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Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.

Type I and type II steroid 5alpha-reductases (5alpha-R) catalyze the conversion of testosterone (T) to dihydrotestosterone (DHT). LY320236 is a benzoquinolinone (BQ) that inhibits 5alpha-R activity in human scalp skin (Ki(typeI)=28.7+/-1.87 nM) and prostatic homogenates (Ki(typeII)=10.6+/-4.5 nM). Lineweaver-Burk, Dixon, and non-linear analysis methods were used to evaluate the kinetics of 5alpha-R inhibition by LY320236. Non-linear modeling of experimental data evaluated V(max) in the presence or absence of LY320236. Experimental data modeled to the following equation 1v=+ fixing the In0c value equal to 1.0 or 0 are consistent with non-competitive or competitive inhibition, respectively. LY320236 is a competitive inhibitor of type I 5alpha-R (In0c=0, Ki=3.39+/-0.38, RMSE = 1.300) and a non-competitive inhibitor of type II 5alpha-R (In0c=1, Ki=29. 7+/-3.4, RMSE = 0.0592). These data are in agreement with linear transformation of the data using Lineweaver-Burk and Dixon analyses. These enzyme kinetic data support the contention that the BQ LY320236 is a potent dual inhibitor with differing modes of activity against the two known human 5alpha-reductase isozymes. LY320236 represents a class of non-steroidal 5alpha-R inhibitors with potential therapeutic utility in treating a variety of androgen dependent disorders.

We evaluated the metabolic inhibitory, antiproliferative, and antisecretory effects of LY300502, a benzoquinolinone human-specific type I-selective steroid 5α-reductase inhibitor in LNCaP human prostatic adenocarcinoma cell cultures. Reductive metabolism of [ 3 H-T] in the LNCaP cells was inhibited in a concentration-dependent manner by LY300502 (IC 50 ≃ 5.77 nM). The proliferative responses of LNCaP cells to LY300502 were examined in the presence of 0.1 nM testosterone (T), a concentration that stimulates maximal LNCaP cellular proliferation. Addition of 0.1 nM T to the culture media significantly stimulated LNCaP cell numbers 40% above control levels. LY300502 significantly antagonized T-induced stimulation of LNCaP cellular proliferation at concentrations greater than 10 nM (P < 0.05), and at 1,000 nM completely blocked the mitogenic effects of T on LNCaP cells. In the absence of androgen, LY300502 had no effect on LNCaP cellular proliferation. In the presence of 100 nM T, an androgen concentration that maximally stimulates in vitro PSA production, LY300502 significantly antagonized T-induced PSA secretion at a concentration equal to or greater than 30 nM (P < 0.05). These studies provide the basis for additional investigations into the pathophysiologic significance of type I 5α-reductase to prostatic cancer and the potential utility of selective inhibitors as therapeutic agents.

The benzothiophene antiestrogen, raloxifene (LY156758), has selective estrogen pharmacological antagonist activity in rats. The PAIII rat prostatic adenocarcinoma model was used to evaluate the effects of this agent on the lymphatic and pulmonary metastasis and survival in tumor-bearing male Lobund-Wistar (LW) rats. Reloxifene was inactive against colony formation of PAIII cells in vitro. Similarly, following subcutaneous (s.c.) implantation of 106 PAIII cells in the tail, s.c. administration of raloxifene (2.0, 10.0, or 20.0 mg/kg/day) for 30 days failed to demonstrate cytoreductive activity against primary tumor growth in the tail. However, in these same animals, raloxifene administration produced significant (P < 0.05) inhibition of PAIII metastasis from the primary tumor in the tail to the gluteal and iliac lymph nodes (maximal responses = 89% and 81% from control values, respectively). PAIII metastasis to the lungs was significantly inhibited by raloxifene treatment. Numbers of pulmonary foci in PAIII-bearing rats were significantly (P < 0.05) reduced by raloxifene administration in a dose-related manner (maximal reduction = 97% from control values). In these animals, maximal regression of 20% for ventral prostate and 21% for seminal vesicle were also seen after raloxifene administration (P < 0.05 for both). Coadministration of E2B and raloxifene had no consistent antagonistic effect upon the antitumor responses produced by raloxifene. Raloxifene (40.0 mg/kg/day for 28 days) produced marked decreases in PAIII metastasis in the lymphatic and pulmonary components. Continued administration of the compound produced significant (P < 0.05) extension of survival of PAIII-bearing rats. Further studies are needed to define the maximal antitumor efficacy and the mechanism of action of raloxifene in urogenital solid tumor animal models. These data support the contention that raloxifene represents a class of active antimetastatic agents with potential efficacy in the treatment of hormone-insensitive human prostatic cancer. © 1995 Wiley-Liss, Inc.

The benzothiophene anti-estrogen, raloxifene [LY156758; (6-hydroxy-2-(4-hydroxyphenyl) benzo(b)thien-3-yl)(4-(2-1-piperidinyl)ethoxy)phenyl methanone hydrochloride] has selective estrogen pharmacological antagonist activity in female rats. The present studies were done in the male rat to assess activity of raloxifene related to inhibition of prostatic growth and effects on the hypothalamic-pituitary-gonadal axis. Raloxifene did not compete for binding of the androgen, [3H]-methyltrienolone (R1881) in cytosolic extracts of ventral prostate. Similarly, the compound did not inhibit prostatic 5 alpha-reductase or testicular 17 alpha-hydroxy/C17,20-lyase activities. Raloxifene had no effect on the ventral prostatic uptake of [3H]-R1881 in vivo. Administration of estradiol to castrated male rats stimulated fourfold increases of in vitro ventral prostatic binding of [3H]-R1881. Raloxifene was devoid of agonist activity in castrated animals, because the compound had no stimulatory effect on prostatic androgen receptor binding activity. When raloxifene was coadministered with estradiol, the compound markedly antagonized the estrogen-induced increase of prostatic [3H]-R1881 binding, confirming its antiestrogenic properties in male rats. Serum prolactin was also elevated significantly (P < 0.05) with a single injection of raloxifene (20.0 mg/kg). In these same animals, serum FSH was significantly (P < 0.05) decreased by one dose (10.0 mg/kg) of the compound. Luteinizing hormone levels in castrated male rats were unaffected by raloxifene administration. Raloxifene treatment of castrated males significantly (P < 0.05) antagonized the stimulatory response of the ventral prostate (VP) to exogenous androgens in a dose-dependent manner. Raloxifene treatment of intact male rats for 14 and 28 days produced significant (P < 0.05) dose-dependent regression of the VP and seminal vesicles (SV). The VP regressive responses to raloxifene were associated with a decline in serum testosterone levels. Histological analysis of the VPs in raloxifene-treated rats was consistent with an androgen-deprived state. These findings support the contention that raloxifene is a pure estrogen antagonist and a physiological antagonist of androgen action in male rats. These pharmacological properties provide support for further structure-activity and mechanistic investigations with benzothiophenes in the medical management of prostatic neoplasia.

LY207320 is an in vitro inhibitor (estimated IC50 = 0.06 microM) of steroid 5 alpha-reductase that catalyzes the conversion of testosterone (T) to dihydrotestosterone (DHT). In contrast, LY207320 was only moderately active against rat prostatic 5 alpha-reductase in vivo (32% inhibition at 50.0 mg/kg single dose). LY207320 did, however, inhibit the in vivo uptake of [3H]-T by the prostate. The antiprostatic and endocrine effects of this agent were evaluated following daily (21 days) administration to castrated, androgen-supplemented castrate, and intact rats. LY207320, which has modest progestational competitive binding activity, does not bind to rat prostatic androgen or uterine estrogen cytosolic receptors. In the castrated male rat, subcutaneously (s.c.) administered LY207320 had no androgen agonist activity, as evidenced by a lack of accessory sex organ weight gains. Administration of s.c. LY207320 to intact rats for 21 days at doses greater than 5.0 mg/kg-day produced significant (P0.05) reductions of seminal vesicle and ventral prostatic weights (maximal regression = -65% and -40% from control values, respectively at 50.0 mg/kg-day). The compound had no regressive activity on male accessory sex organs when administered orally. LY207320 did not alter circulating prolactin, LH, or corticosterone levels, but at high doses (or = 50.0 mg/kg-day), lowered circulating T[-67% from intact control levels (P0.05)]. Histological analysis of the rat ventral prostates (RVPs) in LY207320-treated rats was consistent with an androgen-deprived state. Decreased circulating androgens and prostatic regression are associated with inhibition of testicular 17 alpha-hydroxy/C17,20-lyase enzyme activity (IC50 = 0.06 microM). These findings support the contention that LY207320 is a physiological antagonist of androgen action in male rats, and that its effects are mediated primarily through inhibition of testicular androgen production rather than accessory sex organ 5 alpha-reductase.

Mark E. Stearns (Chairperson),1* Joy L. Ware (Rapporteur),2 David B. Agus,3 Ching-Jer Chang,4 Isaiah J. Fidler,5 Rose S. Fife,6 Robin Goode,7 Eric Holmes,8 Michael S. Kinch,9 Donna M. Peehl,10 Thomas G. Pretlow II,11 and George N. Thalmann12 1Department of Pathology, Medical College of Pennsylvania and Hahnemann University, Philadelphia, Pennsylvania 2Department of Pathology, Virginia Commonwealth University, Richmond, Virginia 3Memorial Sloan-Kettering Cancer Center, New York, New York 4Department of Medicinal Chemistry, Purdue University, West Lafayette, Indiana 5Department of Cell Biology, University of Texas M.D. Anderson Cancer Center, Houston, Texas 6Indiana University School of Medicine, Indianapolis, Indiana 7Lilly Research Laboratories, Indianapolis, Indiana 8Pacific Northwest Cancer Foundation, Seattle, Washington 9Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 10Department of Urology, Stanford University, Stanford, California 11Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 12Universitat Bern, Bern, Switzerland

Activation of protein kinase Cβ (PKCβ) has been repeatedly implicated in tumor-induced angiogenesis. The PKCβ-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses angiogenesis and was advanced for clinical development based upon this antiangiogenic activity. Activation of PKCβ has now also been implicated in tumor cell proliferation, apoptosis, and tumor invasiveness. Herein, we show that Enzastaurin has a direct effect on human tumor cells, inducing apoptosis and suppressing the proliferation of cultured tumor cells. Enzastaurin treatment also suppresses the phosphorylation of GSK3βser9, ribosomal protein S6S240/244, and AKTThr308. Oral dosing with Enzastaurin to yield plasma concentrations similar to those achieved in clinical trials significantly suppresses the growth of human glioblastoma and colon carcinoma xenografts. As in cultured tumor cells, Enzastaurin treatment suppresses the phosphorylation of GSK3β in these xenograft tumor tissues. Enzastaurin treatment also suppresses GSK3β phosphorylation to a similar extent in peripheral blood mononuclear cells (PBMCs) from these treated mice. These data show that Enzastaurin has a direct antitumor effect and that Enzastaurin treatment suppresses GSK3β phosphorylation in both tumor tissue and in PBMCs, suggesting that GSK3β phosphorylation may serve as a reliable pharmacodynamic marker for Enzastaurin activity. With previously published reports, these data support the notion that Enzastaurin suppresses tumor growth through multiple mechanisms: direct suppression of tumor cell proliferation and the induction of tumor cell death coupled to the indirect effect of suppressing tumor-induced angiogenesis.

Trioxifene (LY133314) is a selective estrogen receptor modulator (SERM) with competitive binding activity against estradiol for estrogen receptor alpha (ERalpha) and antagonistic activity against ERalpha-mediated gene expression. The PAIII rat prostatic adenocarcinoma (PCa) is an androgen receptor-negative, ERalpha- and ERbeta-positive, spontaneously metastatic rodent tumor cell line. After s.c. implantation of 10(6) PAIII cells in the tail, s.c. administration of trioxifene (2.0, 4.0, 20.0, or 40.0 mg/kg-day) for 30 days produced significant (P0.05) inhibition of PAIII metastasis from the primary tumor in the tail to the gluteal and iliac lymph nodes (maximum nodal weight decreases, 86% and 88% from control values, respectively). PAIII metastasis to the lungs was significantly inhibited by trioxifene treatment. Numbers of pulmonary foci in PAIII-bearing rats were significantly (P0.05) reduced by trioxifene administration in a dose-related manner (maximal reduction, 98% from control values). Continual administration of the compound significantly (P0.05) extended survival of PAIII-bearing rats. Trioxifene inhibited the proliferation of PAIII cells at micromolar levels in vitro but did not slow growth of the primary tumor growth in the tail. Trioxifene administration also produced regression of male accessory sex organs. In PAIII-tumor-bearing animals, trioxifene administration produced a maximal regression of 76% for ventral prostate and 64% for seminal vesicle (P0.05 for both). SERMs may be preferable to estrogens given their efficacy in experimental PCa models and relative lack of side effects observed in clinical trials. Our data support the contention that trioxifene represents a SERM with potential antimetastatic efficacy for the treatment of androgen-independent, metastatic PCa.

Background Prostate-specific antigen (PSA) is a serine protease that can cleave insulin-like growth factor-binding protein-3 (IGFBP3), thereby decreasing its affinity for insulin-like growth factor-I (IGF-I). Dissociation of the IGF-I-IGFBP3 complex renders IGF-I available to bind to its receptor and stimulates cellular proliferation. We evaluated the potential for PSA to modulate the effects of IGF-I and IGFBP3 on the proliferation of human benign prostatic hyperplasia (BPH)-derived fibromuscular stromal cells in primary cultures. Methods We cultured BPH-derived stromal cells for 48 hours in serum-free RPMI-1640 medium supplemented with 0.2% bovine serum albumin and studied the effects of IGF-I, IGFBP3, PSA, and ZnCl(2) at varying concentrations. Differences in cell growth between control and treated cultures were evaluated by use of Dunnett's test. Concentration-related trends were evaluated by linear regression of log-transformed concentrations of test reagents on BPH-derived stromal cell number responses. Statistical tests were two-sided. Results We observed a concentration-dependent proliferative response of BPH-derived stromal cells to IGF-I. IGFBP3 inhibited this response in a concentration-dependent fashion. IGFBP3 alone had no effect on stromal cell proliferation. When stromal cells were incubated with PSA alone or with PSA, IGF-I, and IGFBP3, an increase in stromal cell numbers that was dependent on PSA concentration was evident in both instances. Zinc, an endogenous inhibitor of PSA enzymatic activity, was able to attenuate the stimulatory effect of PSA at intraprostatic physiologic concentrations. Conclusions These results are consistent with the idea that PSA can modulate in vitro interactions between IGF-I and IGFBP3 and suggest that PSA may play a role in the regulation of human prostatic fibromuscular cell growth.

The conversion of testosterone (T) to dihydrotestosterone (DHT) has been demonstrated to be catalysed by at least two isoforms of human steroid 5 alpha-reductase, designated types I and II. Type II 5 alpha-reductase expression predominates in human accessory sex tissues, localized to the fibromuscular stromal compartment. The type I isoform predominates in skin, prostatic epithelia and, to a lesser extent, in prostatic fibromuscular stroma. The significance of the type I isoform to prostatic cellular growth and function remains undefined. In cultured DU145 cells, we evaluated the metabolism of [14C]-T and demonstrated the time-dependent formation of [14C]-DHT. Oxidative metabolism (conversion of [14C]-T to [14C]-androstenedione) and the formation of conjugated androgen metabolites occurred at a relatively low rate in the DU145 cells. Using human type I 5 alpha-reductase cDNA, Northern blot analysis of DU145 cell mRNA revealed high levels of type I isoform expression. Analogous probing of the DU145 cells with a human 5 alpha-reductase II cDNA failed to reveal expression of the type II isoform. The expression of functional type I activity has been confirmed pharmacologically using isoform-selective 5 alpha-reductase inhibitors. Reductive metabolism of [3H]-T in the DU145 cells was inhibited in a concentration-dependent manner by LY306089, a potent non-steroidal type I-selective inhibitor (IC50 = 10.0 nM). SKF105657, a steroidal type II-specific inhibitor was distinctly less active at inhibiting [3H]-DHT formation. LY306089 was a non-competitive inhibitor of type I 5 alpha-reductase in DU145 cellular homogenates with an apparent Ki value of 4.0 nM. These studies have identified and pharmacologically defined type I 5 alpha-reductase activity in an androgen-insensitive prostatic cancer cell line and provide the basis for additional investigations into the significance of type I 5 alpha-reductase to human prostatic pathophysiology.

LY181984 is a compound in a series of orally active diarylsulfonylureas with broad spectrum in vivo activity against syngeneic rodent and human xenograft tumor models. The PAIII rat prostatic adenocarcinoma model was used to evaluate the effects of this antitumor agent on the lymphatic and pulmonary metastasis of the tumor in male Lobund Wistar rats. LY181984 was inactive against the proliferation of PAIII cells in vitro. Following subcutaneous implantation of 10(6) PAIII cells in the tail, oral administration of LY181984 (25.0, 50.0, or 100.0 mg./kg./day) for 30 days had no significant effects on body weight gain. LY181984 treatment produced significant (p less than 0.05) dose-dependent inhibition of primary tumor growth in the tail (max. inhibition = 46% from untreated control levels). In these same animals, LY181984 administration produced significant (p less than 0.05) dose-dependent inhibiton of PAIII metastasis from the primary tumor in the tail to the gluteal and iliac lymph nodes (maximal responses = 79% and 80% from control values, respectively). PAIII metastasis to the lungs was significantly inhibited by oral LY181984 treatment. Numbers of pulmonary foci in PAIII-bearing rats were significantly (p less than 0.05) reduced by LY181984 administration in a dose-dependent manner (maximal reduction = 78% from control values). While the non-toxic doses (less than 100.0 mg./kg./day for 28 days) of LY181984 produced marked decreases in tumor growth and metastasis, administration of the compound had no effect on the survival of PAIII-bearing rats. These data support the contention that LY181984 represents a new class of orally active antitumor and antimetastatic agents with potential efficacy in the treatment of hormone-insensitive prostatic cancer. Further studies are needed to define maximal efficacy of LY181984 and other sulfonylurea agents in urogenital solid tumor animal models.

An in vivo assay for steroid 5 alpha-reductase in rat ventral prostate has been developed and used to compare the inhibitory activity of N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and 6-methylene-4-pregnene-3,20-dione (LY207320). Immature rats (70-80 g) received test compounds 30 min prior to s.c. injection of [3H]-T. The rats were sacrificed 30 min later and the ventral prostates were analyzed for [3H]-T metabolites. Intraprostatic [3H]-T and [3H]-DHT reached peak levels within 5 min after injection of [3H]-T and declined to about 25% of peak levels after 2 hr. 4-MA was a very potent inhibitor of [3H]-DHT formation with an estimated IC50 of 0.2 mg/kg. LY207320, an inhibitor of 5 alpha-reductase in vitro, was weakly active in vivo and did not achieve greater than 45% inhibition at high doses (greater than 200 mg/kg, s.c.). Tissue uptake of [3H]-T was also inhibited by LY207320, which may contribute to its inhibitory activity on accessory sex organ growth in the rat.

Dose response uterotrophic and antiuterotrophic activity of antiestrogens was examined in immature rats, immature mice and adult ovariectomized mice. LY117018 was the most active antagonist and the least estrogenic, while tamoxifen induced the greatest uterine growth and the weakest antagonism. The reported estrogenic activity of tamoxifen in mice (1) was found to be related to maturity. All compounds caused uterotrophic changes in immature mice similar to those observed in immature rats. However, in adult mice tamoxifen was devoid of antagonism, and trioxifene was active only at a very high dose as both were extremely estrogenic in this model. LY117018 activity in adult mice was comparable to that observed in immature rats and mice. Results depict significant agonist and antagonist advantages of LY117018 over tamoxifen and trioxifene.

The PAIII rodent metastatic prostatic adenocarcinoma model was employed to evaluate the effects of dietary warfarin, a prototypic antagonist of thrombin generation on the lymphatic and pulmonary metastases of the tumor from the tail site of subcutaneous transplantation in male Lobund Wistar (LW) rats. In addition, the anticoagulant effects of warfarin were determined in the same animals. Warfarin, administered in the diet at concentrations equivalent to 0.063, 0.125 or 0.250 mg./kg. b.w. for 30 days had no effect on final body weight, gluteal or iliac lymph node weights. Significant (p less than 0.05) dose-dependent extensions of whole blood prothrombin (WBPT), activated partial thromboplastin (WBAPTT) and clotting times (WBCT) over control values were observed with warfarin treatment. Preliminary studies demonstrated that the 0.500 mg./kg. dose produced 50 per cent mortality at +14 days. Warfarin produced significant (p less than 0.05) dose-dependent decreases in the number of PAIII pulmonary metastases as indicated by reductions in dry lung weights and lung colony numbers when compared to untreated tumor-bearing controls. While the therapeutic index of warfarin is a limiting factor in clinical use as an antimetastatic agent, these results suggest that compounds capable of altering hemostatic mechanisms may be potential inhibitors of tumor metastasis. The PAIII prostatic adenocarcinoma model may be a useful system to quantitatively evaluate potential antimetastatic and cytotoxic agents.

Interaction of tamoxifen, trioxifene and LY117018 with cytosol-estrogen receptors from immature rat uteri was compared. Determination of relative binding affinity (RBA) by competition with [3H] estradiol under various assay conditions revealed that the RBA of LY117018 increased with temperature while that of trioxifene declined. Furthermore, the RBA values of tamoxifen and trioxifene observed after 24 h of incubation at 4 degrees C were significantly lower than those obtained with 1-h incubations. However RBA values obtained with 1- or 24-h incubations of LY117018 at 4 degrees C were similar. The complex formed by estradiol or LY117018 at 4 degrees was relatively stable for 24 h, while significant dissociation of tamoxifen and trioxifene was detected under these conditions. At 30 degrees C estradiol displayed a biphasic pattern of dissociation, but tamoxifen and trioxifene dissociated rapidly and little evidence of a stable phase was apparent. By contrast, the complex formed by LY117018 exhibited greater stability than that of estradiol at 30 degrees C. These results establish a relationship between shifts in competition curves (RBA) and rates of dissociation relative to estradiol; and clearly reveal that LY117018 has different binding characteristics than tamoxifen and trioxifene.

Using separated epithelium (SVE) and fibromuscular stroma (SVM) of guinea pig seminal vesicle, the antihormonal effects of daily subcutaneous administration (14 and 28 days) of the benzothiophene keoxifene (LY156758; [6-hydroxy-2-(4-hydroxyphenyl)benzo(b) thien-3-yl] [4-(2-1-piperidinyl) ethoxyl] phenyl) methanone hydrochloride) in intact, castrate, and androgen/estrogen-maintained castrate animals was evaluated. The compound was devoid of agonist activity in castrated males, in that the compound had no stimulatory effect on SVM wet weight or DNA content. In vitro cytosolic binding of [3H]estradiol (E2) in the SVM was decreased in a concentration-dependent manner by keoxifene, but the compound did not perturb the binding of [3H]dihydrotestosterone (DHT) in the SVM or SVE. Likewise, keoxifene administration to castrated males treated with exogenous steroids antagonized the estrogen-induced hyperplastic response of the SVM, whereas no interference with androgen-induced growth of the SVM or SVE was observed. Keoxifene treatment of intact male guinea pigs produced regression of the androgen-sensitive SVE as well as the androgen/estrogen-sensitive SVM. Keoxifene-induced decreases in guinea pig serum testosterone levels were associated with this activity. Histological analysis of the seminal vesicle under these conditions suggests androgen deprivation. These findings indicate that keoxifene is a physiological antagonist of androgen action in the intact male guinea pig. The pure estrogen antagonist properties of keoxifene and its ability to decrease accessory sex organ epithelium and fibromuscular stroma in vivo suggest potential applications of the benzothiophenes in the medical management of prostatic neoplasia.

The influence of LY117018 and tamoxifen on established uterotropic influence of estradiol was examined in immature ovariectomized rats. When LY117018 was introduced on the fourth day of estradiol treatment, it regressed the uterotropic effect of estradiol. However, tamoxifen did not attenuate estradiol activity under these conditions. Injection of estradiol, commencing on the fourth day of LY117018 treatment, elicited no increase in uterine weight, while tamoxifen caused substantial uterotropic action in the presence of established, continuous LY117018 treatment. The uterotropic influence of tamoxifen also occurred when it was administered concomitantly with LY117018, and a similar result was observed following the injection of 4-hydroxytamoxifen with LY117018. These observations demonstrate that LY117018 can block or regress uterotropic effects of estradiol, but it cannot antagonize the action of tamoxifen or its hydroxylated metabolite. This suggests that these antiestrogens might act at separate sites or by different molecular mechanisms.

Fibrin formation has been hypothesized to be an element of the metastatic process in cancer, and pharmacological interference with such fibrin formation has been proposed as a means of antimetastatic therapy. We have tested this hypothesis through an in vivo study of warfarin in two independent rat disease models--a model of chemical-injury-induced arterial thrombosis, and a model of spontaneous metastasis. We found 0.50 mg/kg-day warfarin to be uniformly lethal after two weeks treatment. The chronic dose of 0.25 mg/kg-day was non-toxic and produced effective anticoagulation and marked antithrombotic and antimetastatic activity. The 0.125 mg/kg-day dose produced a reduction in factor IIc (50%) and factor VIIc (70%), and resulted in statistically significant antithrombotic and antimetastatic activity. The 0.0625 mg/kg-day dose failed to reduce the vitamin K-dependent clotting factors, and failed to produce any antithrombotic or antimetastatic effects. The substantial correlation (very similar dose-response effects) among the anticoagulant, antithrombotic and antimetastatic efficacies of warfarin in the rat suggests that anticoagulation provides the pharmacological mechanism underlying both the antithrombotic and the antimetastatic effects. The poor therapeutic index we observed in the rat may be the attribute which limits the efficacy of warfarin in the treatment of human cancer.

The spontaneous metastatic spread of a suspension of PAIII prostatic adenocarcinoma cells from the tail site of implantation was analyzed over a period of 5 weeks in male Lobund-Wistar (LW) rats. Following subcutaneous injection of the PAIII cells, the tumor metasta-sized through the primary lymphatic drainage. PAIII microfoci were evident in the gluteal and iliac lymph nodes prior to colonization of the lungs. Growth of the primary tumor was evidenced by significant weight differences of the tails of PAIII-bearing and control rats 1 week after tumor implantation. Time-dependent sequential spread of the adenocarcinoma was quantitated. Significant differences were noted between PAIII-bearing and control animals with respect to the gluteal lymph node weights (+2 weeks), iliac lymph node weights (+3 weeks), dry lung weights, and lung colony numbers (+4 weeks) after tumor implantation. During the course of these studies, the whole blood prothrombin, activated partial thromboplastin, and recalcification times for the PAIII-bearing animals were similar to those of the control group. These findings indicate that there were no gross changes in systemic blood coagulation accompanying the metastasis of PAIII cells from the primary tumor. The tumor in LW rats produced a consistent pattern of growth and metastasis that is suitable for quantitation. The PAIII prostatic adenocarcinoma is a sensitive and reproducible system that may be useful to evaluate potential antimetastatic and cytotoxic agents for the treatment of hormone-insensitive prostatic cancer.