Estradiol absorption from vaginal tablets in postmenopausal women.

OBJECTIVE: To evaluate absorption of estradiol (E2) and compare two low doses of 17 beta-E2 (25 microgram and 10 microgram) in postmenopausal women with atrophic vaginitis. METHODS: In a double-masked, randomized, parallel-group study, 58 postmenopausal women were treated with 25 microgram or 10 microgram of 17 beta-E2 for 12 weeks. We report data for 42 eligible subjects who had serum E2 concentrations below 20 pg/mL at baseline and complete data available at the baseline visit (30 minutes before tablet insertion) and weeks 2 and 12. Serum E2 and FSH concentrations were measured at specified intervals. The area under the curve, maximal concentration, and time to maximal concentration were measured for serum E2 concentrations. Maturation values of vaginal epithelial cells were assessed as indicators of change in vaginal epithelium condition in response to treatment. RESULTS: After 12 weeks of treatment, the area under the curve, maximal and average over 24-hour E2 concentration were higher in the 25-microgram (563 pg. hour/mL, 49 and 23 pg/mL) than in the 10-microgram (264 pg. hour/mL, 22 and 11 pg/mL) group. Seventy-four percent in the 25-microgram and 96% in the 10-microgram groups had low systemic absorption of E2, that is, area under the curve (0-24 hour) less than 500 pg/mL. All but three women who received 25 microgram had mean FSH levels below 35 mIU/mL. CONCLUSION: Treatment with 25 or 10 microgram of 17 beta-E2 vaginal tablets resulted in low absorption of estrogen without systemic effects often associated with hormone replacement therapy. After 12 weeks of therapy for atrophic vaginitis, absorption patterns remained consistent, and women did not have accumulations of circulating E2.

Effects of continuous estrogen and estrogen-progestin replacement regimens on cardiovascular risk markers in postmenopausal women.

OBJECTIVE: To evaluate the influence of 2 continuous combined estrogen-progestin replacement products, compared with unopposed estrogen and placebo, on cardiovascular risk markers in postmenopausal women in a randomized, double-blind, placebo-controlled trial. METHODS: Two hundred seventy healthy postmenopausal women were randomly assigned to 1 of 4 treatment groups: placebo, unopposed 17-beta estradiol (1 mg), 1 mg of 17-beta estradiol with 0.25 mg of norethindrone acetate, or 1 mg of 17-beta estradiol with 0.5 mg of norethindrone acetate. The primary outcome variable was change from baseline in low-density lipoprotein cholesterol concentration. Additional outcome variables included changes in other serum lipid levels, hemostatic variables, and indicators of carbohydrate metabolism. RESULTS: The low-density lipoprotein cholesterol level was reduced to a similar degree in all groups receiving active treatment (10%-14% from baseline; P =.001 for 17-beta estradiol with 0.5 mg of norethindrone acetate, P =.004 for 17-beta estradiol with 0.25 mg of norethindrone acetate, and P =. 001 for 1 mg of 17-beta estradiol vs placebo). Compared with unopposed 17-beta estradiol, 17-beta estradiol with 0.5 mg of norethindrone acetate enhanced the reductions in total cholesterol and apolipoprotein B levels (P<.01 vs 17-beta estradiol). 17-beta Estradiol plus norethindrone blunted or reversed the increases in levels of high-density lipoprotein cholesterol, apolipoprotein A-I, and triglycerides produced by 17-beta estradiol alone. Effects of 17-beta estradiol plus norethindrone on hemostatic variables were similar to those of 17-beta estradiol except for factor VII activity, which was significantly reduced with 17-beta estradiol combined with 0.25 mg (P<.01) and 0.5 mg (P<.05) of norethindrone acetate. 17-beta Estradiol plus norethindrone appeared to blunt reductions in C-peptide and insulin levels produced by unopposed 17-beta estradiol but did not elevate these values compared with placebo. CONCLUSIONS: 17-beta Estradiol plus norethindrone produced favorable changes in most cardiovascular risk markers evaluated and has a profile distinct from that of unopposed 17-beta estradiol. The impact of these differences on cardiovascular events warrants investigation. Arch Intern Med. 2000;160:3315-3325.

Norethindrone acetate and estradiol-induced endometrial hyperplasia.

OBJECTIVE: To identify the lowest effective continuous dose of norethindrone acetate that significantly reduces 12-month incidence of endometrial hyperplasia associated with unopposed 17beta-estradiol (E2), 1 mg. METHODS: In a double-masked, randomized, multicenter study, 1176 healthy postmenopausal women 45 years of age or older without evidence of endometrial abnormalities were given 12 months of treatment with unopposed E2, 1 mg, or continuous-combined regimens of E2, 1 mg, and norethindrone acetate, 0.1 mg, 0.25 mg, or 0.5 mg. Endometrial histology was evaluated at the end of the treatment period. RESULTS: Continuous-combined E2-norethindrone acetate regimens significantly reduced 12-month incidence of endometrial hyperplasia compared with unopposed E2 1 mg (P <.001). Endometrial hyperplasia occurred in 14.6% of women treated with unopposed E2 1 mg, whereas in all continuous-combined groups, the rate decreased to less than 1%. Among patients who received E2-norethindrone acetate 0.1 mg, incidence was 0.8%; among those who received 0.25 mg and 0.5 mg, it was 0.4%. CONCLUSION: Continuous norethindrone acetate at doses as low as 0.1 mg combined with E2 1 mg effectively negated risk for endometrial hyperplasia associated with unopposed E2 1 mg, at least for the first year of therapy.

Initial 17beta-estradiol dose for treating vasomotor symptoms.

OBJECTIVE: To compare the efficacy of different doses of 17beta-estradiol (E2) for relief of vasomotor symptoms in menopausal women. METHODS: This was a randomized, double-masked, placebo-controlled, 12-week study in which 333 menopausal women with moderate or severe hot flushes were assigned to treatment with 0.25 mg, 0.5 mg, 1 mg, or 2 mg oral micronized 17beta-E2, or placebo. The number and severity of hot flushes were recorded daily. RESULTS: There was a significant linear correlation between increased dosage of 17beta-E2 and decreased moderate to severe hot flushes per week (P <.001). Rapid reduction in moderate to severe hot flushes was only achieved with 1 and 2 mg, showing a significant difference from placebo at week 4 (P <.05). At week 4, half the women on placebo had reduced moderate to severe hot flushes of at least 52%; the corresponding figures were 56%, 69%, 86%, and 91% for 0.25, 0.5, 1, and 2 mg, respectively. At week 12, all doses except 0.25 mg were significantly better than placebo for reducing moderate to severe hot flushes (P <.001). Although there were no significant differences, twice as many women in the 2-mg group discontinued treatment due to adverse events, compared with the placebo group. CONCLUSION: Oral micronized 17beta-E2 showed a dose-response effect for reducing moderate and severe hot flushes in menopausal women. 17beta-E2 1 mg appeared to be the most useful initial dose.

Uterine bleeding in postmenopausal women on continuous therapy with estradiol and norethindrone acetate. Endometrium Study Group.

OBJECTIVE: To investigate the incidence of uterine bleeding during 12 months of treatment with 17beta-estradiol (E2) 1 mg, unopposed or in combination with three doses of norethindrone acetate. METHODS: This study was a prospective, double-masked, randomized, multicenter trial. A total of 1176 healthy postmenopausal women age 45 years and older without evidence of endometrial abnormalities were randomly assigned to receive either unopposed E2 1 mg, or continuous-combined formulations of E2 1 mg and norethindrone acetate 0.1 mg, 0.25 mg, or 0.5 mg. Any spotting or bleeding episodes during the treatment period were recorded in a daily diary and reported by weekly telephone calls. RESULTS: The incidence of bleeding was low in the combination groups, even during the initial 3 months of treatment (24-28%), after which it decreased with increasing doses of norethindrone acetate. Conversely, the incidence of bleeding increased over time with unopposed E2 1 mg. After the initial 3 months, the incidence of bleeding among the combination groups was lowest in the norethindrone acetate 0.5 mg group. Among women initiating therapy close to menopause, fewer reported bleeding with norethindrone acetate 0.5 mg than with the other combination groups. There was a significantly (P<.05) lower discontinuation rate due to bleeding in the norethindrone acetate 0.5 mg group compared with all other treatment groups. CONCLUSION: Continuous-combined formulations of E2 1 mg with norethindrone acetate 0.1, 0.25, or 0.5 mg are associated with a low incidence of uterine bleeding. After the initial 3 months of treatment, bleeding profiles improved with increasing doses of norethindrone acetate.

Identification and distribution in the rat of acidic metabolites of tamoxifen.

Chromatographic analysis of acidic fractions of urinary radioactivity from immature female rats which had received the triarylethylene antiestrogen tamoxifen (TAM), labeled with 14C, resulted in the identification of two new metabolites. These were tamoxifen acid (TA), in which the side chain of TAM was changed to an oxyacetic acid moiety, and 4-hydroxytamoxifen acid (4-HTA), which had a similarly altered side chain plus a phenolic hydroxy group in its structure. In contrast to other TAM metabolites and other arylacetic acids, neither TA nor 4-HTA were eliminated as glucuronic acid or glycine conjugates in urine. Only small amounts of acidic radioactivity were found in liver tissue 24 and 48 hr after dosing, and none was detected in uterine tissue. However, TA plus 4-HTA accounted for 2.8% and 2.9% of the administered dose eliminated within 24 hr in urine and feces, respectively. These results suggest that TA and 4-HTA are important final products of TAM metabolism and that these, unlike other hydroxylated and/or dealkylated metabolites of this drug, may not contribute to the antiuterotrophic effects of TAM.

Phenolic metabolites of clomiphene: [(E,Z)-2-[4-(1,2-diphenyl-2-chlorovinyl)phenoxy]ethyl]diethylamine. Preparation, electrophilicity, and effects in MCF 7 breast cancer cells.

The triarylethylene antiestrogen clomiphene was previously shown to undergo biotransformation to an active metabolite, 4-hydroxyclomiphene, and to 3-methoxy-4-hydroxyclomiphene plus the respective regioisomers of these, 4 and 5. We now report the synthesis and further chemical and biochemical studies on 3-5. Coupling of 4-[2-(diethylamino)ethoxy]benzophenone with either 4-(benzyloxy)benzaldehyde or its 3-methoxy analogue 11b in the presence of titanium, followed by chlorination and deprotection of the intermediate triarylethylenes, gave 4 and 5, respectively. Condensation of benzylmagnesium chloride with the (2-methoxyethoxy)methyl (MEM) ether of 4-[2-(diethylamino)ethoxy]-3'-methoxy-4'-hydroxybenzophenone, followed by mild acid treatment, afforded deschloro 3 due to facile MEM ether hydrolysis. Acetylation of this, followed by chlorination and deacetylation, gave 3. Compounds 4 and 5 reacted readily with nucleophiles. In particular, 2-mercaptoethanol reacted with 4 to afford deschloro vinyl thioether 13 as suggested by NMR spectral studies, a result that implicated allene-quinone 14 as the electrophilic species produced in solution from 4. Antiestrogen binding sites and estrogen receptors from MCF 7 human breast cancer cells interacted with 3 and 5 with affinities comparable to those of tamoxifen and 1, respectively; 5 was shown not to bind irreversibly with these sites. Inhibition of MCF 7 cell proliferation by 3-5 at 5 microM concentrations (76%, 57%, and 49%, respectively, relative to drug-free controls) compared favorably to that observed with 5 microM 1 (80%). These results suggest that 3-5 as well as 2 may contribute to the antiestrogenic effects of 1.

Synthesis and estrogen receptor selectivity of 1,1-bis(4-hydroxyphenyl)-2-(p-halophenyl)ethylenes.

A series of triarylethylenes (1a-e) were synthesized and evaluated for their ability to compete with [3H]estradiol for high-affinity estrogen receptors (ER) in immature rat uterine cytosol. All compounds showed affinity comparable to that of estradiol, with 1c having the highest affinity and the lowest calculated nonspecific binding of the para-halogenated members. Compound 1a had a higher affinity than did its chlorovinyl counterpart 1b, indicating that a vinyl hydrogen was suitable for high ER affinity in this series. Compound 1c was labeled with 3H ortho to one or both of its hydroxyls. Its ratio of specific to nonspecific binding in rat uterine cytosol, 3.2, was 140% of that of a related triarylethylene, 4-hydroxytamoxifen, and was 24% that of estradiol. Administration of [3H]-1c to immature female rats resulted in accumulation of 3H in uterine tissue which was decreased 39% when [3H]-1c was coadministered with estradiol. The major site of accumulation 1, 4, and 8 h after administration was in the intestinal tract. Chromatographic analysis showed that levels of 1c were less than those of 1c glucuronide in blood plasma, liver, and intestinal contents of rats 1 h after administration of 1c. Uterine 3H was comprised of 85% of 1c and 11% of 1c glucuronide. These results indicate that 1c undergoes ER-mediated uptake in the immature female rat, but selectivity is reduced due to nonspecific accumulation of free and conjugated 1c in uterine tissue.

Synthesis and biological evaluation of poly-gamma-glutamyl metabolites of 10-deazaaminopterin and 10-ethyl-10-deazaaminopterin.

The chemical synthesis of a series of poly-gamma-glutamyl metabolites of the experimental anticancer drugs 10-deazaaminopterin (10-DAAM) and 10-ethyl-10-deazaaminopterin (10-EDAAM) has been carried out by the solid-phase procedure. The synthetic products were identical with the poly-gamma-glutamyl metabolites of radiolabeled 10-DAAM and 10-EDAAM produced by normal mouse tissues with regard to elution volume from [(diethylamino)ethyl]cellulose columns and susceptibility to hydrolysis by human plasma folylpolyglutamate hydrolase. Poly-gamma-glutamyl metabolites with a glutamate chain length of up to four glutamate residues were detected in the tissues. The antifolate activity was evaluated with methotrexate (MTX) sensitive and MTX-resistant strains of Lactobacillus casei and Streptococcus faecium. In general, inhibitory potency decreases with increasing Glu chain length. However there are two exceptions. Addition of one Glu residue to 10-DAAM enhances its potency for MTX-resistant L. casei and addition of one Glu residue to 10-EDAAM enhances its potency for the MTX-sensitive L. casei. As shown earlier for MTX polyglutamates, polyglutamylation greatly enhances the inhibitory potency of 10-DAAM and 10-EDAAM for L. casei thymidylate synthase. MTX polyglutamates are 15-30 times more inhibitory than the corresponding 10-DAAM derivatives and 30-60 times more inhibitory than the corresponding 10-EDAAM derivatives. Polyglutamylation of 10-DAAM had little influence on its ability to inhibit L. casei dihydrofolate reductase; however, with 10-EDAAM, addition of one or two Glu residues enhanced its inhibitory potency 2.3-fold.

Biotransformation of the antiestrogen clomiphene to chemically reactive metabolites in the immature female rat.

Novel metabolites of clomiphene (CLO), an antiestrogen effective in experimental breast cancer models, were characterized in studies using immature female rats. After i.p. administration, CLO was eliminated unchanged in feces and as two components which were chromatographically identical to synthetic CLO analogues bearing a m-methoxy-p-hydroxyphenyl (guaiacol) moiety in place of one or the other of its phenyl rings. These components were also found in liver tissue. In the presence of liver microsomal homogenate, CLO underwent p-hydroxylation of either of its phenyl rings, affording 4-hydroxy-CLO and 4'-hydroxy-CLO. These in turn underwent liver microsomal mediated conversion to the respective guaiacol metabolites. 4'-Hydroxy-CLO and its 3'-methoxy analogue, but not positional isomers of these, had arylating ability as shown by chemical and spectral studies, apparently due to spontaneous conversion to electrophilic allene-quinones. Reproductive tract abnormalities produced in neonatal rats by CLO were suggested not to be mediated via such metabolites, since similar such effects were caused by 4'-fluoro-CLO. However, the latent arylating potential of the 4'-hydroxylated metabolites of CLO suggests that these compounds may be useful in biochemical studies of breast cancer cell growth inhibition.

Folate analogues. 26. Syntheses and antifolate activity of 10-substituted derivatives of 5,8-dideazafolic acid and of the poly-gamma-glutamyl metabolites of N10-propargyl-5,8-dideazafolic acid (PDDF).

The poly-gamma-glutamyl derivatives of n10-propargyl-5,8-dideazafolic acid (PDDF) with a chain length of up to five glutamate residues were synthesized from N10-propargyl-5,8-dideazapteroic acid by the solid-phase procedure. These compounds were evaluated for their antifolate activity using folate-requiring microorganisms and intact and permeabilized L1210 cells and as inhibitors of dihydrofolate reductase and thymidylate synthase derived from L. casei. The polyglutamylated derivatives of PDDF (1) were more active than the parent compound in inhibiting the growth of L. casei, thymidylate synthesis in permeabilized L1210 cells, and L. casei thymidylate synthase. Two analogues of 5,8-dideazafolic acid (2 and 3), one with a 2-butyne and another with a cyclopropylmethyl substituent at N10, were also synthesized and evaluated for their antifolate activities using the above-mentioned test systems. They were considerably less active than PDDF or its polyglutamylated derivatives. N10-Propargyl-5,8-dideazapteroyl tri-, tetra-, and pentaglutamates were equipotent with 5-fluorodeoxyuridylate as inhibitors of thymidylate synthesis in permeabilized L1210 cells. The polyglutamyl metabolites of PDDF were shown to be the most potent antifolate inhibitors of L. casei and L1210 thymidylate synthases yet described.

Mesoionic purinone analogues as inhibitors of cyclic-AMP phosphodiesterase: a comparison of several ring systems.

Several simple alkyl and aralkyl derivatives of mesoionic thiazolopyrimidines (1) and mesoionic 1,3,4-thiadiazolopyrimidines (2) were found to possess theophylline-like activity as inhibitors of cyclic-AMP phosphodiesterase (PDE). Reduction of the C2-C3 double bond of 1 or replacement of the sulfur atom of 1 or 2 with an N-methyl group nearly abolishes activity. Optimal activity appears to be associated with a hydrophobic substituent at the N8 position. The five-membered ring of 1 can be replaced by a pyridine or isoquinoline nucleus without untoward effects. Preliminary kinetic data suggest that the type of enzyme inhibition produced by the mesoionic derivatives is similar to that observed for theophylline. Thus, several novel mesoionic ring systems display activity as inhibitors of cyclic-AMP PDE and can serve as lead compounds for further investigation.