 for signal strength) or contrast-enhanced MRI to measure uterine arterial blood flow, and use a uterine pressure catheter implanted in the uterus to measure uterine smooth muscle contraction strength (Intrauterine pressure curve Area (AUC) measurement '3-D ultrasound or ischemic biomarkers. Study the uterine blood flow and uterine muscle contraction at various times before or after the administration of the disease drug. Also visible at 10 cm A similar scale (VAS) was used to record patients' lower abdominal pain from `` painless' 'to `` most painful' '. Example 3: Synergistic effect of rat uterine smooth muscle as angiotensin, receptor antagonist and cyclooxygenase inhibitor 96335.doc -53- 200528095 preparation The purpose of this study is to show the cyclooxygenase inhibitor (COXi) And vasopressin 1A (V1A) receptor antagonists, whether there is a synergistic effect of reducing uterine muscle high contraction activity. This novel combination treatment may be useful in the treatment of primary dysmenorrhea. Ideally, such experiments should be performed on uterine muscles obtained by a non-pregnant woman during hysterectomy. Not only because this is the target muscle, but also because this muscle expresses Via receptors [Kawamata, M., Mitsui-Saito, M., Kimura, T., Takayanagi, Y., Yanagisawa, T. & Nishimori, K. (2003 )]. In rats, vasopressin-induced uterine contractions are mainly promoted by oxytocin receptors, but not in human shellfish 1J, Eur J Pharmacol, 472, 229-34. However, such tissues are in short supply and cannot control the hormonal status of patients. Uterine tissue obtained from other species, including mice (Kawamata et al., 2003), has been characterized and has been shown to contain mainly oxytocin (OT) receptors, as opposed to humans, which are predominantly Via receptors. This can be problematic in confirming dysmenorrhea models in animals because the number of receptors expressed in the womb varies from animal to animal. But because both OT and Via receptors come from the same G-protein coupled receptor system and have the same pathway of intracellular signal system [Barberis, C ·, Morin, D ·, Durroux, T ·, Mouillac, B · , Guillon, G., Seyer, R., Hibert, M.T., Ribollet, E. & Manning, M. (1999a), Molecular Phamacology of AVP and OT receptors and therapeutic potential. Drug News & Perspectives, 12 279-292], OT receptors in mouse uterine muscle tissue are functionally the same as V! A receptors in human uterine muscle tissue. Therefore, when using mouse uterine muscle tissue to study whether these two components have a synergistic effect, it is necessary to use COXi and OT receptor antagonists. 96335.doc -54- 200528095 METHODS AND MATERIALS All animal studies are subject to local insights and conducted in accordance with UK Home Office regulations. Preparation of Rat Uterine Muscle Tissue-Rats (Sprague-Dawley's, 250_300 g) were pre-administered with estradiol (1 ml / kg 0.5 mg / ml corn oil solution, subcutaneously) 24 hours before selection to reduce estrus [Engstrom, T., Bratholm, P.? Christensenm NJ & Vilhardt, H. (1999), Up-regulation of oxytocin receptors in non-pregnant rat myometrium by isoproterenol: effects of steroids. J Endocrinol. 161, 403-11] 〇 Yi The rat was killed by impact and then severed from the cervix. A midline incision was made, and the uterus was removed, and the uterus was placed in standard Krebs buffer at room temperature, pH 7.4. Cut the two uterine horns in half and make a longitudinal incision. Place the tissue in 5 ml of organ baths, and connect it to force displacement isometric transducers under 1 g of resting tension. Continue to infiltrate the tissue with the modified Krebs buffer, Maintain at 37 ° C and let in 95% 02 and 5% C02 gas to equilibrate for 1 hour. Functional Mouse Synergy Study-Take initial baseline readings and construct a cumulative dose-response curve corresponding to OT (0.1-300 nM). Immerse the tissue in Krebs buffer until a stable baseline is generated. Take a second baseline reading and give a single dose corresponding to the OT receptor antagonist, L-368899 (3, 10, or 30 nM), or COXi, indomethacin (30, 100, or 300 μM) or vehicle (DMSO). A second cumulative dose-response curve corresponding to 0T (0.1-300 ηM) was reconstructed for all tissues. Tissues were washed continuously with Krebs buffer until 96335.doc -55- 200528095 was restored. Then take the third baseline and give L_368899 (3, i (^ 30m) and indomethacin (30, 100 or 300 μM) or vehicle (with time control). Repeat the third accumulation of OTOM nM_3GG capsule in all tissues Dose-response curve).

Functional human synergy studies. If human uterine muscles are used for research, the tissue should be prepared from the uterus of a non-pregnant woman. The tissue should be cut to a width of 10 m and a length of 10 mm and placed in a 5 ml organ bath as described above. This study should be carried out as described above, but with arginine vasopressin (AVP) instead of τ and vm antagonists such as SR49059 instead of OT antagonists. Studies should be performed with paired tissues incapable of obtaining repeated concentration response curves to AVP. When using a human uterine muscle tissue strip without endometrium, the basal prostate content must be stimulated externally. Data collection and analysis-ADAnet (pfizer h〇use pr0gramme) is used to collect raw data. This ADAnet can automatically collect recorded readings into an excellent worksheet. The ADAnet reading is the i-response measurement; it is the area under the curve measured at 3 minutes when the baseline was set at 0

(AUC). The crude starting response measurement of the cumulative dose response is then transformed in two steps: 1) Subtract the baseline reading from all subsequent readings. 2) Data are expressed as a percentage corresponding to the maximum contractile response caused by OT. Labstats (Excel) dose-response curve matching function was used to fit the s-shaped curve. This s-shaped curve was limited by 0 to 100% and the measured ec25 value of each curve. Calculate the out-of-synergy index to determine if the indomethacin or ^ 368899 combination has a synergy 'or just add or have 25% of the effect antagonism (producing 25% anti-96335.doc -56- 200528095 should correspond to οτ stimulus Agent dose). Materials-The drugs and chemicals used and their sources are: oxytocin acetate (Sigma); indomethacin (Sigma); Krebs buffer (Sigma); dimethylsulfite (Fisher Scientfic); L-368899 [Pettibone, DJ ·, Clineschmidt, BV, Guidotti, EV, Lis, EV ·, Reiss, DR ·, Woyden, CJ ·, Bock, MG Evans, BE ·, Freidinger, RM · & Hobbs, DW (1993), L-368899 , a Potent Orally Active Oxytocin Antagonist for Potential Use in Preterm Labor. Drug Development Research, 30, 129-142] was synthesized by Pfizer Global Research and Development, Sandwich, dissolved in 100% DMSO and diluted to 10% with DMSO. All other drugs were dissolved in and diluted with distilled water. Drug concentration is the final bath concentration. Results Effect of L-3 68899 alone and indomethacin on rat uterine contraction. Uterine strips obtained from estrone-treated rats were constructed with or without OT receptor antagonist (L-368899) or COXi (indomethacin). Cumulative dose response curve corresponding to OT. Increasing the concentration of OT receptor antagonist L-368899 caused a dose-dependent right shift on the OT dose-response curve, and there was no maximum contraction corresponding to the OT response (Figure 1A and Table 1). COXi, indomethacin, also caused a dose-dependent right shift in the dose-response curve corresponding to OT and a decrease in dependence on the maximum contractile response corresponding to OT (Figure 1B and Table 2). Table 1: EC50 values of OT in rat uterine smooth muscle in the absence or presence of L-368899 96335.doc -57- 200528095 curve OTEC50's soil sem (nM) η curve 1-3668899 EC50's soil sem (nM) η curve indomethacin ec50丨 s sem (nM) η 1 4.62 ± 0.48 5 1 4.62 ± 0.48 5 1 4.62 ± 0.48 5 2 3.27 ± 0.33 5 2 8.15 ± 0.80 5 2 7.25 ± 0.76 5 3 3.81 ± 0.46 4 3 13.0 ± 1 · 34 5 3 16.8 ± 4.52 4 4 18.5 ± 1.81 5 4 10.5 ± 1.23 4 Effect of L-368899 and indomethacin combination on rat uterine contraction All L-368899 (3, 10, 30 nM) and indomethacin (30, 100, 3 (00 μΜ) to evaluate whether the two drugs have a synergistic effect. For example, the synergistic effect of 30 μM indomethacin and 3 nML-368899 is shown in FIG. 2. At individual administrations, 30 μM indomethacin and 3 nM L-368899 produced EC25 corresponding to 0 butan of 1.11 nM and 1.2 nM, respectively. When given in combination, its qt ec25 reached 1.89 nM, which means that the combination of ττ contraction response inhibition Mingtou was greater than that of indomethacin or L-368899 alone (Figure 2). A more detailed description of the systematic study of all combinations .... All combinations produced a dose-dependent inhibition of OT-induced contractile responses (Figure 3). Synergy studies were used with indomethacin (30, 100, 300 μM). And L-3 68899 (3, 10, 30 nM) in the presence (Table 2) respectively or in the presence of a combination of OT agonist produces a 25% response (EC25) dose to calculate the synergy index. The 25% response was chosen (rather than 500 / 〇) because some indomethacin and the combination dose observed a decrease in the maximum OT contractile response 0 96335.doc -58- 200528095 indomethacin (Um) ec25p L-368899 (ηΜ) ec25p 30 1.11E- 09 3 1.20E- 09 100 3.67E- 09 10 1.96E- 09 300 9.89E- 09 30 2.35E- 09 Table 2: In the presence of L-368899 (3, 10, 30 nM) or anti-inflammatory The EC25 value corresponding to the OT dose response curve in the presence of pain (30, 100, 300 μM). For the study of indomethacin and L-368899 combination, at 25 ° / of 4 combinations. Synergistic effects were observed in the effect amounts (Table 4). L-368899 dose synergy index at 35% of 25% effective amount 3nM 10ηM 30 ηM 30 μM Synergy Synergy Fail to measure indomethacin dose 100 μM Synergy Synergy Fail to measure 300 μM Fail to measure Fail to measure Fail to measure Note : The synergistic effect can only be quantified when the highest dose of one or two doses given alone achieves a 25% contractile response to oxytocin. The inhibition of the maximum response of some combinations is so large that it cannot be measured. The synergy index of the combination of COXi (indomethacin) is used to determine whether the dose combination has a synergistic, additive or antagonistic effect on the administered effect. ί < 1, Synergy 'Synergy Index = —I— Addition> AB ^ > 1, Antagonistic ic 96335.doc -59- 200528095 where A and B are drugs A (individual) and B (individual) that produce specific effects Dose and (a, b) are the combined doses that produce this effect. All analyses in this study used 25% of the maximum OT agonistic response. Example of synergy using a 30 μM indomethacin / 3 nM L-3 68899 dose combination: For three different each indomethacin doses (30, 100, 300 μM) and L-368899 (3, 10, 30 ηM) The ECU response alone is plotted with a horizontal line of 189 nM EC25 response of the combination of 30 μM indomethacin / 3 nM L_368899 (Figure 4). The dose-response curve interpolation method (interp〇iati〇n) can be used in Figure 4 It can be seen that 9nM L-368899 alone and 50 μM indomethacin alone can achieve a response of 1-89E-09EC25. Substitute this equivalent into the lower synergy index equation. Synergy refers to deafness f = L-dose 30 3 in the indomethacin dose in the combination, 7 in L, indomethacin dose, m = o · 9 Synergy <1; Addition = 1; Antagonistic effect is 25% effective The dose-time-dose combination (30 indomethacin, 3 nM L 368899) is synergistic (because the synergy index < 1). These calculations can also be used for other indomethacin in combination with L_368899 at a 25% first-use index (Table 5). The effective dose of L-368899 at a 25% monthly dose index is 3nM 10nM 30 nM 30 μM 0.9 < 0.6 < 1.2 Indomethacin dose 100 uM < 0.9 < 0.7 < 1.4 ~~ ----- μΜ < 1 · 1 < 1 · 3 < 2.0 96335.doc 200528095 Note · Some synergy indexes can only be quantified as "less than "Because the combination cannot be achieved when given alone at one or two of the highest doses. Table 5. Synergy index of indomethacin combined with L-368899 at 25% effect. Conclusion From Table 4, it can be seen that 30 μM or 100 μM indomethacin combined with 3 nM or 10 nM L-368899 at 25% effect amount produces synergy. effect. From the data obtained from dentition animals, uterine relaxation effects of cox inhibitors and V1 a receptor antagonists can also be observed in women, because v i a receptors rather than qt receptors cause uterine contractions. This pre-clinical synergy will translate into more beneficial clinical effects in the treatment of uterine muscles with excessive contractions such as dysmenorrhea. [Brief description of the figure] Figures 1A and 1B show the presence or absence of mouse uterine smooth muscle a: L-368899 (3, 10, 30 nM), B: indomethacin (30, 100, 300 μM) corresponding to Ττ dose-response curve. Figure 2 shows the EC25 response of L_368899 alone (30 μM) and indomethacin alone (3 nM) and the dose combination (30 μM indomethacin and 3 nM L-368899). Figures 3A, 3B, and 3C show the presence or absence of a rat's uterine smooth muscle: a: 3 nM L-3 68899 and Gado's indomethacin (30, 100, 300 μM) combination, B: 10 nM L-368899 and G The multiple indomethacin (30, 100, 300 μM) combination corresponds to a cumulative dose response of OT. Figure 4 shows the EC25 response of each individual dose of L-368899 (dotted dots), indomethacin alone (square dots), and combination (30 μM indomethacin and 3 nM L-368899), plotted on a horizontal line. 96335.doc -61-

Claims (1)

200528095 10. Scope of patent application: 1. Combination of (A) vasopressin receptor antagonist or its pharmaceutically acceptable derivative with (B) COX inhibitor or its pharmaceutically acceptable derivative It is used as a medicine for treating or preventing dysmenorrhea. 2. A use for the treatment or prevention of dysmenorrhea according to a combination as defined in claim 1. 3. · On request! The combination of the defined and (B) is used to make a medicine for treating or preventing dysmenorrhea by administering (A) and (B) simultaneously, sequentially or separately. 4. The use according to any one of claims 1 to 3, wherein (B) is a cox2 inhibitor. 5. The use according to any one of claims 1 to 3, wherein (B) is a COX2 selective inhibitor. 6. The use according to claim 5, whose COX2 inhibitor is selected from the group consisting of: celecoxib, parecoxib, deracoxib 'valdecoxib, Lu Miliracoxib, etoricoxib, rofecoxib, or a pharmaceutically acceptable salt or solvate thereof. . 7. The use according to any one of claims 1 to 3, wherein (A) is a Via receptor antagonist. 8. Use according to any one of claims 1 to 3, wherein (A) is selected from the group consisting of: SR49049 (Relcovaptan), atosiban (Tractocile®), and conivaptan (YM- 087); OPC21268, 8-Ga-5-methyl-l- (3,4,5,6-tetrahydro-2H- [1,2,] dihydrocarbyl-4-yl) -5,6- Dihydro-4H- 96335.doc 200528095 2,3,5,1〇b_tetraaza-benzyl 0] glucan and 8-chloropyrimidine_2_yl_ hexahydropyridyl-4-yl) Coincidentally, 6_dichloro.2,3, full triazine_benzidine and bad menstruation or a pharmaceutically acceptable salt or solvate thereof. 9. 10. 11. 12. 13 .: According to any one of the items 1 to 3, _Dysmenorrhea is primary pain = According to the use of any one of the items 1 to 3, where dysmenorrhea is Secondary soreness ::: Uses' Among them, secondary dysmenorrhea is an increase in uterine tonicity, a fibroid or an intrauterine contraceptive device. Product Γ pack 3 μ Seek any one of items 1 to 3-(1) and (B) of the medical products: as a combined preparation for simultaneous, separate or sequential use: two and to prevent dysmenorrhea. σ 一种 A pharmaceutical composition, 1 person 4 items / 3 effective summer according to any one of the claims 1 to 3-a mixture of (A) and (B), as the meaning of the vehicle, for preventive administration or Give at the beginning of pain. ’, Can access the party 96335.doc