Studies on the Antiprostatic Action of Estracyt, a Nitrogen Mustard of Estradici1

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[CANCER RESEARCH 34, 1031-1037, May 1974] Studies on the Antiprostatic Action of Estracyt, a Nitrogen Mustard of Estradici1

R. Y. Kirdani, J. MÃ¼ntzing,M. J. Varkarakis, G. P. Murphy, and A. A. Sandberg Roswell Park Memorial Institute, Buffalo, New York 14203

SUMMARY androgens and estrogens may play a role in prostatic cells and possibly in those of other organs. Estracyt, a chemical ester of a nitrogen mustard with Estrogens have been shown to affect the prostate in a estradiol-17/3, was tested for its antiprostatic effects in dogs number of animals, including the human (14). The synthesis and rats. Estracyt was shown to interfere with the uptake of of a nitrogen mustard of E2 was thought to be of promise in labeled estriol by the dog prostate, without affecting that of the treatment of cancer of the prostate, if the E2 moiety of testosterone and dihydrotestosterone. Similarly, it affected the molecule could be used as a carrier ("piggy back") for the uptake of estradiol-170 by the prostates of castrated the antimetabolite into the cells and if important groups rats, although in this animal it also interfered with the necessary for E2 to associate with receptor proteins in the uptake of dihydrotestosterone. Estracyt greatly reduced the prostatic cells were not affected by the nitrogen mustard prostatic secretion in dogs. The results of these short-term moiety. Recently, a nitrogen mustard of E2, Estracyt experiments indicate that Estracyt affects a number of (Chart 1), has been shown to be effective, at least pallia- different parameters in the prostates of dogs and rats. lively, in cancer of the prostate in a number of clinical Possible mechanisms of action of Estracyt were discussed. studies in Europe (1, 5, 6, 12, 13) and at our Institute (11). The purpose of the present study was to ascertain the possible mechanism of action and the prostatic effects of INTRODUCTION Estracyt in dogs and rats, in order to understand more fully the effects of the drug in human subjects. The concept behind the development of chemotherapeutic The plan of study was very similar to that we have utilized agents consisting of chemical conjugates, usually esters, of in ascertaining the effects of other antiprostatic agents steroid hormones and antimetabolites is based on relatively (20-22) and included the following. recent findings concerning the possible mechanism of action 1. The effects of Estracyt on the prostatic uptake of of these hormones, particularly the estrogens, at the cellular labeled testosterone, DHT, E3, and E2 in the dog and rat. level. Thus, it has been demonstrated that special receptor proteins for E22and other estrogens are present in the target These steroids have been shown to localize in the prostates of these animals (3, 7, 20, 21) and, hence, it was thought organs of these hormones, e.g., uterus, breast (4). Similar worthwhile to establish the effects of Estracyt on the receptor proteins for androgens (testosterone, DHT) are prostatic uptake of these steroids. present in male target organs, e.g., prostate, seminal vesi 2. Effect on prostatic fluid excretion and composition in cles (25). Furthermore, in the prostate of several species, the dog. the presence of intracellular receptor proteins for both 3. Effect on rat and dog prostatic 5a-reductase and estrogens and androgens has been demonstrated recently arginase activities. The 5a-reductase is essential in the (2, 3, 7). reduction of testosterone to DHT, and DHT being neces It has been inferred from the above that the interaction of sary for prostatic integrity and function. Although the exact the cytoplasmic receptor protein with a steroid leads to role of arginase is uncertain, it and 5a-reductase are modification of this protein, which then associates with the profoundly under the influence of androgens and possibly nucleus, thus influencing transcription of the DNA of the estrogens (18, 26). nuclear chromatin. This process is essential for the control of cellular metabolism. It is also inferred that the presence of receptor proteins in MATERIALS AND METHODS a cell may indicate that the steroids with high affinity for these proteins play an important role in the integrity and For tissue deposition studies, male mongrel dogs weigh metabolism of such cells. Hence, it would appear that both ing 10 to 15 kg (2 to 4 years old) were used. Mixtures of differently labeled steroids (50 ÃŸd DHT-3H and 10 /iCi E3-14Cor 50 MCi E3-3H and 10 /iCi testosterone 14C)were 'This study has been supported in part by NIH Grants AM-01240, injected as previously described (9, 20, 21). Biopsies of AM-11754, and CA-15436. 2The abbreviations used are: E2, estradiol-17/8; DHT, dihydrotestoster various tissues were taken at 15, 30, 45, 60, 90, and 120 min one; E3, estriol; E2-phosphate, estradiol-17/3, 17-phosphate. following the i.v. injection of the steroids. Details of the Received November 5. 1973; accepted January 28. 1974. preparation of tissue samples for the determination of

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1974 American Association for Cancer Research. Kirdani, MÃ¼ntzing, Varkarakis, Murphy, and Sandberg radioactivity and the methods utilized with the tissue liquid as possible. Homogenate equivalent to 300 mg of oxidizer have been previously described (8, 9, 21). wet weight of tissue was taken for 5Â«-reductase activity. The effects of Estracyt on the deposition of labeled Incubation with testosterone-14C for 1 hr at 37Â°was per testosterone, DHT, and E3 in the prostate and other tissues formed following the procedure of Shimazaki et al. (18). of the dog were examined following the i.v. administration After addition of alcohol to the incubation medium, of a commercial preparation of the drug (A. B. Leo, standard testosterone, DHT, and androstenedione were Helsingborg, Sweden; Batch X4645 of Estracyt), which was added; the mixture was extracted according to the method given twice daily (2.5 mg/kg) for 2 days and then 2 to 3 hr of Shimazaki et al. (18); and an initial paper chromatog prior to the injection of the labeled steroids on the 3rd day. raphy was performed on the extract in 80% methanol in Since Estracyt is phosphorylated at position 17 of the water and benzene. Two peaks separated with typical RK estradiol, the effects of E2-phosphate on the deposition of values of 0.41 (testosterone) and a minor peak with an labeled steroids (DHT and E3) were determined in dogs. The RF of 0.63 (DHT and androstenedione). The 2nd peak E2-phosphate was given to 3 dogs (1.7 mg/kg/day i.v. twice was then eluted, acetylated, and rechromatographed in daily in 0.9% NaCl solution) for 2.5 days, and then 50 nC\ of the system 80% methanol in water and heptane. This DHT-3H and 10 /iCi of E3-14C were injected i.v. 2 to 3 hr system separated androstenedione (RF 0.39) from acetyl after the last dose of the estrogen phosphate. The results ated DHT (RK 0.94). The DHT acetate and androstene were compared with those of untreated normal male dogs of dione were eluted and together with the testosterone peak similar age and weight. from the Bush-3 chromatogram were quantitated for re DHT-1,2-3H (48 mCi/^M), E2-4-14C (50 Â¿iCi/MM),E3-4- covery by determining the UV absorption for androstene 14C (37 juCi/MM), E3-6,7-3H (50 mC\/n\i, and testos dione and testosterone. Arginase activity was determined by terone 4-14C (50 /uCi/jiM) were purchased from New Eng the method of Yamanaka et al. (26). land Nuclear, Boston, Mass., and checked for purity by paper chromatography. The A. B. Leo Co. kindly supplied us with nonlabeled Estracyt, dephosphorylated Estracyt, RESULTS and E2-phosphate. The influence of Estracyt on the deposition of labeled The Effects of Estracyt on the Prostatic Uptake of testosterone and E2 in intact and castrated rats was Labeled DHT, Testosterone, and E3 in the Dog. The changes determined in 2 separate experiments. In 1 set of experi with time in the prostatic uptake of radioactivity associated ments Estracyt was given p.o. by gavage for 2 days (25 with injected labeled steroids in untreated and Estracyt treated dogs are shown in Charts 2 to 4. The steroids were mg/kg) and 30 min before the i.v. injection of the labeled injected i.v. in pairs (DHT-3H plus E3-14C and E3-3H plus steroids on the 3rd day to intact and castrated rats. The testosterone-14C), but for graphic presentation the results animals were anesthetized 15 min after the injection of the labeled steroids, and the various tissues and organs were for each steroid are shown individually. The results indicate excised. The Estracyt was given to the castrated rat 2 days a definite inhibition of uptake of the labled E3 (Charts 1 and after the surgery. In another set of experiments the Estra 3) in the prostate of the dogs following the administration of cyt was given to intact and castrated rats for 4 days, start Estracyt for only 2 days. On the other hand, the uptake of ing on the day of castration. the labeled DHT (Chart 2) was significantly increased, Prostatic fluid from dogs was obtained through a cysto- whereas that of labeled testosterone was not affected by the preputiostomy-type fistula after i.v. injection with pilocar Estracyt treatment. pine (0.7 mg/kg) (10). Fluid collections were made every 10 The levels of radioactivity shown in Charts 2 to 4 had a min after injection of the labeled steroid mixture. We found standard error of the mean that did not exceed 10% of the that radioactivity in these samples is best determined by the counts shown for any point in the figures and usually was in use of the sample oxidizer; otherwise, variable results were the range of Â±5%of the dpm. obtained when direct determinations of radioactivity were Statistical analysis of the differences between the values shown for control and Estracyt-treated dogs in Charts 2 to 4 made. The effects of Estracyt on prostatic secretion in dogs were revealed p values of < 0.001 for all points in Charts 2 and 4 determined after the animals were treated with the drug for 7 days (2.5 mg/kg i.v. twice daily). In addition to various chemical determinations on the fluids, the excretion of radioactivity following the injection of labeled DHT-3H and E3-14C was determined. Measurement of prostatic 5a-reductase activity was made using a modification of the method of Shimazaki et 0 al. (18). A 50% homogenate of prostatic tissue was made (CI-CH2-CH2)2-N-C-0X by grinding the specimen by hand in an all-glass Potter- Chart 1. Chemical formula of Estracyt, showing it to be an ester of a Elvehjem homogenizer to which had been added a volume nitrogen mustard and estradiol-17/3, the former being attached to the latter equal to the weight of the tissue of 50 mM Tris-HCl at position 3. A phosphate group is present at position 17. Estramustine buffer, pH 7.4. The homogenate was filtered through phosphate is manufactured by the A. E. Leo Co. and consists of gauze, and the gauze was squeezed to drain out as much estradiol-3-bis(2-chloroethyl)carbamate-17-dihydrogen phosphate.

1032 CANCER RESEARCH VOL. 34

2000 Charts 2 and 3. Mean radioactivity levels in the canine 1800' prostate following treatment with Estracyt (2.5 mg/kg/ 1600- 16,000 day i.v. for 2.5 days to 3 dogs). The pair of steroids estriol-"C and DHT-3H was injected i.v., 10/iCi and 50 1400 14,000 1200' 12,000 fiCi, respectively. The mean levels of untreated dogs are CO shown for comparison. Estracyt caused a markedly lOOO- 10,000 decreased uptake of the E3-"C by the prostate. There 800 ESTRACYT TREATED 2 8000 was no decrease in the uptake of DHT-3H and. if < anything, a somewhat increased concentration of radio- , 600 g 6OOO i activity in the prostate. The values for the 2 separate 400 Â£ 4000 groups of 3 untreated dogs are shown for comparison in Chart 3. 200 2OOO

30 45 60 90 120 15 30 45 6O60 90 120 TIME IN MINUTES TIME IN MINUTES

10,000 vesicles of both intact and castrated animals, and in the pan creas of intact rats; testosterone in the prostate of castrated rats, and the levator ani, testes, and pancreas of intact rats. In all organs and tissues, except the pancreas, of the rats, both intact and castrated, the 3H/'"C far exceeded the 6000 injected one (5/1) and indicates more rapid excretion of the labeled E2 than of testosterone. In the pancreas the ratio was actually decreased and is in keeping with the avid uptake of E2 by pancreatic tissue previously described by us ESTRACYT TREATED (9). Parenthetically, the 3H/14C in the pancreas was not 20OO affected by Estracyt. Effects of Estracyt on Prostatic Eluid Excretion and

15 30 45 60 90 120 Composition in the Dog. Following 1 week of treatment of 4 TIME IN MINUTES dogs with Estracyt (2.5 mg/kg i.v. twice daily), the secretion Chart 4. Mean radioactivity levels in the prostates of 2 dogs treated with of prostatic fluid totally stopped in 2 animals, and in the Estracyt (2.5 mg/kg/day i.v. for 2.5 days) following the i.v. injection of other 2 animals the volume decreased to 43 and 35% of the 50 Â¿iCiE3-3H(injected with testosterone-"C. but. since no effect was seen controls, respectively. The chemical composition of the fluid on the "C levels in the prostate, the results are not shown). The values in 6 before and after the administration of Estracyt and the untreated animals are shown for comparison. It is evident that Estracyl radioactivity excreted following the injection of the pair of decreases the uptake of the labeled E3 by the canine prostate. steroids DHT-3H (50 Â¿Â¿Ci)andE3-14C(10 Â¿/Ci)areshown in Table 2. and p values of <0.005 in Chart 3 and indicate a very Estracyt definitely decreased the concentration of acid significant effect of Estracyt on the localization of the phosphatase (p < 0.001) in the prostatic fluid without radioactivity of administered E3 and DHT in the prostate. significantly (p > 0.5) affecting those of the other parame Since the E2 in Estracyt is phosphorylated at position 17, ters determined. The 3H/14C observed in the prostatic fluid we next examined the effect of E2-phosphate on the following the i.v. injection of DHT-3H and E3-UC was deposition of DHT-3H (50 fid) and E3-UC (10 ftCi). The increased following Estracyt, primarii) due to a decreased effects on the uptake of the labeled E3 by the prostate were excretion of radioactivity associated with E3. This is in similar to those of Estracyt or E2. No significant effects on keeping with the lesser amounts of radioactivity of E3 the uptake of the labeled E3 and DHT were seen in other present in the prostatic gland following treatment with tissues, however, including the pancreas. The latter organ Estracyt (see Charts 2 to 4). Thus, the canine prostatic has been shown to concentrate significant amounts of E3 secretion is definitely affected by the Estracyt administered. and E2 (15). This contrasts with data obtained following the Effects of Estracyt on Rat Prostatic Weight and 5Â«- administration of E2 or E3 to dogs (20), which does lead to a Reductase and Arginase Activities. In order to determine the decreased deposition of labeled E2 or E3 in the pancreas, antiprostatic effect in the rat, groups of 7 animals were each apparently the unlabeled steroids competing with the la given various doses of Estracyt i.p. daily for 4 days and the beled ones for intracellular sites (16). weight of the prostate glands and kidney was determined. The Effects of Estracyt on the Uptake of Labeled The 5Â«-reductase and arginase activities were also estab Testosterone and I by Rat Tissues, Including the Prostate. lished. The results are shown in Charts 5 to 8. The enzyme When Estracyt was administered p.o. to male adult intact or results are expressed in 2 ways: per g of tissue and per total castrated rats for 2 days (Table 1), a markedly decreased gland. It is evident that the weight of ventral and dorsolat- deposition of radioactivity following the i.v. injection of the eral glands decreased significantly, particularly when a very pair of steroids (testosterone-3H and E2-14C) was observed large dose of Estracyt was given (Chart 5). The body weight as follows: E2 in the prostate of castrated rats, in the seminal of the animals did not change, nor was there a significant

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Table I Effects of Estracyt on the in vivo deposition of injected testosterone-3H (20 p.Ci) and E2-\IC (4 ÃŸd) in intact and castrated male rats

Effect of Estracyt (dpm/g tissue x 10

Tissue Group 1Â° Group 2 Group 3 Group 4

Ventralprostate3H"C"H/"CDorsolateral 55.0Â± Â±23.3 (4.4)(3.0)118.0(4.8)6.5Â±7.4 (3.0.3 Â±25.3 2.3Â± Â±18.9 Â±23.5 (1.1.844.0(4.2.8 Â±0.9 1.6Â± Â±310.0 Â±279.8 Â±1.0 prostate3H"C3H/"CSeminal 37.5Â± Â±16.1 Â±14.2 Â±79..2 3.5Â± Â±19.5 (2.8)0.819.0 Â±19.9 (2.1.342.1 Â±2..9 1.4Â± Â±157.0 Â±176.6 Â±15 vesicles3H"C3H/"CLevator 19.2Â± Â±7.7 (2.5)0.9 Â±8.2 (32.4(11.49.5 Â±20..2 2.3Â± Â±20.3 (1.4)0,43.1 Â±21.9 Â±2.. 1.4Â± Â±98.2 Â±68.2 1 Â±25..9 ani3H"C"H/"CTestes3HMC"H/"CPancreas3H"C3H/"CKidnev3H"C3H/"CMuscle3H"C3H/"C195.110.917.5324.220.416.2196.412.616.0103.54.816.1153.411.213.7351.683.75.1369.221.817.384.26.014.0Â± 31.3Â± Â±6.8 (1.6)0.7(1.3)1.054.2Â±4.8 (10.4 Â±26.9 0.3Â± Â±14.6 Â±14.0 (10.713.8 Â±1.7 0.4Â±25.1Â± Â±165.8 Â±95.2 Â±0.2

Â±8.6 (11.0(10.612.9 1.8Â± Â±11.0 0.2Â± Â±141.1

65.4Â± Â±43.6 (2.4)16.2 Â±46.2 (24.3 Â±89.7 36.3Â± Â±3.9 (7.1)0.458.3 Â±3.1 (90.219.8 Â±26.8 (9.0).8.8(4.0).5(2.6). 1.6Â±57.1Â± Â±302.5 Â±211.5 Â±0.3

Â±18.2 (4.7)5.2(2.2)2.414.71.61.3174.2Â±13.0 (30.4 Â±22.5 4.8Â± Â±17.7 Â±16.3 (21.119.00.71.62)6)7)9)0)7)2)0).6).8).4).9).6).8)187.92128312221814441096151763732941321754137Â±1.9 1.0Â± Â±65.7 Â±58.9 Â±1.1 1.3.4.3

12.4Â± Â±5.8 Â±4.8 Â±13.1 0.4Â± Â±11.7 Â±1.4 Â±2.9 0.5(2.3)"(1.8)(3.9)(3.5)(2.3)â€¢(2.1)(1.2)(0.8)(1.8)(4.2)(13.9)(4.4)(3.6)287.8Â±100.1 Â±19.4 Â± 08(2.6)7(1.7)72(3.6)8(2.2)38(2.2)5(1.3)92(1.5)3(1.3)8.0(2.3).4 Â°Group I, control intact; Group 2, control castrated; Group 3, intact pretreated with Estracyt; Group 4, castrated pretreated with Estracyt. " Numbers in parentheses, ratios of dpm in a g of tissue to those in a g of muscle.

Table 2 Proslalic fluid composition before and after treatment of dogs with Estracyt

Av. values of determinations in 2 dogs (shown in parentheses)

protein(mg/100 phosphatase-(IU/mt)27.0(26.2,Acid ml)565.0(556.0, value)4.7(4.6,4.8)8.1(8.0,

BeforetreatmentAfter 120.0)136.0(135.1, 28.6)145.5(142.5,433.0)Total574.0)430.0(427.0,433.0)Alkalinephosphate27.8)20.0(19.2, 139.1)25.0(24.6, treatmentChloride(mEq/1)114.0(108.0, 136.9)Sodium(mEq/1)7.6(7.4,7.8)3.8(3.7,3.9)Potassium(mEq/1)128.0(127.4,20.8)(lU/ml)133.0131.9,25.4)3H/"C(mean 8.2) change in the weight of the kidney. The latter organ was activity was observed in the kidney, although a decrease in included, since it has considerable 5a-reductase and argi- the arginase activity was found. nase activities, subject to control by androgens and possibly Effect of Estracyt on Sa-Reductase of Dog Prostate. by estrogens. There was some reduction in testicular weight The 5a-reductase in the prostate of dogs pretreated with (pre-Estracyt 1745 Â± 21 mg versus 1594 Â± 78 post- Estracyt (5 mg/day i.v. for 2.5 days) was determined in 3 Estracyt). The 5a-reductase and arginase activities declined animals. The canine prostatic 5a-reductase usually ranges significantly in the prostatic glands, particularly in the between 4 to 8% in control animals in our laboratory. The ventral one (Charts 6 to 8). No change in the 5Â«-reductase values found in the 3 dogs treated with Estracyt were 0.25,

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1974 American Association for Cancer Research. Antiprostatic Action of Es tracy t drolysis of the Estracyt molecule. The results in the dog and baboon are significantly different from each other and from those in man. A double nitrogen mustard ester of E2 (NSC 11259), i.e., alkylating agents attached at both the 3 and 17 positions of this steroid, has been shown to have antitumor activity in a number of experimental tumors in rats (23, 24). Since E2per se was not effective in all the tumor systems studied, whereas the E2 mustard was, it was inferred that antitumor O.I 1.0 100 activity must have originated in the mustard moiety, even ESTRACYT (mg/kg) though definite estrogenic effects were seen in the animals. Chart 5. Weight of prostate glands and kidney in rats treated with The toxicity of the mustard alone was much greater than various doses of Estracyt. The results are expressed as a percentage of that of the E2 mustard. A number of severe toxic symptoms control values. Each point consists of the average of the values obtained on was observed in beagles given this E2 mustard (17) and these 7 rat prostates. Only a slight decrease in the weight of the kidney was apparently precluded its use in humans. This toxicity evident, whereas the 2 prostates showed significant decreases in weight, contrasts with the lack of such following Estracyt adminis particularly the ventral gland. tration to humans (11-13) and may indicate that the

- loo

5 100 II 31 50 VENTRAL PROSTATE 5O

0 O.I 1.0 10 100 EXTRACYT (mg/kg) 0 O.I 1.0 10 100 ESTRACYT(mg/kg) Chart 7. Arginase activity expressed in terms of total organ in the Chart 6. Arginase activity, expressed per g of tissue, in rat prostates and prostate glands and kidney of rats treated with various doses of Estracyt. In kidney following the administration of various doses of Estracyt. In the the case of the ventral prostate and kidney, the combination of decreased weight and decreased arginase activity led to a very significant decrease in dorsolateral gland, except for a decrease at the lowest dose, the arginase activity was increased with the higher doses. The decreased activities in the total organ activity. In the case of the dorsolateral gland, the effects are not ventral gland and kidney, although not very profound, were more very consistent and present only at the lowest dose of Estracyt. consistent.

1.4, and 2.8%. These decreases are probably significant. It is possible that further decreases would have been obtained upon prolonged Estracyt therapy.

DISCUSSION The increasing importance of combinations of chemotherapeutic agents, including chemical conjugates, with steroid hormones in human cancer warrants some discus sion regarding the data obtained with Estracyt. The results obtained with Estracyt in 1 animal and/or its tissues should not be extrapolated regarding its action or effectiveness in another. In one animal the Estracyt may be readily hydrolyzed totally before it reaches the prostate, whereas in 0.1 1.0 10.0 IOO.O Dose Â¡nmg/kg another it may be but insignificantly hydrolyzed, e.g., in the Chart 8. The effects of various doses of Estracyt on 5a-reduclase human (A. A. Sandberg et al., unpublished observation). activity of rat prostates. The decrease in the activity of the ventral prostate Hence, the results presented in the present paper apply to is profound but is less so in the dorsolateral gland. These values, taken in the dog and rat and should be extrapolated to the human or conjunction with the weight reduction of the glands (see Chart 5), indicate other animals with great caution. Thus, we have found that very greatly decreased 5Â«-reductaseactivity in the prostates of the rat. The the metabolism and fate of labeled Estracyt in the human is kidney did not show this decrease and, if anything. 5Â«-reductaseactivity quite different from that of E2 and indicates minor hy was elevated by the administered Estracyt.

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nitrogen mustard moieties of the former drug (NSC 11259) ACKNOWLEDGMENTS become readily hydrolyzed and capable of producing sys temic effects. This is further substantiated by the evident We wish to thank Dr. H. Yamanaka for the arginase determinations: estrogenic effects, including that on the prostate, observed Dr. T. M. Chu for the chemistries on ihe prostatic fluids; Christine Hanson in the animals treated with the double ester of E2 (17). for clerical help; and Joyce Romano, Anne Agacz, Carol Houenstein, and Elek Karsay for technical assistance. The data of the present study indicate that Estracyt has a definite antiprostatic effect as manifested by the decreased weight of the gland in the rat; decreased prostatic secretion REFERENCES in the dog; and the decreased levels of acid phosphatase, 1. Alfthan. O. S., and Rusk, J. Estracyt" in Advanced Prostatic 5a-reductase, and arginase activities in the dog and rat Carcinoma. Ann. Chir. Gynaecol. Fennial, 58: 234 240, 1969. prostates. Since only some of these effects and/or a 2. Armstrong, E. G., Morris, M. H., Young, J. D., and Bashirelahi, N. combination of these can be produced by the administration 17/3-Estradiol Binding by Human Prostate. Abstract 287, Endocrine of E2 or other estrogens to intact animals, and others may Society, 55th Meeting, Chicago, 111.,June 1973, 92: 192, 1973. be due to the nitrogen mustard moiety, the data point to a 3. Ghanadian. R.. and Fotherby, K. Interaction between Steroids in rather complicated mechanism of antiprostatic action of Regard to Their Uptake by Rat Prostate. Steroids Lipids Res., 3: Estracyt. Thus, if in fact some of the antiprostatic effects 363-371. 1972. are due to the E2 moiety, these indicate a capacity on the 4. Jensen, E. V.. and DeSombre, E. R. Mechanism of Action of the part of the prostate to hydrolyze in situ both the phosphate Female Sex Hormones. Ann. Rev. Biochem., 41: 203 230, 1972. 5. JÃ¶nsson, G. Behandling av Cancer Prostate Med ett Kombinerat and nitrogen mustard moieties, since only the free E2 is Estrogen-Cytostaticum. Nord. Med., 81: 618 619, 1968. biologically effective. It is possible that such hydrolyses 6. JÃ¶nsson, G., and HÃ¶gberg, B. Treatment of Advanced Prostatic occurred in the body at a site other than the prostate and Carcinoma with Estracyt". A Preliminary Report. Scand. J. Urol. that the E2 thus released is then available for the production Nephrol., 5: 103 107, 1971. of the antiprostatic effects observed either through inhibi 7. Jungblut. P. W., Hughes, S. F., GÃ¶rlich, L.. Gowers, U., and Wagner, tion of interstitial cell-stimulating hormone release from the R. K. Simultaneous Occurrence of Individual Estrogen- and Androgen pituitary and/or direct effect on testosterone secretion by Recptors in Female and Male Taeget Organs. Z. Physiol. Chem., 352: the testes, or testosterone effects in the prostate per se. This 1603 1610, 1971. indeed remains a distinct possibility, at least in some 8. Kirdani, R. Y.. Slaunwhite, W. R.. Jr.. and Sandberg, A. A. Studies on Phenolic Steroids. XI. The Biosynthesis and Proof of Structure of animals, although there is suggestive evidence in the present Estriol-3,16-Diglucosiduronate. Steroids, 12: 171 182. 1968. study that the phosphate may remain attached to the E2 and 9. Kirdani, R. Y., Varkarakis, M. J., Murphy, G. P., and Sandberg, A. subsequently be hydrolyzed in the prostate. This is borne A. Distribution of Simultaneously Injected Androgens and Estrogens out by the failure of Estracyt or E2-phosphate to affect the in Animal Tissues. Endocrinology, 90: 1245 1251, 1972. deposition of E2 or E3 in the pancreas of the dog, while 10. Mason, M. M., Keefe, F., and Boria, T. Speciali/ed Surgery of the affecting the uptake in the prostate. It appears, thus, that Canine Bladder and Prostate.Gland. J. Am. Vet. Med. Assoc., 139: dephosphorylation may be a key step in the effects of 1007 1014, 1961. Estracyt and that such hydrolysis is readily accomplished by 11. Miintizing, J., Shukla. S. K.. Chu. M. C., Mittleman, A., and Murphy. the prostates of the dog and rat. G. P. Pharmacoclinical Studies on Oral Estraumustine Phosphate (Estracyt) in Advanced Carcinoma of the Prostate. Invest. Urol., in Were a major part of the E2 in Estracyt to be released at a press. site from which it could become systemically effective, one 12. Nilsson, T.. and Muni/ing, J. Estracyt11 in Advanced Prostatic would expect to see major estrogenic effects in organs and Carcinoma. Case Rept. Scand. J. Urol. Nephrol.. 6: 11-16, 1972. tissues other than the prostate. This has not been the case in 13. Nilsson, T., and Miinti/ing, J. Histochemical and Biochemical Investi humans (11-13) or mice (J. MÃ¼ntzing et al., unpublished gation of Advanced Prostatic Carcinoma Treated with Estramustine observation), and the observations indicate that if Estracyt Phosphate, EstracytÂ». Scand. J. Urol. Nephrol., 7: 18-22, 1973. is hydrolyzed at a site other than the prostate it does not 14. Ofner, P. Effects and Metabolism of Hormones in Normal and leave that site in a form that is biologically effective. Neoplastic Prostate Tissue. Vitamins Hormones, 26: 237-291, 1969. 15. Sandberg, A. A., Kirdani, R. Y., Varkarakis, M. J., and Murphy, G. It has been demonstrated by Tritsch et al. (19) that P. Estrogen Receptor Protein of Pancreas. Steroids, 22: 259 271, human serum phosphatases readily hydrolyze off the 1973. phosphate group of Estracyt. The drug was shown to have 16. Sandberg, A. A., Rosenthal, H.. Schneider. S., and Slaunwhite. W. R.. a high affinity for the enzymes, particularly for alkaline Jr. Proc. United States Japan Joint Conference on "Dynamics of phosphatase, but the highest reaction velocity was ob Steroid Hromones," Tokyo, May 1965. New York: Academic Press. served with acid phosphatase. Inc.. 1966. Some of the observations obtained point to the nitrogen 17. Schaeppi, U., Heyman, I. A., Fleischman, R. W., Rosenkrantz, H., mustard of Estracyt as playing a part in the antiprostatic Ilievski, V., Phelan, R., Cooney, D. A., and Davis R. D. Toxicity in effects observed. The interference of Estracyt with the Beagle Dogs of Three Alkylating Esters with Antitumor Activity: Phenesterin (NSC-104469), Estradici Mustard (NSC-112259), and uptake of DHT by the rat prostate and the reduction of Dehydroepiandrosterone Mustard (NSC-121210). Cancer Chemother 5a-reductase activity in the prostates of the dog and rat apy Rept., 4: 85 95, 1973. indicate a possible direct effect of the nitrogen mustard, 18. Shimazaki, J., Matsushita, I., Furuya, N.. Yamanaka. H.. and Shida, since such a combination of effects has not been observed K. Reduction of Sa-Position of Testosterone in the Rat Ventral with estrogens alone. Prostate. Endocrino!. Japon., Â¡6:453 458, 1969.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1974 American Association for Cancer Research. Antiprostatic Action of Es tracy t 19. Tritsch, G. L.. Shukla, S. K.. Mittelman, A., and Murphy, G. P. 23. Vollmer, E. P., Taylor, D. J., Masnyk, 1. J., Cooney, D.. Levine. B.. Estracyt (NSC-89199) Ã¤saSubstrate for Serum Phosphatases. Invest. and Piczak, C. Estradici Mustard (NSC-112259) Clinical Brochure. Urol., in press. Cancer Chemotherapy Kept., 4: 121 140, 1973. 20. Varkarakis, M. J., Kirdani. R. Y.. Abramczyk, J., Murphy, G. P., and 24. Wall, M. E.. Abernethy, G. S.. Jr.. Carroll, F. !.. el al. The Effects of Sandberg, A. A. Effects of Castration, Androgens and Estrogens on Some Steroidal Alkyluting Agents on Experimental Animal Mam- Steroid Levels in the Dog Prostate. Invest. Urol., //: 106 113, 1973. mary Tumor and Leukemia Systems. J. Med. Chem., 12: 810 818. 21. Varkarakis. M. J., Kirdani, R. Y. Murphy, G. P., and Sandberg, A. A. 1969. Androgen and Estrogen Uptake in the Canine Prostate and Their 25. Wilson. J. D. Recent Studies on the Mechanism of Action of Excretion in Prostalic Fluid. J. Surg. Res., 13: 39 46, 1972. Testosterone. New Engl. J. Med.. 25: 1284 1291, 1972. 22. Varkarkis, M. J., Kirdani, R. Y.. Yamanaka, H., Murphy, G. P., and 26. Yamanaka, H., Mayuzumi. T., Shimazaki, J., and Shida, K. Proper- Sandberg, A. A. Prostatic Effects of a Nonsteroidal Antiandrogen. ties and Androgen-lnduced Changes of Arginase in Kidney and Invest. Urol., accepted for publication. Ventral Prostate of Rats. Endocrinol. Japon.. 18: 487 494. 1971.

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R. Y. Kirdani, J. Müntzing, M. J. Varkarakis, et al.

Cancer Res 1974;34:1031-1037.

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