US 20160058774A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0058774 A1 EL-ALFY et al. (43) Pub. Date: Mar. 3, 2016
(54) SEX STEROID PRECURSORS ALONE OR IN (52) U.S. Cl. COMBINATION WITH SELECTIVE CPC ...... A6 IK3I/5685 (2013.01); A61 K3I/453 ESTROGEN RECEPTORMODULATORS FOR (2013.01) THE PREVENTION AND TREATMENT OF (57) ABSTRACT DYSPAREUNA IN POSTMENOPAUSAL WOMEN Novel methods for treating or reducing the likelihood of acquiring vaginal dysfunctions, more particularly vaginal (71) Applicants:Mohamed EL-ALFY, Quebec (CA); dryness and dyspareunia, leading to sexual dysfunction and Fernand LABRIE, Quebec (CA); low sexual desire and performance, in Susceptible warm Louise BERGER, Quebec (CA) blooded animals including humans involving administration of a sex steroid precursor. Further administration of estrogen (72) Inventors: Mohamed EL-ALFY, Quebec (CA): or selective estrogen receptor modulator, particularly those Fernand LABRIE, Quebec (CA); selected from the group consisting of Raloxifene, Arzoxifene, Louise BERGER, Quebec (CA) Tamoxifen, Droloxifene, Toremifene, Iodoxifene, GW 5638, TSE-424, ERA-923, and lasofoxifene, and more particularly (73) Assignee: Endorecherche, Inc. compounds having the general structure:
(21) Appl. No.: 14/464,536 (22) Filed: Aug. 20, 2014 Related U.S. Application Data (62) Division of application No. 1 1/255,617, filed on Oct. 20, 2005, now Pat. No. 8,835,413. (60) Provisional application No. 60/620.452, filed on Oct. 20, 2004.
Publication Classification is specifically disclosed for the medical treatment and/or inhi bition of development of some of these above-mentioned (51) Int. Cl. diseases. Pharmaceutical compositions for delivery of active A6 IK3I/5685 (2006.01) ingredient(s) and kits) useful to the invention are also dis A6 IK3 L/453 (2006.01) closed. Patent Application Publication Mar. 3, 2016 Sheet 1 of 24 US 2016/0058774 A1
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SEX STEROID PRECURSORS ALONE OR IN Metab., 82: 3498-3505). It was indicated (page 3500) “Vagi COMBINATION WITH SELECTIVE nal cytology was examined as specific parameter of the estro ESTROGEN RECEPTORMODULATORS FOR genic action of DHEA’. This is due to the method of evalu THE PREVENTION AND TREATMENT OF ation, namely vaginal Smear which is limited to the Superficial DYSPAREUNAN POSTMENOPAUSAL and easily removed cells of the epithelium. WOMEN 0006. The present invention describes the effects of DHEA and other components on the three layers of the CROSS-REFERENCE TO RELATED vagina, namely the muscularis, the lamina propria and the APPLICATIONS epithelium with novel benefits at the three levels. The benefi 0001. This is a divisional of U.S. patent application Ser. cial effects of DHEA on the lamina propria and muscularis No. 1 1/255,617, filed Oct. 20, 2005, and entitled SEX STE are believed to be of major importance for the positive action ROID PRECURSORS ALONE OR IN COMBINATION of inhibitors of type 5 c6MP phospho-diesterase, such as WITH A SELECTIVE ESTROGEN RECEPTOR MODU Viagra and other compounds or prostaglandin E1. LATOR AND/OR WITHESTROGENS AND/ORATYPE 5 BACKGROUND OF THE RELATED ART CGMP PHOSPHODIESTERASE INHIBITOR FOR THE PREVENTION AND TREATMENT OF VAGINAL DRY 0007 Vaginal dryness affects about 50% of postmeno NESS AND SEXUAL DYSFUNCTION IN POSTMENO pausal women at the age of 50 to 60 years and 72% after 70 PAUSAL WOMEN, which application claims the benefit of years (Rossin-Amar, 2000, Gynecol Obstet Fertil, 28(3): 245 the priority of U.S. Provisional Application No. 60/620,452 249). Of these women, about 80% experience urogenital dis filed Oct. 20, 2004, the contents of which are specifically orders, especially vaginitis and dyspareunia (Pandit and Ous incorporated by reference herein. Applicants claim priority to lander, 1997, Am J MedSci, 314(4): 228–31). Since these each of the foregoing related applications. problems are believed to be at least partially related to a deprivation of sex Steroids, appropriate local hormonal FIELD OF THE INVENTION replacement therapy should be considered at menopause. It has recently been recognized that postmenopausal women are 0002 The present invention relates to a method for treat not only deprived of all ovarian estrogens but they are also ing or reducing the likelihood of acquiring problems affecting progressively deprived of the androgens originating from the the layer lamina propria or the layer muscularis of the vagina peripheral intracrine transformation of dehydroepiandroster using sex steroid precursors alone or in a novel combination one (DHEA) into both androgens and estrogens (Labrie et al., therapy on Susceptible warm-blooded animals, including 1991, Mol Cell Endocrinol, 78: C113-C118: Labrie et al., humans. In particular, the combinations include administer 1995, Ann NY AcadSci, 774: 16-28; Labrie et al., 2003, End ing a selective estrogen receptor modulator (SERM) and rais Rev. 24(2): 152-182). In fact, serum DHEA and DHEA-S ing the patient’s level of precursors of sex Steroids, said progressively decrease from the age of 30 to 40 years (Labrie precursor being selected from the group consisting of dehy et al., 2003, End Rev. 24(2): 152-182; Orentreich et al., 1984, droepiandrosterone (DHEA), dehydroepiandrosterone sul J Clin Endocrinol Metab, 59:551-555; Labrie et al., 1997, J fate (DHEA-S), and androst-5-ene-3 f6, 17 B-diol (5-diol). Clin Endocrinol Metab, 82: 2396-2402). A series of studies Estrogens may also be administered to conteract the potential indicate that low levels of DHEA and DHEA-S are associated effects of some SERMs on hot flashes and other menopausal with a series of age-related morbidity and diseases (Labrie et symptoms. Type 5 c6MP phosphodiesterase inhibitor may al., 1997, J. Clin. Endocrinol. Metab., 82: 3498-3505; Heiz also be administered to improve sexual activity. The invention isouer et al., 1992, Cancer Res, 52(1): 1-4: Szathmari et al., also relates to kits and pharmaceutical compositions for prac 1994, Osteoporos Int, 4(2): 84-88; Thoman and Weigle, 1989, ticing the foregoing combination. Adv Immununol, 46: 221-261; Barrett-Connor et al., 1999, J 0003. In U.S. Pat. No. 5,843.932, was reported the effect ReprodMed, 44(12): 1012-1020; Barrett-Connoret al., 1999, on vaginal atrophy of one, three or six months treatment with JAm Geriatr Soc, 47(6): 685-691). DHEA administered at a dose of 30 mg twice daily in a 0008. An efficient approach to alleviate vaginal dryness solution of 50% ethanol-50% propylene glycol on an area of and other menopausal symptoms is the use of hormone 2 cm of the dorsal skin in ovariectomized rat. Histopatho replacement therapy (HRT) (Greendale and Judd, 1993, JAm logic examination showed proliferation and murification of Geriatr Soc, 41(4): 426-436; Studdet al., 1980, Pasetto, Pale the vaginal epithelium and reversal of vaginal mucosal atro otti and Ambrus Eds, MT Press, Lancaster, p: 127-139). phy in the rats treated with DHEA Recent clinical studies, however, have indicated that combin 0004. The study performed with the rat when DHEA was ing estrogens and progestins increases the incidence of breast applied on the dorsal skin for 1, 3 and 6 months examined cancer with a potential negative impact on cardiovascular only the effect on the vaginal epithelium (Sourla et al., 1998, events (Colditz et al., 1995, N Engl J Med, 332: 1589-1593; J. Steroid Biochem Mol Biol. 66(3): 137-149) and not on the Ross et al., 2000, J Natl Cancer Inst, 92(4): 328-332: Ros two other layers, namely the lamina propria and the muscu souw et al., 2002, JAMA, 288(3): 321-333). Meanwhile, laris. It was then observed that DHEA was about 10 times there is an increasing interest in the potential of combined more efficient on the vaginal epithelium when applied topi estrogen-androgen replacement therapy (Rosenberg et al., cally than when administered on the skin at a site distant from 1997, J ReprodMed, 42(7): 394-404: Burdet al., 2001, Curr the vagina, thus requiring systemic absorption to exert its Women Health Rep, 1(3):202-205), although the use of the action. estrogenic component is limited by the potential complica 0005. In a previous study on the effect of DHEA admin tions mentioned above. Based upon recent advances in our istered on the skin in postmenopausal women for 12 months understanding of human sex steroid physiology, especially in in a 10% DHEA cream, only the estrogenic activity of DHEA postmenopausal women (Labrie et al., 1991, Mol Cell Endo was evaluated (Labrie et al., 1997, J. Clin. Endocrinol. crinol, 78: C113-C118; Labrie et al., 2003, End Rev. 24(2): US 2016/0058774 A1 Mar. 3, 2016
152-182), the use of DHEA becomes a possibility to provide 0015. In another embodiment, the invention includes addi postmenopausal women with the appropriate levels of andro tional administration of a Type 5 ccjMP phosphodiesterase gens and estrogens synthesised in specific tissues by intrac inhibitor or prostaglandin E1 to improve sexual activity. rine mechanisms, with no systemic effects (Labrie et al., 1997, J. Clin. Endocrinol. Metab., 82: 3498-3505: 16-28; 0016. As used herein, a selective estrogen receptor modu Labrie et al., 2003, End Rev. 24(2): 152-182; Labrie, 2001, lator (SERM) is a compound that either directly or through its Ref Gyn Obstet, 8:317-322; Lasco et al., 2002, 145: 457 active metabolite functions as an estrogen receptor antagonist 461). The restauration of androgen-sensitive elements of (“antiestrogen') in breast tissue, yet provides estrogenic or vaginal function should also help the action of inhibitors of estrogen-like effect on bone tissue and on serum cholesterol type 5 c6MP phosphodiesterase or prostaglandin E1. levels (i.e. by reducing serum cholesterol). Non-steroidal 0009. The selective estrogen receptor modulator (SERM) compounds that function as estrogen receptor antagonists in Acolbifene (EM-652) is a benzopyran derivative originally vitro or in human or rat breast tissue (especially if the com developed for the prevention and treatment of breast cancer pound acts as an antiestrogen on human breast cancer cells) is (Gauthier et al., 1997, J Med Chem, 40: 2117-2122). Acolb likely to function as a SERM. Conversely, steroidal antiestro ifene is the compound having the highest affinity of all known gens tend not to function as SERMs because they tend not to compounds for the ER (Gauthier et al., 1997, J Med Chem, display any beneficial effect on serum cholesterol. Non-ste 40: 2117-2122; Labrie et al., 1999, J Steroid Biochem Mol roidal antiestrogens we have tested and found to function as Biol, 69 (1-6): 51-84: Tremblay et al., 1997, Mol. Endo SERMs include EM-800, EM-01538, Raloxifene, Tamox crinol., 11:353–365) and it exerts its activity on both ERC. and ifen, Droloxifene, Toremifene, Idoxifene, TSE-424, ERA ERB (Tremblay et al., 1998, Endocrinology, 139: 111-118). 923, Lasoxifene (CP336156), Arzoxifene (LY 353.381) and This compound displays a pure and highly potent antiestro GW-5638. We have tested the steroidal antiestrogen ICI 182. genic activity in the mammary gland and endometrium while 780 and found not to function as SERM. SERMs, in accor decreasing serum cholesterol and triglycerides and prevent dance with the invention may be administered in the same ing bone loss, at least in the rat (Labrie et al., 1999, J Steroid dosage as known in the art when these compounds are used as Biochem Mol Biol. 69 (1-6): 51-84). Moreover, it has been antiestrogens. demonstrated that the administration of DHEA, not only does 0017. Without intending to be bound by theory, it is not interfere, but does exert an additive inhibitory effect with believed that SERMs, many of which, preferably, have two the pure antiestrogen Acolbifene on human breast tumour aromatic rings linked by one to two carbon atoms, are growth in the nude mouse (Dauvois et al., 1991, Cancer Res, expected to interact with the estrogen receptor by virtue of the 51: 3131-3135; Luo et al., 1997, Endocrinology, 138: 4435 foregoing portion of the molecule that is best recognized by 4444). Combined treatment of DHEA and Acolbifene has the receptor. Preferred SERMs have side chains which may been proposed as a beneficial chemopreventive and therapeu selectively cause antagonistic properties in breast tissue with tic approach in breast cancer (Labrie, 2001, Ref Gynecol out having significant antagonistic properties in other tissues. Obstet, 8:317-322). In fact, the inhibitory effect of DHEA on Thus, the SERMs may desirably functions as antiestrogens in the growth of human breast cancer xenografts in nude mice the breast while surprisingly and desirably functioning as supports its use as hormone replacement therapy (Dauvois et estrogens (or providing estrogen-like activity) in bone and on al., 1991, Cancer Res, 51: 3131-3135; Couillard et al., 1998, the blood components (where concentrations of lipids and/or J Natl Cancer Inst, 90: 772-778). cholesterol are favorably affected). The favorable effect on 0010 WO99/63974 disclosed medical uses of a selective cholesterol and/or lipids potentially translates to a favorable estrogen receptor modulator in combination with sex steroid effect against atherosclerosis which is known to be adversely precursors affected by improper levels of cholesterol and lipids. 0018. In another embodiment, the invention includes SUMMARY OF THE INVENTION method, pharmaceutical composition and kit wherein the (0011. It is accordingly an object of the present invention to selective estrogen receptor modulator has a molecular for provide effective methods of treatment for vaginal problems, mula with the following features: more particularly vaginal dryness, dyspareunia, and sexual (0.019 a) two aromatic rings spaced by 1 to 2 intervening dysfunction which can lead to decrease in sexual desire and carbon atoms, both aromatic rings being either unsub activity while minimizing undesirable side effects. stituted or substituted by a hydroxyl group or a group 0012. It is another object to provide methods of reducing converted in Vivo to hydroxyl; the risk of acquiring the above problems. 0020 b) a side chain possessing an aromatic ring and a 0013. In one embodiment, the invention pertains to a method of treating or reducing the risk of acquiring vaginal tertiary amine function or salt thereof. dryness comprising increasing levels of a sex steroid precur It is preferred that the side chain is selected from the group sor selected from the group consisting of dehydroepiandros consisting of: terone (DHEA), dehydroepiandrosterone-sulfate (DHEA-S) and androst-5-ene-3B.17B-diol (5-diol), in a subject or patient in need of said treatment or said steroid precursor, and further comprising administering to said patient a therapeutically effective amount of a selective estrogen receptor modulator (SERM) as part of a combination therapy. 0014. In another embodiment, the invention includes addi tional administration of estrogens to conteract the effects of SERMs on hot flashes and other menopausal symptoms. US 2016/0058774 A1 Mar. 3, 2016
-continued
(0029 wherein D is OCHCHN(R)R or —CH=CH-COOH (R and Reither being independently selected from the group consisting of C1-C4 alkyl, or R. R. 0021. It is also preferred that the two aromatic rings are and the nitrogenatom to which they are bound, together being both phenyl and that the side chain possesses a moiety a ring structure selected from the group consisting of pyrro selected from the group consisting of a methine, a methylene, lidino, dimethyl-1-pyrrolidino, methyl-1-pyrrolidinyl, pip —CO. —O—, and —S , an aromatic ring, and a tertiary amine function or salt thereof. eridino, hexamethyleneimino and morpholino); 0022. In another embodiment, the selective estrogen 0030 wherein E and K are independently hydrogen or receptor modulator is selected from the group consisting of a hydroxyl; wherein J is hydrogen or halogen. benzothiophene derivative, triphenylethylene derivative, Particularly, selective estrogen receptor modulator is Tamox indole derivative, benzopyran derivative, 5,6,7,8-tetrahy ifen, OH-tamoxifen, Droloxifene, Toremifene, Iodoxifene, dronaphtalene and centchroman derivative. and GW 5638. 0023. In one embodiment, it is preferred that the selective 0031. In another embodiment, it is preferred that the selec estrogen receptor modulator is a benzothiophene derivative tive estrogen receptor modulator is an indole derivative com compound of the following formula: pound of the following formula: -Oy-O R-O N 2 /* O)- R6 0024 wherein R and Rare independently selected from the group consisting of hydrogen, hydroxyl, and a moiety converted in vivo in hydroxyl: 0032 wherein D is selected from the groups consisting of –OCHCHN(R)Rs, -CH=CH-CON(R)Rs –CC— 0025 wherein R and Rare either independently selected (CH2), N(R-7)Rs (R, and Rs either being independently from the group consisting of C1-C4 alkyl, or wherein R. R. selected from the group consisting of C-C alkyl, or R. Rs and the nitrogen to which they are bound, together are any and the nitrogenatom to which they are bound, together being structure selected from the group consisting of pyrrolidino, a ring structure selected from the group consisting of pyrro dimethyl-1-pyrrolidino, methyl-1-pyrrolidinyl, piperidino, lidino, dimethyl-1-pyrrolidino, methyl-1-pyrrolidinyl, pip hexamethyleneimino and morpholino; eridino, hexamethyleneimino, morpholino, ring); 0026 wherein A is selected from the group consisting of 0033 wherein X is selected from the group consisting of: —CO—, —CHOH, and —CH2—, hydrogen, and C1-C6 alkyl: 0027 wherein B is selected from the group consisting of 0034) wherein R. R. R. R. Rs, and Rare independently phenylene, pyridylidene, and -cycloCHN . selected from the group consisting of hydrogen, hydroxyl, Particularly, the selective estrogen receptor modulator is C-C alkyl, and a moiety converted in vivo in hydroxyl. selected from the group consisting of Raloxifene, Arzoxifene Particularly, the selective estrogen receptor modulator is (LY 353381) and LY 335563. TSE-424 and ERA-923. 0028. In another embodiment, it is preferred that the selec 0035. In another embodiment, it is preferred that the selec tive estrogen receptor modulator is a triphenylethylene tive estrogen receptor modulator is a compound of the fol derivative compound of the following formula: lowing formula: US 2016/0058774 A1 Mar. 3, 2016
0046 wherein G is selected from the group consisting of hydrogen, methyl and ethyl. More particularly, benzopyran derivatives of the following general structure are preferred:
R-O
0036) wherein R and Rare independently selected from the group consisting of hydrogen, hydroxyl, and a moiety converted in vivo in hydroxyl: 0037 wherein Rs and R are independently hydrogen or C-C alkyl; 0038 wherein D is OCHCHN(R)R (R and R. 0047 wherein D is OCHCHN(R)R (R and R. either being independently selected from the group consisting either being independently selected from the group consisting of C-C alkyl, or R. R. and the nitrogenatom to which they of C-C alkyl, or R. R. and the nitrogenatom to which they are bound, together being a ring structure selected from the are bound, together being a ring structure selected from the group consisting of pyrrolidino, dimethyl-1-pyrrolidino, group consisting of pyrrolidino, dimethyl-1-pyrrolidino, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, methyl-1-pyrrolidinyl, piperidino, hexamethyleneimino, morpholino). morpholino, ring). 0039. Wherein X is selected from the group consisting of 0048 wherein R and Rare independently selected from —O— and —CH2—. the group consisting of hydrogen, hydroxyl, and a moiety Particularly, the compound is selected from the group con converted in vivo in hydroxyl. sisting of: (-)-cis-(5R,6S)-6-phenyl-5-4-(2-pyrrolidin-1- 0049. It is also preferred that benzopyran derivatives are ylethoxy)phenyl-5,6,7,8-tetrahydronaphthalen-2-ol, D-(-)- optically active compounds having an absolute configuration tartrate salt (lasofoxifene) and (3,4-trans-2,2-dimethyl-3- S on carbon 2 or pharmaceutically acceptable salts thereof, phenyl-4-4-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl-7- said compounds having the molecular structure: methoxychroman). 0040. In another embodiment, it is preferred that the selec tive estrogen receptor modulator has the following formula:
0050 wherein R and Rare independently selected from 0041 wherein R and R are independently hydrogen, the group consisting of hydroxyl and a moiety convertible in hydroxyl or a moiety which is converted to hydroxyl in vivo; vivo to hydroxyl: wherein Z is a bivalent closing moiety, particularly, Z is I0051 wherein R is a species selected from the group selected from the group consisting of —O— —NH-, consisting of Saturated, unsaturated or Substituted pyrrolidi —S , and —CH2—, nyl, Saturated, unsaturated or Substituted piperidino, Satu 0042 wherein the R100 is a bivalent moiety which dis rated, unsaturated or Substituted piperidinyl, Saturated, unsat tances L. from the B-ring by 4-10 intervening atoms; urated or Substituted morpholino, nitrogen-containing cyclic 0043 wherein L is a bivalent or trivalent polar moiety moiety, nitrogen-containing polycyclic moiety, and NRaRb selected from the group of —SO—, —CON —N<, and (Ra and Rb being independently hydrogen, straight or - SON<; branched C-C alkyl, straight or branched C-C alkenyl, 0044 wherein G is selected from the group consisting of and straight or branched C-C alkynyl). hydrogen, a C to Cs hydrocarbon or a bivalent moiety which 0052. In one embodiment, the benzopyran derivative is a joins G and L to form a 5-to 7-membered heterocyclic ring, salt of an acid selected from the group consisting of acetic and halo or unsaturated derivatives of the foregoing. acid, adipic acid, benzenesulfonic acid, benzoic acid, cam 0045 wherein G is either absent or selected from the phorsulfonic acid, citric acid, fumaric acid, hydroiodic acid, group consisting of hydrogen, a C to Cs hydrocarbon or a hydrobromic acid, hydrochloric acid, hydrochlorothiazide bivalent moiety which joins G and L to form a 5- to 7-mem acid, hydroxy-naphthoic acid, lactic acid, maleic acid, meth bered heterocyclic ring, and halo or unsaturated derivatives of anesulfonic acid, methylsulfuric acid, 1.5-naphthalenedisul the foregoing; fonic acid, nitric acid, palmitic acid, pivalic acid, phosphoric US 2016/0058774 A1 Mar. 3, 2016
acid, propionic acid, Succinic acid, Sulfuric acid, tartaric acid, mg/kg) on an area of 2x2 cm of the dorsal skin, an hyper terephthalic acid, p-toluenesulfonic acid, and Valeric acid. trophic epithelium made of 3-5 layers of mucous cells (m) 0053. The preferred selective estrogen receptor modula was seen overlying a basal layer (b). Several invaginations tOrS are: characterized this epithelium (arrow). 0064 FIG. 2H illustrates that in most areas of the vaginal
epithelium of DHEA+Acolbifene-treated animals, a layer of mucous cells (m) rested on a basal cell layer (b), while in Some areas, many layers of mucous cells overlaid the basal cell layer. Several invaginations characterized this epithelium (arrows). 0065 FIG. 2I illustrates that treatment with DHEA+Pre marin led to a mixed epithelium composed of a three to seven cell layer-thick stratified squamous epithelium(s) overlaid by 3-5 layers of mucous cells (m) in 3 animals. In 2 animals, areas of stratified squamous epithelium were predominant (insert). Bar in insert, 30 Lum. Acolbifene 0066 FIG. 2J illustrates that when DHEA, Premarin and Acolbifene were combined, the epithelium was similar to that Raloxifene, Arzoxifene, Tamoxifen, Droloxifene, of the DHEA+Acolbifenegroup. Toremifene, Iodoxifene, GW 5638, TSE-424, ERA-923, and 0067 FIGS. 3A-3B show the vaginal mucosa of OVX lasofoxifene. animals. FIG. 3A shows severe inflammatory changes char acterized by focal leukocyte infiltration with intraepithelial BRIEF DESCRIPTION OF THE DRAWINGS microabscess (M). FIG. 3B shows focal erosion (E) charac 0054 The patent or application file contains at least one terized by reduced epithelial thickness and ulceration (U) drawing executed in color. Copies of this patent or patent visualized as a complete disappearance of the epithelium. application publication with color drawing(s) will be pro 0068 FIGS. 4A-4D illustrate the estrogen-like effect of vided by the Office upon request and payment of the neces Acolbifene on the vaginal epithelium lining of segment 1 sary fee. (external opening). Comparisons with relevant groups are 0055 FIG. 1 shows the Division along the longitudinal made. In intact rats at estrus Figure A and in Premarin-treated, axis of the rat vagina into seven cross-segments, from the animals FIG. 4D, the epithelium is 10-15 layer-thick and external orifice (ostium) (segment 1) to the cervix level (seg keratinized. In OVX controls FIG. 4B, the epithelial thickness ment 7) (portio vaginalis uteri). is reduced to 4-6 layers and is generally not keratinized (see 0056 FIGS. 2A-2J show the vaginal epithelium histomor the absence of stratum granulosum), while in Acolbifene phology of segment 5 in the nine groups of rats. Barin (J), 40 treated animals FIG. 4C, the thickness is restored to 9-11 lm. layers and keratinized. 0057 FIG. 2A is representative of the estrogenic effect, 0069 FIGS.5A-5D show the thickness (um) of the three the stratified squamous epithelium of cycling rats at estrous different vaginal compartments at the level of the fifth seg consists of four main layers: one cell layer of stratum basale ment of the rat vagina: FIG. 5A epithelium, FIG. 5B lamina (b), 6 to 7 cell layers of stratum spinosum (S) and a stratum propria, FIG. 5C muscularis and FIG.5D total thickness, after granulosum of 5 to 6 layers (g) overlaid by the stratum cor 36 weeks treatment of OVX animals with DHEA, Premarin neum (c) made of tightly packed flattened cornified cells. and Acolbifene, alone or in combination. Intact animals at 0058 FIG. 2B illustrates cycling rats at proestrus which estrus and proestrus are added as reference controls. Groups are under an estrogenic-progestational influence, and is used sharing the same letter are not statistically different at p-0.05. to illustrate mucification. In most segments 2 to 7, one basal 0070 FIGS. 6A-6C show the compactness of collagen cell layer (b) was overlaid by 5 to 6 cell layers of stratum fibers of the lamina propria is in segment 5 illustrated as low spinosum (S), and a stratum mucification (m) consisting of (FIG. 6A), moderate (FIG. 6B), or high (FIG. 6C). 3-4 layers of mucous cells. (0071 FIGS. 7A-7I show microphotographs of the three 0059 FIG. 2C illustrates that in the OVX control, a basal vaginal compartments: (E) epithelium, (L) lamina propria cell layer (b) was overlaid by a layer of atrophic cuboidal cells and (M) muscularis, at the level of the fifth segment of the rat (a). vagina with emphasis on the relative muscularis thickness in 0060 FIG. 2D illustrates the vaginal epithelium of OVX the different groups. Separation of the 3 vaginal wall layers rats Treated with an daily oral dose of Acolbifene, (2.5 mg/kg) with bars is indicated to best estimate the thickness distribu and shows atrophy, but with an outer layer of low columnar tion between the different groups. FIG. 7A Intact, 7B OVX. mucous cells (m) overlying the basal cell layer (b). Also illustrated is OVX treated with FIG. 7C Acolbifene, 0061 FIG. 2E illustrates vaginal epithelium of OVX rats FIG. 7D Premarin, FIG. 7E Premarin-i-Acolbifene, FIG. 7F treated with an daily oral dose of Premarin (0.5 mg/kg). The DHEA, FIG. 7G DHEA+Acolbifene, FIG. 7H DHEA+Pre OVX-induced atrophy was replaced by an estrogenic pattern marin, FIG. 7IDHEA+Premarin-i-Acolbifene. comparable to that found at estrus. 0072 FIG. 8 shows the vaginal weight measured 36 weeks 0062 FIG. 2F illustrates that in OVX animals which after OVX and treatment of OVX animals with Acolbifene, received Premarin-Acolbifene, atrophy predominated with a Premarin and DHEA alone or in combination. Intact animals morphology similar to that of Acolbifene-treated animals, are added as controls. although larger mucous cells were seen. 0073 FIG. 9 shows the Immunohistochemical localiza 0063 FIG.2G illustrates that following treatment of OVX tion of AR, ERC. and PR in the (E) epithelium, (L) lamina animals with a once daily cutaneous application of DHEA(80 propria and (M) muscularis at the level of the fifth vaginal US 2016/0058774 A1 Mar. 3, 2016 segment of the different groups. The bars indicate the sepa Non-mucified epithelial areas are also present. In segment 6. ration between the three vaginal compartments. FIG.17B, the epithelial thickness is reduced and consists of a 0074 FIGS. 10A-10H show a comparison of indicated rat squamous stratified epithelium of 2 to 4 cell layers, Sur vaginal epithelial morphology following treatment with dif mounted by a layer of well-aligned mucous cells. Mucified ferent SERMs (FIGS. 10B-10D and FIGS. 10E-10H) versus areas varied from 20 to 80% of the vaginal surface, in alter control (FIGS. 10A and 10E). nance with non-mucified areas. 0075 FIGS. 11A-11C show a comparison of indicated rat I0084 FIGS. 18A-18E show the effect of DHEA 1 mg/sup vaginal epithelial morphology following treatment with each pository. In segment 4, FIG. 18A, the stratified squamous of three different SERMs. epithelium displays 3 to 9 cell layers. Hypertrophied mucous 0076 FIG. 12 shows the picture of a microscope slide to cells overlying 3 to 4 squamous cell layers cover 50 to 70% of display the positioning of the 8 vaginal segments (the whole the vaginal Surface, while well-aligned mucous cells line 5 to upper row and the section at the right-down corner), followed 30% of the vaginal surface. Non-mucified areas are inter by the 4 uterine segments (1 segment of uterine neck and 3 spersed between mucified cells. In segment 6, FIG. 18B, the segments of the two uterine horns 2 sections per horn) in a epithelial thickness is also reduced and consists of 2 to 4 cell sequence corresponding to their original anatomical position. layers. Hypertrophied mucous cells overlying 1 to 3 Squa Segment 8 includes the lateral vaginal folds neighbouring the mous cell layers cover about 30 to 100% of the vaginal Sur beginning of the uterine cervix. face while well-aligned mucous cells cover 0 to 70%. Few 0077 FIG. 13 A-13D show the effect intact rat: interspersed non-mucified areas are also observed. 0078. At estrus, the vaginal epithelium is 12-18 layer I0085 FIG. 19 is a graph of the effect on body weight of thick in segment 4 (FIG. 13A) and 12-15 layer-thick in seg 14-day treatment with DHEA (suppository) applied intrav ment 6 (FIG. 13C). aginally in OVX rats. Compared with placebo group, the 0079 At metestrus, the vaginal epithelium displays 6 to 10 body weight of the groups given DHEA at doses of 0.33 mg cell layers in segment 4 (FIG. 13B), and 5 to 7 layers in and 0.66 mg slightly decreased, while the decrease was more segment 6 (FIG. 13D). pronounced in the 1 mg group. 0080 FIGS. 14A-14B show the effect OVARIECTO MIZED UNTOUCHED Rats. In segment 4, FIG. 14A, the I0086 FIG. 20 is a graph of the serum concentration of vagina is lined by a squamous stratified epithelium of 2 to 6 DHEA following intravaginal application of 0.33 mg, 0.66 cell layers. In segment 6, FIG. 14B, complete atrophy is mg or 1 mg to OVX rats. Area under the curve (AUC) values observed, with flat 2 to 3 cell layers. Some rare foci of small seven hours following DHEA treatment were 32+4 ngh/ml, cylindrical mucous cells could be seen throughout the whole 50+7 ngh/ml and 131-23 ngh/ml when DHEA was given at Vagina. the doses of 0.33 mg, 0.66 mg or 1 mg, respectively. I0081 FIGS. 15A-15B show the effect of placebo/supposi tory. In comparison with the OVX untouched group, the vagi DETAILED DESCRIPTION OF THE INVENTION nal squamous stratified epithelium of the placebo group was I0087. After cessation of estrogen secretion by the ovaries slightly thicker, with segment 4, FIG. 15A, displaying 4 to 8 at menopause, practically all androgens and estrogens are cell layers. In segment 6, FIG. 15B, the same morphology as synthesized in peripheral target tissues by intracrine mecha that of OVX untouched group was observed, with 2-3 epithe nisms from dehydroepiandrosterone (DHEA) of adrenal ori lial cell layers. Foci of small cylindrical mucous cells could be gin. In fact, in the absence of ovarian estrogens, the gradual found throughout the vagina, slightly more when compared decrease in serum DHEA is likely to play an important role in with the OVX untouched animals. the vaginal dryness, inflammation, dyspareunia and sexual 0082 FIGS. 16A-16B show the effect of DHEA 0.33 dysfunction frequently associated with menopause. In order mg/Suppository. In segment 4, FIG.16A, the vaginal epithe to assess the specific estrogenic and/or androgenic effects of lium is stratified squamous with 3 to 7 cell layers. About 20 to a potential novel hormone replacement therapy that could, 60% of the vaginal lining consists of well-aligned mucous among other beneficial effects, alleviate vaginal dryness, we cells in alternance with 15 to 50% of hypertrophied mucous have examined the morphology of the rat vagina eight months cells, overlying one or more squamous stratified epithelium after ovariectomy (OVX) and treatment of OVX animals with layers, and with non-mucified areas. In segment 6, FIG. 16B, DHEA, conjugated estrogens (Premarin) and the selective the epithelial thickness is reduced to almost the thickness estrogen receptor modulator Acolbifene, administered alone found in OVX untouched animals: 2-5 cell layers overlaid by or in combination. In intact animals at estrus and in OVX rats cylindrical mucous cells. A large variation in the occurrence treated with Premarin, the typical vaginal estrogenic pattern of mucous cells was observed, these cells covering from 5 to is a keratinized stratified squamous epithelium. OVX led to a 75% of the vaginal surface, in alternance with non-mucified general atrophy associated with inflammatory changes while aaS. Acolbifene reduced the inflammation incidence and 0.083 FIGS. 17A-17B show the effect of DHEA 0.66 increased the number and size of mucous cells in the vaginal mg/Suppository. In segment 4, FIG. 17A, the stratified squa epithelium. At the doses used, Premarin completely reversed mous epithelium is 3 to 6 cell layer-thick. About 5 to 20% of the OVX-induced epithelial atrophy, while DHEA partially the vaginal lining consists of well-aligned mucous cells in reversed the atrophy. In fact, the vaginal epithelium of OVX alternance with 10 to 75% of hypertrophied mucous cells, animals treated with DHEA became hyperplastic with 3-5 overlying one or more squamous stratified epithelium layers. layers of columnar mucous and goblet cells typical of an US 2016/0058774 A1 Mar. 3, 2016
androgenic effect. The addition of Premarin to DHEA led to an epithelium thicker than in intact animals. Moreover, com pactness of the collagen fibers in the lamina propria was increased by DHEA. On the other hand, treatment with Acol bifene alone showed a tendency for an increase in the lamina propria thickness, which reached intact values when com bined with DHEA. 0088. After OVX, the vaginal muscular layer was decreased by 46%, an effect which was 41% and 100% reversed by DHEA and Premarin, respectively. On the other hand, the 50% decrease in total vaginal wall thickness fol lowing OVX was 42% and 93% reversed by DHEA and is the hydrochloride salt of the potent antiestrogen EM-652. Premarin, respectively, while the combination of DHEA and Compared to EM-800, EM-1538 is a safter, simpler, and Acolbifene reversed total vaginal wall thickness to a value not easier salt to synthesize. In administering either EM-800 or significantly different from intact control animals. EM-1538, it is believed to result in the same active compound 0089. Immunohistochemistry revealed strong androgen in vivo. receptor (AR) labeling in all DHEA-treated groups. On the other hand, estrogen receptor alpha (ERC) and progesterone (0094. Another preferred SERM is Lasoxifene (CP-336, receptor (PR) labeling were not detected in any of the Acol 156; (-)-cis-(5R,6S)-6-phenyl-5-4-(2-pyrrolidin-1- bifene-treated groups. ylethoxy)phenyl-5,6,7,8-tetrahydronaphthalen-2-ol, D-(-)- 0090. In conclusion, treatment with DHEA or the combi tartrate salt) (available from Pfizer Inc., USA). nation of DHEA and Acolbifene partially or completely pre vents the OVX-induced atrophic changes observed in various (0095. Other preferred SERMs of the invention include layers of the vaginal wall through a predominant androgenic Tamoxifen ((Z)-2-[4-(1,2-diphenyl-1-butenyl)-N,N-dim effect, as revealed by epithelial mucification and AR up ethylethanamine) (available from Zeneca, UK), Toremifene regulation. The present data also show particularly interesting (available from Orion-Farmos Pharmaceutica, Finland, or effects of DHEA on the three layers of the vaginal wall, Schering-Plough), Droloxifene, and Raloxifene (Eli Lilly namely a highly mucified epithelium, an increased compact and Co., USA), Arzoxifene (LY 335563) and LY353381 (Eli ness of the collagen fibers in the lamina propria as well as an Lilly and Co., USA), Ospemifene (FC 1271) (available from increase of the muscularis thickness. DHEA thus exerts both Orion-Farmos Pharmaceutica, Finland), Iodoxifene (Smith androgenic and estrogenic effects on the vaginal mucosa, thus Kline Beecham, USA), Levormeloxifene (3,4-trans-2,2-dim providing a more physiological replacement therapy. While ethyl-3-phenyl-4-4-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl each of the examined compounds has potential beneficial 7-methoxychroman) (Novo Nordisk, A/S, Denmark) which is effects on vaginal function, DHEA alone or in combination could well optimally relieve Vaginal dryness and restore glo disclosed in Shalmiet al. WO97/25034, WO 97/25035, WO bal Vaginal physiology and help correcting sexual dysfunc 97/25037, WO 97/25038; and Korsgaard et al. WO tion associated with menopause. DHEA and other compo 97/25036), GW-5638 (described by Willson et al., 1997, nents could be administered locally or systemically. Endocrinol, 138(9): 3901-3911), SERM 3339 in develop 0091. A selective estrogen receptor modulator of the ment by Aventis (France) and indole derivatives (disclosed by invention has a molecular formula with the following fea Miller et al. EP 0802183A1) and TSE-424 and ERA-923 tures: a) two aromatic rings spaced by 1 to 2 intervening developed by Wyeth Ayers (USA) and disclosed in carbon atoms, both aromatic rings being either unsubstituted JP10036347 (American home products corporation) and or Substituted by a hydroxyl group or a group converted in nonsteroidal estrogen derivatives described in WO97/32837. Vivo to hydroxyl; and b) a side chain possessing an aromatic ring and a tertiary amine function or salt thereof. 0096. Any SERM used as required for efficacy, as recom 0092. One preferred SERM of the invention is EM-800 mended by the manufacturer, can be used. Appropriate dos reported in PCT/CA96/00097 (WO 96/26201) The molecular ages are known in the art. Any other non steroidal antiestro Structure of EM-800 is: gen commercially available can be used according to the invention. Any compound having activity similar to SERMs (example: Raloxifene can be used). OCOC(CH3)3 0097 SERMs administered in accordance with the inven tion are preferably administered in a dosage range between 0.01 to 10 mg/kg of body weight per day (preferably 0.05 to 1.0 mg/kg), with 10 mg per day, especially 20 mg per day, in (HC)3CCOO two equally divided doses being preferred for a person of average body weight when orally administered, or in a dosage range between 0.003 to 3.0 mg/kg of body weight per day (preferably 0.015 to 0.3 mg/ml), with 1.5 mg per day, espe 0093. Another preferred SERM of the invention is acolb cially 3.0 mg per day, in two equally divided doses being ifene (EM-1538, also called EM-652.HCl) reported in U.S. preferred for a person of average body weight when paren Pat. No. 6,710,059 B1 tally administered (i.e. intramuscular, Subcutaneous or percu US 2016/0058774 A1 Mar. 3, 2016
taneous administration). Preferably the SERMs are adminis 0103 Twenty-four hours after the last dosing, overnight tered together with a pharmaceutically acceptable diluent or fasted animals were sacrificed under isofluraneanesthesia by carrier as described below. exsanguination at the abdominal aorta (9 animals per group) or by intracardiac perfusion with 10% neutral buffered for 0098 Preferred type 5 c6MPphosphodiesterase inhibitor malin (5 animals per group). Vaginae from non-perfused ani are Sildenafil marketed under the Tradename “Viagra” by mals were collected and weighed, while the vaginae collected Pfizer USA, Tadalafil marketed under the Tradename "Cialis' from perfused animals were marked with black ink on the by Eli Lilly USA, Vardenafil marketed under the Tradename ventral side, and then trimmed as described below. “Levitra’ by Bayer (Germany). It is also preferred type 5 cGMPphosphodiesterase inhibitor which are presently in Histological Procedures development: DA-8159 by Dong-A Pharm Tech (South 0104. The entire vagina of each perfused animal was post Korean), EMR-62203 by Merck (Germany), TA-1790 by fixed in 10% neutral buffered formalin. Each vagina was then Tanabe Seiyaku (Japan), SCH-446132 by Schering-Plough divided into seven equal cross-segments as illustrated in FIG. (U.S.A.), and UK-371800 by Pfizer (U.S.A.). 1, routinely processed and embedded altogether in the same 0099. One preferred prostaglandin is alprostadi1 marketed paraffin block. Within the paraffin block, the seven vaginal under the Tradename “Alprox-TD” by NexMed (USA). cylindrical segments were positioned in a sequence corre 0100. With respect to all of the dosages recommended sponding to their original anatomical position and oriented herein, the attending clinician should monitor individual perpendicular to the surface of the block, thus allowing the patient response and adjust dosage accordingly. segments to be cut in cross-sections. For each animal, a 4 um-thick paraffin section was right and stained with haema EXAMPLE OF EFFICACY OF THE INVENTION toxylin-eosin for morphological examination. Histomorphometry Example 1 0105 Measurements of the different vaginal layers were performed on the fifth segment (FIG. 1), which is approxi Materials and Methods mately halfway between the middle region and the portio vaginalis uteri (segment 7). This fifth segment was found to Animals and Treatments display a representative epithelial Surface and a sufficient thickness of Smooth muscle. Images were captured with a 0101 Ten to twelve week-old female Sprague-Dawley DC-330 3CCD color camera (Dage-MTI, Michigan City, rats (Crl:CDR(SD)Br VAF/PlusTM) (Charles River Labora Ind., USA) and quantified using Image-Pro Plus 3.0 software tory, St-Constant, Canada) weighing approximately 220-270 (Media Cybernetics, Silver Spring, Md., USA). Thus, using a g at Start of the experiment were used. The animals were x5 objective (Leica Microsystems, Willowdale, Ont., acclimatized to the environmental conditions (temperature: Canada), 3 to 4 thickness measurements per layer were 22+3° C.; humidity: 50+20%; 12-hlight-12-h dark cycles, obtained from representative areas of the epithelium and mus lights on at 07:15 h) for at least one week before starting the cularis, and for the three vaginal layers together. The total experiment. The animals were housed individually and were vaginal thickness, the thickness of the epithelium and the allowed free access to water and rodent food (Lab Diet 5002, thickness of the muscularis were thus measured. The thick Ralston Purina, St-Louis, Mo.). The experiment was con ness of the lamina propria was obtained by Subtracting the ducted in accordance with the CCAC Guide for Care and Use thickness of the epithelium and muscularis from total vaginal of Experimental Animals in an animal facility approved by thickness. the Canadian Council on Animal Care (CCAC) and the Asso ciation for Assessment and Accreditation of Laboratory Ani Immunohistochemistry mal Care (AAALAC). 0106 Immunostaining was performed using Zymed SP 0102) A total of 126 female rats were randomly distributed kits (San Francisco, Calif.). Paraffin sections (4 um) were into 9 groups of 14 animals each as follows: 1) Intact control; deparaffinized in toluene and rehydrated through ethanol. 2) Ovariectomized control (OVX); 3) OVX+Acolbifene (2.5 Endogenous peroxidase activity was eliminated by preincu mg/kg); 4) OVX--Premarin (0.5 mg/kg); 5) OVX+Premarin-- bation with 3% HO, in methanol for 30 min. A microwave Acolbifene; 6) OVX+DHEA (80 mg/kg); 7) OVX+DHEA+ retrieval technique using citrate buffer (Tacha and Chen, Acolbifene; 8) OVX+DHEA+Premarin; 9) OVX+DHEA+ 1994) was applied. After cooling the slides, non-specific Acolbifene+Premarin. On the first day of the study, the binding was blocked using 10% goat serum for 20 min. Sec animals of all groups (except group one) were bilaterally tions were then incubated for 1.5 hat room temperature with ovariectomized (OVX) under isoflurane-induced anesthesia. ERC. (AB-1, Calbiochem, California), AR (N-20, Santa Cruz Premarin and Acolbifene were administered by oral gavage Biotechnology, California) or PR (Ab-4, NeoMarkers, Cali (0.5 ml/rat) as suspensions in 0.4% methylcellulose while fornia) antibodies, at 1:200, 1:250 and 1:250 respectively. DHEA in 50% ethanol-50% propylene glycol (0.5 ml/rat), After washing in PBS buffer, sections were incubated with was topically applied on a shaved area of 2x2 cm of the dorsal biotinylated anti-rabbit secondary antibody for 10 min and skin. Dosage selection for Premarin corresponds to the mini thereafter with streptavidin-peroxidase for another 10 min. mal dose sufficient to reverse OVX-induced uterine atrophy, Diaminobenzidine was used as the chromogen to visualize while Acolbifene was administered at a dose sufficient to the biotin/streptavidin-peroxidase complex, under micro cause uterine atrophy similar to OVX after its administration Scope monitoring. Counterstaining was performed using #2 to Premarin-treated OVX animals. The dose of DHEA used Gill's hematoxylin for 30 sec. For controls, immunoabsorp gave DHEA blood levels at 70-100 nmol/L. Treatments were tion with an excess of the peptide used to raise the antibody, initiated on day 2 of the study and the compounds were or substitution with non immune rabbit IgG, was performed. administered once daily for 36 weeks. Animals from the Semiquantitative evaluation of the number and intensity of intact and OVX control groups received the vehicle alone. immunostained nuclei was performed as indicated in Table 2. US 2016/0058774 A1 Mar. 3, 2016
TABLE 1. Histological evaluation of the epithelium, lamina propria and muscularis in the seven rat vaginal segments. Vaginal Segment Segment Segment Segment Segment Segment Segment Group walls 2 3 4 5 6 7
INTACT estruS E KS KS KS KS KS KS KS
M S S M T T T proestrus E KS MSM-KS MSM MSM MSM-KS MSM-KS MSM-KS TM -M -M -M t-M M-M M-M M S S M M T T OVX Control E A. A. A. A. A. A. A.
M S S t t M Acolbifene E KS SCM-A SCM-A SCM-A SCM-A SCM-A SCM-A
M S S S S t t t Premarin E KS KS-MSM KS-MSM KS-MSM KS-MSM KS KS
M S S M T T T Premarin + E KS A-SCM SCM-A SCM-A SCM-A SCM-A SCM-A Acolbifene M-M -M -H -H M-M M-M M-M M S S t t M DHEA E KS-LHM LHM LHM LHM LHM LHM-SCM LHM-SCM
M S S S M M M DHEA- E KS-LHM-SCM SCM-LHM SCM-LHM SCM-LHM SCM-LHM SCM-LHM LHM-SCM Acolbifene M-M -M -H -H M-H M-H M-H M S S S t M M DHEA- E KS MSM-KS MSM-KS MSM-KS MSM-KS KS KS Premarin M-M -L -M -M M-H M-M M-H M S S M T T T DHEA- E KS SCM-LHM-KS SCM-LHM SCM-LHM SCM-LHM SCM-LHM LHM-SCM Premarin + L M-M M-H -H M-H t-H M-H M-M Acolbifene M S S S M M M
E = epithelium morphology: KS (keratinized stratified squamous), LHM (large hypertrophied mucous cells), SCM (small aligned columnar or cuboidal mucous cells), MSM (mixed stratified squamous overlaid by mucous cells), A (atrophy: small cuboidal cells), Two different abreviations for a given segment indicate two different patterns and that the first one is the predominant. L = lamina propria thickness; T (thick), MT (moderately thick), t (thin); 2 compactness of collagen fibers: H(high), M (moderate), L (low); M = muscularis thickness; T (thick), MT (moderately thick), t (thin), s (scarce).
TABLE 2 Semiquantitative evaluation of the number and intensity of immunostained nuclei for AR, ER alpha and PR in the epithelium, lamina propria and muscularis in the fifth segment of the rat vagina. Sex Steroid receptor
AR ERC PR Vaginal layer
Group E L M E L M E L. M
Intact Estrus 2 2 1 1 1 3 2 3 3 Reference ------of low ERC. and high PR Diestrus 3 3 3 3 1 1 O 2 2 Reference ------of high ERC. and ow PR OVX control 1 1 1 2 1 1 O O O ------Acolbifene 2 1 1 O O O O O O ------Premarin 2 2 1 2 3 3 2 2 3 ------Premarin + 2 1 1 O O O O O O Acolbifene ------DHEA 3 3 3 2 2 3 O O 1 ------US 2016/0058774 A1 Mar. 3, 2016
TABLE 2-continued Semiquantitative evaluation of the number and intensity of immunostained nuclei for AR, ER alpha and PR in the epithelium, lamina propria and muscularis in the fifth segment of the rat vagina. Sex steroid receptor
AR ERC Vaginal layer
Group E L M E L M E
DHEA- 3 3 3 O O O O O Acolbifene ------DHEA- 2 3 3 2 2 3 2 3 Premarin ------DHEA- 3 3 3 O O O O O Premarin + ------Acolbifene Androgen receptor (AR), estrogen receptor alpha (ERC), progesterone receptor (PR), epithelium (E), lamina propria (L), muscularis (M), Numbers represent the semi-quantitative evaluation of labeled nuclei: 0 = none, 1 = low, 2 = moderate, 3 = high Labeling intensity is indicated as low; +, moderate: ++ and high: +++
Statistical Analysis animals. In the OVX-Acolbifenegroup, only three out offive 0107 Data are presented as means-SEM of 8-9 animals animals showed signs of minimal inflammation while mod per group for vaginal weight or 5 animals per group for erate to severe inflammatory changes were general findings in vaginal layer thickness determinations. Statistical signifi all OVX animals. cance was determined according to the multiple-range test of 0111. In all groups, segment 1 showed a comparable strati Duncan-Kramer (Kramer, 1956, Biometrics, 12: 307-310). fied squamous epithelium, except in the animals of the OVX group where atrophy was a predominant characteristic (FIG. Results 4). Moreover, in the OVX--DHEA and OVX--DHEA+Acol bifene groups, mucous cells frequently accompanied the Morphology and Thickness of the Different Layers of Rat dominant stratified squamous epithelium of segment 1 (not Vagina shown). 0108. To examine with precision and detail the three layers 0112 The seven segments of the vaginal epithelium of of the rat vaginal wall, namely the epithelium, lamina propria OVX animals which received DHEA were composed of large and muscularis, seven segments obtained along the longitu multilayered columnar mucous cells with distended cytoplas dinal axis (FIG. 1) were first examined. While important mic vacuoles, a feature typical of an androgenic effect (Table morphological differences were observed between the 1, FIG. 2G). Several large invaginations characterized the groups, in general, the differences were uniforminal animals epithelium after DHEA treatment. A similar epithelial mor of the same group and segments 2, 3, 4, 5, 6 and 7 have shown phology was found in all segments of the OVX--DHEA+ a similar epithelial morphology. The few exceptions observed Acolbifenegroup, except that the size of the mucous cells and will be mentioned later. Segment 5 was thus used to illustrate the number of their layers were decreased (Table 1, FIG. 2H). the effect of the various treatments on the vaginal epithelium. In the DHEA+PREMARIN-treated animals, a thick stratified 0109. In the OVX group, the absence of ovarian stimula epithelium of a “mixed' type composed of different ratios of tion led to an atrophy of the vaginal epithelium which char squamous epithelium covered by layers of mucous cells acterized all segments (Table 1 and FIG. 2C) with the pres (Table 1, FIG. 2I) was observed from the second to the fifth ence of Some Small mucous cell areas. Most importantly, segment, thus revealing combined estrogenic and androgenic moderate to severe inflammation with many foci of intraepi effects. In this group, three animals displayed mucification thelial microabcesses (Small areas of agglomerated leuco over the above-mentioned segments, while in the other two cytes within the epithelium) were a frequent finding. These animals, a stratified squamous epithelium was observed in all changes were frequently accompanied by focal erosion segments (Table 1). When PREMARIN was combined with (Zones where the epithelial layer partially disappears) and DHEA and Acolbifene, the epithelium was similar to that of ulceration (complete disappearance of the epithelial layer) the DHEA+Acolbifenegroup, thus indicating a blockade of (FIG. 3). the estrogen-induced squamous cell proliferation by Acolb 0110. In OVX animals treated with Acolbifene, segment 1 ifene (Table 1 and FIG. 2J). showed a keratinized stratified squamous epithelium similar 0113. The administration of DHEA to OVX animals to that of the intact group, except that it included 9-11 cell increased thickness of the vaginal epithelium to 38-4 um, layers compared to the 10-15 layers found in animals at estrus while the addition of Acolbifene to DHEA did not change the or when OVX animals received Premarin (FIGS. 4A-4D). epithelial thickness which remained at 37+5um. When Pre The epithelial thickness of Acolbifene-treated animals in seg marin and DHEA were coadministered to OVX animals, a ment 1 was thus higher than that of OVX rats, which com high thickness of 84-6 um was observed, a value not signifi prised only 4-6 cell layers. In the other segments (Table 1), the cantly different from that of the group of animals which basal layer was covered by a layer of low columnar mucous received Premarin alone. Finally, the combination of Pre cells (FIG. 2D), which were more developed than in OVX marin, DHEA and Acolbifene resulted in an epithelium of US 2016/0058774 A1 Mar. 3, 2016
32+3 um-thick, a value similar to that of the groups which 0118 When total vaginal wall thickness was measured received DHEA alone or DHEA combined with Acolbifene. (FIG.5D), the outer thin layer of connective tissue composing the adventitia was not included. OVX led to a marked (51%) Lamina Propria vaginal wall atrophy (128+3 lum, versus 262+39 um in the intact) and the addition of Acolbifene to OVX had no effect 0114. The degree of compactness of collagen fibers in the (108+8 um) (FIGS. 7A, 7B and 7C). Premarin treatment vagina was categorized as low, moderate and high (FIGS. maintained the total thickness to a value (253-10 um) similar 6A-6C, respectively), as observed in the area proximal to the to that of intact animals while the addition of Acolbifene to epithelium.
and HRT are the treatments of choice. However, as new an effect which could mask any potential coexisting minor information on sex steroid physiology in women strongly estrogenic effect at the epithelial level. A previous study has Suggests an important role of androgens (Labrie et al., 2003, shown the same mucification effect in the rat vagina (Sourla End Rev. 24(2): 152-182), the present study compares the et al., 1998, J Steroid Biochem. Mol. Biol. 66(3): 137-149): overall hormonal effects of an alternative to HRT or ERT, In that previous study, the intravaginal application of DHEA namely DHEA alone or in association with the pure anties achieved a significant effectata dose tentimes lower than that trogen Acolbifene and also with Premarin on rat vaginal found to be active following application of DHEA on the morphology and sex steroid receptor expression. DHEA was dorsal skin. administered percutaneously to avoid first pass through the 0.126 The present data indicate co-existing major andro liver (Labrie et al., 1996, Endocrinol, 150: S107-S118). genic and minorestrogenic actions of DHEA in the rat vagina. 0122 Finally, our results have shown a significant vaginal Moreover, the present results are well supported by the obser weight increase with Premarin treatment, when compared to vation of the vaginal epithelium of OVX animals which OVX animals, the value being similar to that observed in received the same topical application of DHEAthan that used DHEA-treated group. It is appropriate to recall that, although in our study, plus a Subcutaneous dose of the antiandrogen vaginal and uterine weight increases in rodents are commonly Flutamide (FLU) (unpublished results). Indeed, the andro used as measures of estrogenicity, these organs can also genic effect of mucification produced by the androgenic com respond to progesterone and testosterone, among other com ponent of DHEA was completely reversed by FLU and pounds (Emmens and Martin, 1964, Dorfman Ed, Ed Aca resulted in a stratified squamous epithelium, typical of an demic Press NY: 1). estrogenic effect (data not shown). If DHEA would have an 0123. In the potential triple combination, the equine estro exclusive androgenic effect (mucification) in the rat vagina, gen Premarin is aimed at acting in the brain to relieve the this effect would be reversed by FLU and an atrophic epithe vasomotor symptoms. In fact, the benefits of co-administra lium similar to that of OVX animals should then be observed. tion of the pure selective antiestrogen Acolbifene with an Previous studies in gonadectomized male and female rats estrogen in order to neutralize the unwanted peripheral effects have clearly established that treatment with DHEA leads to of the estrogen have been well described by Labrie et al. stimulatory androgenic and/or estrogenic effects on the pros (Labrie et al., 2003, Endocrinol, 144 (11): 4700-4706). In the tate, seminal vesicle and uterus, depending upon the target present study, when Acolbifene was given to OVX animals, tissue under investigation (Labrie et al., 1988, Endocrinol, the most typical morphological feature induced by the SERM 123: 1412-1417). was the appearance of a Superficial layer of well-aligned 0127. The combination of DHEA+Acolbifene, on the Small mucus cells overlying a basal cell layer, a pattern which other hand, shows a reduction in the extent of epithelial muci was slightly more pronounced in the Premarin-i-Acolbifene fication, thus displaying the alternance of a well-aligned group and which remained preponderant in all groups treated mucus cell layer—an Acolbifene effect—and invaginations with Acolbifene, including when the SERM was added to of multilayered hypertrophied mucous cells, which corre DHEA. Vaginal epithelium mucification under treatment spond to the androgenic effect of DHEA. Accordingly, Acol with an antiestrogen has been reported in immature (Ander bifene inhibits the partial estrogenic effect of DHEA, while son and Kang, 1975, Am J anat, 144(2): 197-207) and adult the majorandrogenic counterpart of DHEA is maintained and (Yoshida et al., 1998, Cancer Lett, 134(1): 43-51) rats. a small estrogen-like effect of Acolbifene is added, the latter Although this morphological pattern has been compared to being best seen at the ostium level. The vaginal weight the progesterone-induced mucification (Anderson and Kang, decrease observed after the addition of Acolbifene to DHEA 1975, Am Janat, 144(2): 197-207), the molecular mechanism is smaller than that obtained after addition of Acolbifene to by which an antiestrogen induces epithelial mucification Premarin, thus illustrating the Smaller estrogenic component remains unknown. of DHEA which is reversed by the antiestrogen, while the (0.124 Many beneficial effects of DHEA have been major androgenic component is not affected. In addition, the reported in postmenopausal women (Labrie et al., 1997, J. increases in lamina propria thickness and in compactness of Clin. Endocrinol. Metab., 82: 3498-3505; Labrie et al., 1991, the collagen fibers, when compared to those of OVX and Mol Cell Endocrinol, 78: C113-C118). Since no specific intact animals respectively, in the DHEA+Acolbifenegroup, receptor for DHEA has been characterized, the morphologi Suggest, again, a potential role of androgens in increasing cal changes observed in the rat vagina after DHEA treatment collagen compactness and thickness, since the estrogenic reflect its intracrine conversion into active sex steroids having component of DHEA is blocked by Acolbifene. androgenic and/or estrogenic action through intracrine 0128. Other studies have demonstrated that the action of mechanisms (Labrie et al., 1991, Mol Cell Endocrinol, 78: DHEA in the rat mammary gland (Sourla et al., 1998, Endo C113-C118). Those changes comprise intense epithelial crinol, 139: 753-764), skin sebaceous glands (Sourla et al., mucification, high compactness of delicate, finely woven 2000, J. Endocrinol, 166(2): 455-462) and bone mineral den lamina propria collagen fibers and moderate muscularis sity (Martelet al., 1998, J Endocrinol, 157:433-442) is almost thickness increase when compared to OVX animals. The two exclusively androgenic. Nevertheless, the presence of an first morphological changes are typical of androgenic effects estrogenic action of DHEA in the rat vagina has been previ while the third shows an estrogen-like activity, which is fur ously demonstrated through induction of vaginal opening and ther Supported by a concomitant increase in progesterone precocious ovulation in immature rats treated with this com receptor expression in the muscularis layer. pound, while DHT, an androgen not aromatizable to estro 0.125 Since DHEA is transformed into either or both gens, did not produce Such effects (Knudsen and Mahesh, androgens and estrogens in peripheral tissues, the thick muci 1975, Endocrinol, 97(2): 458-468). The capacity of rat vagi fied multilayered epithelium observed in the present study nal tissue to aromatize androgens, especially Testo, is likely after treatment of OVX animals with DHEA, suggests a pre to account for the major part of the estrogenic effect of DHEA dominant androgenic effect of mucification in the rat vagina, in this organ (Lephart et al., 1989, Biol Reprod, 40(2): 259 US 2016/0058774 A1 Mar. 3, 2016
267). Androst-5-ene-3?,17? 3-diol (5-diol), a DHEA metabo mized postmenopausal women, ERT is still used but with the lite known to bind the estrogen receptor (Shao et al., 1950, J well-known increased risk of developing breast cancer. More Biol Chem, 250: 3095-3100; Poortman et al., 1975, J. Clin over, ERT decreases serum androgen levels by increasing sex Endocrinol Metab, 40(3): 373-379; Van Doom et al., 1981, hormone binding globulin, which may induce a relative ova Endocrinol, 108: 1587-1594; Adams et al., 1981, Cancer Res, rian and adrenal androgen deficiency, thus creating an addi 41: 4720-4726), could also contribute to the estrogenic effect tional rationale for concurrent physiologic androgen replace (Poulin and Labrie, 1986, Cancer Res, 46: 4933-4937). The ment (Casson et al., 1997, ObstetGynecol, 90(6): 995-998). proposed combination of the antiestrogen Acolbifene with DHEA would thus prevent any unwanted stimulatory effect Example 2 of 5-diol. On the other hand, acolbifene would show addi tional benefits by preventing bone loss (Martel et al., 2000, J Methods Steroid Biochem Mol Biol. 74 (1-2): 45-56). 0.134. Animals and Treatment 0129. To the best of our knowledge, no previous study has 0.135 Ten to twelve week-old female Sprague-Dawley shown the stimulatory effects of DHEA on the compactness rats (Crl:CD(SD)Br) (Charles River Laboratory, St-Constant, and morphology of the lamina propria's collagen fibers and, Canada) weighing 215-265 g at time of ovariectomy were to a lesser extent, on the muscularis. Such actions of DHEA used. The animals were housed individually in an environ derived androgens and estrogens could have beneficial effects mentally-controlled room (temperature: 22+3°C.; humidity: on vaginal function in postmenopausal women and should 50+20%; 12-hlight-12-h dark cycles, lights on at 07:15 h). provide the substrates required for the action of inhibitors of The animals were allowed free access to tap water and a type 5 phosphodiesterase Such as Sildenafil or tadalafil, pos certified rodent feed (Lab Diet 5002 (pellet), Ralston Purina, sibly via androgen or estrogen-induced endothelial nitric St-Louis, Mo.). The experiment was conducted in an animal oxide synthase (eNOS)-mediated facilitation of vaginal facility approved by the Canadian Council on Animal Care smooth muscle relaxation (reviewed in Munarriz et al., 2003, (CCAC) and the Association for Assessment and Accredita J Urol, 170 (2 Pt 2): S40-S44, Discussion S44-S45). In fact, tion of Laboratory Animal Care (AAALAC) in accordance Acolbifene has been found to induce eNOS in human and rat with the CCAC Guide for Care and Use of Experimental endothelial cells (Simoncini et al., 2002, Endocrinol, 143(6): Animals. 2052-2061). 0.136 Different experiments were performed: two 20-day 0130. The results obtained in all the groups involving and two 8-month studies. In each of the two first experiments, DHEAtreatment reveal that AR expression is up-regulated by the animals were randomly distributed between 3 groups of the androgens derived from the intracrine transformation of 13 or 14 animals as follows: 1) Intact control; 2) Ovariecto DHEA in the rat vagina, in a similar fashion in the three tissue mized (OVX) control; 3) OVX+raloxifene (0.5 mg/kg), or, 3) compartments. OVX--LY 363381 (0.5 mg/kg). In one 8-month study, the 0131 The beneficial morphological changes, observed animals were randomly distributed between 5 groups of 7-8 concomitantly with the strong modulation of rat vaginal AR animals as follows: 1) Intact control; 2) Ovariectomized by androgens, Suggest that decreased serum levels of DHEA (OVX) control; 3) OVX+Acolbifene (0.5 mg/kg); 4) OVX+ derived androgens in postmenopausal women could contrib ERA-923 (0.5 mg/kg); 5) OVX+TSE-424 (0.5 mg/kg). In the ute to the decreased vaginal health and eventually to the loss other 8-month study, the animals were randomly distributed of libido and sexual enjoyment observed in this age group. between 3 groups of 10-11 animals as follows: 1) Intact Decreased serum total Testo, free Testo and DHEA-S were control; 2) Ovariectomized (OVX) control; 3) OVX--lasofox indeed found in women who consulted for decreased sexual ifene (0.5 mg/kg). On the first day of the study, the animals of desire (Guay and Jacobson, 2002, J Sex Marital Ther, 28 the appropriate groups were bilaterally ovariectomized Suppl 1: 129-142). (OVX) under isoflurane anesthesia. The tested compounds 0.132. In the present study, we have shown that treatment were then given once daily by oral gavage as a suspension in of OVX female rats with DHEA or the combination of DHEA 0.4% methylcellulose (0.5 ml/rat) from day 1 to day 20 of the with Acolbifene and of DHEA with Acolbifene and Premarin study or from day 1 to week 32 of the study. reversed the OVX-induced atrophic changes found in the 0.137 Twenty-four hours after the last dosing, the animals vagina through a predominant androgenic effect, via AR, were sacrificed under isoflurane anesthesia by exsan which was reflected by intense epithelial mucification and guination. The vaginae were collected and immersed in 10% increased compactness of the collagen fibers in the lamina neutral buffered formalin immediately after resection. The propria. Treatment with DHEA alone also moderately vaginae were then either cut in 7 cross-segments (for Acolb increased muscularis thickness. Such data underline the ifene, ERA-923 and TSE-424) or cut to sample only the importance of sex steroids synthesized locally from DHEA middle cross-segment. (raloxifene, lasofoxifene or LY and DHEA-S in peripheral target tissues. In addition to these 363381). The segments were then routinely processed and beneficial effects, Acolbifene reduced the inflammation inci embedded in paraffin blocks. When appropriate, all the seg dence, possibly through an estrogen-like induction of a pro ments were positioned in the paraffin block in a sequence tective keratinized squamous epithelium at the ostium level corresponding to their original anatomical position and ori and an antiestrogenic effect of mucification at the internal ented perpendicular to the surface of the block, thus allowing level. the segments to be cut in cross-sections. For each animal, a 4 0133. Various attempts have been made to solve the prob um-thick paraffin section was cut and stained with haema lem of vaginal dryness, often linked to dyspareunia and loss toxylin-eosin for morphological examination. of sexual enjoyment. As an example, local vaginal estrogen 0.138. In FIGS. 10A-10H and FIGS. 11A-11C is shown a preparations are often prescribed to provide relief but the comparison between different SERMs: vaginal epithelium endometrium may be stimulated by the unopposed estrogen morphology, in the OVX rat, at magnification 200x. FIGS. (Mattson et al., 1989, Maturitas, 11:217-222). In hysterecto 10A-10D show the epithelial morphology of the first segment US 2016/0058774 A1 Mar. 3, 2016 of the vagina (ostium). While the epithelium of the OVX amount of Suppository base pellets to weight in order to animals is thin (4-6 cell layers), atrophic and does not display obtain the final volume needed was calculated based on the a visible granular layer in FIG. 10A, the same epithelium is previously measured density of the Suppository base, which thicker in the 3 SERMs (8-11 cell layers) and displays a was 47.5 mg per 50 ul. The Suppository base was put in a multilayered granular compartment, in FIGS. 10B, 10C and small beaker, melted in microwave at power 50% for about 3 10D. In FIGS. 10E-10H and FIGS. 11A-11C, a segment min and 30 sec and then transferred on a heating plate, which situated in the middle of the vagina is illustrated. In the OVX was adjusted at the lowest power in order to keep the Supposi animals, the vaginal epithelium is thin and atrophic (2-3 cell tory base at a temperature of 50+3°C. The DHEA micronized layers), in FIG.10E, while the thickness is increased in all the powder (obtained from Scheweizerhall Inc. at a purity of treated groups (FIGS. 10E-10H and FIGS. 11A-11C), due to 100%) was then added in the base, all at once, the whole the presence of one (Acolbifene, ERA-923 and TSE-424) or clump left to sink and to wet for 3 min before being meticu many (raloxifene, lasofoxifene or LY 363381) mucous cell lously and slowly crushed and dissolved with a smooth layer(s) overlying the basal cell layer. rounded-end glass rod. Finally, the base with the dissolved DHEA (the placebo was the base without DHEA) was Example 3 poured, using a 1000 ul-Gilson pipette with a pre-heated pipette tip, in tube racks containing a series of 50 moulds. Materials and Methods These moulds were made from microcentrifuge tubes, each of them previously calibrated with 50 ul of water and cut at the 0139 Ten to twelve week-old female Sprague-Dawley level of the line drawn at the water surface. After 30 min of rats (Crl:CDR(SD)Br VAF/PlusTM) (Charles River Labora tory, St-Constant, Canada) weighing approximately 250-275 cooling at room temperature, the Suppositories were put at 4 g at Start of the experiment were used. The animals were C. for another 30 min. Finally, the excess of base was trimmed acclimatized to the environmental conditions (temperature: with a razor blade and the suppositories were removed from 22+3° C.; humidity: 50+20%; 12-hlight-12-h dark cycles, their moulds and stored in closed glass bottles at 4°C. until lights on at 07:15 h) for at least one week before starting the use. They were allowed to reach room temperature before experiment. The animals were housed individually and were being applied to the animals. allowed free access to water and rodent food (Lab Diet 5002, Histological Procedures Ralston Purina, St-Louis, Mo.). The experiment was con ducted in accordance with the CCAC Guide for Care and Use 0143. The organs were immersed in 10% neutral buffered of Experimental Animals in an animal facility approved by formalin immediately after resection. Each vagina and uterus the Canadian Council on Animal Care (CCAC) and the Asso was divided into 8 equal cross-segments and 4 cross-seg ciation for Assessment and Accreditation of Laboratory Ani ments including two from the cervix and two from each of the mal Care (AAALAC). two proximal and distal horns, respectively. The segments 0140. A total of 25 female rats were randomly distributed were then routinely processed and embedded all together in into 5 groups as follows: 1) Three ovariectomized untouched the same paraffin block. Within the paraffin block, all the control (OVX); 2) Four OVX-placebo suppository; 3) Six segments were positioned in a sequence corresponding to OVX+0.33 mg DHEA suppository; 4) Six OVX+0.66 mg their original anatomical position and oriented perpendicular DHEA suppository; 5) Six OVX-1.00 mg DHEA supposi to the surface of the block, thus allowing the segments to be tory. On the first day of the study, the animals of all groups cut in cross-sections. For each animal, a 4 um-thick paraffin were bilaterally ovariectomized (OVX) under isoflurane-in section was cut and stained with haematoxylin-eosin for mor duced anesthesia. The insertion into the vagina of one Sup phological examination. pository began on day 5 of the study and was continued once daily until day 18 of the study, for a total of 14 days of Results treatment. The Suppository was gently placed in the vaginal opening and then delicately pushed about 2-3 mm inside the Morphology of the Epithelium vagina with the help of a small, Smooth glass stick, disin 0144. After examination of all vaginal segments, it was fected in 70% alcohol and dried between each animal. found that segments 4 and 6 are representative to compare 0141 Twenty-four hours after the last dosing, the animals between treatments. Segment 4 was the most proximal to the were sacrificed under isoflurane anesthesia by exsan ostium which did not display features of the external skin, and guination. Vagina—including the urethra—and uterus were was not or to a lesser extent affected by the slight to moderate collected, and then trimmed as described below. For further inflammatory changes usually found in the external area of analysis, two pieces of inguinal mammary gland were the vaginal mucosa in control animals. Segment 6 represented sampled, as well as a square piece of about 2X2 cm of shaved the deeper vaginal mucosa and is separated from the uterine dorsal skin, the latter being put and gently flattened on a piece cervix by only one segment. of carton. In general, the Ventral side of the Vagina displays a thicker and a more mucified epithelium when compared to the dorsal Preparation of the Suppositories side.
0142. DHEA suppositories were prepared using Whitep Intact sol H-15 base (Medisca, Montreal, Canada). Any other lipo philic base such as Fattibase, Wecobee, cocoa butter, theo 0145 At estrus, which is under major estrogenic influ broma oil or other combinations of Whitepsol bases could ence, the vaginal epithelium is thick and keratinized. It dis also be used. The properamounts of DHEA were weighed to plays from 12 to 18 cell layers in segment 4 (FIG. 13A), and give final concentrations of 0.33 mg or 0.66 mg or 1.00 mg per 12 to 15 layers in segment 6 (FIG. 13C). Contrary to the thick suppository of a final volume of 50 ult3 ul. The proper stratified squamous epithelium seen at the ostium, the granu US 2016/0058774 A1 Mar. 3, 2016
lar layers are not obvious in the internal vaginal epithelium. alternance with 10 to 75% of hypertrophied mucous cells, At metestrus, under minimal hormonal influence, the vaginal overlying one or more squamous stratified epithelium layers. epithelium is thinner. It displays 6 to 10 cell layers in segment Non-mucified epithelial areas are also present. In segment 6 4 (FIG. 13B), and 5 to 7 layers in segment 6 (FIG. 13D). (FIG. 17B), the epithelial thickness is reduced and consists of a squamous stratified epithelium of 2 to 4 cell layers, Sur Ovariectomized Untouched mounted by a layer of well-aligned mucous cells. The amount of mucified areas varied from 20 to 80% of the vaginal sur 0146 In segment 4 (FIG. 14A), the vagina is lined by a face, in alternance with non-mucified areas. Of the six treated squamous stratified epithelium of 2 to 6 cell layers. The animals, two displayed mild inflammation in the three exter thickness of this segment is quite variable between animals. nal segments. In segment 6 (FIG. 14B), complete atrophy is observed, with DHEA 1.00 mg/Suppository 2 to 3 cell layers of flat cells. Some rare foci of small cylin 0150. In this group, the effect of DHEA is also discernible drical mucous cells could be seen throughout the whole in segment 2, but it increases significatively in segments 3-4 vagina of these animals. Of the three animals of this group, and 5 where the vast majority of hypertrophied mucous cells two displayed mild inflammatory changes in the external are observed, and very slowly decreases in segments 6-7 and three segments. 8, where the vast majority of well-aligned mucous cells are observed. Thus, in segment 4 (FIG. 18A), the stratified squa Placebo mous epithelium displays 3 to 9 cell layers. Hypertrophied 0147 In comparison with the OVX untouched group, the mucous cells overlying 3 to 4 squamous cell layers cover 50 vaginal squamous stratified epithelium of the placebo group to 70% of the vaginal surface, while well-aligned mucous was slightly thicker, with segment 4 (FIG.15A), displaying 4 cells cover 5 to 30% of the vaginal surface. Non-mucified to 8 cell layers. However, in segment 6 (FIG. 15B), the same areas are interspersed between mucified cells. In segment 6 morphology found in the OVX untouched group was (FIG. 18B), the epithelial thickness is also reduced and con observed, with 2-3 epithelial cell layers. As in OVX sists of 2 to 4 cell layers. Hypertrophied mucous cells over untouched animals, foci of Small cylindrical mucous cells lying 1 to 3 squamous cell layers cover about 30 to 100% of could be found throughout the vagina, slightly more when the vaginal surface while well-aligned mucous cells cover 0 to compared with the OVX untouched animals. Of the four 70%. Few interspersed non-mucified areas are also observed. animals of this group, three displayed mild inflammatory Of the six treated animals, four of them displayed mild changes in the external three segments. inflammation in the four external segments. DHEA 0.33 mg/Suppository 0148. In general, at low doses, the morphological effect of CONCLUSIONS DHEA is characterized by well-aligned cylindrical mucous 0151 1. The vehicle used for rat intravaginal treatment cells, while at higher doses, the mucous cells enlarge (hyper has desirable characteristics: trophy), vacuolate and pile up. In the present group, the effect 0152 a. Easily prepared in calibrated mould: of DHEA is slightly discernible in segment 2 and gradually 0153 b. Excellent capability to completely dissolve increases in segments 3-4, where there is an alternance of the DHEA micronized powder, thus assuring a stan well-aligned and hypertrophied mucous cells. The DHEA dardized dosage; effect slowly decreases in segments 5 and 6 where almost 0154 c. No significant tissue reaction and morpho only well-aligned mucous cells can be observed. The effect logical alterations of the vaginal mucosa: only a slight becomes very little in segment 7 and disappears in the lateral thickness increase of the epithelium was observed in vaginal folds of segment 8 (which includes the beginning of the first 3 to 4 segments, possibly due to the manipu the cervix). Thus, in segment 4 (FIG. 16A), the vaginal epi lations accompanying the insertion of the Supposi thelium is stratified squamous with 3 to 7 cell layers. About 20 tory. to 60% of the Vaginal lining consists of well-aligned mucous 0.155 2. DHEA produced an effect on the vaginal epi cells in alternance with 15 to 50% hypertrophied mucous thelial morphology at the lowest tested dose (0.33 cells, overlying one or more squamous stratified epithelium mg/suppository). The effect is maximal from the 3" to layers, and with non-mucified areas. In segment 6 (FIG.16B), the 5" segment, then decreases gradually to disappear at the epithelial thickness is reduced to almost the thickness found in OVX untouched animals. It consists of a squamous the level of the 7" segment. stratified epithelium of 2 to 5 cell layers, covered by a layer of 0156 3. At the DHEA dose of 0.66 mg/suppository, the well-aligned mucous cells. A large variation in the occurrence effect is also present at the3" segment and remains quite of mucous cells was observed, these cells covering from 5 to strong in the 6" segment. 75% of the vaginal surface, in alternance with non-mucified (O157 4. At the DHEA dose of 1.00 mg/suppository, the areas. Of the six treated animals, four of them displayed mild effect is strong from segment 2-3 to the deep vaginal inflammation, unevenly distributed over the whole vagina. folds neighbouring the uterine cervix. DHEA 0.66 mg/Suppository PHARMACEUTICAL COMPOSITION 0149. In this group, the effect of DHEA is discernible in segment 2 and increases significatively in segments 3-4 and 5 EXAMPLES where a majority of hypertrophied mucous cells are observed. 0158 Set forth below, by way of example and not of limi The number of hypertrophied mucous cells decreases in seg tation, are several pharmaceutical compositions utilizing pre ments 6 and 7 where the majority of cells are well-aligned ferred active SERMA.colbifene, preferred active sex steroid mucous cells. Thus, in segment 4 (FIG. 17A), the stratified precursor DHEA, preferred estrogen 173-estradiol or pre squamous epithelium is 3 to 6 cell layer-thick. About 5 to 20% marin, preferred type 5 c6MP phosphodiesterase inhibitor of the vaginal lining consists of well-aligned mucous cells in Sildenafil or Tadalafil. The concentration of active ingredient US 2016/0058774 A1 Mar. 3, 2016
may be varied over a wide range as discussed herein. The Example E amounts and types of other ingredients that may be included are well known in the art. (0163 Example A Tablet 0159) Weight% Ingredient (by weight of total composition)
Tablet Acolbifene S.O DHEA 1S.O Weight% Premarin O.S Ingredient (by weight of total composition) Gelatin S.O Lactose 63.0 DHEA 1S.O Starch 16.5 Gelatin S.O Lactose 63.5 Starch 16.5 Example F (0164 Example B
(0160 Gelatin capsule Weight% Gelatin capsule Ingredient (by weight of total composition) Weight% Acolbifene S.O Ingredient (by weight of total composition) DHEA 1S.O Premarin O.S DHEA 1S.O Lactose hydrous 64.5 Lactose hydrous 7O.O Starch 4.8 Starch 4.8 Cellulose microcrystalline 9.8 Cellulose microcrystalline 9.8 Magnesium stearate 0.4 Magnesium stearate 0.4
Example G Example C (0165 (0161
Tablet Tablet Weight% Weight% Ingredient (by weight of total composition) Ingredient (by weight of total composition) Sildenafil Acolbifene S.O DHEA DHEA 1S.O Gelatin S.O Gelatin S.O Lactose 48.5 Lactose 58.5 Starch 16.5 Starch 16.5
Example H Example D (0166 (0162 Gelatin capsule Gelatin capsule Weight% Weight% Ingredient (by weight of total composition) Ingredient (by weight of total composition) Sildenafil Acolbifene S.O Acolbifene DHEA 1S.O DHEA Lactose hydrous 6S.O Lactose hydrous Starch 4.8 Starch Cellulose microcrystalline 9.8 Cellulose microcrystalline Magnesium stearate 0.4 Magnesium stearate US 2016/0058774 A1 Mar. 3, 2016
Example I KIT EXAMPLES (0167 0171 Set forth below, by way of example and not of limi tation, are several kits utilizing preferred active SREM Acol bifene, preferred active a sex steroid precursor DHEA, pre Tablet ferred estrogens 17B-estradiol or conjugated estrogens, Weight% preferred type 5 c6MP phosphodiesterase inhibitor Sildena Ingredient (by weight of total composition) fil or Tadalafil. The concentration of active ingredient may be varied over a wide range as discussed herein. The amounts Acolbifene S.O DHEA 1S.O and types of other ingredients that may be included are well Sildenafil 1S.O known in the art. Gelatin S.O Lactose 43.5 Example A Starch 16.5 (0172. The SERM, estrogens and type 5 cQMP phosphodi esterase inhibitor are orally administered while the sex ste Example J roid precursor is applied locally or percutaneously adminis tered. (0168 SERM composition Gelatin capsule for oral administration (capsules Weight% Weight% Ingredient (by weight of total composition) Ingredient (by weight of total composition) Acolbifene S.O Acolbifene S.O DHEA 1S.O Lactose hydrous 80.0 Sildenafil 1S.O Starch 4.8 Lactose hydrous SO.O Cellulose microcrystalline 9.8 Starch 4.8 Magnesium stearate 0.4 Cellulose microcrystalline 9.8 Magnesium stearate 0.4
SERM + estrogens composition Example K for oral administration (capsules (0169 Weight% Ingredient (by weight of total composition) Acolbifene S.O Vaginal cream Premarin O.S Lactose hydrous 79.5 Weight % Starch 4.8 Ingredient (by weight of total composition) Cellulose microcrystalline 9.8 Magnesium stearate 0.4 DHEA 1.O acobifene O.2 Emulsifying Wax, NF 18.0 Light mineral oil, NF 12.0 Benzyl alcohol 1.O Ethanol 95% USP 33.8 SERM + estrogens + type 5 c6MP phosphodiesterase Purifed water, USP 34.0 inhibitor composition for Oral administration (capsules Weight% Ingredient (by weight of total composition) Example L Acolbifene S.O Premarin O.S 0170 Sildenafil 1S.O Lactose hydrous 64.5 Starch 4.8 Vaginal Suppository Cellulose microcrystalline 9.8 Magnesium stearate 0.4 Weight % Ingredient (by weight of total composition) DHEA O.66 to 2.0 Sex Steroid precursor composition for topical administration (gel Whitepsol H-15 base 98 to 99.34 Weight% Ingredient (by weight of total composition) DHEA suppositories were prepared using Whitepsol H-15 DHEA 2.O base (Medisca, Montreal, Canada). Any other lipophilic base Caprylic-capric Triglyceride S.O such as Fattibase, Wecobee, cocoa butter, theobroma oil or (Neobee M-5) other combinations of Whitepsol bases could used. US 2016/0058774 A1 Mar. 3, 2016
-continued of either or both of the layer lamina propria and/or the layer muscularis of the vagina in postmenopausal women, wherein Sex Steroid precursor composition for topical administration (gel said disease or condition is dyspareunia, said method com prising administering a sex Steroid precursor selected from Weight% the group consisting of dehydroepiandrosterone, dehydroe Ingredient (by weight of total composition) piandrosterone sulfate, androst-5-ene-3?,17(3-diol and 4-an Hexylene Glycol 1S.O drosten-3,17-dione to a patient in need of said treatment. Transcutol (diethyleneglycol S.O monomethyl ether) 33. The method of claim 32 further comprising the step of Benzyl alcohol 2.0 administering to said patient a therapeutically effective Cyclomethicone (Dow corning 345) S.O amount of a selective estrogen receptor modulator for uterine Ethanol (absolute) 64.O Hydroxypropylcellulose (1500 cps) 2.0 and mammary gland protection against cancer as part of a (KLUCEL) combination therapy. 34. The method of claim 33, wherein the selective estrogen receptor modulator is: Example B
(0173 The SERM and the sex steroid precursor are orally administered
Non-Steroidal Antiestrogen composition for oral administration capsules Weight% Ingredient (by weight of total composition) Acolbifene S.O Lactose hydrous 80.0 Acolbifene Starch 4.8 Cellulose microcrystalline 9.8 Magnesium stearate 0.4 35. The method of claim 32, wherein the sex steroid pre cursor is dehydroepiandrosterone. 36. The method of claim 33, wherein the sex steroid pre cursor is dehydroepiandrosterone. Sex Steroid precursor composition for oral administration 37. The method of either of claim 32 or 33, wherein the sex Gelatin capsule steroid precursor is administered intravaginally. Weight% 38. The method of either of claim 32 or 33, wherein the sex Ingredient (by weight of total composition) steroid precursor is orally or percutaneously administered. DHEA 1S.O 39. The method of claim 33, wherein the selective estrogen Lactose hydrous 7O.O Starch 4.8 receptor modulator is orally, percutaneously or intravaginally Cellulose microcrystalline 9.8 administered. Magnesium stearate 0.4 40. The method of claim 34, wherein the selective estrogen receptor modulator is orally, percutaneously or intravaginally (0174) Other SERMs (Toremifene, Ospemifene, Ralox administered. ifene, Arzoxifene, Lasofoxifene, TSE-424, ERA-923, 41. A method of treating dyspareunia in postmenopausal EM-800, SERM 3339, GW-5638) may be substituted for women said method comprising administering a sex steroid Acolbifene in the above formulations, as well as other sex precursor selected from the group consisting of dehydroepi steroid inhibitors may be substituted for DHEA, other estro androsterone, dehydroepiandrosterone Sulphate, androst-5- gens may be substituted for Premarin and other type 5 c6MP ene-3 ?.17 B-diol and 4-androsten-3,17-dione in association phosphodiesterase inhibitor may be substituted for Sildenafil with a therapeutically effective amount of a selective estrogen or Tadalafil or by prostaglandin E1. More than one SERM or receptor modulator for uterine and mammary gland protec more than one precursor or estrogens or type 5 c6MP phos tion against cancer to a patient in need of Such treatment. phodiesterase inhibitor may be included in which case the 42. The method of claim 41, wherein the sex steroid pre combined weight percentage is preferably that of the weight cursor and the selective estrogen receptor modulator are percentage for the single precursor or single SERM given in administered intravaginally, orally or percutaneously. the examples above. 43. The method of either of claim 33 or 41, wherein the 0.175. The invention has been described in terms of pre selective estrogen receptor modulator has a molecular for ferred embodiments and examples, but is not limited thereby. mula with the following features: Those of skill in the art will readily recognize the broader a) two aromatic rings spaced by 1 to 2 intervening carbon applicability and scope of the invention which is limited only atoms, both aromatic rings being either unsubstituted or by the patent claims herein. Substituted by a hydroxyl group or a group converted in 1.-31. (canceled) vivo to hydroxyl; and 32. A method of treating or reducing the likelihood of b) a side chain possessing an aromatic ring and a tertiary acquiring a vaginal disease or condition caused by theatrophy amine function or salt thereof. US 2016/0058774 A1 Mar. 3, 2016
44. The method of claim 43, wherein the selective estrogen Saturated, unsaturated or Substituted piperidino, Satu receptor modulator has the following formula: rated, unsaturated or Substituted piperidinyl, Saturated, unsaturated or substituted morpholino, nitrogen-con taining cyclic moiety, nitrogen-containing polycyclic moiety, and NRaRb, Ra and Rb being independently hydrogen, straight or branched C-C alkyl, straight or branched C-C alkenyl, and straight or branched C-C, alkynyl. 46. The method of claim 45, wherein said compound or salt Substantially lacks (2R)-enantiomer. 47. The method of claim 45, wherein the benzopyran derivative is a salt of an acid selected from the group consist ing of acetic acid, adipic acid, benzenesulfonic acid, benzoic wherein R and R are independently hydrogen, hydroxyl acid, camphorsulfonic acid, citric acid, fumaric acid, or a moiety which is converted to hydroxyl in vivo; hydroiodic acid, hydrobromic acid, hydrochloric acid, hydro wherein Z is a bivalent closing moiety; chlorothiazide acid, hydroxy-naphthoic acid, lactic acid, wherein the Roo is a bivalent moiety which distances L. maleic acid, methanesulfonic acid, methylsulfuric acid, 1.5- from the B-ring by 4-10 intervening atoms; naphthalenedisulfonic acid, nitric acid, palmitic acid, pivalic wherein L is a bivalent or trivalent polar moiety selected acid, phosphoric acid, propionic acid, Succinic acid, Sulfuric from the group of —SO , —CON<, N<, and acid, tartaric acid, terephthalic acid, p-toluenesulfonic acid, - SON<; and Valeric acid. wherein G is selected from the group consisting of hydro 48. The method of either of claim 33 or 41, wherein the gen, a C to Cs hydrocarbon or a bivalent moiety which selective estrogen receptor modulator is selected from the joins G and L to form a 5- to 7-membered heterocyclic group consisting of Raloxifene, Arzoxifene (LY353381), LY ring, and halo or unsaturated derivatives of the forego 335563, Tamoxifen, OH-Tamoxifen, Droloxifene, 1ng Toremifene, Idoxifen, Ospemifene, GW 5638, SERM 3339, wherein G is either absent or selected from the group TSE-424, ERA-923, Lasofoxifene (CP 336156), consisting of hydrogen, a C to Cs hydrocarbon or a Levormeloxifene, Acolbifene (EM-1538), EM-652 and bivalent moiety which joins G and L to form a 5- to EM-800. 7-membered heterocyclic ring, and halo or unsaturated 49. The method of claim 41 wherein the selective estrogen derivatives of the foregoing; receptor modulator is: wherein G is selected from the group consisting of hydro
gen, methyl and ethyl. 45. The method of claim 43, wherein the selective estrogen receptor modulator is an optically active compound having an absolute configuration S on carbon 2 or pharmaceutically acceptable salt thereof, said compound having the molecular Structure:
5
5 4 3' R Acolbifene 6 N 3 1' 2 R- 21 ... 1" 50. The method of any one of claims 39, 40, 41 and 42, wherein the sex steroid precursor is dehydroepiandrosterone. 7 8 O1 2 O R3 51. The method of claim 37, wherein the sex steroid pre 6" 4" 1N1 cursor is dehydroepiandrosterone. 52. The method of claim 38, wherein the sex steroid pre cursor is dehydroepiandrosterone. wherein R and R are independently selected from the group consisting of hydroxyland a moiety convertible in 53. The method of either of claim 33 or 41 wherein the vivo to hydroxyl: selective estrogen receptor modulator is Acolbifene and the wherein R is a species selected from the group consisting sex steroid precursor is dehydroepiandrosterone. of Saturated, unsaturated or substituted pyrrolidinyl, k k k k k