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Background: For screening prostate cancer (CAP) using prostate-specific antigen (PSA), the indications of biopsies for patients showing slightly elevated PSA values are still controversial. Furthermore, the dependence of total PSA, free-to-total PSA ratio (FIT) and PSA density (PSAD) on prostatic volume in gray zone cases is unclear. Methods: By analyzing 1913 patients with a serum total PSA ranging from 2.0 to 20 ng/ml, we evaluated the correlation between total PSA and age or prostatic volume and positive predic­ tive value (PPV) in each range for total PSA, c;lge and prostatic volume. Then suitable reference values for total PSA, FIT and PSAD were decided according to prostatic volume. Results: There was no close correlation between PSA and age or volume. The PPV was high in the group with a prostatic volume of 10-30 ml. Prostatic volume was categorized into three groups

High pressure liquid chromatography (HPLC, Toyo Soda TSK-GEL G3000 SW column) was used to analyse the properties of Estramustine binding protein (EBP) in the cytosol of rat dorsal prostate. There exist in the cytosol of rat dorsal prostate two binding components having a high affinity for Estramastine. When estimated by HPLC, the molecular weights of these Estramustine binding components are 45, 000-50, 000 and 25, 000-30, 000 daltons, respectively. The binding of 3H-Estramustine to a macromolecule with a molecular weight of 25, 000-30, 000 is more heat labile than binding of 3H-Estramustine to a macromolocule with a molecular weight of 45, 000-50, 000. The present study also demonstrates that the HPLC method offers higher resolution, smaller sample size and faster analysis than other methods used in binding studies.

A synthetic nonapeptide agonist of luteinizing hormone-releasing hormone (LH-RH) des-Gly10-[D-Leu6]-LH-RH ethylamide, was entrapped in a nonbiodegradable copolymer of hydrophobia diethylene glycol dimethacrylate and hydrophilic polyethylene glycol #600 dimethacrylate having a sandwich-type structure prepared by radiation-induced polymerization. The two types of controlled release formulations prepared in this study have average release rates of 7 and 320 μg/day in the in vivo experiments. In this case, the controlled release formulations implanted subcutaneously in the backs of male Wistar rats were maintained over a period of 10 weeks. Serum drug levels were found to be 2.6 and 30 ng ml−1 respectively and were constant throughout the experimental period. The serum testosterone level and serum LH level were significantly reduced to castrated values at 2 weeks after implantation. At the same time, the ventral prostates, seminal vesicles, and dorsolateral prostates also showed a considerable decrease in weight. These data show excellent therapeutic potential of LH-RH agonist in controlled release formulations.

The antiandrogenic properties of a new nonsteroidal antiandrogens, AA560 (N-2-chloromethyl-2-hydroxypropionyl)-3, 4, 5-trichloroaniline) were investigated. The ventral prostate, dorselateral prostate and coagulating gland weights in rats given AA560 at 1-9 mg/head were significantly less than those in the intact rats. The seminal vesicle weights in rats given 3-9 mg/head were significantly less than those of the intact group. In intact animals given daily 3 or 9 mg of AA560 there were significantly increases of serum FSH, LH and testosterone concentrations. In the in vivo experiment, the pretreatment with AA560 decreased the uptake of 3H-androgens in the nuclear fraction of the ventral prostate. On the other hand, a significant increase in the uptake of 3H-radioactivity in the cytosol fraction was observed. It was proved by the in vitro displacement study that AA560 inhibited the binding of 5 alpha-dihydrotestosterone with a receptor protein in the prostatic cytosol.

AA 560, one of potent nonsteroidal antiandrogens, was implanted in the arcuate nucleus of the hypothalamus of intact male rats through a stainless double cannula. The prostate weight and serum LH level significantly increased on the 5th day after the implantation of AA 560 when compared with those in the sham-operated animals. There existed a definite correlation between the weight of the ventral prostate and the serum LH level in AA 560 implanted animals.

The binding of 5alpha-dihydrotestosterone to the hypophyseal and hypothalamic cytosol macromolecules prepared from castrated male rats was observed. The effects of antiprostatic agents on 5alpha-dihydrotestosterone binding to both hypophyseal and hypothalamic cytosol macromolecules was examined. Cyproterone acetate and chlormadionone acetate showed the significant inhibiting effects on 5alpha-dihydrotestosterone binding to 7-8 S macromolecules of cytosol from both hypophysis and hypothalamus. SCH 13521 and AA 560 did also affect 5alpha-dihydrotestosterone binding to 7-8 S macromolecules of cytosol from both tissues.

Estradiol-17β, estrone, estriol or diethylstilbesterol showed the increase in arginase activity of the dorsolateral prostate of intact adult male rats. Neither castration nor adrenalectomy modified the response of arginase activity by administration of estrogen. However, the arginase activity of the dorsolateral prostate of hypophysectomized male rats was significantly reduced regardless of estrogen treatment. On the other hand, almost the similar decrease was observed in the arginase activity of the ventral prostate from castrated, adrenalectomized or hypophysectomized rats, as well as from intact ones.From these results, it was concluded that the increase in arginase activity of the dorsolateral prostate after estrogen treatment might be due to some indirect effects of estrogen, and that pituitary the gland seemed to be involved in this estrogenic effect.

In Vivo Release of Testosterone from Hydrophobic–Hydrophilic Copolymer Composites in Long Term Delivery Systems

Incubations of rat ventral prostate with testosterone-14C and 5a-dihydrotestosterone (5α-androstane-17β-ol-3-one)-3H revealed the gradual increase in uptake of both steroids into the tissue. Ratios of 3H/14C of the incorporated steroids were similar during the incubation periods, and this indicated that incorporation of both the steroids proceeded in the same pattern. Radioactivity in purified 5α-dihydrotestosterone from the incubated whole tissues showed a gradual increase in both 14C and 3H, and 3H/'4C ratio was also almost constant throughout the incubation period. Therefore, rate of the conversion of testosterone to 5α-dihydrotestosterone seems to be constant during the incubation.A considerable difference in 3H/14C ratios of ethyl acetate extracts was observed between the various subcellular components after 30 min incubation. 3H/14C ratio in 5α-dihydrotestosterone, however, showed a small difference in all subcellular components. Most of the radioactivity incorporated into purified nuclei was attached to 5adihydrotestosterone.

The rate of 5α-reduction and 17β-dehydrogenation of testosterone added in vitro, the activity of Na+, K+-insensitive ATPase, and the content of citrate were determined in the ventral prostate of rats of various ages and of castrated animals with and without testosterone replacement therapy.The rate of 5α-reduction of testosterone changed significantly with aging of the gland, while no significant changes were observed in the rate of 17β-dehydrogenation of testosterone and ATPase activity in all animals used in this experiment. Although the rate of 5α-reduction in the old rat was very low, testosterone treatment increased the rate markedly. Castration caused a significant decrease in the rate of 5α-reduction, and the administration of testosterone restored the rate to the control level. This rate of 5α-reduction recovered to the control level on the 4th day after a testosterone treatment had been started prior to complete growth of the tissue in the castrated animals. These changes of the rate of 5α-reduction proceeded those of the citrate content of the gland.Physiological significance of 5α-reduction of testosterone in the rat ventral prostate was discussed.

Increase of RNA synthesis in the kidney of castrated rats was observed following testosterone administration. Using whole homogenates, the newly synthesized RNA in the kidney was found most abundantly three hrs. after testosterone administration, followed by a decrease, and returned to the previous level 24 hrs. later.Such newly synthesized RNA consists of both ribosomal RNA and DNA-like RNA, while scarcely any increase of synthesis was observed in soluble RNA. Before the increase in the new synthesis of RNA found in the kidney homogenates, increased synthesis of RNA was observed in the nuclei, mainly in ribosomal RNA. These facts might suggest a selective synthesis of ribosomal RNA in the nuclei in a certain stage, following testosterone administration.

Treatment of the rats with actinomycin D or actidione for 48 hr prior to sacrificing animals resulted in a significant reduction of DNA synthesis and the activity of DNA polymerase in testosterone-stimulated ventral prostate on the 4th day after testosterone administration. Under the same treatment of inhibitors, increase of the rate of 5α-reduction of testosterone on the 4th day after testosterone treatment was lower than that in the from the animals treated with testosterone alone. However, some elevation of the of 5α-reduction was observed between the 2nd and 4th day after testosterone treatment, regardless of the administration of actinomycin D or actidione. Treatment of mitomycin C did not make a significant difference in the increase of the rate of 5α-reduction DNA polymerase activity.It was concluded that the mode of increase of rate of 5α-reduction was different from that of DNA polymerase activity on the 4th day after testosterone treatment, and that some part of elevation of the rate of 5α-reduction was independent of the treatment of metabolic inhibitors.

Polymer-testosterone composites with long periods of controlled slow release were made by radiationinduced polymerization in a supercooled state at low temperature using glass-forming monomers. The in vitro release of testosterone from various vinyl polymer composites was found to follow a matrixcontrolled process ( Q-t 1 2 ). The rate of drug delivery was accelerated with increasing water content of polymers. In experiments in vivo, the composites were implanted subcutaneously in the back of castrated rats during the 30 day test period. The in vivo release rate of testosterone was a little smaller than in vitro. This difference between two releases also increased with the increase of hydrophilicity of polymer. The physiological response in rats was investigated by measuring the weight of ventral prostate and serum testosterone concentration with testosterone-containing composites. The weight of ventral prostate increased linearly with increasing rate of drug release and the serum testosterone concentration could be correlated with the release and with the weight increase of ventral prostate. It was found from microscopic observation that the used polymer carriers had relatively good biocompatibility to cause little foreign body reaction.

Daily treatment with 5 or 500 mug of estradiol benzoate of male adult rats for 7 days did not change the activity of testosterone 5alpha-reductase in the ventral prostate, while the activity decreased slightly in the dorsolateral prostate. The activity of alkaline phosphatase was increased by 60% over the respective control in the ventral prostate from rats treated with the larger dose of estrogen, but the estrogen treatment did not affect the alkaline phosphatase activity in the dorsolateral prostate. On the contrary, the estrogen treatment evoked three-fold elevation in the arginase activity of the dorsolateral prostate in contrast to the decreased arginase activity in the ventral prostate following estrogen administration. From these results, it was concluded that the alterations in some enzyme activity of the ventral and the dorsolateral prostates evoked by estrogen treatment were different from those observed in the respective lobes from castrated animals, although both estrogen administration and castration induced atrophy of the tissue. Furthermore, it might be also worth-while to mention that the ventral and the dorsolateral prostates of rats responded in a different manner to estrogen administered.

Hypolipidemia caused by Simfibrate feeding for 3 weeks reduced responses of weight and nucleic acid contents to testosterone administration in the ventral and dorsolateral prostates of castrated rats. However, the effect of testosterone on the contents of citrate, fructose, zinc and the activity of testosterone 5alpha-reductase in the glands of castrated animals was not modified by Simfibrate feeding.

The metabolic fate of chlormadinone acetate (CMA), which is a new anti-androgenic agent, has been investigated with rats, rabbits, dogs and humans. Fourteen unconjugated metabolites and three conjugated metabolites were isolated from urine, feces and bile after oral administration of CMA. The structures of these metabolites were deduced from physico-chemical data and definitely characterized by direct comparison with the authentic samples (see Chart 1). There was a marked species difference in the metabolic pattern. Especially, configuration of 2-hydroxy group in the 2-hydroxylated metabolites was different among species. Further, the occurrence of 1-hydroxylation and 15-hydroxylation, new metabolic pathways in steroidal agents, was found in the rats and humans. The anti-androgenic activities of the metabolites were listed in Table III. 3β-Hydroxy CMA, one of the main metabolites in humans and rats, and its acetate were 0.7 time as active as CMA, whereas other main metabolites, 2α, 3β-dihydroxy CMA and 2α-acetoxy CMA, were not effective.

To examine the content and the composition of free amino acids in the intraluminal fluid of rat epididymis, the fluids were obtained by light pressure on the dissected tissues. The amount of the total free amino acids in the pressed fluid from the caput epididymis was significantly higher than those of the cauda epididymis and the testis. Glu and Gln were predominant amino acids in the caput, and their amounts occupied more than half of the total ninhydrin reactive compounds. Such a high concentration of Glu and Gln was not observed either in the cauda or in the testis. Castration decreased Glu and increased Gln in amount. Testosterone treatment to castrated animals did not restore Glu and Gln contents in the pressed fluid from the caput epididymis to the level observed in intact rats completely. Therefore, it was assumed that a large amount of Glu in the caput was due to many factors; secretion and metabolism of epithelial cells of the gland which might be regulated by androgen, inflow of rete testis fluid, and sperm metabolism of amino acids in the epididymis. The results obtained from the caput epididymis to which the efferent duct of the testis was ligated also supported this interpretation.

Hydrophilic vinyl polymer-protein composites containing testosterone were made by means of thermal denaturation of albumin after radiation-induced polymerization of 2-hydroxyethylmethacrylate (HEMA) at −78°C. The albumin-HEMA mixed polymer can be considerably digested with trypsin. The degree of digestion was smaller than that expected from calculation. It was deduced that the digestion of the albumin component was retarded in the presence of HEMA. The same tendency was observed in in vivo experiments. At the same time, in vivo release of testosterone was depressed in albumin-HEMA mixed polymer composite in accordance with the weight decrease of polymer composite resulting from digestion. The effect of testosterone on the weight of ventral prostate was investigated using composites in castrated Wistar rats. The effect was larger in the controlled slow release from implanted composites rather than that of dosage by injection. The microscopic observation showed that the inflammation and foreign body reaction in rat tissue were retarded in albumin-HEMA mixture polymer composite compared with 100% albumin composite.

Effects of estradiol -17β on testosterone-induced growth and increase in enzyme activities were studied in the ventral prostate of castrated rats. Estrogen administration did not affect testosterone induced growth of the atrophied tissue as determined by rates of increase in weight, the content of nucleic acids and DNA synthesis. However, cytoplasmic alkaline phosphatase activity was markedly increased by estrogen administration over the enzyme level of rats treated with testosterone alone. In rats treated simultaneously with androgen and estrogen throughout the experimental period, the synergistic effect on increase in the enzyme activity was observed to begin abruptly on the 6th day after initiation of the hormone treatment. This synergistic effect was found to be observed very clearly only in rats that received estrogen treatment during an early period of testosterone-induced recovery of the gland. Moreover, the observed synergism was found to be quite specific, since no effects of estrogen were detected on the enzyme levels of lactate, malate and isocitrate dehydrogenases or acid phosphatase in the tissue of testosterone-treated rats.

It was found that more than half of the activity of testosterone 5α-reduction was attached to nuclear fraction of rat ventral prostate, and some properties of this reaction were investigated using purified nuclear fraction of the gland.The optimum pH for reduction of testosterone was about 7.2 and required NADPH as a donor of reducing equivalent. Apparent km of nuclear 5α-reductase was 3.2×10-5M for testosterone and 3.0×10-4M for NADPH. Apparent Km for 19-nortestosterone was similar to that for testosterone, but the value for androst-4-ene-3, 17-dione was higher than that for testosterone.Natural and synthetic estrogenic compounds showed their inhibitory effect on the rate of testosterone 5α-reduction, but one of the gestagen and of anti-androgen did not inhibit the reaction. Estradiol-17β exhibited a most profound inhibition among the compounds examined, and the double reciprocal plots revealed that type of the inhibition is competitive.The reverse reaction of 5α-reductase using NADP or phenazine dimethyl sulfate as electron acceptor was not detected in purified nuclear fraction from rat ventral prostate.

The activity of testosterone 5α-reductase in purified nuclei of rat ventral prostate was inhibited by addition of estradiol-17β, estrone or estriol in vitro. Rate of the inhibition of the enzyme activity by these estrogens was of similar degree. Therefore, this inhibitory effect does not seem to be correlated to their estrogenic potency.Effect of three anti-androgens (Ro 2-7239, SK & F 7690 and TSAA 291) on the activity of testosterone 5α-reductase was also examined and it was revealed that SK & F 7690 and TSAA 291 showed the inhibitory effect on the enzyme activity.

It was confirmed that the nuclear testosterone 5α-reductase was attached to membrane fraction as observed by Moore and Wilson (1972) in rat ventral prostate. Testosterone 5α-reductase of the nuclear membrane fraction was partially solubilized by phospholipase A, and overall 20 fold purification was attained. Optimum pH, pyridine nucleotide requirement and time course of the reaction of the solubilized enzyme were examined.

Arginase activity in the kidney and the prostate of rats was found in both supernatant and precipitate after centrifugation at 12, 000×g for 20min. Ratios of enzyme activity in supernatant to precipitate varied in tissues examined; ratios of the liver and the ventral prostate were 5.5 and 2.3, respectively, however that of the kidney was 0.3. Arginase in the precipitate seems to be attached to membraneous element, because the activities of arginase are lost from nucleus after Triton X-100 treatment or heavy sucrose purification.Arginases from supernatant and precipitate in kidney and ventral prostate showed similar Km for substrate, but the rate of activation by Mn++ was slightly higher in supernatant than in precipitate.Castration caused a marked decrease of arginase activity in the precipitate of ventral prostate, while the change of the activity in supernatant was quite moderate. On the other hand, no significant changes of enzyme activity were observed in both supernatant and precipitate of the kidney. Testosterone treatment on castrated rats evoked an increase of the enzyme activity in the precipitate of kidney and of ventral prostate, and the elevation of the arginase activity in the supernatant was followed by the increase of the activity in the precipitate. The increase in the enzyme activity of ventral prostate was more prompt than in kidney after testosterone administration.