WO2005039565A1 - Combinations of cox and vasopressin inhibitors for the treatment of dismenorrhea - 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 the treatment or prophylaxis of dysmenorrhoea.

Description

PC26183 1

COMBINATIONS OF COX AND VASOPRESSIN INHIBITORS FOR THE TREATMENT OF DISMENORRHE

This invention relates to a synergistic combination of antagonists of the vasopressin receptor family with cyclooxygenase inhibitors, the use of such combinations in the treatment of dysmenorrhoea, methods of treating dysmenorrhoea using such combinations and medicaments containing such combinations.

There is a high unmet need in the area of menstrual disorders and it is estimated that up to 90% of all menstruating women are affected to some degree. Up to 42% of women miss work or other activities due to menstrual pain and it has been estimated that around 600 million work hours a year are lost in the US as a result {Coco, A.S. (1999). Primary dysmenorrhoea. [Review] [30 refs]. American Family Physician, 60, 489-96.}. Dysmenorrhoea can be divided into two classes, primary and secondary. Primary dysmenorrhoea is generally defined as cramping pain in the lower abdomen occurring at the onset of menstruation, in the absence of any identifiable pelvic disease. This affects approximately 50% of the female population {Coco, A.S. (1999). Primary dysmenorrhoea. [Review] [30 refs]. American Family Physician, 60, 489-96.; Schroeder, B. & Sanfilippo, J.S. (1999). Dysmenorrhoea and pelvic pain in adolescents. [Review] [78 refs]. Pediatric Clinics of North America, 46, 555- 71}.

Secondary dysmenorrhoea is described as painful menstruation associated with specific pathological conditions such as endometriosis, pelvic inflammatory disease, fibroids, intra-uterine contraceptive devices etc. The pathogenesis of dysmenorrhoea is unknown, although there appears to be a close association between myometrial hyperactivity and reduced uterine blood flow with the pain felt by these women. Secondary dysmenorrhoea is diagnosed in only approximately 25% of women suffering from dysmenorrhoea. Dysmenorrhoea can occur in conjunction with menorrhagia. PC26183 2

In healthy women uterine contractility varies during the menstrual cycle {Akerlund, M. (1997), Contractility in the non-pregnant uterus. [Review] Annals of the New York Academy of Sciences, 828, 213-22.}. The changes do not follow the fluctuations in plasma concentrations of ovarian hormones, but may be related to tissue levels because there are time lags between the two. During the first few days of the menstrual cycle uterine contractility is coordinated throughout the whole uterus, with contractions that are regular and of comparatively high amplitude, with well-demarked relaxations between the contractions. During the follicular phase, in particular around the time of ovulation a more uncoordinated uterine contractility occurs, with contractions that are of relatively high frequency, low amplitude and high basal tone. This continues through the luteal phase until 2-3 days before the onset of menstruation when the uterine activity becomes more coordinated again. At this time the cyclic propagation of contractions occurs, both in the direction of the cervix and towards the fundus. The direction can change for a patient within a few minutes. Propagation towards the cervix may be important for the expulsion of endometrium and blood at menstruation.

In comparison, women with dysmenorrhoea have pronounced uterine hyperactivity. Their contractile patterns are irregular. Also uterine blood flow is reduced and is mainly ischaemic in nature {Akerlund, M. (1997), Contractility in the non-pregnant uterus. [Review] Annals of the New York Academy of Sciences, 828, 213-22.}. The reduction in blood flow is probably an effect of both: •Compression of vessels caused by increased uterine pressure - it is believed this may be associated with the colicky pain experienced by these women ^»An influence of vasoactive agents on the smooth muscle of arterial walls causing longer lasting reduction in blood flow - this may be cause of the continuous aching pain experienced by these women.

Of the currently available treatments for dysmenorrhoea, non-steroidal anti- inflammatory drugs (NSAID's) tend to be the first line choice unless birth control is also desired, in which case oral contraceptives are used. PC26183 3

Primary dysmenorrhoea has been associated with increased endometrial prostaglandin F_2α (PGF_2α) at the time of menstruation {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, 185-.}, but not perimenstruation {Lundstrom, V. & Green, K. (1978). Endogenous levels of prostaglandin F2alpha and its main metabolites in plasma and endometrium of normal and dysmenorrheic women. American Journal of Obstetrics & Gynecology, 130, 640-6.}. PGF_2α is known to increase uterine contractility and cause dysmenorrhoeic like pain {Roth-Brandel, U., Bygdeman, M. & Wiqvist, N. (1970). Effect of intravenous administration of prostaglandin E1 , and F2 on the contractility of the non-pregnant human uterus in vivo. Ada Obstetricia et Gynecologica Scandinavica - Supplement, 5, 19-25.}. Prostaglandins are also known to have direct pain-producing properties by sensitizing pain receptors, which may also be involved in the pain felt at the time of menstruation {Ferreira, S. (1976). Pain and Fever. In Prostaglandin and Thromboxanes: NATO advanced study institute on advances on prostaglandins. pp. 433-442. New York: Plenum Press.}. NSAID's have been shown in clinical trials to alleviate pain and restore uterine motility in some dysmenorrhoeic patients {Pulkkinen, M.O. & Csapo, A.I. (1978). The effect of ibuprofen on the intrauterine pressure and menstrual pain of dysmenorrheic patients. Prostaglandins, 15, 1055-62.}. However, they are not effective in all dysmenorrhoeic sufferers, in particular those with severe dysmenorrhoea. Furthermore, they are associated with side effects including upper gastrointestinal tract symptoms, drowsiness and tinnitus. These agents do have the advantage over oral contraceptives of only being administered for 2-3 days per month and they reduce some of the side effects associated with dysmenorrhoea (dizziness, nausea and vomiting).

U.S. Patent No. 5,466,823 discloses pyrazolyl cyclooxygenase-2 inhibitors useful in treating inflammation and inflammation related disorders, including menstrual cramps. U.S. Patent No. 5,932,598 discloses prodrugs of cyclooxygenase-2 inhibitors useful in treating inflammation and inflammation related disorders. Morrison et al. describe a study where the cyclooxygenase-2 inhibitor, rofecoxib, is PC26183 4

used to treat primary dysmenorrhoea (Onstet. Gynecol., 94(4), 504-508 (1999)). Compounds that selectively inhibit cyclooxygenase-2 and are useful in treating menstrual cramps have also been described in U.S. Patent No. 5,521 ,207 and U.S. Patent No. 5,633,272. The various classes of compounds that are selective inhibitors of cyclooxygenase-2 have been reviewed by J. Talley in Prog. Med. Chem., 36, 201-234 (1999). Compounds that selectively inhibit cyclooxygenase-2 have also been described in the following individual publications: U.S. Patent No.'s 5,380,738, 5,344,991 , 5,393,790, 5,434,178, 5,474,995 and 5,510,368; International Publication No.'s 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.

The combination of NSAIDs and oral contraceptives has been used in cases where neither treatment alone was effective in treating primary dysmenorrhoea (Coco, A., American Family Physician, 60(2), 489-496 (1999)).

U.S. Patent No. 5,811 ,416 discloses the combination of an endothelin antagonist and/or an endothelin synthase inhibitor with at least one of a progestin, an estrogen, a combination of a progestin and estrogen, a cyclooxygenase inhibitor, a nitric oxide donor or a nitric oxide substrate for the treatment of menstrual disorders including dysmenorrhoea. U.S. Patent No. 5,912,006 discloses the combination of an omega fatty acid and a cyclooxygenase inhibitor for the reduction or alleviation of uterine or vaginal pain associated with the onset of menstruation. WO 02/062391 discloses a combination therapy method for the treatment and prevention of dysmenorrhoea comprising a COX-2 inhibitor and sex steroids.

Oral contraceptives are a second line therapy for most women unless birth control is also desired. They have to be taken continuously throughout the cycle and it may take up to 3 cycles for menstrual pain to noticeably diminish. In comparison to NSAID's, oral contraceptives prevent menstrual pain by reducing menstrual fluid volume {Nakano, R. & Takemura, H. (1971 ). Treatment of functional dysmenorrhoea; a double-blind study. Ada Obstetrica et Gynaecologica Japonica, PC26183 5

18, 41-4.}, suppressing ovulation and decreasing endometrial volume. Thus resulting in a decrease in prostaglandin production {Chan, W.Y. & Hill, J.C. (1978). Determination of menstrual prostaglandin levels in non-dysmenorrheic and dysmenorrheic subjects. Prostaglandins, 15, 365-75.}.

However, it is a recognized problem that there is a persistent failure rate with NSAID's and oral contraceptives (10-15%), particularly in patients with severe dysmenorrhoea {Coco, A.S. (1999). Primary dysmenorrhoea. [Review]. American Family Physician, 60, 489-96; Schroeder, B. & Sanfilippo, J.S. (1999). Dysmenorrhoea and pelvic pain in adolescents. [Review]. Pediatric Clinics of North America, 46, 555-71.}. Newer, less well-characterized treatments are now being investigated, but they are not yet available as a current therapy option.

The physiological response of the uterus to vasopressin changes throughout the menstrual cycle, with a maximal sensitivity observed pre-menstrually {Bossmar, T., Akerlund, M., Szamatowicz, J., Laudanski, T., Fantoni, G. & Maggi, M. (1995). Receptor-mediated uterine effects of vasopressin and oxytocin in non-pregnant women. British Journal of Obstetrics & Gynaecology, 102, 907-12.}. Plasma vasopressin levels do not alter significantly during the menstrual cycle of control patients {Forsling, M.L., Akerlund, M. & Stromberg, P. (1981 ). Variations in plasma concentrations of vasopressin during the menstrual cycle. Journal of Endocrinology, 89, 263-6.}. However, vasopressin levels are raised in dysmenorrhoeic women both pre- and during menstruation {Hauksson, A., Akerlund, M., Forsling, M.L. & Kindahl, H. (1987). Plasma concentrations of vasopressin and a prostaglandin F2 alpha metabolite in women with primary dysmenorrhoea before and during treatment with a combined oral contraceptive. Journal of Endocrinology, 115, 355-61.}. This occurs in association with increased pain, myometrial hyperactivity and reduced 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 Journal of Obstetrics & Gynaecology, 99, 680-4.}. The exact details of the contribution of PC26183 6

vasopressin in the mechanisms of menstruation and dysmenorrhoea are still unknown. However, the peptide has pronounced constrictor effects on smooth muscle activity of both myometrium {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 arteries {Kostrzewska, 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. Ada Obstetricia et Gynecologica Scandinavica, 77, 3-7,} via the vasopressin Vι._A receptor, which is different from the V₂ type regulating kidney function.

There is clinical evidence that an orally active VIA antagonist has a significant therapeutic benefit in the prevention of dysmenorrhoea {Akerlund, M. (1987). Can primary dysmenorrhoea be alleviated by a vasopressin antagonist? Results of a pilot study. Ada Obstetricia et Gynecologica Scandinavica, 66, 459-61.}. In a double blind, randomised placebo controlled cross over trial, Relcovaptan (SR49059) dose dependently reduced the intensity of pain in women with dysmenorrhoea, without effecting the mechanisms regulating 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 dysmenorrhoea. BJOG: an International Journal of Obstetrics & Gynaecology, 107, 614-9.}. This compound also displays activity at the Oxytocin receptor. The peptide vasopressin V_1A antagonist/Oxytocin receptor antagonist 1 - deamino-2-D-Tyr (Oet)-4-Thr-8-Om-oxytocin, when given intravenously is also effective in the treatment of dysmenorrhoea {Akerlund, M. (1987). Can primary dysmenorrhoea be alleviated by a vasopressin antagonist? Results of a pilot study. Ada Obstetricia et Gynecologica Scandinavica, 66, 459-61.}.

To address the continuing need to find safe and effective agents for the prophylaxis and treatment of dysmenorrhoea, combination therapies of therapeutic agents are herein described. Surprisingly, it has been found that PC26183 7

combination therapy with an antagonist of the vasopressin receptor family and a COX inhibitor results in unexpected and synergistic improvement in the treatment of dysmenorrhoea. When administered simultaneously, sequentially or separately, the vasopressin antagonist and COX inhibitor may have the advantage that, due to a synergistic interaction between the active ingredients, they are more potent, have a longer duration of action, more effectively reduce disease progression and, therefore, the requirement for surgical intervention, have a broader range of activity, are more stable, have fewer side effects or are more selective (in particular they may have beneficial effects in dysmenorrhoea) or have other more useful properties than the compounds and combinations of the prior art.

The combination of the present invention not only provides a treatment of myometrial hypercontractility, uterine arterial vasoconstriction and subsequent pain, but also provides a treatment to reduce the basal tone of myometrium and uterine arteries, allowing them to remain in a more relaxed state. Although the dysmenorrhoea is cyclical, if the myometrium and uterine arteries maintain a more relaxed state each month then, in the long term, we hypothesis that this could lead to a reduction in the treatment required for future symptom relief.

Thus, in accordance with the invention, there is provided 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, in the manufacture of a medicament for the treatment or prophylaxis of dysmenorrhoea.

Preferably (B) is a COX-2 inhibitor. More preferably (B) is a COX-2 selective inhibitor.

Further there is provided a use of a combination of (A) and (B) as defined above for the treatment or prophylaxis of dysmenorrhoea. PC26183 8

Still further, there is provided a use of a combination of (A) and (B) as defined above for the manufacture of a medicament for combination therapy by simultaneous, sequential or separate administration of (A) and (B) in the treatment or prophylaxis of dysmenorrhoea.

Alternatively, there is provided a method for the treatment or prophylaxis of dysmenorrhoea comprising administering to a subject in need of such treatment amounts of (A) and (B) as defined above, which are together effective.

Further, there is provided a therapeutic composition comprising (A) and (B) as defined above for use in the treatment or prophylaxis of dysmenorrhoea. Still further, there is provided a pharmaceutical product containing (A) and (B) as defined above, and a pharmaceutically acceptable carrier, as a combined preparation for simultaneous, separate or sequential use in the treatment or prophylaxis of dysmenorrhoea.

The invention involves the preventive management of painful uterine cramps, dysmenorrhoea, in women. The treatment may be given at the on-set of pain, before pain and after pain. A key improvement over existing technologies is that moderate to severe pain is not experienced prior to initiating treatment, but that it can be pre-empted, providing a much more satisfactory outcome. Another advantage is that by employing this regimen, lower doses of analgesic medication may be required.

The administration of a combination of a vasopressin receptor family antagonist and a COX inhibitor for the treatment or prophylaxis of dysmenorrhoea is superior to the use of either compound alone. It is believed that the effects provided by the combination of a vasopressin receptor family antagonist in combination with a COX inhibitor in the treatment or prophylaxis of dysmenorrhoea are superior to the effects that would be expected of the combination on the basis of the effects provided by use of the vasopressin receptor family antagonist or the COX inhibitor separately. PC26183 9

Combination therapies comprising vasopressin receptor family antagonists and COX inhibitors are useful not only for improving the symptoms of dysmenorrhoea, but also for reducing the dosages of the compounds administered. The administration of lower dosages provides a reduction in side effects that may be associated with the compounds.

The dysmenorrhoea may be primary or secondary dysmenorrhoea. The secondary dysmenorrhoea may be a consequence of increased uterine tone, such as uterine fibroids or intra-uterine contraceptive devices.

As used herein, the terms "treating" or "to treat", mean to alleviate symptoms, eliminate the causation either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms. The term "treatment" includes alleviation, elimination of causation of, or prevention of symptoms and disorders associated with dysmenorrhoea.

The vasopressin receptor family comprises Via, V1 b, V2 and Oxytocin receptors {Thibonnier M., Exp. Opin. Invest. Drugs (1998) 7(5), 729-740}. The vasopressin receptor antagonist for use with the invention is preferably selective for the Via receptor and the closely related oxytocin receptor. Activity at the oxytocin receptor may be beneficial. More preferably, the vasopressin receptor antagonist for use with the invention is selective for the V1 a receptor.

Examples of vasopressin receptor family antagonists, suitable for use in the present invention are disclosed in US 6,090,818; EP0873309; WO 98/25901 ; WO 02/083685; JP 2000-63363; and WO 02/32864.

Examples of Via receptor antagonists for use with the invention are: SR49049 (Relcovaptan), atosiban (Tractocile®), conivaptan (YM-087) and OPC21268. Additionally, the Via receptor antagonists described in WO 01/58880 are suitable for use in the invention. PC26183 10

Further examples of Via antagonists for use with the invention are disclosed in WO 2004/037809, i.e. compounds of formula (I),

or pharmaceutically acceptable salts or solvates thereof, wherein R¹ represents C C₆ alkyl, -(CH₂)c-[C₃-C8 cycloalkyl]-, -(CH₂)_C-W or -(CH₂)_C-Z-

(CH₂)_d-W; W represents C C₆ alkyl, Cι-C₆ alkyloxy, -C0₂[CrC₆ alkyl], -CONR⁴R⁵, a phenyl group, NR⁴R⁵, het² or het³, the phenyl group being optionally substituted with one or more groups independently selected from halogen, CF₃, OCF₃, R³, OR³, C0₂R³, CONR⁴R⁵, CN, S0₂NR⁴R⁵ and NR³S0₂Me; Z represents O or S(0)_g; g represents 0, 1 or 2; R² represents a phenyl group, optionally fused to a 5- or 6- membered aryl or heterocyclic group which may contain one or more heteroatoms selected from N, O or S; the phenyl group and the optionally fused group being optionally substituted with one or more groups independently selected from the list defined below; Ring A represents a 4-, 5- or 6- membered saturated heterocyclic group containing at least one N; Ring B represents a phenyl group or het¹, each group being optionally substituted with one or more groups independently selected from the list defined below; R⁷ independently represents H, C C₆ alkyl, OR³, -(CH₂)_e- ³ or -(CH₂ -0- (CH₂)e-R³; at each occurrence R³ independently represents H, C-i-Cβ alkyl optionally substituted by Y, -(CH₂)_g-[C₃-C₈ cycloalkyl], phenyl, benzyl, pyridyl or pyrimidyl; at each occurrence R⁴ and R⁵ independently represent H, C-i-Cβ alkyl (optionally substituted with C C₆ alkyloxy), (CH₂)gC0₂-[Cι-C6 alkyl], -S0₂Me, - PC26183 11

(CH₂)g-[C3-C₈ cycloalkyl], S0₂Me, phenyl, benzyl, pyridyl or pyrimidyl; or R⁴ and R⁵ together with the N atom to which they are attached represent a heterocyclic group of from 3 to 8 atoms; Y independently represents a phenyl group, NR⁴R⁵ or het⁴, the phenyl group being optionally substituted with one or more groups independently selected from halogen, CF_3l OCF₃, R⁴, OR⁴, C0₂R⁴, CONR⁴R⁵, CN, S0₂NR⁴R⁵, NR⁴S0₂Me and -NR⁴R⁵; het¹ represents a 4-, 5- or 6- membered saturated, or unsaturated, heterocyclic group containing at least one N (but which may also contain one or more O or S atoms); het² represents a 4-, 5-, 6- or 7- membered saturated, or unsaturated, heterocyclic group containing at least one N (but which may also contain one or more O or S atoms), optionally substituted with one or more groups independently selected from the list defined below; het³ represents a 4-, 5-, 6- or 7- membered saturated, or unsaturated, heterocyclic group containing at least one O (but which may also contain one or more N or S atoms), optionally substituted with one or more groups independently selected from the list defined below; het⁴ represents a 4-, 5-, 6- or 7- membered saturated or unsaturated heterocyclic group containing at least one N (but which may also contain one or more O or S atoms), optionally substituted with one or more groups independently selected from the list defined below; substituents for R², Ring B, het¹, het², het³ and het⁴ are independently selected from the following list: halogen, CF₃, OCF₃, 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; at each occurrence R⁶ independently represents H, Cι-C₆ alkyl optionally substituted by Y, -(CH₂)_g-[C3-C₈ cycloalkyl], phenyl, benzyl, pyridyl or pyrimidyl; a and b independently represent 0 or 1 ; c, d, e and g independently represent 0, 1 , 2, 3 or 4; f independently represents 1 , 2, 3 or 4; provided that: PC26183 12

(i) a + b cannot equal 0; and (ii) provided that when R¹ represents -(CH2)_c-Z-(CH₂)_d-W and W represents NR⁴R⁵ or any N linked heterocyclic group then d must not be 0 or 1 ; and (iii) provided that when R² represents a phenyl group substituted by a group of formula -(CH₂)_eOR³, -(CH₂)_e-C0₂R³ or - (CH₂)_eOCOR³; or het¹ and/or het² are substituted by a group of formula - (CH₂)_eOR³, -(CH₂)_e-C0₂R³ or -(CH₂)_eOCOR³; or when R⁷ represents -OR³ or -(CH₂)rO-(CH₂)e-R³ and e is 0; or when W represents a phenyl group substituted with -OR³ or - C0₂R³; and R³ represents an alkyl group substituted with Y, and Y represents NR R⁵ or an N-linked het³; then R³ must represent C₂-C₆ alkyl substituted with Y.

Further examples of Via antagonists for use with the invention are disclosed in WO 2004/074291 , i.e. compounds of formula (1),

(I) or pharmaceutically acceptable derivatives thereof, wherein V represents -(CH₂) (0)_β-, -CO-, or -CH(Cι_-6 alkyl)-; W is -O-, -S(0)_a-, or -N(R¹)- R¹ represents H, Cι_-6 alkyl, (CH₂)_bCOR², CO(CH₂) NR²R³, S0₂R², (CH₂)_cOR², (CH₂)_CNR²R³, or (CH₂) het¹; het¹ represents a saturated or unsaturated heterocycle of from 3 to 8 atoms containing one or more heteroatoms selected from O, N, or S, optionally substituted with Cι_-8 alkyl; PC26183 13

X and Y independently represent H, Cι_₆ alkyl, halogen, OH, CF₃, OCF₃, OR⁴; Z represents -(CH₂)_f(0)_g-, -CO- or -CH(Cι_-6 alkyl)-; Ring A represents a 4-7 membered, saturated N-containing heterocycle, optionally substituted with OH, and in which optionally at least one ring N is substituted with O; Ring B represents phenyl or a 4-7 membered unsaturated N-containing heterocycle, optionally substituted with OH, halogen, CN, CONH₂, CF₃, OCF₃, and in which optionally at least one ring N is substituted with O; R² and R³ independently represent H, Cι_-6 alkyl [optionally substituted with

OH, halogen, N(Cι_-6 alkyl)₂, or C_1-6 alkyloxy], Cι_-6 alkyloxy, N(C_1-6 alkyl)₂, or [C_3-8 cycloalkyl]; or R² and R³, together with the nitrogen atom to which they are attached independently represent a heterocycle of from 3 to 8 atoms, optionally substituted with C1-6 alkyl; R⁴ represents straight or branched Cι-β 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.

In particular 8-chloro-5-Methyl-1-(3,4,5,6-ietrahydro-2H-[1 ^bipyridinyM-y -S^- dihydro-4H-2,3,5,1 Ob-tetraazo-benzo[e]azulene and 8-chloro-1 -(1 -pyrimidin-2-yl- piperidin^-y -δ.δ-dihydro^H^ IOb-triaza-benzotejazulene, or pharmaceutically acceptable salts (in particular the dibesylate salt) or solvates thereof are preferred.

The vasopressin receptor antagonists for use in the invention may be tested in the screens set out below:

1.0 VIA Filter Binding Assay

1.1 Membrane Preparation

Receptor binding assays were performed on cellular membranes prepared from CHO cells stably expressing the human VIA receptor, (CHO-hV_1A). The CHO-hV-ι_A PC26183 14

cell line was kindly provided under a licensing agreement by Marc Thibonnier, Dept. of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio. CHO-hVι_A cells were routinely maintained at 37°C in humidified atmosphere with 5% C0₂ in DMEM/Hams F12 nutrient mix supplemented with 10 % fetal bovine serum, 2 mM L-glutamine, 15 mM HEPES and 400 μg/ l G418. For bulk production of cell pellets, adherent CHO-hV _A cells were grown to confluency of 90-100% in 850 cm² roller bottles containing a medium of DMEM/Hams F12 Nutrient Mix supplemented with 10 % fetal bovine serum, 2 mM L-glutamine and 15 mM HEPES. Confluent CHO-hV_1A 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 homogenised in membrane preparation buffer consisting of 50 mM Tris- HCl, pH 7.4, 5 mM MgCl₂ and supplemented with a protease inhibitor cocktail, (Roche). The cell homogenate was centrifuged at 1000 rpm, 10 min, 4°C and the supernatant was removed and stored on ice. The remaining pellet was homogenised and Gentrifuged as before. The supematants were pooled and centrifuged at 25,000 x g for 30 min at 4°C. The pellet was resuspended in freezing buffer consisting of 50 mM Tris-HCI, pH 7.4, 5 mM MgCI₂ and 20 % glycerol and stored in small aliquots at -80°C until use. Protein concentration was determined using Bradford reagent and BSA as a standard.

1.2 VIA Filter binding

Protein linearity followed by saturation binding studies were performed on each new batch of membrane. Membrane concentration was chosen that gave specific binding on the linear portion of the curve. Saturation binding studies were then performed using various concentrations of [³H]-arginine vasopressin, [³H]-AVP (0.05 nM - 100 nM) and the K_ά and B_max determined.

Compounds were tested for their effects on [³H]-AVP binding to CHO-hV-i_A membranes, (³H-AVP; specific activity 65.5 Ci / mmol; NEN Life Sciences). Compounds were solubilised in dimethylsulfoxide (DMSO) and diluted to working concentration of 10% DMSO with assay buffer containing 50 mM Tris-HCL pH 7.4, 5 mM MgCI₂ and 0.05% BSA. 25 μl compound and 25 μl [³H]-AVP, (final PC26183 15

concentration at or below Kd determined for membrane batch, typically 0.5 nM - 0.6 nM) were added to a 96-well round bottom polypropylene plate. The binding reaction was initiated by the addition of 200 μl membrane and the plates were gently shaken for 60 min at room temperature. The reaction was terminated by rapid filtration using a Filtermate Cell Harvester (Packard Instruments) through a 96-well GF/B UniFilter Plate which had been presoaked in 0.5% polyethyleneimine to prevent peptide sticking. The filters were washed three times with 1 ml ice cold wash buffer containing 50 mM Tris-HCL pH 7.4 and 5 mM MgCl₂. The plates were dried and 50 μl Microscint-0 (Packard instruments) was added to each well. The plates were sealed and counted on a TopCount Microplate Scintillation Counter (Packard Instruments). Non-specific binding (NSB) was determined using 1 μM unlabelled d(CH2)5Tyr(Me)AVP ([β-mercapto-β,β-cyclopentamethylenepropiony[,

) (βMCPVP), (Sigma). The radioligand binding data was analysed using a four parameter logistic equation with the min forced to 0%. The slope was free fitted and fell between -0.75 and -1.25 for valid curves. Specific binding was calculated by subtracting the mean NSB cpm from the mean Total cpm. For test compounds the amount of ligand bound to the receptor was expressed as % bound = (sample cpm - mean NSB cpm)/specific binding cpm x100. The % bound was plotted against the concentration of test compound and a sigmoidal curve was fitted. The inhibitory dissociation constant (K_\) was calculated using the Cheng-Prusoff equation:

where [L] is the concentration of ligand present in the well and _d is the dissociation constant of the radioligand obtained from Scatchard plot analysis.

2.0 VIA Functional Assay: Inhibition of AVP / VIA-R mediated Ca²* mobilization by FLIPR (Fluorescent Imaging Plate Reader) (Molecular Devices)

Intracellular calcium release was measured in CHO-hVι_A cells using FLIPR, which allows the rapid detection of calcium following receptor activation. The CHO-hV_1A cell line was kindly provided under a licensing agreement by Marc Thibonnier, Dept. of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio. CHO-V-IA cells were routinely maintained at 37°C in humidified PC26183 16

atmosphere with 5% C0₂ in DMEM/Hams F12 nutrient mix supplemented with 10 % fetal bovine serum, 2 mM L-glutamine, 15 mM HEPES and 400 μg/ml G418. On the afternoon before the assay cells were plated at a density of 20,000 cells per well into black sterile 96-well plates with clear bottoms to allow cell inspection and fluorescence measurements from the bottom of each well. Wash buffer containing Dulbecco's phosphate buffered saline (DPBS) and 2.5 mM probenecid and loading dye consisting of cell culture medium containing 4 μM Fluo-3-AM (dissolved in DMSO and pluronic acid),(Molecular Probes) and 2.5 mM probenecid was prepared fresh on the day of assay. Compounds were solubilised in DMSO and diluted in assay buffer consisting of DPBS containing 1% DMSO, 0.1% BSA and 2.5 mM probenecid. The cells were incubated with 100 μl loading dye per well for 1 hour at 37°C in humidified atmosphere with 5% C0₂. After dye loading the cells were washed three times in 100 μl wash buffer using a Denley plate washer. 100 μl wash buffer was left in each well. Intracellular fluorescence was measured using FLIPR. Fluorescence readings were obtained at 2s intervals with 50 μl of the test compound added after 30s. An additional 155 measurements at 2s intervals were then taken to detect any compound agonistic activity. 50 μl of arginine vasopressin (AVP) was then added so that the final assay volume was 200 μl. Further fluorescence readings were collected at 1s intervals for 120s. Responses were measured as peak fluorescence intensity (Fl). For pharmacological characterization a basal Fl was subtracted from each fluorescence response. For AVP dose response curves, each response was expressed as a % of the response to the highest concentration of AVP in that row. For IC₅₀ determinations , each response was expressed as a % of the response to AVP. IC50 values were converted to a modified _b value using the Cheng-Prusoff equation which takes into account the agonist concentration, [A], the agonist EC₅₀ and the slope: K_b=IC₅o/(2+[A]/A₅o]^π)^1/n-1 where [A] is the concentration of AVP, A₅₀ is the EC₅₀ of AVP from the dose response curve and n=slope of the AVP dose response curve.

The terms "cyclooxygenase-2 inhibitor", or "COX2 inhibitor", which can be used interchangeably, embrace compounds which inhibit the COX2 enzyme regardless of the degree of inhibition of the cyclooxygenase-1 enzyme, and include pharmaceutically acceptable salts of those compounds. Thus, for the purposes of PC26183 17

the present invention, a compound is considered a COX2 inhibitor although the compound may inhibit the COX2 enzyme to an equal, greater, or lesser degree than the COX1 enzyme.

Examples of COX2 inhibitor compounds are non-steroidal anti-inflammatory drugs (NSAIDs). Thus, compounds that can serve as the COX2 inhibitor of the present invention include non-steroidal anti-inflammatory drug compounds, a pharmaceutically acceptable salt thereof or a pure (-) or (+) optical isomeric form thereof. Non-limiting examples of NSAID compounds include ibuprofen, naproxen, benoxaprofen, flurbiprofen, fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen, oxaprozin, prapoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, tiaprofenic acid, fluprofen, bucloxic acid, indomethacin, sulindac, tolmetin, zomepirac, diclofenac, fenclofenec, alclofenac, ibufenac, isoxepac, furofenac, tiopinac, zidometacin, acetyl salicylic acid, indometacin, piroxicam, tenoxicam, nabumetone, ketorolac, azapropazone, mefenamic acid, tolfenamic acid, diflunisal, podophyllotoxin derivatives, acemetacin, droxicam, floctafenine, oxyphenbutazone, phenylbutazone, proglumetacin, acemetacin, fentiazac, clidanac, oxipinac, mefenamic acid, meclofenamic acid, flufenamic acid, niflumic acid, flufenisal, sudoxicam, etodolac, piprofen, salicylic acid, choline magnesium trisalicylate, salicylate, benorylate, fentiazac, clopinac, feprazone, isoxicam and 2-fluoro-a-methyl[1 ,1'-biphenyl]-4-acetic acid, 4-(nitrooxy)butyl ester (See Wenk, et al., Europ. J. Pharmacol. 453:319-324 (2002)). Preferred compounds of the above examples include ibuprofen, naproxen, sulindac, ketoporfen, fenoprofen, tiaprofenic acid, suprofen, etodolac, carprofen, ketrolac, piprofen, indoprofen, salicylic acid, and flurbiprofen.

It is preferred that the COX2 inhibitor is a COX2 selective inhibitor. As used herein, the terms "cyclooxygenase-2 selective inhibitor" or "COX2 selectvie inhibitor", which can be used interchangeably, embrace compounds which selectively inhibit COX2 over COX1 , and include pharmaceutically acceptable salts of those compounds. Thus, for the purposes of the present invention, the term "COX2 inhibitor" encompasses compounds that are "COX2 selective inhibitors", plus all other compounds that inhibit the COX2 enzyme. PC26183 18

In practice, the selectivity of a COX2 inhibitor varies depending upon the condition under which the test is performed and on the inhibitors being tested. However, for the purposes of this specification, the selectivity of a COX2 inhibitor can be expressed as a ratio of the in vitro or in vivo IC₅o value for inhibition of COX1 , divided by the 1C₅₀ value for inhibition of COX2 (COX1 IC₅₀/COX2 IC₅₀). A COX2 selective inhibitor is any inhibitor for which the ratio of COX1 IC50 to COX2 IC50 is greater than 1. In preferred embodiments, this ratio is greater than 2, more preferably greater than 5, yet more preferably greater than 10, still more preferably greater than 50, and more preferably still greater than 100. As used herein, the term "IC₅₀" refers to the concentration of a compound that is required to produce 50% inhibition of cyclooxygenase activity. Preferred COX2 selective inhibitors of the present invention have a COX2 IC₅₀ of less than about 1 μM, more preferred of less than about 0.5 μM, and even more preferred of less than about 0.2 μM. Preferred cycloxoygenase-2 selective inhibitors have a cyclooxygenase-1 IC50 of greater than about 1 μM, and more preferably of greater than 20 μM. Such preferred selectivity may indicate an ability to reduce the incidence of common NSAlD-induced side effects.

The COX inhibitors for use in the invention may be tested in the screens set out below:

3.0 Evaluation of COX activity in vitro

3.1 preparation of recombinant COX baculoviruses Recombinant COX1 and COX2 are prepared as described by Gierse et al. (J,. Biochem.. 305. 479-484 (1995)). A 2.0 kb fragment containing the coding region of either human or murine COX1 or human or murine COX2 is cloned into a BamH1 site of the baculovirus transfer vector pVL1393 (Invitrogen) to generate the baculovirus transfer vectors for COX1 and COX2 in a manner similar to the method of D.R. O'Reilly et al (Baculovirus Expression Vectors: A Laboratory Manual (1992)). PC26183 19

Recombinant baculoviruses are isolated by transfecting 4μg of baculovirus transfer vector DNA into SF9 insect cells (2x10 e8) along with 200 ng of linearised baculovirus plasmid DNA by the calcium phosphate method. See M.D. Summers and G.E. Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures. Texas Agric. Exp. Station Bull. 1555 (1987). Recombinant viruses are purified by three rounds of plaque purification and high titer (10⁷-10⁸ pfu/mL) stocks of virus are prepared. For large scale production, SF9 insect cells are infected in 10 litre fermentors (0.5x10⁶/mL) with the recombinant baculovirus stock such that the multiplicity of infection is 0.1. After 72 hours the cells are centrifuged and the cell pellet homogenised in Tris/Sucrose (50 mM; 25%, pH 8.0) containing 1 % 3-[(3-cholamidopropyI)dimethylammonio]-1-propanesulfonate (CHAPS). The homogenate is centrifuged at 10,000xG for 30 minutes, and the resultant supernatant is stored at -80°C before being assayed for COX activity.

3.2 Assay for COX1 and COX2 activity.

COX activity is assayed as PGE₂ formed/ μg protein/time using an ELISA to detect the prostaglandin released. CHAPS-solubilised insect cell membranes containing the appropriate COX enzyme are incubated in a potassium phosphate buffer (50 mM, pH 8.0) containing epinephrine^', phenol, and heme with the addition of arachidonic acid (10 μM). Compounds are pre-incubated with the enzyme for 10- 20 minutes prior to the addition of arachidonic acid. Any reaction between the arachidonic acid and the anzyme is stopped after ten minutes at 37°C/room temperature by transferring 40 μl of reaction mix to 160 μl ELISA buffer and 25 μM indomethacin. The PGE₂ formed is measured by standard ELISA technology (Cayman Chemical).

3.3 Rapid Assay for COX1 and COX2 Activity

COX activity is assayed as PGE₂ formed/μg protein/time using an ELISA to detect the prostaglandin released. CHAPS-solubilized insect cell wall membranes containing the appropriate COX enzyme are incubated in a potassium phosphate buffer (50 mM potassium phosphate, pH 7.5, 300μM epinephrine, 2μM phenol, lμM heme) with the addition of 20 μL of 100μM arachidonic acid (10μM). Compounds are pre-incubated with the enzme for 10 minutes at 37°C prior to the PC26183 20

addition of arachidonic acid. Any reaction between the arachidonic acid and the enzyme is stopped after 2 minutes at 37°C/room temperature by transferring 40μL of reaction mix into 160μL ELISA buffer and 25μM indomethacin. The PGE₂ formed is measured by standard ELISA technology (Cayman Chemical).

Also included within the scope of the present invention are compounds that act as prodrugs of COX2 selective inhibitors. As used herein in reference to COX2 selective inhibitors, the term "prodrug" refers to a chemical compound that can be converted into an active COX2 selective inhibitor by metabolic or simple chemical processes within the body of the subject. One example of a prodrug for a COX2 selective inhibitor is parecoxib, which is a therapeutically effective prodrug of the tricy ic COX2 selective inhibitor valdecoxib. An example of a preferred COX2 selective inhibitor prodrug is parecoxib sodium. A class of prodrugs of COX2 inhibitors is described in U.S. Patent No. 5,932,-598.

Non-limiting examples of COX2 selective inhibitors suitable for use in the present invention are illustrated in (i) - (liv) below:

(i) Formula B-1 , meloxicam, (CAS registry number 71125-38-7; described in U.S. Patent No. 4,233,299), or a pharmaceutically acceptable salt or prodrug thereof;

(ii) Formula B-2, RS 57067, 6-[[5-(4-chlorobenzoyl)-1 ,4-dimethyl-l H-pyrrol-2- yl]methyl]-3(2H)-pyridazinone, (CAS registry number 179382-91-3; EP714895), or a pharmaceutically acceptable salt or prodrug thereof; PC26183 21

(iii) Substituted benzopyran derivatives that are described in U.S. Patent No. 6,271,253. Also benzopyran derivatives described in U.S. Patent Nos. 6,034,256 and 6,077,850 along with International Publication Nos WO 98/47890 and WO 00/23433;

(iv) Chromene COX2 selective inhibitors illustrated in Table 1. below: Table 1: Examples of Chromene COX2 Selective Inhibitors

26183 22

26183 23

26183 24

26183 25

PC26183 26

(v) The following specific compounds: 1-methylsulfonyl-4-[1 ,1-dimethyl-4-(4-fluorophenyl)cyclopenta-2,4-dien- 3-yl]benzene (described in WO 95/30656, WO 95/30652, WO 96/38418 and WO 96/38442); and 2-(4-methoxyphenyl)-4-methyl-1-(4-sulfamoylphenyl)-pyrrole (described in European Patent Application Publication No. 799823); or pharmaceutically acceptable salts or prodrugs thereof;

(vi) The group of compounds, illustrated in Table 2, which includes celecoxib (B- 18), valdecoxib (B-19), deracoxib (B-20), rofecoxib (B-21), etoricoxib (MK- 663; B-22), JTE-522 (B-23), or a pharmaceutically accepatable salt or prodrug thereof; 3 27

Table 2: Examples of Tricyclic COX-2 Selective Inhibitors

PC26183 28

Preferably the COX2 selective inhibitor is selected from the group consisting of celecoxib, rofecoxib and etoricoxib;

(vii) Parecoxib (described in U.S. Patent No. 5,932,598), having the structure shown in B-24, which is a therapeutically effective prodrug of the tricyclic Cox-2 selective inhibitor valdecoxib, B-19, (described in U.S. Patent No. 5,633,272); PC26183 29

A preferred form of parecoxib is sodium parecoxib;

(viii) The compound ABT-963 having the formula B-25, described in International Publication number WO 00/24719;

(ix) Phenylacetic acid derivative^" Cox-2 selective inhibitors disclosed in U.S.2003/0013739 and represented by the general structure of Formula VI:

wherein: R²⁷ is methyl, ethyl, or propyl; R ,28 is chloro or fluoro; R ι29 is hydrogen, fluoro, or methyl; R³⁰ is hydrogen, fluoro, chloro, methyl, ethyl, methoxy, ethoxy or hydroxy; R³¹ is hydrogen, fluoro, or methyl; and 83 30

R³² is chloro, fluoro, trifluoromethyl, methyl, or ethyl, provided that R²⁸, R²⁹, R³⁰ and R³¹ are not all fluoro when R²⁷ is ethyl and R³⁰ is H;

A phenylacetic acid derivative COX2 selective inhibitor suitable for use in the present invention is described in WO 99/11605 and has the designation of COX189 (CAS RN 346670-74-4) corresponds to a compound of Formula VI, wherein: R²⁷ is ethyl; R²⁸ and R³⁰ are chloro;

R²⁹ and R³¹ are hydrogen; and R³² is methyl;

A further phenylacetic acid derivative COX2 selective inhibitor suitable for use in the present invention is a compound described in U.S.2003/0013739 having a Formula VI, wherein:

R²⁷ is propyl;

R²⁸ and R³⁰ are chloro; R²⁹- and R³ are methyl; and

R³² is ethyl;

A further phenylacetic acid derivative COX2 selective inhibitor suitable for use in the present invention is described in WO 02/20090 and is a compound referred to as COX-189 (also termed lumiracoxib), having CAS

Reg. No. 220991-20-8;

Further compounds having a structure similar to that of Formula VI, which can serve as COX2 selective inhibitor suitable for use in the present invention, are described in U.S. Patent Nos. 6,310,099, 6,291 ,523, and

5,958,978; PC26183 31

(x) Compounds having the general structure shown in formula VIII, where the J group is a carbocycle or a heterocycle. Preferred embodiments have the structure below:

nimesulide (described in US 3,840,597 and discussed in J. Carter, Exp.Qpin.Ther.Patents, 8(1). 21-29 (1997)) - wherein: X is O; J is 1 -phenyl; R³³ is 2-NHS0₂CH₃; R³⁴ is 4-N0₂; and there is no R³⁵ group; flosulide (discussed in J. Carter, Exp.Qpin.Ther.Patents. 8(1 ), 21 -29 (1997)) - wherein X is O; J is 1-oxo-inden-5-yl; R³³ is 2-F; R³⁴ is 4-F; and R³⁵ is 6- NHS0₂CH₃; NS-398 (CAS RN 123653-11-2; disclosed in U.S.Patent No. 4,885,367 and discussed in J. Carter,. Exp.Qpin.Ther.Patents, 8(1 ). 21-29 (1997); Yoshimi, N. et al., in Japanese J. Cancer Res., 90(4):40β - 412 (1999); Falgueyret, J.-P. et al, in Science Spectra; Iwata, K. et al., in Jpn. J. Pharmacol., 75(2J:191 - 194 (1997)) - wherein X is O; J is cyclohexyl; R³³ is 2-NHS0₂CH₃; R³⁴ is 5-N0₂; and there is no R³⁵ group; L-745337 (disclosed in U.S. Patent No. 5,604,260 and Kirchner et al., in J Pharmacol Exp Ther282, 1094-1101 (1997)) - wherein X is S; J is 1-oxo-inden-5-yl; R³³ is 2-F; R³⁴ is 4-F; and R³⁵ is 6-N^" S0₂CH₃ • Na⁺; RWJ-63556 (disclosed in U.S. Patent No. 5,783,597) - wherein PC26183 32

X is S; J is thiophen-2-yl; R³³ is 4-F; there is no R³⁴ group; and R³⁵ is 5-NHS0₂CH₃; L-784512 (disclosed in U.S. Patent No's 6,020,343; 5,981 ,576; 6,140,515) - wherein X is O; J is 2-oxo-5(R)-methyl-5-(2,2,2-trifluoroethyl)furan-(5H)-3- yl; R³³ is 3-F; R³⁴ is 4-F; and R³⁵ is 4-(p-S0₂CH₃)C₆H4;

(xi) Diarylmethylidenefuran derivatives described in U.S. Patent No. 6,180,651 ; Particular derivatives that are included in this family of compounds, and which can be used as the COX2 selective inhibitor in the present invention, include N-(2-cyclohexyloxynitrophenyl)methane sulfonamide, and (E)-4-[(4- methylphenyl) (tetrahydro-2-oxo-3-furanylidene) methyI]benzenesulfonamide;

(xii) Darbufelone (Pfizer, described in U.S. Patent 5,143,928), CS-502 (Sankyo), LAS 34475 (Almirall Profesfarma), LAS 34555 (Almirall Profesfarma), S- 33516 (Servier), SD 8381 (Pharmacia, described in U.S. Patent No. 6,034,256), BMS-347070 (Bristol Myers Squibb, described in U.S. Patent No. 6,180,651), MK-966 (Merck, described in U.S. Patent No. 5,968,974), L- 783003 (Merck), T-614 (Toyama, described in German Patent Publication No. 38/34204), D-1367 (Chiroscience), L-748731 (Merck, described in U.S. Patent No. 5,968,974), CT3 (Atlantic Pharmaceutical, described in U.S. Patent No. 533,783), CGP-28238 (Novartis, described in EP88282 and EP56956), BF-389 (Biofor/Scherer, described in U.S. Patent No's 4,892,870 and 512,330), GR-253035 (Glaxo Wellcome), 2,6-dioxo-9H-purin-8-yl- cinnamic acid (Glaxo Wellcome), and S-2474 (Shionogi, described in European Patent Publication No. 595546); Information about S-33516, mentioned above, can be found in Current Drugs Headline News, at http://www.current-drugs.com/NEWS/lnflam1.htm, 10/04/2001 , where it was reported that S-33516 is a tetrahydroisoinde PC26183 33 derivative which has IC₅o values of 0.1 and 0.001 mM against COX1 and COX2, respectively;

(xiii) Multibinding compounds containing from 2 to 10 ligands covalently attached to one or more linkers, as described in U.S. Patent No. 6,395,724;

(xiv) Conjugated linoleic acids described in U.S. Patent No. 6,077,868;

(xv) Heterocyclic aromatic oxazole compounds described in U.S. Patents 5,994,381 and 6,362,209;

(xvi) Compounds and pharmaceutically acceptable derivatives described in U.S. Patent Nos. 6,080,876 and 6,133,292;

(xvii) Pyridines described in 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, along with International Patent Application Publication Nos WO 96/03392 and WO 96/24585;

(xviii)Diarylbenzopyran derivatives disclosed in U.S. Patent No. 6,340,694;

(xix) 1-(4-Sulfamylaryl)-3-substituted-5-aryl-2-pyrazolines described in U.S. Patent No. 6,376,519;

(xx) Heterocycles described in U.S. Patent No. 6,153,787;

(xxi) 2,3,5-Trisubstituted pyridines described in U.S. Patent No. 6,046,217;

(xxii) Diaryl bicyclic heterocycles described in U.S. Patent No. 6,329,421 ; (xxiii)Salts of 5-amino or substituted amino 1 ,2,3-triazole compounds described in U.S. Patent No. 6,239,137;

(xxiv) Pyrazole derivatives described in U.S. Patent No. 6,136,831 ; PC26183 34

(xxv) Substituted derivatives of benzosulphonamides described in U.S. Patent No. 6,297,282;

(xxvi) 3-Phenyl-4-(4(methylsulfonyl)phenyl)-2-(5H)-furanones described in U.S. Patent No. 6,239,173;

(xxvii) Bicycliccarbonyl indole compounds described in U.S. Patent No. 6,303,628;

(xxviii) Benzimidazole compounds described in U.S. Patent No. 6,310,079;

(xxix) Indole compounds described in U.S. Patent No. 6,300,363;

(xxx) Aryl phenylhydrazides described in U.S. Patent No. 6,077,869;

(xxxi) 2-Aryloxy, 4-aryl furan-2-ones described in U.S. Patent No. 6,140,515;

(xxxii) Bisaryl compounds described in U.S. Patent No. 5,994,379;

(xxxiii) 1 ,5-Diarylpyrazoles described in U.S. Patent No. 6,028,202;

(xxxiv) 2-Substituted imidazoles described in U.S. Patent No. 6,040,320;

(xxxv) 1 ,3- and 2,3-diarylcycloalkano and cycloalkeno pyrazoles described in U.S. Patent No. 6,083,969;

(xxxvi) Esters derived from indolealkanols and amides derived from indolealkylamides described in U.S. Patent No 6,306,890;

(xxxvii) Pyridazinone compounds described in U.S. Patent No. 6,307,047;

(xxxviii) Benzosulphonamide derivatives described in U.S. Patent No. 6,004,948; PC26183 35

(xxxix) Compounds described in U.S. Patent Nos. 6,169,188, 6,020,343, 5,981 ,576 ((methylsulfonyl)phenyl furanones); U.S. Patent No. 6,222,048 (diaryl-2-(5H)-furanones); U.S. Patent No. 6,057,319 (3,4- diaryl-2-hydroxy-2,5-dihydrofurans); U.S. Patent No. 6,046,236 (carbocyclic sulfonamides); U.S. Patent Nos. 6,002,014 and 5,945,539 (oxazole derivatives); and U.S. Patent No. 6,359,182 (C-nitroso compounds);

(xl) 1 ,5-Diphenyl-3-substituted pyrazoles described in International Patent Application Publication No. WO 97/13755;

(xii) Radicicol and pharmaceutically acceptable derivatives thereof, described in International Patent Application Publication No. WO 96/25928 and discussed in Kwon et al., Cancer Res. (1992), 52, 6296);

(xlii) Compound TP-72 and pharmaceutically acceptable derivatives thereof, which is discussed in Cancer Res. (1998), 58, 4, 717-723;

(xliii) Indomethacin-derived indolalkanoic acid derivatives described in International Patent Application Publication No. WO 96/374679;

(xliv) Pyrazoles described in Interntational Patent Application Publication Nos WO 95/15316, WO 95/15315 and WO 96/03385;

(xlv) Thiophene analogues described in International Patent Application Nos. WO 95/00501 and WO 94/15932;

(xlvi) Oxazoles according to International Patent Application Publication Nos WO 95/00501 and WO 94/27980;

(xlvii) Isoxazoles described in International Patent Application Publication No. WO 96/25405; PC26183 36

(xlviii) Imidazoles according to International Patent Application Publication Nos WO 96/03388 and WO 96/03387;

(xlix) Cyclopentene derivatives described by U.S. Patent No. 5,344,991 and International Patent Application Publication No. WO 95/00501 ;

(I) Terphenyl compounds according to International Patent Application Publication No. WO 96/16934;

(li) Thiazole derivatives described by International Patent Application Publication No. WO 96/03392;

(lii) Benzopyranopyrazolyl compounds disclosed by International Patent Application Publication No. WO 96/09304;

(liii) Benzopyran derivatives according to International Patent Application Publication No. WO 98/47890; and

(liv) Arylpyridazinones disclosed by International Patent Application Publication No. WO 00/24719.

Preferred COX2 selective inhibitor compounds are those compounds selected from the group comprising celecoxib, parecoxib, deracoxib, valdecoxib, etoricoxib, meloxicam, 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-33516, SD-838 , prodrugs of any of them, and mixtures thereof.

More preferred is that the COX2 selective inhibitor is selected from the group comprising celecoxib, parecoxib, deracoxib, valdecoxib, lumiracoxib, etoricoxib, rofecoxib, prodrugs of any of them, and mixtures thereof.

Still further preferred is that the COX2 selective inhibitor comprises celecoxib. PC26183 37

COX2 inhibitors that are useful in present invention can be supplied by any source as long as the COX2 inhibitor is pharmaceutically acceptable. Likewise, COX2 inhibitors that are useful in the present invention can be synthesized, for example, according to the description in Example 1. Several COX2 inhibitors that are suitable for use with the compositions and methods of the present invention may be synthesized by the methods described in, for example, U.S. Patent No. 5,466,823 to Talley, et. al. COX2 inhibitors can also be isolated and purified from natural sources. For purposes of the present invention, the COX2 inhibitors should be of a quality and purity that is conventional in the trade for use in pharmaceutical products.

Any combination of the Via antagonists and COX2 inhibitors described above can be used in novel compositions, pharmaceutical compositions and kits of the present invention. For example, a COX2 inhibitor such as celecoxib cab be combined with any of the aforementioned Via antagonists described above, including 8-chloro-5-Methyl-1 -(3,4,5,6-tetrahydro-2H-[1 ,2']bipyridinyl-4-yl)-5,6-di hydro-4H-2,3,5,10b-tetraazo-benzo[e]azulene and 8-chloro-1-(1-pyrimidin-2-yl- piperidin-4-yl)-5,6-dihydro-4H-2,3,10b-triaza-benzo[e]azulene.

Pharmaceutically acceptable derivatives of the compounds (A) and/or (B) according to the invention include salts, solvates, complexes, polymorphs, prodrugs, stereoisomers, geometric isomers, tautomeric forms, and isotopic variations of compounds (A) and/or (B). Preferably, pharmaceutically acceptable derivatives of compounds (A) and/or (B) comprise salts, solvates, esters and amides of the compounds (A) and/or (B). More preferably, pharmaceutically acceptable derivatives of compounds (A) and/or (B) are salts and solvates.

The compounds for use in the present combination invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms, including hydrated forms, which may contain isotopic substitutions (e.g. D20, d6-acetone, d6-DMSO), are equivalent to unsolvated forms and are encompassed within the scope of the present invention. PC26183 38

The pharmaceutically acceptable salts of the compounds for use in the present combination invention include the acid addition and base salts thereof.

Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, D- and L-lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, palmoate, phosphate, hydrogen phosphate, dihydrogen phosphate, saccharate, stearate, succinate, sulphate, D- and L- tartrate, tosylate and trifluoroacetate salts. A particularly suitable salt is the besylate derivative of the compounds of the present invention.

Suitable base salts are formed from bases, which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.

For a review on suitable salts see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Wiley-VCH, Weinheim, Germany (2002).

A pharmaceutically acceptable salt of a compound for use in the present combination invention may be readily prepared by mixing together solutions of the compound and the desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the salt may vary from completely ionised to almost non-ionised.

The compounds for use in the present invention possess may one or more chiral centers and each center may exist in the R(D) or S(L) configuration. The present PC26183 39

invention includes all enantiomeric and epimeric forms as well as the appropriate mixtures thereof. Separation of diastereoisomers or cis and trans isomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of a compound of the invention or a suitable salt or derivative thereof.

Also included within the present scope of the compounds for use in the invention are polymorphs thereof.

In a further embodiment there is provided a pharmaceutical composition comprising a mixture of effective amounts of (A) as hereinbefore defined and (B) as hereinbefore defined, optionally together with a pharmaceutically acceptable carrier, for administration either prophylactically or when pain commences. ^*

In the pharmaceutical compositions of the present invention, (A) is present in an amount of from 1 mg up to 1000 mg per dose, and (B) is present in an amount of from 1 mg up to 1000 mg per dose. The physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.

The pharmaceutical compositions of the present invention can be administered alone but will generally be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term "excipient" is used herein to describe any ingredient other than the compound of the invention. The choice of excipient will to a large extent depend on the particular mode of administration.

The compounds for use in the invention may be administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compound enters the blood stream directly from the mouth. PC26183 40

Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids or powders, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, films (including muco- adhesive), ovules, sprays and liquid formulations.

Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example water, ethanol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.

The compounds for use in the invention may also be used in fast-dissolving, fast disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, 11 (6), 981 -986 by Liang and Chen (2001 ).

A typical tablet may be prepared using standard processes known to a formulation chemist, for example, by direct compression, granulation (dry, wet or melt), melt congealing, or extrusion. The tablet formulation may comprise one or more layers and may be coated or uncoated.

Examples of excipients suitable for oral administration include carriers, for example, cellulose, calcium carbonate, dibasic calcium phosphate, mannitol and sodium citrate, granulation binders, for example, polyvinylpyrrolidine, hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC) and gelatin, disintegrants, for example, sodium starch glycollate and silicates, lubricating agents, for example, magnesium stearate and stearic acid, wetting agents, for example, sodium lauryl sulphate, preservatives, anti-oxidants, flavours and colourants.

Solid formulations for oral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled dual-, targeted and programmed release. Details PC26183 41

of suitable modified release technologies such as high energy dispersions, osmotic and coated particles are to be found in Verma ef al, Pharmaceutical Technology On-line, 25(2), 1-14 (2001). Other modified release formulations are described in US Patent No. 6,106,864.

The compounds for use in the invention may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intreperitoneal, intrathecal, intraventricular, intraurethral, intrastemal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.

Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free, water.

The preparation of parenteral formulations under sterile conditions, for example, by lyophilisation, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.

The solubility of compounds for use in the present combination invention used in the preparation of parenteral solutions may be increased by suitable processing, for example, the use of high energy spray-dried dispersions (see WO 01/47495) and/or by the use of appropriate formulation techniques, such as the use of solubility-enhancing agents.

Formulations for parenteral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled dual-, targeted and programmed release. PC26183 42

The compounds for use in the invention may also be administered topically to the skin or mucosa, either dermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin and propylene glycol. Penetration enhancers may be incorporated - see, for example, J. Pharm. Sci., 88(10), 955-958 by Finnin and Morgan (October 1999).

Other means of topical administration include delivery by iontophoresis, electroporation, phonophoresis, sonophoresis and needle-free or microneedle injection.

Formulations for topical administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled dual-, targeted and programmed release. Thus compounds for use in the invention may be formulated in a more solid form for administration as an implanted depot providing long-term release of the active compound.

The compounds for use in the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phosphoiipids) from a dry powder inhaler or as an aerosol spray from a pressurised container, pump, spray, atomiser (preferably an atomiser using electrohydrodynamics to produce a fine mist), or nebuliser, with or without the use of a suitable propellant, such as dichlorofluoromethane.

The pressurised container, pump, spray, atomizer, or nebuliser contains a solution or suspension of the active compound comprising, for example, ethanol (optionally, aqueous ethanol) or a suitable alternative agent for dispersing, solubilising, or extending release of the active, the propellant(s) as solvent and an optional surfactant, such as sorbitans trioleate or an oligolactic acid. PC26183 43

Prior to use in a dry powder or suspension formulation, the drug product is micronised to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenisation, or spray drying.

A suitable solution formulation for use in an atomiser using electrohydrodynamics to produce a fine mist may contain from 1μg to 10mg of the compound of the invention per actuation and the actuation volume may vary from 1μl to 1001 μi. A typical formulation may comprise a compound for use in the present combination invention, propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol.

Capsules, blisters and cartridges (made, for example, from gelatin or HPMC) for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as Meucine, mannitol, or magnesium stearate.

Formulations for inhaled/intranasal administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled dual-, targeted and programmed release.

The compounds for use in the invention may be administered rectally, vaginally or via the intrauterine route, for example, in the form of a suppository, pessary, or enema. Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.

Formulations for rectal/vaginal administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled dual-, targeted and programmed release. PC26183 44

The compounds for use in the invention may also be administered directly to the eye or ear, typically in the form of drugs of a micronised suspension or solution in isotonic, pH-adjusted, sterile saline. Other formulations suitable for ocular and andial administration include ointments, biodegradable (e.g. absorbable gel sponges, collagen) and non-biodegradable (e.g. silicone) implants, wafers, lenses and particulate or vesicular systems, such as niosomes or liposomes. A polymer such as crossed-linked polyacrylic acid, polyvinylalcohol, hyaluronic acid, a cellulosic polymer, for example, hydroxypropylmethylcellulose, hydroxyethylcellulose, or methyl cellulose, or a heteropolysaccharide polymer, for example, gelan gum, may be incorporated together with a preservative, such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.

Formulations for ocular/andial administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled dual-, targeted, or programmed release.

The compounds for use in the invention may be combined with soluble macromolecular entities such as cyclodextrin or polyethyleneglycol-containing polymers to improve their solubility, dissolution rate, taste-masking, bioavailability and/or stability.

Drug-cyclodextrin complexes, for example, are found to be generally useful for most dosage forms and administration routes. Both inclusion and non-inclusion complexes may be used. As an alternative to direct complexation with the drug, the cyclodextrin may be used as an auxiliary additive, i.e. as a carrier, diluent, or solubiliser. Most commonly used for these purposes are alpha-, beta- and gamma-cyclodextrins, examples of which may be found in International Patent Applications Nos. WO 91/11172, WO 94/02518 and WO 98/55148.

The compositions of the present invention may be administered by direct injection. For some applications, preferably the agent is administered orally. For some applications, preferably the agent is administered topically. PC26183 45

Pharmaceutical compositions according to the invention may contain 0.1%-95% of the compounds of this invention, preferably 1%-70%.

"Effective amounts" as used herein is an amount of (A) and (B) that will elicit the biological or medical response being sought. The daily dose of (A) and (B) employed in the method of treatment is similar to the doses described for use in the pharmaceutical compositions hereinbefore described. In the method of treatment according to the present invention (A) and (B) can be administered together combined in a single dosage form, or they can be administered separately, essentially concurrently, each in its own dosage form but as part of the same therapeutic treatment program, and it is envisaged that (A) and (B) may be separately administered, at different times and by different routes.

EXAMPLES:

Examplel : Preparation of the Cox-2 inhibitor, celecoxib.

Step 1: Preparation of 1-(4-methylphenyl)-4,4,4-trifluorobutane-1 ,3-dione. Following the disclosure provided in U.S. Patent No. 5,760,068, 4'- Methylacetophenone (5.26 g, 39.2 mmol) was dissolved in 25 mL of methanol under argon and 12 mL (52.5 mmol) sodium methoxide in methanol (25%) was added. The mixture was stirred for 5 minutes and 5.5 mL (46.2 mmdl) ethyl trifluoroacetate was added. After refluxing for 24 hours, the mixture was cooled to room temperature and concentrated. 100 mL 10% HCI was added and the mixture extracted with 4 x 75 mL ethyl acetate. The extracts were dried over MgS0₄, filtered and concentrated to afford 8.47 g (94%) of a brown oil which was carried on without further purification.

Step 2: Preparation of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1- yljbenzenesulfonamide.

To the dione from Step 1 (4.14 g, 18.0 mmol) in 75 mL absolute ethanol, 4.26 g (19.0 mmol) 4-sulphonamidophenylhydrazine hydrochloride was added. The reaction was refluxed under argon for 24 hours. After cooling to room temperature PC26183 46

and filtering, the reaction mixture was concentrated to afford 6.13 g of an orange solid. The solid was recrystallized from methylene chloride/hexane to give 3.11 g (8.2 mmol, 46%) of the product as a pale yellow solid, having a melting point (mp) of 157°-159°C; and a calculated composition of C17 H₁₄ N₃ 0₂ SF₃ ; C, 53.54; H, 3.70; N, 11.02. The composition that was found by analysis was: C, 53.17; H, 3.81; N, 10.90.

Example 2:

Clinical studies to investigate synergy in the myometrium and uterine arteries:

Both the individual components and the combination therapy are tested clinically using oral therapies in women suffering from primary dysmenorrhoea. In a randomised, double blind placebo controlled study, utilising 12 women with a history of primary dysmenorrhoea, the effects of a COX inhibitor or V-ι_A antagonist (e.g. SR49059) on their own or in combination are examined. Experiments are performed on three occasions within the first three days of three, usually consecutive, menstrual cycles. Uterine artery blood flow is measured using either 3-D Doppler velocimetry, 2-D colour Doppler (measured as the power Doppler signal intensity) or contrast enhanced MRl and uterine smooth muscle contractility by either the implantation of intrauterine uterine pressure catheters (measured as area under the intrauterine pressure curve (AUC)), 3-D ultrasonography or ischaemic biomarkers. Both uterine blood flow and myometrial contractility are studied at time intervals before and after drug administration to the patients. Lower abdominal pain can also be continuously recorded on a 10 cm visual analogue scale (VAS) graded from "no pain" to "maximal pain. PC26183 47

Example 3:

Synergistic Study of the Effects between a Vasopressinι_A Receptor Antagonist and a Cyclooxygenase Inhibitor in Rat Uterine Smooth Muscle: The purpose of this study was to demonstrate whether or not there is synergy between a cyclooxygenase inhibitor (COXi) and a vasopressin-iA ( I_A) receptor antagonist to reduce myometrial hypercontractility. This novel combination therapy may have utility in the treatment of primary dysmenorrhoea.

Ideally these experiments would have been conducted in human myometrium from non-pregnant women undergoing hysterectomies. Not only is it the target tissue, but also it expresses the V_1A receptor [Kawamata, M., Mϊtsui-Saito, M., Kimura, T., Takayanagi, Y., Yanagisawa, T. & Nishimori, K. (2003)]. Vasopressin-induced contraction of uterus is mediated solely by the oxytocin receptor in mice, but not in humans. Eur J Pharmacol, 472, 229-34. This tissue however is in short supply and it is impossible to control the hormonal status of patients.

Uterine tissue from various other animal species, including rat (Kawamata et al., 2003), has been characterised and shown to contain predominantly oxytocin (OT) receptors, unlike the human where Vι_A receptors predominate. This poses a problem when identifying an animal model for dysmenorrhoea, as there is a species difference in the receptor populations expressed in the uterus. However, as the OT and V_1A receptor both come from the same G-protein coupled receptor family and have the same intracellular signalling pathways [Barberis, O, Morin, D., Durroux, T., Mouillac, B., Guillon, G., Seyer, R., Hibert, M.T., Ribollet, E. & Manning, M. (1999a), Molecular pharmacology of AVP and OT receptors and therapeutic potential. Drug News & Perspectives, 12, 279-292], the OT receptors in rat myometrial tissue are functionally equivalent to the Via receptors in human myometrial tissue. Therefore, when using rat myometrial tissue, the study to demonstrate whether or not there is synergy between the two components needs to be conducted with a COXi and an OT receptor antagonist.. PC26183 48

Methods and Materials

All animal studies were subjected to local ethical review and performed in accordance with UK Home Office regulations.

Rat myometrial tissue preparation - Female rats (Sprague-Dawley's, 250-300g) were pre-dosed with oestradiol (1 ml/kg of a 0.5mg/ml solution, in corn oil, injected subcutaneously) 24 hours prior to culling, in order to induce oestrus [Engstrom, T., Bratholm, P., Christensen, N.J. & Vilhardt, H. (1999), Up-regulation of oxytocin receptors in non-pregnant rat myometrium by isoproterenol: effects of steroids. J Endocrinol, 161, 403-11]. The rats were killed by concussion followed by cervical dislocation. Midline incisions were made followed by removal of the uterus, which was placed in a standard Krebs buffer at room temperature at pH 7.4. The two uteri horns were cut in half and dissected longitudinally. The tissues were mounted in 5ml organ baths and connected to force displacement isometric transducers, under a resting tension of 1 g. The tissues were continuously perfused with modified Krebs buffer maintained at 37°C and gassed with 95% 0₂ and 5% C0₂for an equilibration period of 1h.

Functional rat synergy studies - An initial baseline reading was taken prior to constructing a cumulative dose response curve to OT (0.1-300nM). The tissues were then perfused with Krebs buffer until a stable baseline was achieved. A second baseline reading was taken and single doses of an OT receptor antagonist, L-368899 (3, 10 or 30nM), or a COXi, indomethacin (30,100 or 300 μM) or vehicle (DMSO) were administered. A second cumulative dose response curve to OT (0.1-300nM) was then constructed in all tissues. The tissues were continually washed with Krebs buffer until contractions returned to basal levels. A third baseline reading was taken, and combinations of L-368899 (3, 10 or 30nM) and indomethacin (30,100 or 300 μM) or vehicle were administered (time matched control). A third cumulative dose response curve to OT (0.1nM-300nM) was then repeated in all tissues.

Functional human synergy studies - Should studies with human myometrium be conducted the tissue would be prepared from non-pregnant women undergoing PC26183 49

hysterectomy. The tissue would be dissected into strips, 2-3mm wide and 10mm long and mounted as above in 5ml organ baths. The studies would be conducted as above, but substituting OT for arginine vasopressin (AVP) and the OT antagonist for a V_1A antagonist such as SR49059. Studies would be conducted in paired tissue due to the inability to obtain repeat concentration responses curves to AVP. When utilising human myometrial tissue strips that have no endometrium, basal prostaglandin levels will need to be stimulated exogenously.

Data collection and Analysis - The raw data was captured using ADAnet (Pfizer house programme), which automatically collects recorded readings into an excel worksheet. The ADAnet reading is a measurement of 1 response; which is the area under the curve (AUC) measured over 3 minutes with the baseline set to zero. The raw response measurements for cumulative dose response curves are then transformed in 2 steps: 1) The baseline reading is subtracted from all subsequent readings. 2) Data is then expressed as a percentage of the maximum contractile response to the OT. The dose response curve-fitting function in Labstats (Excel) was used to fit a sigmoidal curve constrained through 0 and 100% and an EC₂₅ value determined for each curve.

A synergy index was calculated to determine whether the dose combinations of indomethacin or L-368899 were synergyistic, additive or antagonistic for the 25% effect level (dose of OT agonist required to give a 25% response).

Materials - The drugs and chemicals used and their sources were: Oxytocin acetate salt (Sigma): Indomethacin (Sigma): Krebs buffer (Sigma): Dimethyl sulphoxide (Fisher Scientific): L-368899 [Pettibone, D.J., Clineschmidt, B.V., Guidotti, EN., Lis, EN., Reiss, D.R., Woyden, C.J., Bock, M.G., Evans, B.E., Freidinger, R.M. & Hobbs, D.W. (1993), L-368899, a Potent Orally Active Oxytocin Antagonist for Potential Use in Preterm Labor. Drug Development Research, 30, 129-142.] was synthesized at Pfizer Global Research and Development, Sandwich and is dissolved in 100% DMSO and diluted in 10% DMSO. All other the drugs PC26183 50

were dissolved and diluted in distilled water. Drug concentrations are given as final bath concentrations.

Results

Effects of L-368899 and Indomethacin alone on rat uterine contractility

Three cumulative dose response curves to OT were constructed in uterine strips from oestradiol-treated rats in the absence and presence of either an OT receptor antagonist (L-368899) or a COXi (Indomethacin). Increasing concentrations of the OT receptor antagonist, L-368899, caused dose dependant rightward shifts in the OT dose response curve with no suppression in the maximum contractile response to OT (Figure 1A & Table 1). The COXi, Indomethacin, also caused a dose dependant rightward shift in the OT dose response curve as well as a dose dependant decrease in the maximum contractile response to OT (Figure 1 B & Table2).

Table 1: EC ₅₀ values for OT in the absence and presence of L-368899 or indomethacin in rat uterine smooth muscle.

Effects of combinations of L-368899 and Indomethacin on rat uterine contractility All possible combinations of L-368899 (3, 10, 30nM) and indomethacin (30, 100, 300 /M) were tested in order to assess whether or not there is a synergistic effect between the two drugs. As an example, the synergistic effect between 30//M PC26183 51

indomethacin and 3nM L-368899 is shown in Figure 2. When administered individually, 30 M indomethacin and 3nM L-368899 gave an EC₂₅ to OT of 1.11nM & 1.2nM respectively. When given in combination an OT EC₂₅ of 1.89nM was achieved i.e. a significantly greater inhibition of the OT contractile response was observed with the combination than when either indomethacin or L-368889 alone (figure 2).

More Detailed Description of the Systematic Study of all the Combinations:

All of the combinations produced a dose dependant inhibition of the OT induced contractile response (Figure 3).

Synergy study

The synergy index was calculated using a dose of OT agonist that gave a 25% response (EC₂₅) in the presense of indomethacin (30, 100, 300 /M) and L-368899 (3, 10, 30nM) on their own (table 2) and also for the combination of interest. The 25% response (rather than the 50%) was chosen because of the decrease in maximum OT contractile response observed for some of the indomethacin and combination doses.

Table 2: EC₂₅ val

(3, 10, 30nM) or indomethacin (30, 100, 300 /M). PC26183 52

For the combinations of indomethacin and L-368899 studied, synergy was observed at the 25% effect level for 4 combinations (Table 4).

Note: synergy can only be quantified as being when the 25% contractile response to oxytocin was reached with the highest dose of one or both agent when given alone. For some of the combinations the suppression in the maximum response was so great this was not achievable Table 4: Synergy at the 25% (EC₂₅) effect level for the combinations of indomethacin with L-368899.

Synergy Analysis - The synergy index was calculated for each combination of the OT antagonist (L-368889) and COXi (indomethacin) to determine whether the dose combination was synergistic, additive or antagonistic for a given effect level. < 1 , synergy Synergy index α- JL = 1, additive A B > 1 , antagonist ic

Where A and B are the doses of drug A (alone) and B (alone) that give a specified effect, and (a, 6) is the combination dose that produces this effect level. For these studies the 25% effect of the maximum OT agonist response was used for all analyses.

Example of synergy using the 30μM indomethacin/ 3nM L-368899 dose combination: PC26183 53

The three different EC₂₅ responses for each dose of indomethacin (30, 100, 300uM) and L-368899 (3, 10, 30nM) alone were plotted, with the horizontal line being the combination 30 /M indomethacin/ 3nM L-368899 EC₂₅ response of 1.89nM (Figure 4).

Using interpolation on the dose response curves in Figure 4, it can be seen that an EC25 response of 1.89E-09 would be achieved using 9nM L-368899 alone and 50 /M indomethacin alone. These values can then be plugged into the synergy index equation as follows. dose of indo in combo dose of L - in combo 30 3

Synergy index + = — + - = 0.9 dose of indo alone dose of - alone 50 9

Synergy <1; Additive = 1; Antagonistic >1

So the dose combination (30uM indo, 3nM L-) at the 25% effect level was synergistic (since the synergy index was < 1).

These calculations can be used for the other combinations of indomethacin with L- 368899 to give the synergy indexes at the 25% effect level (Table 5).

Note: Some synergy indexes can only be quantified as being 'less than' since the 25% for the combination was not reached with the highest dose of one or both when given alone.

Table 5: Synergy indexes at the 25% effect level for the combinations of Indomethacin with L-368899. PC26183 54

Conclusions

From Table 4 it can be seen that 30 /M or 100/;M indomethacin in combination with 3nM or 10nM L-368899 produced a synergistic response at the 25% effect level.

As a consequence of this rodent data, in women one would observe a synergy in uterine relaxant effects between a COX inhibitor and a V_1A receptor antagonist since VIA receptors rather than OT receptors mediate uterine contractility.

This preclinical synergy will translate into a greater benefical clinical effect in treating conditions with increased myometrial contractility, such as dysmenorrhoea.

Claims

PC26183 55CLAIMS:

1. 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, in the manufacture of a medicament for the treatment or prophylaxis of dysmenorrhoea.

2. The use of a combination of (A) and (B) as defined in claim 1 for the treatment or prophylaxis of dysmenorrhoea.

3. The use of a combination of (A) and (B) as defined in claim 1 for the manufacture of a medicament for combination therapy by simultaneous, sequential or separate administration of (A) and (B) in the treatment or prophylaxis of dysmenorrhoea.

4. The use according to any of claims 1 to 3, wherein (B) is a COX2 inhibitor.

5. The use according to any of claims 1 to 3, wherein (B) is a COX2 selective inhibitor.

6. The use according to claim 5, wherein the COX2 inhibitor is selected from: celecoxib, parecoxib, deracoxib, valdecoxib, lumiracoxib, etoricoxib, rofecoxib, or a pharmaceutically acceptable salt or solvate thereof.

7. The use according to any of claims 1 to 6, wherein (A) is a Via receptor antagonist.

8. The use according to any of claims 1 to 7, wherein (A) is selected from: SR49049 (Relcovaptan), atosiban (Tractocile®), conivaptan (YM-087); OPC21268, 8-chloro-5-Methyl-1-(3,4,5,6-tetrahydro-2H-[1 ,2"]bipyridinyl-4-yl)-5,6- dihydro-4H-2,3,5,10b-tetraazo-benzo[e]azulene and 8-chloro-1-(1-pyrimidin-2-yl- piperidin-4-yl)-5,6-dihydro-4H-2,3, 10b-triaza-benzo[e]azulene, or a pharmaceutically acceptable salt or solvate thereof. PC26183 56

9. The use according to any of claims 1 to 8 wherein the dysmenorrhoea is primary dysmenorrhoea.

10. The use according to any of claims 1 to 8 wherein the dysmenorrhoea is secondary dysmenorrhoea.

11. The use according to claim 10 wherein the secondary dysmenorrhoea is a consequence of increased uterine tone, such as uterine fibroids or intra-uterine contraceptive devices.

12. A pharmaceutical product containing (A) and (B) as defined in claims 1 to 8, as a combined preparation for simultaneous, separate or sequential use in the treatment or prophylaxis of dysmenorrhoea.

13. A pharmaceutical composition comprising a mixture of effective amounts of (A) and (B) as defined in claims 1 to 8, optionally together with a pharmaceutically acceptable carrier, for administration either prophylactically or when pain commences.

14. A method of treatment or prophylaxis of dysmenorrhoea comprising administering to a subject in need of such treatment amounts of (A) and (B) as defined in claims 1 to 8 which are together effective.