Cloning, expression and characterization of rhesus macaque types 1 and 2 5alpha-reductase: evidence for mechanism-based inhibition by finasteride.

The rhesus macaque types 1 and 2 5alpha-reductase (5aR1 and 5aR2) were cloned and expressed in COS cells to facilitate comparison of rhesus and human 5aRs. The deduced protein sequences of the rhesus SaRs shared 94% and 96% identity with the human type 1 and 2 isozymes, respectively. Despite a four amino acid insertion at the N-terminal region of rhesus 5aR1, the biochemical properties of rhesus and human homologs are very similar with respect to pH optimum, Km values for testosterone and progesterone, and inhibition by a variety of inhibitors. As expected, the biochemical properties of the human and rhesus 5aR2 are also very similar. The mechanism of inhibition of the rhesus 5aR1 and 5aR2 by finasteride was investigated in more detail. Finasteride displays time dependent inhibition of the rhesus 5aR1 and 5aR2 with second order rate constants of 4 x 10(3) M(-1) s(-1) and 5.2 x 10(5) M(-1)s(-1). Inhibition of rhesus 5aR2 with 3H-finasteride resulted in 3H bound to the enzyme which is not released by dialysis. Heat denaturation of the [rhesus SaR2:inhibitor] complex releases dihydrofinasteride, a breakdown product presumably related to the NADP+-adduct previously identified with the human SaRs (Bull et al., Mechanism-based inhibition of human steroid 5alpha-reductase by finasteride: Enzyme catalyzed formation of NADP-dihydrofinasteride, a potent bisubstrate analog inhibitor. J. Amer. Chem. Soc., 1996, 118, 2359-2365). Taken together, these results provide good evidence that the rhesus macaque is a suitable model to evaluate the pharmacological properties of finasteride and other 5aR inhibitors.

Effects of finasteride, a type 2 5-alpha reductase inhibitor, on fetal development in the rhesus monkey (Macaca mulatta).

In genetic male fetuses, dihydrotestosterone (DHT) plays an important role in normal prostatic and external genital differentiation. The enzyme steroid 5-alpha reductase (5 alpha R) catalyzes the conversion of testosterone (T) to DHT. The importance of 5 alpha R in sexual differentiation is evident from the study of human genetic males who congenitally lack this enzyme and consequently develop ambiguous genitalia. These individuals are specifically deficient in the type 2 isozyme, whereas the normal type 1 isozyme activity has been found. The purpose of this study was to determine 1) the suitability of the rhesus monkey for testing the safety of 5 alpha R inhibitors when administered during pregnancy and 2) the potential risk of administering a known type 2 5 alpha R inhibitor, finasteride, during the critical period of internal and external genital differentiation in rhesus monkeys. In vitro studies were also performed on selected rhesus monkey tissues to determine the distribution of the 5 alpha R isozymes. Gravid monkeys were treated once daily from gestational days (GD) 20 to 100. Sonographic monitoring was performed during the course of gestation to monitor viability, growth, and organ system development. Detailed fetal evaluations for developmental abnormalities were performed at term (GD 152 +/- 2). A group of 13 pregnant monkeys ("positive control") were given a high oral dose (2 mg/kg/day) of finasteride to demonstrate that inhibiting type 2 5 alpha R results in specific external genital abnormalities in male fetuses. Thirty-two pregnant monkeys were administered an intravenous (i.v.) formulation of finasteride at doses of 8, 80, or 800 ng/day. The highest i.v. dose selected was at least 60-750 times the semen levels of finasteride in man given orally 5 or 1 mg/day, respectively. Seventeen vehicle-control pregnant monkeys were also included. Administration of a high oral dose (2 mg/kg/day) of finasteride resulted in external genital abnormalities characterized by hypospadias, preputial adhesions to the glans, a small underdeveloped scrotum, a small penis, and a prominent midline raphe in male fetuses; however, no developmental abnormalities were seen in female fetuses. Similarly, no abnormalities were observed in either male or female fetuses of mothers given iv doses (8, 80, or 800 ng/day) of finasteride during pregnancy. The in utero sonographic findings in fetuses correlated with the gross findings at term. These studies have shown that external genital abnormalities can be produced in male monkey fetuses when exposed to a high oral dose (2 mg/kg/day) of finasteride, whereas no abnormalities were observed in fetuses exposed to the i.v. formulation of finasteride. Detailed in vitro studies demonstrated that the rhesus monkey also has two 5 alpha R isozymes (types 1 and 2) with a tissue distribution similar to that seen in man and, furthermore, that finasteride is a potent, mechanism-based inhibitor with selectivity for both human and rhesus type 2 5 alpha R. These studies have demonstrated that the monkey is a suitable model for assessing the safety of 5 alpha R inhibitors administered during pregnancy.