Characterization of the in vitro biotransformation of the HIV-1 reverse transcriptase inhibitor nevirapine by human hepatic cytochromes P-450.

Nevirapine (NVP), a non-nucleoside inhibitor of HIV-1 reverse transcriptase, is concomitantly administered to patients with a variety of medications. To assess the potential for its involvement in drug interactions, cytochrome P-450 (CYP) reaction phenotyping of NVP to its four oxidative metabolites, 2-, 3-, 8-, and 12-hydroxyNVP, was performed. The NVP metabolite formation rates by characterized human hepatic microsomes were best correlated with probe activities for either CYP3A4 (2- and 12-hydroxyNVP) or CYP2B6 (3-and 8-hydroxyNVP). In studies with cDNA-expressed human hepatic CYPs, 2- and 3-hydroxyNVP were exclusively formed by CYP3A and CYP2B6, respectively. Multiple cDNA-expressed CYPs produced 8- and 12-hydroxyNVP, although they were produced predominantly by CYP2D6 and CYP3A4, respectively. Antibody to CYP3A4 inhibited the rates of 2-, 8-, and 12-hydroxyNVP formation by human hepatic microsomes, whereas antibody to CYP2B6 inhibited the formation of 3- and 8-hydroxyNVP. Studies using the CYP3A4 inhibitors ketoconazole, troleandomycin, and erythromycin suggested a role for CYP3A4 in the formation of 2-, 8-, and 12-hydroxyNVP. These inhibitors were less effective or ineffective against the biotransformation of NVP to 3-hydroxyNVP. Quinidine very weakly inhibited only 8-hydroxyNVP formation. NVP itself was an inhibitor of only CYP3A4 at concentrations that were well above those of therapeutic relevance (K(i) = 270 microM). Collectively, these data indicate that NVP is principally metabolized by CYP3A4 and CYP2B6 and that it has little potential to be involved in inhibitory drug interactions.

Novel nonnucleoside inhibitors of HIV-1 reverse transcriptase. 7. 8-Arylethyldipyridodiazepinones as potent broad-spectrum inhibitors of wild-type and mutant enzymes.

Like other nonnucleoside inhibitors of HIV-1 reverse transcriptase, the dipyridodiazepinone nevirapine (Viramune, 1) selects for drug resistant variants of HIV-1, both in cell culture and in patients. In particular, the mutation of residue 181 from tyrosine to cysteine (Y181C) is associated with resistance to most reported nonnucleoside inhibitors. Introduction of an arylethyl substituent at the 8-position of the tricyclic dipyridodiazepinone skeleton confers enhanced potency against Y181C RT. Several analogues of this series display good broad spectrum potency against a panel of mutant enzymes.

Novel nonnucleoside inhibitors of HIV-1 reverse transcriptase. 8. 8-Aryloxymethyl- and 8-arylthiomethyldipyridodiazepinones.

Nevirapine (I) is the first human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitor to reach regulatory approval. As a result of a second generation program around the tricyclic core system of nevirapine, 2-chloro-5, 11-dihydro-11-ethyl-5-methyl-8-(2-(pyridin-4-yl)ethyl)-6H-dipyrido[3, 2-b:2',3'-e][1,4]diazepin-6-one (II)1a and 2-chloro-5, 11-dihydro-11-ethyl-5-methyl-8-phenylethyl-6H-dipyrido[3,2-b:2', 3'-e][1,4]diazepin-6-one (III)1a were identified as broad spectrum HIV-1 RT inhibitors. A detailed examination of replacing either of the methylenes of the 8-ethyl linker of II or III is presented. It was found that 8-aryloxymethyl and 8-arylthiomethyl are the preferred pattern of substitution for potency against RT. The most potent compounds were further evaluated against a panel of clinically significant mutant RT enzymes (K103N, V106A, G190A, P236L) and in cytotoxicity and in vitro metabolism assays. The most potent compound was 2-chloro-8-phenylthiomethyl analogue 37 which displayed sub-100 nM activity against all HIV-1 RT enzymes tested.

The distribution, elimination, and in vivo biotransformation of aldicarb in the rainbow trout (Oncorhynchus mykiss).

The distribution and elimination of [14C]aldicarb, administered orally and by intraperitoneal (ip) injection, was examined in the rainbow trout (Oncorhynchus mykiss). Tissue residues were determined by monitoring radioactivity at various time periods up to 96 hr in trout administered [14C]aldicarb orally. Periodic water samples and a single tissue residue radioactivity level were obtained after 24 hr in free swimming and spinally transected fish which received [14C]aldicarb via intraperitoneal injection. Aldicarb appears to be absorbed rapidly (99% within 3 hr) and distributed to all tissues. Elimination profiles from both dosage groups demonstrate a rapid alpha phase (oral 24 hr; ip 3 hr) probably due to branchial excretion (96% after ip injection) followed by a slower beta phase (oral 107 hr; ip 28 hr) suggesting a deeper compartment such as muscle. [14C]Aldicarb and/or its metabolites were slowly being transported to the bile after 24 hr. The in vivo biotransformation of [14C]aldicarb was examined in spinally transected trout 24 hr after ip injection. The major metabolite found was aldicarb sulfoxide (7.6%) along with lesser amounts of aldicarb oxime (5.4%).

The effect of piperonyl butoxide on hepatic cytochrome P-450-dependent monooxygenase activities in rainbow trout (Salmo gairdneri).

Although piperonyl butoxide (PBO) is commonly used both in vivo and in vitro as an inhibitor of cytochrome P-450-dependent monooxygenase (MO) activity in a wide variety of species, the effect of PBO on the hepatic MO of fishes has never been characterized. The MO activity in hepatic microsomes from rainbow trout exposed to either 1 or 2 ppm PBO for 24 hr in a static system was induced to a similar level in both treatment groups. Conversely, when PBO was administered in a flow-through system to trout, the hepatic microsomes of treated animals contained MO activities that were induced in a dose-dependent manner. Furthermore, total cytochrome P-450 was significantly increased in the livers of trout treated in a flow-through system with 1 ppm or more of PBO. The in vitro inhibition kinetics of PBO toward the 7-ethoxycoumarin-O-deethylase (ECOD) and 7-ethoxyresorufin-O-deethylase (EROD) activities of hepatic microsomes from trout treated with beta-naphthoflavone (BNF) (100 mg/kg, ip) or PBO (4 ppm by flow-through) and untreated trout were compared with Dixon plots. With respect to ECOD activity, the slopes of Dixon plots from control, BNF- and PBO-treated animals were similar. However, the slopes of Dixon plots of EROD inhibition by PBO in microsomes from BNF- and PBO-treated trout were significantly different from each other. Treatment of trout with PBO in a flow-through system resulted in an increase in ECOD and EROD activity in hepatic microsomes while simultaneously decreasing their activity toward [14C]rotenone oxidation. These data suggest that the cytochrome P-450 isozyme composition in hepatic microsomes from PBO-treated rainbow trout may be qualitatively different from that of BNF-treated trout. Also, the activity of hepatic microsomes from PBO-treated trout toward a specific substrate may be either inhibited or induced.

The effects of noise upon human hearing sensitivity from 8000 to 20 000 Hz.

High-frequency (8 to 20 kHz) hearing sensitivity was compared in thirty-six, 20 to 29-year-old military veterans with histories of steady-state or impulsive noise exposure. Threshold shifts were prominent for the steady-state noise subjects from 13 to 20 kHz. Mean thresholds from 8 through 12 kHz were maximally 20 dB poorer than a sample of young adult normals. Audiometric configurations for this group were generally smooth and symmetrical above 8000 Hz. For the impulsive noise group, substantial shifts in sensitivity were seen from 2 to 20 kHz and the high-frequency audiometric configurations were often jagged and/or asymmetrical. The variability of subjects in this group was greater than that seen in the steady-state noise exposed sample. Several case studies are presented to illustrate these characteristics. Measurement of auditory sensitivity from 8 to 20 kHz extends the mapping of basal cochlear function, providing information which often is not predictable from conventional audiometric measurement. This additional information provides for more comprehensive inter- and intra-subject comparison of the degree and extent of threshold changes present.

The effects of impulsive noise upon human hearing sensitivity (8 to 20 kHz).

The effects of impulsive noise exposure upon hearing sensitivity from 8000 to 20000 Hz were determined for a sample of 23 young military veterans. The subjects' histories consisted primarily of major incidents of weapons fire. Based on audiometric configuration, the subject sample was divided in two groups characterized by predominantly unilateral or bilateral shifts in threshold sensitivity. This division was consistent with history information. The main finding was extensive threshold shifts from 8000 to 20000 Hz which was highly individual-specific and unpredictable. High frequency audiometry frequently revealed extensive changes not evident in the 250 through 8000 Hz range.

Effects of steady-state noise upon human hearing sensitivity from 8000 to 20 000 Hz.

Young adults with steady-state noise exposure histories were evaluated with a high frequency (8--20 kHz) test system. High frequency hearing threshold shifts were most prominent from 12-20 kHz and were often highly individual-specific. Good standard range (.25-8 kHz) sensitivity does not assure good hearing above this region. Mild through severe threshold changes may be present. High frequency testing expands monitoring audiometry and has potential special application to hearing conservation programs.

A system for evaluating auditory function from 8000--20 000 Hz.

A system for the measurement of auditory function from 8000--20 000 Hz is described. This system introduces advances in: (a) maximum power output, (b) signal fidelity, and (c) transducer characteristics. Two case studies are presented to illustrate the clinical information gained from the measurement of high-frequency auditory sensitivity, which is not readily apparent in conventional threshold assessment.

2AFC versus standard clinical measurement of high frequency auditory sensitivity (8--20 KC/S).

A two-alternative forced choice (2AFC) psychophysical method of assessment was employed to judge the validity of using a standard clinical test procedure for obtaining thresholds in the frequency region 8--20 kc/s. Close agreement was demonstrated between "same-day" thresholds obtained with a standard clinical test procedure and the 2AFC method, for 6 well trained normal-hearing adults. In addition, close agreement was shown between the 2AFC sensitivity measure and the mean of thresholds obtained at 4 separate times during this investigation with the standard method. The standard clinical test method demonstrated good between-test reliability. It is concluded that a standard clinical test technique is a valid and feasible clinical measurement procedure for this high frequency region.