Publications

A microdialysis technique was used in male rats to directly assess the postulate that pubertal maturation is associated with accelerated GnRH pulsatility. Juvenile male rats, postnatal d 43 or 45 (n = 4) were stereotaxically fitted with guide cannulas directed toward the lateral median eminence, and repeated microdialysis experiments were conducted over 4-6 d. In each session, samples were collected continuously over 12 h (0900-2100 h) at 5-min intervals Results from individual peripubertal animals were pooled into two time bins for postnatal d 45-47 and 48-50, respectively, and GnRH characteristics were compared between the two epochs. The GnRH pulse frequency and mean GnRH concentration were significantly elevated at 48-50 d compared with 45-47 d. The GnRH pulsatility characteristics for 45-47 d vs. 48-50 d were as follows: pulse frequency, 0.74 +/- 0.16 vs. 1.79 +/- 0.19 pulses/h (P < 0.05); pulse amplitude, 254.1 +/- 22.3 vs. 347.2 +/- 15.8 deltapg/ml (difference in value from trough to peak); and mean release, 0.55 +/- 0.03 vs. 2.04 +/- 0.04 pg/5 min (P < 0.05). An additional two rats were dialyzed only once on postnatal d 50 to assess the effects of repeated sampling; the GnRH pulse characteristics in these animals were similar to those in rats sampled for a third or fourth time on postnatal d 48-50. To further assess the possible effects of repeated sampling on GnRH release profiles, a group of adult male rats (postnatal d 95-105; n = 3) was also dialyzed on four consecutive days. In these rats no significant alteration in GnRH pulse generator activity was observed over the four sessions. Moreover, the increase in GnRH pulse frequency observed in the peripubertal rats was found to be sustained in adult animals. To better understand the temporal relationship of GnRH pulse generator activity to reproductive maturation, groups of male rats were killed from postnatal d 45-56 along with an adult group at 95-105 d (n = 5/group) and examined for physiological signs of reproductive development. Gradual increases in serum levels of LH and testosterone and decreases in FSH and inhibin B were seen from postnatal d 45-56 to adulthood. Mature spermatozoa were found in the vas deferens by postnatal d 53. Our results demonstrate that in the late juvenile stage of male rat development, GnRH pulse generator activity is gradually accelerated over the course of consecutive days. This acceleration occurs over a period during which serum LH and testosterone are rising to adult levels, and it precedes the presence of mature spermatozoa in the vas deferens by 3 d. Our observations provide direct support for the hypothesis that an acceleration of GnRH pulsatility is the critical neural stimulus for the initiation of pubertal maturation in males. The peripheral and central cues that prompt the pubertal activation of the GnRH pulse generator remain to be characterized.

CYP3A9 is an estrogen-inducible ortholog of human liver CYP3A4 with 76.5% sequence identity to CYP3A4. Unlike CYP3A4, it is a very poor testosterone 6beta- and 2beta-hydroxylase, but a relatively better catalyst of progesterone monohydroxylation largely at 6beta, 16alpha, and 21 positions with negligible 6beta, 21-dihydroxylation. We reasoned that such differences in substrate catalyses must be due to differences in the active site architecture of each CYP3A enzyme. Indeed, alignment of CYP3A4 substrate recognition sites (SRSs) with the corresponding regions of CYP3A9 sequence revealed that of the 22 fully divergent residues, 4 reside in SRS regions [P107N (SRS-1), M371G (SRS-5), and L479K and G480Q (SRS-6)]. Accordingly, we substituted these and other divergent CYP3A9 SRS residues with the corresponding residues of CYP3A4 and/or CYP3A5. Our findings of the influence of these site-directed mutations of the CYP3A9 active site on its catalysis of testosterone and three other established but structurally different CYP3A substrates (progesterone, imipramine, and carbamazepine) are described. These findings revealed that some mutations (N107P, N107S, V207T, G371M, and Q480G) not only improved the ability of CYP3A9 to hydroxylate testosterone at the 6beta and 2beta positions, but also converted it into a robust progesterone 6beta, 21-dihydroxylase. The latter in the case of CYP3A9N107P was accompanied by a shift from sigmoidal to hyperbolic enzyme-substrate kinetics. In contrast, the catalytic potential of CYP3A9 mutants K206N, K206S, M240V, and K479L/Q480G was either relatively unchanged or negligible to nonexistent. Together these findings attest to the unique substrate-active site fit of each CYP3A enzyme.

Cyclosporine (CyA) is an immunosuppressant metabolized primarily by the liver and small intestine. The pharmacokinetics (PK) of CyA-were studied in 6 patients prior to and 1 to 3 months after liver transplantation (tx). Sixteen blood samples were collected over 24 hours following a 2-3 mg/kg intravenous dose of CyA. PK parameters, presented as mean +/- SD, were estimated using noncompartmental techniques. Pre-tx AUCs (14,540 +/- 5200 micrograms.h/L) were found to be significantly higher than during the post-tx phase (8120 +/- 2870 micrograms.h/L, p = 0.04). CyA clearance values were lower pre-tx as compared to post-tx (0.21 +/- 0.06 L/h/kg vs. 0.38 +/- 0.14 L/h/kg, respectively). There was no change in volume of distribution. End-stage liver disease can markedly decrease hepatic clearance of CyA relative to patients with stable hepatic function post-liver tx. The degree of impairment in clearance is not consistent or predictable based on liver function tests.

With advances in antiretroviral therapy, many HIV+ individuals are living longer lives and some are developing end-stage renal and/or hepatic disease requiring transplantation. These patients require concomitant use of immunosuppressants (e.g., cyclosporine [CsA]) and antiretrovirals (e.g., protease inhibitors [PIs]), which exhibit narrow therapeutic windows and are substrates and inhibitors of cytochrome P450 3A enzymes and the cellular transporter P-glycoprotein. In this pilot study, HIV+ subjects on either oral nelfinavir (NFV) or indinavir (IND) with nondetectable viral loads and normal renal and hepatic function had 12 hour pharmacokinetic (PK) studies on 3 separate days: PIs alone, PIs+intravenous CsA, and PIs+oral CsA to determine the extent of PK interactions between these medications. PIs and CsA concentrations were measured by LC/MS in plasma and whole blood, respectively. Nine subjects (n=7 on NFV, n=2 on IND) completed the study. Only the results of those subjects taking NFV are reported. Oral co-administration of CsA increased NFV T(max) from 2.6+/-0.9 to 3.2+/-0.8 h (p<0.05), and AUC(0-infinity) from 27.9+/-15.2 to 43.2+/-27.1 mg(*)h/mL (p=0.06). Intravenous CsA did not appreciably alter oral pharmacokinetics of NFV. Both CsA and NFV PK parameters exhibited a high degree of intersubject variability, underscoring the need for routine therapeutic drug monitoring of both CsA and PIs in HIV+ subjects undergoing transplantation.