Kenneth Bachmann

Human Pregnane X Receptor Antagonists and Agonists Define Molecular Requirements for Different Binding Sites

The pregnane X receptor (PXR) is an important transcriptional regulator of the expression of xenobiotic metabolism and transporter genes. The receptor is promiscuous, binding many structural classes of molecules that act as agonists at the ligand-binding domain, triggering up-regulation of genes, increasing the metabolism and excretion of therapeutic agents, and causing drug-drug interactions. It has been suggested that human PXR antagonists represent a means to counteract such interactions. Several azoles have been hypothesized to bind the activation function-2 (AF-2) surface on the exterior of PXR when agonists are concurrently bound in the ligand-binding domain. In the present study, we have derived novel computational models for PXR agonists using different series of imidazoles, steroids, and a set of diverse molecules with experimental PXR agonist binding data. We have additionally defined a novel pharmacophore for the steroidal agonist site. All agonist pharmacophores showed that hydrophobic features are predominant. In contrast, a qualitative comparison with the corresponding PXR antagonist pharmacophore models using azoles and biphenyls showed that they are smaller and hydrophobic with increased emphasis on hydrogen bonding features. Azole antagonists were docked into a proposed hydrophobic binding pocket on the outer surface at the AF-2 site and fitted comfortably, making interactions with key amino acids involved in charge clamping. Combining computational and experimental data for different classes of molecules provided strong evidence for agonists and antagonists binding distinct regions on PXR. These observations bear significant implications for future discovery of molecules that are more selective and potent antagonists.

Cyclooxygenase‐2 inhibition by rofecoxib reverses naturally occurring fever in humans

Cyclooxygenase (COX) exists as constitutive (COX‐1) and inducible (COX‐2) isoforms. Nonsteroidal anti‐inflammatory drugs (NSAIDs) such as ibuprofen and diclofenac inhibit both COX‐1 and COX‐2. The role of COX‐2 in the genesis of fever in monkeys and humans was examined with use of the specific COX‐2 inhibitor rofecoxib. Rofecoxib was administered to monkeys made febrile by 6 μg/kg intravenous lipopolysaccharide. Induced pyrexia was followed by oral rofecoxib (1 or 3 mg/kg), diclofenac (3 mg/kg), or vehicle. Rofecoxib and diclofenac rapidly reversed the elevated temperature (P < .05 versus vehicle for 3 mg/kg rofecoxib and diclofenac at 70 to 90 minutes after dosing). A single‐dose, parallel‐group, double‐blind randomized trial was conducted in 94 patients with fever caused by a viral‐type illness. Mean baseline temperature was similar for all groups (~38.5°C). Patients received oral doses of 12.5 mg rofecoxib, 25 mg rofecoxib, 400 mg ibuprofen, or placebo and the mean ± SE change in oral temperature at 4 hours after dosing was −0.97°C ± 0.11°C, −1.19°C ± 0.09°C, −1.20°C ± 0.11°C, and 0.01°C ± 0.17°C, respectively (P < .001 for active treatments versus placebo). Specific inhibition of COX‐2 by rofecoxib results in antipyretic activity in monkeys and humans comparable to dual COX‐1/COX‐2 inhibitors such as diclofenac or ibuprofen. The data support the hypothesis that it is the COX‐2 isoform that is primarily involved in the genesis of fever in humans.

Impact of ciprofloxacin on theophylline clearance and steady-state concentrations in serum.

The effect of a multiple-dose regimen of oral ciprofloxacin (750 mg every 12 h for 11 doses) on the clearance and steady-state concentrations of theophylline in trough (predose) serum was evaluated in nine healthy male subjects, each serving as his own control. Theophylline was taken as a sustained release tablet per os in a dose of 200 mg every 12 h for 19 doses. Theophylline concentrations in serum were measured immediately before each theophylline dose. Ciprofloxacin was administered on study day 4 through the first dose of study day 8. Theophylline concentrations in serum were also measured on study days 3, 6, 8, and 10 at the following times after the first dose of each day: 0, 0.25, 0.50, 1, 2, 4, 6, 8, 10, and 12 h. Steady-state theophylline concentrations in trough serum were significantly higher during ciprofloxacin treatment (day 8) than before (day 3) or after (day 10) ciprofloxacin administration (P less than 0.01). Likewise, theophylline clearance was significantly slower (P less than 0.01) during ciprofloxacin treatment (day 8) than before it (day 3) or after it (day 10). The magnitude of ciprofloxacin-induced changes was approximately 30%. These results suggest that a multidose regimen of ciprofloxacin significantly slows the clearance of theophylline and elevates theophylline concentrations in serum.

Computational Discovery of Novel Low Micromolar Human Pregnane X Receptor Antagonists

Very few antagonists have been identified for the human pregnane X receptor (PXR). These molecules may be of use for modulating the effects of therapeutic drugs, which are potent agonists for this receptor (e.g., some anticancer compounds and macrolide antibiotics), with subsequent effects on transcriptional regulation of xenobiotic metabolism and transporter genes. A recent novel pharmacophore for PXR antagonists was developed using three azoles and consisted of two hydrogen bond acceptor regions and two hydrophobic features. This pharmacophore also suggested an overall small binding site that was identified on the outer surface of the receptor at the AF-2 site and validated by docking studies. Using computational approaches to search libraries of known drugs or commercially available molecules is preferred over random screening. We have now described several new smaller antagonists of PXR discovered with the antagonist pharmacophore with in vitro activity in the low micromolar range [S-p-tolyl 3′,5-dimethyl-3,5′-biisoxazole-4′-carbothioate (SPB03255) (IC50, 6.3 μM) and 4-(3-chlorophenyl)-5-(2,4-dichlorobenzylthio)-4H-1,2,4-triazol-3-ol (SPB00574) (IC50, 24.8 μM)]. We have also used our computational pharmacophore and docking tools to suggest that most of the known PXR antagonists, such as coumestrol and sulforaphane, could also interact on the outer surface of PXR at the AF-2 domain. The involvement of this domain was also suggested by further site-directed mutagenesis work. We have additionally described an FDA approved prodrug, leflunomide (IC50, 6.8 μM), that seems to be a PXR antagonist in vitro. These observations are important for predicting whether further molecules may interact with PXR as antagonists in vivo with potential therapeutic applications.

A Comprehensive in Vitro and in Silico Analysis of Antibiotics That Activate Pregnane X Receptor and Induce CYP3A4 in Liver and Intestine

We have investigated several in silico and in vitro methods to improve our ability to predict potential drug interactions of antibiotics. Our focus was to identify those antibiotics that activate pregnane X receptor (PXR) and induce CYP3A4 in human hepatocytes and intestinal cells. Human PXR activation was screened using reporter assays in HepG2 cells, kinetic measurements of PXR activation were made in DPX-2 cells, and induction of CYP3A4 expression and activity was verified by quantitative polymerase chain reaction, immunoblotting, and testosterone 6β-hydroxylation in primary human hepatocytes and LS180 cells. We found that in HepG2 cells CYP3A4 transcription was activated strongly (>10-fold) by rifampin and troleandomycin; moderately (≥7-fold) by dicloxacillin, tetracycline, clindamycin, griseofulvin, and (≥4-fold) erythromycin; and weakly (>2.4-fold) by nafcillin, cefaclor, sulfisoxazole, and (>2-fold) cefadroxil and penicillin V. Similar although not identical results were obtained in DPX-2 cells. CYP3A4 mRNA and protein expression were induced by these antibiotics to differing extents in both liver and intestinal cells. CYP3A4 activity was significantly increased by rifampin (9.7-fold), nafcillin and dicloxacillin (5.9-fold), and weakly induced (2-fold) by tetracycline, sufisoxazole, troleandomycin, and clindamycin. Multiple pharmacophore models and docking indicated a good fit for dicloxacillin and nafcillin in PXR. These results suggest that in vitro and in silico methods can help to prioritize and identify antibiotics that are most likely to reduce exposures of medications (such as oral contraceptive agents) which interact with enzymes and transporters regulated by PXR. In summary, nafcillin, dicloxacillin, cephradine, tetracycline, sulfixoxazole, erythromycin, clindamycin, and griseofulvin exhibit a clear propensity to induce CYP3A4 and warrant further clinical investigation.

Predicting Inhibitory Drug—Drug Interactions and Evaluating Drug Interaction Reports Using Inhibition Constants

OBJECTIVE: To review the use of inhibitory constants (Ki) determined from in vitro experiments in the prediction of the significance of inhibitory drug—drug interactions (DDIs). DATA SOURCES: Searches of MEDLINE (1966—August 2004) and manual review of journals, conference proceedings, reference textbooks, and Web sites were performed using the key search terms cytochrome P450, drug—drug interaction, inhibition constant, and Ki. STUDY SELECTION AND DATA EXTRACTION: All articles identified from the data sources were evaluated, and information deemed relevant was included for this review. DATA SYNTHESIS: The cytochrome P450 isoenzymes factor prominently in the explanation of numerous DDIs. Although the regulation of these enzymes by one drug can affect the pharmacokinetics of other drugs, the consequences may not necessarily be significant either in terms of pharmacokinetic or clinical outcomes. Yet, many DDI monographs originate as unconfirmed case reports that implicate the influence of one drug on the CYP-mediated metabolism of another, and these often uncorroborated mechanisms can eventually become regarded as dogma. One consequence of this process is the overprediction of potentially important DDIs. The pharmaceutical industry, Food and Drug Administration, and pharmaceutical scientists have developed a strategy for predicting the significance of inhibitory DDIs at the earliest possible stages of drug development based on a new chemical entitys Ki value, determined in vitro. CONCLUSIONS: We suggest that the use of Ki values of drugs purported to behave as CYP inhibitors be incorporated in the assessment of case reports that ascribe DDIs to inhibition of metabolism of one drug by another.

The use of in vitro methods to predict in vivo pharmacokinetics and drug interactions.

With the dramatic change underway in the process of drug discovery and development it has become increasingly important to define, both qualitatively and quantitatively, the dispositional features of new chemical entities (NCEs) as early in the process as possible. To that end strategies have emerged that are designed to enable reasonable predictions about a NCEs absorption from the gastrointestinal tract, systemic bioavailability and likelihood for significant pre-systemic clearance, character of metabolic processing both within the gastrointestinal tract and the liver, in vivo pharmacokinetics (PK), and likelihood for clinically significant interactions with other drugs. To some extent these strategies have embraced interspecies allometric scaling in which findings in animals are extrapolated to predict outcomes in humans. However, a greater emphasis in recent years has been placed on predicting human PK and the likelihood of clinically significant drug-drug interactions for NCEs solely from in vitro experiments. These general strategies have been methodologically streamlined so that hundreds or even thousands of experiments on a given NCE can be conducted within several days. Dispositional data from these pre-clinical experiments is useful for rapidly identifying potential marketing advantages for NCEs, and for screening out those substances that should not be placed into more expensive and labor-intensive animal experiments or brought to clinical trial. The key issue in these strategies is the accuracy with which pre-clinical findings predict clinical outcomes. Based largely on retrospective analyses the current state of the art exhibits a high percentage of useful predictions. However, there are many examples in which the prediction of either human PK or clinical drug-drug interactions from pre-clinical data has failed. The reasons for inaccurate predictions are manifold, and may include the actual in vitro methodology used, inappropriate model selection, and errant scale-up factors. Additionally, in vitro methods may fail to account for complex hepatobiliary processing including transport phenomena and Phase II metabolism. Progress has been made in establishing humanized methodologies that accurately describe these processes, with a view toward reconstituting the contributions of each into a more complex and accurate depiction and prediction of in vivo PK and drug-interaction potential.

The Influence of Anesthetic Agents on Rat Hepatic Cytochromes P450 in vivo

The objective of this study was to identify which anesthetics when used acutely will affect cytochrome P450 (CYP) activity in male Sprague-Dawley rats in vivo. The anesthetics tested were fentanyl citrate, alpha-chloralose, ketamine, urethane (ethyl carbamate), halothane, and ether. CO2 anesthesia was used as the control comparator. Theophylline was used as a probe for CYP1A activity, phenobarbital for CYP2B/2C, flecainide for CYP2D1, and ethosuximide for CYP3A activity. All probes were administered via tail vein injection after anesthetic-induced loss of the righting reflex. Single sample probe clearances were estimated, and used as an index of CYP activity. Fentanyl citrate, alpha-chloralose, halothane, and ether did not have statistically significant effects on any of the CYP activities. Ketamine did not significantly affect CYP1 or CYP2B/2C activity. However, it decreased the clearance of flecainide (i.e. CYP2D1 activity) by 13.4% (p < 0.001) and the clearance of ethosuximide (i.e. CYP3A activity) by 17.6% (p < 0.0001). Urethane increased the clearance of theophylline by 91.5% (p < 0.0001), and decreased the clearance of ethosuximide by 40.5% (p < 0.0001) though it did not affect CYP2B/2C or CYP2D1 activities significantly. From this data, we conclude that a single dose of ketamine mildly inhibits the activity of CYP2D1 and CYP3A, and a single dose of urethane strongly inhibits CYP3A but increases CYP1A activity.

Use of single sample clearance estimates of cytochrome P450 substrates to characterize human hepatic CYP status in vivo

1. Single sample clearance estimates (CL/F) of orally administered ethosuximide were obtained in four groups of healthy adult subjects. One group was treated with phenobarbital to induce CYP2B/2C and CYP3A activity; one group was treated with rifampin to induce CYP3A activity; one group consisted of cigarette smokers (increased CYP1A activity), and one group was untreated (controls). Ethosuximide CL/F values were slightly, though not significantly, increased among cigarette smokers (12.5% increase) and the phenobarbital group (25% increase), but rifampin treatment resulted in a significant increase (65%). 2. The influence of rifampin treatment on the single sample oral clearances of antipyrine, theophylline, phenytoin, carbamazepine, ethosuximide, quinidine, valproic acid, and lorazepam was investigated to determine whether rifampin induces only CYP3A. Rifampin treatment significantly increased the oral clearance of each drug from 1.4-fold (valproic acid) to 3.4-fold (quinidine). These findings indicate that the inductive effect of rifampin extends well beyond CYP3A.

The Effect of Erythromycin on the Disposition Kinetics of Warfarin

Erythromycin is generally regarded as innocuous in regard to adverse interactions with other drugs. Recently, however, its potentiation of theophylline and warfarin has been reported. The present investigation defined more specifically the kinetics of the interaction between erythromycin and warfarin. Warfarin kinetics were evaluated in 12 normal subjects who took a single 1 mg/kg dose of warfarin with and without erythromycin. Erythromycin (250 mg p.o.) every 6 h for 8 days decreased warfarin clearance by 14% (p less than 0.001). Warfarins apparent volume of distribution was not affected. Further, the effect of erythromycin was greatest among subjects whose control phase warfarin clearance was relatively slow, and least among those whose control phase warfarin clearance was relatively fast. The magnitude of the decrease in warfarin clearance correlated negatively with control warfarin clearance (r = -0.89, p less than 0.005). These data are consistent with the interpretation that erythromycin can potentiate warfarin-induced hypoprothrombinemia by slowing warfarin clearance.