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Humans in industrialized areas are continuously exposed to phthalate plasticizers, prompting concerns of their potential toxicities. Previous studies from our laboratory and others have shown that various phthalates activate several mammalian nuclear receptors, in particular the constitutive androstane receptor (CAR), the pregnane X receptor (PXR), and the peroxisomal proliferator-activated receptors (PPARs), although often at concentration levels of questionable relevance to human exposure. We discovered that di(2-ethylhexyl) phthalate (DEHP) and di-isononyl phthalate (DiNP), two of the highest volume production agents, were potent activators of human CAR2 (hCAR2), a unique human CAR splice variant and, to a lesser degree, human PXR (hPXR). These diphthalates undergo rapid metabolism in mammalian systems, initially to their major monophthalate derivatives MEHP and MiNP. Although MEHP and MiNP are reported activators of the rodent PPARs, with lower affinities for the corresponding human PPARs, it remains unclear whether these monophthalate metabolites activate hCAR2 or hPXR. In this investigation, we assessed the relative activation potential of selected monophthalates and other low molecular weight phthalates against hCAR, the most prominent hCAR splice variants, as well as hPXR and human PPAR. Using transactivation and mammalian two-hybrid protein interaction assays, we demonstrate that these substances indeed activate hCARs and hPXR but to varying degrees. MEHP and MiNP exhibit potent activation of hCAR2 and hPXR with higher affinities for these receptors than for the hPPARs. The rank order potency for MEHP and MiNP was hCAR2 > hPXR > hPPARs. Results from primary hepatocyte experiments also reflect the MEHP and MiNP upregulation of the respective human target genes. We conclude that both di- and monophthalates are potently selective hCAR2 activators and effective hPXR activators. These results implicate these targets as important mediators of selective phthalate effects in humans. The striking differential affinities for these compounds between human and rodent nuclear receptors further implies that biological results obtained from rodent models may be of only limited relevance for interpolating phthalate-mediated effects in humans., Competing Interests: The authors declare no competing financial interest. more...

We hypothesized that treatment of pregnant rat dams with a dual reactive monoclonal antibody (mAb4G9) against (+)-methamphetamine [METH; equilibrium dissociation rate constant (KD) = 16 nM] and (+)-amphetamine (AMP; KD = 102 nM) could confer maternal and fetal protection from brain accumulation of both drugs of abuse. To test this hypothesis, pregnant Sprague-Dawley rats (on gestational day 21) received a 1 mg/kg i.v. METH dose, followed 30 minutes later by vehicle or mAb4G9 treatment. The mAb4G9 dose was 0.56 mole-equivalent in binding sites to the METH body burden. Pharmacokinetic analysis showed baseline METH and AMP elimination half-lives were congruent in dams and fetuses, but the METH volume of distribution in dams was nearly double the fetal values. The METH and AMP area under the serum concentration-versus-time curves from 40 minutes to 5 hours after mAb4G9 treatment increased >7000% and 2000%, respectively, in dams. Fetal METH serum did not change, but AMP decreased 23%. The increased METH and AMP concentrations in maternal serum resulted from significant increases in mAb4G9 binding. Protein binding changed from ∼15% to > 90% for METH and AMP. Fetal serum protein binding appeared to gradually increase, but the absolute fraction bound was trivial compared with the dams. mAb4G9 treatment significantly reduced METH and AMP brain values by 66% and 45% in dams and 44% and 46% in fetuses (P < 0.05), respectively. These results show anti-METH/AMP mAb4G9 therapy in dams can offer maternal and fetal brain protection from the potentially harmful effects of METH and AMP., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.) more...

The CAR (constitutive androstane receptor; NR1I3) is a critical xenobiotic sensor that regulates xenobiotic metabolism, drug clearance, energy and lipid homoeostasis, cell proliferation and development. Although constitutively active, in hepatocytes CAR is normally held quiescent through a tethering mechanism in the cytosol, anchored to a protein complex that includes several components, including heat-shock protein 90. Release and subsequent nuclear translocation of CAR is triggered through either direct binding to ligand activators such as CITCO {6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime} or through indirect chemical activation, such as with PB (phenobarbital). In the present study, we demonstrate that proteasomal inhibition markedly disrupts CAR function, repressing CAR nuclear trafficking, disrupting CAR's interaction with nuclear co-activators and inhibiting induction of CAR target gene responses in human primary hepatocytes following treatment with either PB or CITCO. Paradoxically, these effects occur following accumulation of ubiquitinated hCAR (human CAR). Furthermore, a non-proteolytic function was indicated by its interaction with a SUG1 (suppressor for Gal1), a subunit of the 26S proteasome. Taken together, these data demonstrate that the proteasome complex functions at multiple levels to regulate the functional biology of hCAR activity. more...

This lead candidate selection study compared two anti-(+)-methamphetamine (METH) monoclonal antibodies (mAb) to determine their ability to reduce METH-induced locomotor effects and redistribute METH and (+)-amphetamine (AMP) in a preclinical overdose model. Both mAbs have high affinity for METH, but mAb4G9 has moderate and mAb7F9 has low affinity for AMP. In the placebo-controlled behavioral experiment, the effects of each mAb on the locomotor response to a single 1 mg/kg intravenous (IV) METH dose were determined in rats. The doses of mAb binding sites were administered such that they equaled 1, 0.56, 0.32, and 0.1 times the molar equivalent (mol-eq) of METH in the body 30 min after the METH dose. METH disposition was determined in separate animals that similarly received either a 1 or 0.32 mol-eq dose of mAb binding sites 30 min after a 1 mg/kg METH dose. Total METH-induced distance traveled was significantly reduced in rats that received the highest three doses of each mAb compared with saline. The duration of METH effects was also significantly reduced by mAb7F9 at the highest dose. The disposition of METH was altered dose-dependently by both mAbs as shown in reductions of volume of distribution and total clearance, and increases in elimination half-life. These data indicate that both mAbs are effective at reducing METH-induced behavior and favorably altering METH disposition. Both were therefore suitable for further preclinical testing as potential human medications for treating METH use; however, due to results reported here and in later studies, mAb7F9 was selected for clinical development. more...

Regulation of gene transcription is controlled in part by nuclear receptors that function coordinately with coregulator proteins. The human constitutive androstane receptor (CAR; NR1I3) is expressed primarily in liver and regulates the expression of genes involved in xenobiotic metabolism as well as hormone, energy, and lipid homeostasis. In this report, DAX-1, a nuclear receptor family member with corepressor properties, was identified as a potent CAR regulator. Results of transaction and mutational studies demonstrated that both DAX-1's downstream LXXLL and its PCFQVLP motifs were critical contributors to DAX-1's corepression activities, although two other LXXM/LL motifs located nearer the N terminus had no impact on the CAR functional interaction. Deletion of DAX-1's C-terminal transcription silencing domain restored CAR1 transactivation activity in reporter assays to approximately 90% of control, demonstrating its critical function in mediating the CAR repression activities. Furthermore, results obtained from mammalian two-hybrid experiments assessing various domain configurations of the respective receptors showed that full-length DAX-1 inhibited the CAR-SRC1 interaction by approximately 50%, whereas the same interaction was restored to 90% of control when the DAX-1 transcription silencing domain was deleted. Direct interaction between CAR and DAX-1 was demonstrated with both alpha-screen and coimmunoprecipitation experiments, and this interaction was enhanced in the presence of the CAR activator 6-(4-chlorophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). Results obtained in primary human hepatocytes further demonstrated DAX-1 inhibition of CAR-mediated CITCO induction of the CYP2B6 target gene. The results of this investigation identify DAX-1 as a novel and potent CAR corepressor and suggest that DAX-1 functions as a coordinate hepatic regulator of CAR's biological function. more...

Illicit drug use during pregnancy is a serious social and public health problem inflicting an array of deleterious effects on both mother and offspring. We investigated the hypothesis that a murine anti-phencyclidine (PCP) monoclonal antibody (mAb6B5; K(D)=1.3 nM) can safely protect mother and fetus from PCP-induced adverse health effects in pregnant rats. Pregnant Sprague-Dawley rats (n=4-5) were intravenously administered bolus injections of PCP (1mg/kg) on multiple days during pregnancy. They were also chronically treated with anti-PCP mAb6B5 at 45 mg/kg as a PCP antagonist. This dose provided one mAb-PCP binding site for every four PCP molecules. Therapeutic and safety study endpoints included pregnancy outcome (litter size, number of live vs. dead pups), maternal hemodynamic status and locomotor activity. Maternal hemodynamic changes (i.e., blood pressure and heart rate) and locomotor activity were measured in dams from gestation days 6-21 (one day antepartum) using a radiotelemetry-tracking device with a femoral arterial pressure catheter. This mAb6B5 treatment regimen significantly (p=0.008) reduced the number of PCP-induced in utero fetal deaths (odds ratio=3.2; 95%CI 1.3 to 7.9) and significantly (p<0.05) reduced acute PCP-induced maternal locomotor effects in the second trimester. Maternal hemodynamic responses to PCP were not significantly affected by mAb6B5 treatment. In conclusion, these data suggest that anti-PCP mAb treatments administered during pregnancy can safely protect a mother and her fetus(es) from PCP-related morbidity and mortality even when the mAb dose is too low to significantly prevent other PCP-induced maternal pharmacological effects., (Copyright © 2011 Elsevier B.V. All rights reserved.) more...

The constitutive androstane receptor (CAR; NR1I3) is a member of the nuclear receptor superfamily and functions as an important xenochemical sensor and transcriptional modulator in mammalian cells. Upon chemical activation, CAR undergoes nuclear translocation and heterodimerization with the retinoid X receptor subsequent to its DNA target interaction. CAR is unusual among nuclear receptors in that it possesses a high level of constitutive activity in cell-based assays, obscuring the detection of ligand activators. However, a human splice variant of CAR, termed CAR3, exhibits negligible constitutive activity. In addition, CAR3 is activated by ligands with similar specificity as the reference form of the receptor. In this study, we hypothesized that similar CAR3 receptors could be constructed across various mammalian species' forms of CAR that would preserve species-specific ligand responses, thus enabling a more sensitive and differential screening assessment of CAR response among animal models. A battery of CAR3 receptors was produced in mouse, rat, and dog and comparatively evaluated with selected ligands together with human CAR1 and CAR3 in mammalian cell reporter assays. The results demonstrate that the 5-amino acid insertion that typifies human CAR3 also imparts ligand-activated receptor function in other species' CAR while maintaining signature responses in each species to select CAR ligands. These variant constructs permit in vitro evaluation of differential chemical effector responses across species and coupled with in vivo assays, the species-selective contributions of CAR in normal physiology and in disease processes such as hepatocarcinogenesis. more...

Phthalates and other endocrine-disruptive chemicals are manufactured in large quantities for use as plasticizers and other commercial applications, resulting in ubiquitous human exposure and thus, concern regarding their toxicity. Innate defense against small molecule exposures is controlled in large part by the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). The human CAR gene undergoes multiple alternative splicing events resulting in the CAR2 and CAR3 variant receptors. Recent studies from our laboratory show that CAR2 is potently and specifically activated by di(2-ethylhexyl) phthalate (DEHP). We hypothesized that alternative splicing is a mechanism for increasing CAR's functional diversity, broadening the human receptors' repertoire of response to environmental xenobiotics. In these studies, we examine the interaction of alternatively spliced CARs and PXR with a range of suspected endocrine disruptors, including phthalates, bisphenol A (BPA), and 4-N-nonylphenol (NP). Transactivation and two-hybrid studies in COS-1 cells revealed differential selectivity of endocrine-disrupting chemicals for the variant CAR and PXR. Ex vivo studies showed DEHP and di-isononyl phthalate potently induced CYP2B6 and CYP3A4 expression in human hepatocytes. Mutation analysis of CAR2, in silico modeling, and ligand docking studies suggested that the SPTV amino acid insertion of CAR2 creates a unique ligand-binding pocket. Alternative gene splicing results in variant CAR receptors that selectively recognize phthalates and BPA. The interaction of phthalates with CAR and PXR suggests a xenobiotic response that is complex and biologically redundant. more...

During preclinical development of neuroprotective antiaddiction therapeutic monoclonal antibodies (mAbs) against phencyclidine (PCP) and (+)-methamphetamine, we discovered novel, gestation stage-specific changes in mAb disposition spanning the entire reproductive cycle of female rats. Each pharmacological change was independent of mAb dose and antigen target but was precisely coincident with transitions between the gestational trimesters, parturition, and lactation periods of the female reproductive cycle. Whereas anti-PCP mAb6B5 terminal elimination half-life (t(1/2λz)) in nonpregnant females was 6.6 ± 1.6 days, the mAb6B5 t(1/2λz) significantly changed to 3.7 ± 0.4 days, then 1.4 ± 0.1 days, then 3.0 ± 0.4 days in the second trimester, third trimester, and postpartum periods, respectively (p < 0.05 for each change). Initially, these evolving changes in mAb6B5 clearance (3.3-fold), distribution volume (1.8-fold), and elimination half-life (4.7-fold) affected our ability to sustain sufficient mAb6B5 levels to sequester PCP in the bloodstream. However, understanding the mechanisms underlying each transition allowed development of an adaptive mAb-dosing paradigm, which substantially reduced PCP levels in dam brains and fetuses throughout pregnancy. These mAb functional studies also revealed that antidrug mAbs readily cross the placenta before syncytiotrophoblast barrier maturation, demonstrating the dynamic nature of mAb pharmacokinetics in pregnancy and the importance of maintaining maternal mAb levels. These studies provide the first preclinical pregnancy model in any species for chronic mAb dosing and could have important implications for the use of antibody therapies involving blood organ barriers (such as addiction) or other chronic diseases in women of childbearing age (e.g., irritable bowel diseases, multiple sclerosis, breast cancer, rheumatoid arthritis). more...

Inflammatory signaling plays a key role in tumor progression, and the pleiotropic cytokine interleukin-6 (IL-6) is an important mediator of protumorigenic properties. Activation of the aryl hydrocarbon receptor (AHR) with exogenous ligands coupled with inflammatory signals can lead to synergistic induction of IL6 expression in tumor cells. Whether there are endogenous AHR ligands that can mediate IL6 production remains to be established. The indoleamine-2,3-dioxygenase pathway is a tryptophan oxidation pathway that is involved in controlling immune tolerance, which also aids in tumor escape. We screened the metabolites of this pathway for their ability to activate the AHR; results revealed that kynurenic acid (KA) is an efficient agonist for the human AHR. Structure-activity studies further indicate that the carboxylic acid group is required for significant agonist activity. KA is capable of inducing CYP1A1 messenger RNA levels in HepG2 cells and inducing CYP1A-mediated metabolism in primary human hepatocytes. In a human dioxin response element-driven stable reporter cell line, the EC(25) was observed to be 104nM, while in a mouse stable reporter cell line, the EC(25) was 10muM. AHR ligand competition binding assays revealed that KA is a ligand for the AHR. Treatment of MCF-7 cells with interleukin-1beta and a physiologically relevant concentration of KA (e.g., 100nM) leads to induction of IL6 expression that is largely dependent on AHR expression. Our findings have established that KA is a potent AHR endogenous ligand that can induce IL6 production and xenobiotic metabolism in cells at physiologically relevant concentrations. more...

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in the regulation of multiple cellular pathways, such as xenobiotic metabolism and Th17 cell differentiation. Identification of key physiologically relevant ligands that regulate AHR function remains to be accomplished. Screening of indole metabolites has identified indoxyl 3-sulfate (I3S) as a potent endogenous ligand that selectively activates the human AHR at nanomolar concentrations in primary human hepatocytes, regulating transcription of multiple genes, including CYP1A1, CYP1A2, CYP1B1, UGT1A1, UGT1A6, IL6, and SAA1. Furthermore, I3S exhibits an approximately 500-fold greater potency in terms of transcriptional activation of the human AHR relative to the mouse AHR in cell lines. Structure-function studies reveal that the sulfate group is an important determinant for efficient AHR activation. This is the first phase II enzymatic product identified that can significantly activate the AHR, and ligand competition binding assays indicate that I3S is a direct AHR ligand. I3S failed to activate either CAR or PXR. The physiological importance of I3S lies in the fact that it is a key uremic toxin that accumulates to high micromolar concentrations in kidney dialysis patients, but its mechanism of action is unknown. I3S represents the first identified relatively high potency endogenous AHR ligand that plays a key role in human disease progression. These studies provide evidence that the production of I3S can lead to AHR activation and altered drug metabolism. Our results also suggest that prolonged activation of the AHR by I3S may contribute to toxicity observed in kidney dialysis patients and thus represent a possible therapeutic target. more...

Increasingly, research suggests that for certain systems, animal models are insufficient for human toxicology testing. The development of robust, in vitro models of human toxicity is required to decrease our dependence on potentially misleading in vivo animal studies. A critical development in human toxicology testing is the use of human primary hepatocytes to model processes that occur in the intact liver. However, in order to serve as an appropriate model, primary hepatocytes must be maintained in such a way that they persist in their differentiated state. While many hepatocyte culture methods exist, the two-dimensional collagen "sandwich" system combined with a serum-free medium, supplemented with physiological glucocorticoid concentrations, appears to robustly maintain hepatocyte character. Studies in rat and human hepatocytes have shown that when cultured under these conditions, hepatocytes maintain many markers of differentiation including morphology, expression of plasma proteins, hepatic nuclear factors, phase I and II metabolic enzymes. Functionally, these culture conditions also preserve hepatic stress response pathways, such as the SAPK and MAPK pathways, as well as prototypical xenobiotic induction responses. This chapter will briefly review culture methodologies but will primarily focus on hallmark hepatocyte structural, expression and functional markers that characterize the differentiation status of the hepatocyte. more...

These studies examined the in vivo pharmacokinetics and efficacy of five anti-methamphetamine monoclonal antibodies (mAbs, K(D) values from 11 to 250 nM) in rats. While no substantive differences in mAb systemic clearance (t(1/2)=6.1-6.9 days) were found, in vivo function was significantly reduced within 1-3 days for four of the five mAbs. Only mAb4G9 was capable of prolonged efficacy, as judged by prolonged high methamphetamine serum concentrations. MAb4G9 also maintained high amphetamine serum concentrations, along with reductions in methamphetamine and amphetamine brain concentrations, indicating neuroprotection. The combination of broad specificity for methamphetamine-like drugs, high affinity, and prolonged action in vivo suggests mAb4G9 is a potentially efficacious medication for treating human methamphetamine-related medical diseases. more...

Chronic (+)-methamphetamine (METH) use during pregnancy increases the health risk for both mother and fetus. To provide insights into these risks, the relationship between changes in METH disposition and METH-induced pharmacological effects were studied in Sprague-Dawley rat dams and litters. Timed-pregnant rats (n = 5-6) were given saline or METH (5.6-17.8 mg/kg/day) by continuous sc infusion from gestational day (GD) 7 (before organogenesis) until GD21 (0-2 days before delivery). By GD11, all rats in the 17.8-mg/kg/day group died or were sacrificed for humane reasons. There were significant (p < 0.05) dose- and gestational time-dependent decreases in maternal body weight in the 10- to 13.2-mg/kg/day groups, which slowly recovered to near normal by GD21. Continued METH dosing in the surviving groups did not affect the mean pups/litter weight at the end of the experiment on GD21. While maternal and fetal METH and (+)-amphetamine serum concentrations were similar on GD21, brain concentrations were significantly greater in the dams (p < 0.05). Importantly, brain-to-serum ratios in the dams were 9:1 and 3:1 in the pups. METH systemic clearance (Cl(S)) in dams significantly (p < 0.05) decreased from 52 +/- 14 ml/min/kg on GD10 to 28 +/- 6 ml/min/kg on GD21 in all dose groups, indicating late-gestational stage reductions in METH Cl(S). Overall, these findings suggest that there were two periods of increased susceptibility for dams and fetuses during chronic METH treatment. First was the period after the start of METH dosing in which neuroadaptation and tolerance to METH occurs in the adult. The second was at the end of pregnancy when METH clearance was significantly reduced. more...

Chronic or excessive (+)-methamphetamine (METH) use often leads to addiction and toxicity to critical organs like the brain. With medical treatment as a goal, a novel single-chain variable fragment (scFv) against METH was engineered from anti-METH monoclonal antibody mAb6H4 (IgG, kappa light chain, K(d) = 11 nM) and found to have similar ligand affinity (K(d) = 10 nM) and specificity as mAb6H4. The anti-METH scFv (scFv6H4) was cloned, expressed in yeast, purified, and formulated as a naturally occurring mixture of monomer ( approximately 75%) and dimer ( approximately 25%). To test the in vivo efficacy of the scFv6H4, male Sprague-Dawley rats (n = 5) were implanted with 3-day s.c. osmotic pumps delivering 3.2 mg/kg/day METH. After reaching steady-state METH concentrations, an i.v. dose of scFv6H4 (36.5 mg/kg, equimolar to the METH body burden) was administered along with a [(3)H]scFv6H4 tracer. Serum pharmacokinetic analysis of METH and [(3)H]scFv6H4 showed that the scFv6H4 caused an immediate 65-fold increase in the METH concentrations and a 12-fold increase in the serum METH area under the concentration-time curve from 0 to 480 min after scFv6H4 administration. The scFv6H4 monomer was quickly cleared or converted to multivalent forms with an apparent t(1/2lambdaz) of 5.8 min. In contrast, the larger scFv6H4 multivalent forms (dimers, trimers, etc.) showed a much longer t(1/2lambdaz) (228 min), and the significantly increased METH serum molar concentrations correlated directly with scFv6H4 serum molar concentrations. Considered together, these data suggested that the scFv6H4 multimers (and not the monomer) were responsible for the prolonged redistribution of METH into the serum. more...

Previously, our laboratory produced a high affinity, anti-phencyclidine (PCP) murine monoclonal antibody (mAb6B5) that also binds other PCP-like arylcyclohexylamines. In this project, mAb6B5 is engineered into a mouse/human chimera (ch-mAb6B5) to assess the feasibility of developing it into a medication for PCP and PCP-like drug abuse. To create ch-mAb6B5, the light and heavy chain constant regions of mAb6B5 were replaced with human kappa and IgG(2) constant regions in order to decrease its potential immunogenicity in humans. To be an effective anti-PCP medication, ch-mAb6B5 must retain the critical immunochemical binding properties of mAb6B5. Expression vectors containing ch-mAb6B5 light chain and heavy chain cDNA were constructed and expressed in the murine myeloma cell line P3X63-Ag8.653. Immunoassays confirm that ch-mAb6B5 is indeed a chimera, composed of mAb6B5's PCP-binding variable domains and human kappa and IgG constant regions. Radioimmunoassays show that ch-mAb6B5 has the same drug-binding profile as mAb6B5. Ch-mAb6B5 and mAb6B5 bind PCP with a K(D) of 0.67 nM and 1.17 nM (respectively) and bind PCP-like arylcyclohexylamines 1-[1-(2-thienyl)cyclohexyl]piperidine and N-ethyl-1-phenylcyclohexylamine with similar specificity. Additionally, ch-mAb6B5 and mAb6B5 have the same calculated isoelectric points and molecular weights, critical properties in antigen-antibody interactions. These data demonstrate that mouse/human ch-mAb6B5, a "more human" version of murine mAb6B5, retains mAb6B5's unique drug-binding properties. This work supports our continued efforts to develop ch-mAb6B5 into a medication for PCP and PCP-like drug abuse - introducing the intriguing possibility of using a single therapeutic mAb for treating a class of abused drugs. more...

(+)-Methamphetamine (METH) and (+)-amphetamine (AMP) are structurally similar drugs that are reported to induce similar pharmacological effects in rats of the same sex. Because pharmacokinetic data suggest female rats should be more affected than males, the current studies sought to test the hypothesis that the behavioral and temporal actions of METH and AMP should be greater in female Sprague-Dawley rats than in males. Using a dosing regimen designed to reduce the possibility of tolerance and sensitization, rats were administered 1.0 and 3.0 mg/kg intravenous drug doses. Distance traveled, rearing events and focal stereotypies (e.g., head weaving, sniffing) were measured. Female rats traveled significantly greater distances and displayed a greater number of rearing events than males after both doses. Analysis of stereotypy ratings after 3.0 mg/kg revealed that focal stereotypies were more pronounced and lasted longer in females. The second study compared the potencies of METH and AMP in inducing locomotor activity and focal stereotypies in each sex. No differences in potency were found when METH and AMP effects were compared within males or females. In summary, these studies showed female rats displayed greater and longer-lasting locomotor activity and more stereotypic behaviors, supporting earlier evidence of significant sexual dimorphism in pharmacokinetics. more...

The purpose of these studies was to determine if a high-affinity, anti-(+)-methamphetamine (METH) monoclonal antibody (mAb6H4; KD=11 nM) protects against METH-induced central nervous and cardiovascular system effects in rats. Rats (n=5 per group) received one of three anti-METH mAb6H4 doses, equal to 0.32, 0.56 or 1 times the mole equivalent (mol-eq) amount of METH in the body following a 1 mg/kg i.v. METH dose. Each rat was challenged with METH (1 mg/kg, i.v.) 1 and 4 days after the anti-METH mAb dose. The 1 mol-eq anti-METH mAb dose significantly reduced the duration of METH-induced locomotor activity (horizontal locomotion and rearing events), heart rate and blood pressure effects from 2 to 3 h to about an hour. This resulted in a significant reduction in total locomotor activity and the area under the hemodynamic effect vs. time curve for heart rate and blood pressure. In addition, the time to peak locomotor activity was decreased after the 1 mol-eq mAb dose vs. the lower doses. These changes were limited to the first METH challenge. The responses to the second METH challenge were not different from baseline. The peak hemodynamic and locomotor activity values were unchanged after both challenges. These results indicate anti-METH mAb6H4 can safely reduce the hemodynamic and locomotor effects of METH given one day after anti-METH IgG, and that the mAb is safe when administered in the absence of METH. These results are important because they indicate these antibody medications have simultaneous beneficial effects in multiple organ systems. more...

The effectiveness of a high-affinity monoclonal antibody (mAb) antagonist against chronic phencyclidine (PCP) use has been demonstrated in rats. In this study, we tested the hypothesis that intravenous doses of PCP in excess of the binding capacity of an anti-PCP mAb cannot easily surmount the beneficial effects of the mAb, even in the presence of a high body burden of the drug. One day after steady-state PCP concentrations were achieved in male rats by continuous s.c. infusion (18 mg/kg/day), a single i.v. dose of saline or the anti-PCP mAb (KD = 1.3 nM; at one-third the molar dose of the PCP body burden), treatment was administered. In an attempt to further surmount the effects of the mAb, rats were challenged with a single 1.0 mg/kg i.v. bolus PCP dose (along with a [3H]PCP tracer) 3 days after the mAb or saline treatment. Total (i.v. bolus + s.c. infusion) PCP concentrations were measured in serum, brain, and testis by radioimmunoassay before and after the challenge, and [3H]PCP concentrations were measured by liquid scintillation spectrometry. The anti-PCP mAb protected against adverse health effects, significantly increased the serum total and bolus PCP concentrations (p < 0.05), and significantly decreased brain total and bolus PCP concentrations (p < 0.05) after the i.v. challenge. These results showed the antibody can counteract extreme and potentially fatal PCP challenges and disproved the hypothesis that attempts to surmount the effects of the antibody with extremely high PCP doses would have immediate adverse health effects. more...

These studies investigated how (+)-methamphetamine (METH) dose and rat sex affect the pharmacological response to METH in Sprague-Dawley rats. The first set of experiments determined the pharmacokinetics of METH and its pharmacologically active metabolite (+)-amphetamine (AMP) in male and female Sprague-Dawley rats after 1.0 and 3.0 mg/kg METH doses. The results showed significant sex-dependent changes in METH pharmacokinetics, and females formed significantly lower amounts of AMP. While the area under the serum concentration-time curve in males increased proportionately with the METH dose, the females showed a disproportional increase. The sex differences in systemic clearance, renal clearance, volume of distribution, and percentage of unchanged METH eliminated in the urine suggested dose-dependent pharmacokinetics in female rats. The second set of studies sought to determine the behavioral implications of these pharmacokinetic differences by quantifying locomotor activity in male and female rats after saline, 1.0, and 3.0 mg/kg METH. The results showed sex- and dose-dependent differences in METH-induced locomotion, including profound differences in the temporal profile of effects at higher dose. These findings show that the pharmacokinetic and metabolic profile of METH (slower METH clearance and lower AMP metabolite formation) plays a significant role in the differential pharmacological response to METH in male and female rats. more...

The roles of monoclonal antibody affinity and treatment time of (+)-methamphetamine-induced pharmacological effects in rats were studied using two anti-(+)-methamphetamine monoclonal antibodies. These studies tested the preclinical protective effects of monoclonal antibody antagonists in (+)-methamphetamine overdose and pretreatment scenarios. The higher affinity antibody (mAb6H4; KD=11 nM for (+)-methamphetamine) more effectively antagonized (+)-methamphetamine-induced behavioral effects (distance and rearing) than the low affinity antibody (designated mAb6H8; KD=250 nM) and had a longer duration of action. Both antibodies more effectively reduced (+)-methamphetamine effects in the overdose model than in the pretreatment model. (+)-Methamphetamine pharmacokinetic studies showed the mAb6H4 significantly reduced brain concentrations over time in both models. However, while mAb6H4 immediately reduced brain concentrations in the overdose model, it did not prevent the initial distribution of (+)-methamphetamine into the brain in the pretreatment model. Thus, anti-(+)-methamphetamine monoclonal antibody affinity and administration time (relative to (+)-methamphetamine dosing) are critical determinants of therapeutic success. more...

These studies examined the role of (+)-methamphetamine ((+)METH) administration route on spontaneous behavioral activity vs. time relationships, and pharmacokinetic mechanisms for differences in effects. Male Sprague-Dawley rats (n=6 per administration route) received saline and three doses (0.3, 1.0 and 3.0 mg/kg) of (+)METH in a mixed-sequence design by intravenous (iv), subcutaneous (sc) or intraperitoneal (ip) administration. Locomotion and stereotypy were quantified by video-tracking analysis. The effects of (+)METH on spontaneous behavior were dose- and route-dependent. In particular, total locomotor activity was greatest following 3.0 mg/kg intraperitoneally (P<0.05) and stereotypy ratings were greatest following 3.0 mg/kg subcutaneously (P<0.05). In addition, the duration of locomotor effects was greatest after 3.0 mg/kg subcutaneously (P<0.05). Serum pharmacokinetic parameters were determined in separate rats given 3.0 mg/kg by subcutaneous and intraperitoneal administration (n=4 per administration route). The (+)METH elimination half-life was not different between the routes, but the (+)METH AUC was greater (P<0.05), and the (+)METH and (+)-amphetamine (AMP) maximum concentrations occurred later following subcutaneous than after intraperitoneal dosing (P<0.05), increasing and prolonging drug exposure. In conclusion, the overall pattern of (+)METH effects on locomotor activity depend on dose and the route of administration, which affects serum concentration and the time course of behavioral effects. more...

The aim of this paper was to develop LC/MS/MS methodology for the determination of methamphetamine (METH) and amphetamine (AMP) using low microliter volumes (20-150 microl) of rat serum and demonstrate the use of this method for the study of serum pharmacokinetics in the rat. The analytes were extracted from rat serum using solid-phase extraction followed by an isocratic separation on a narrow-bore Hypersil C(18) column. Lower limits of quantitation for METH and AMP were 0.3 ng/ml using positive ion electrospray tandem mass spectrometry. The accuracy of the method was within 20% of the actual values over a wide range of serum concentrations. The within-day and between-day precision was better than 20% (R.S.D.). Ion-suppression matrix effects on electrospray ionization were evaluated for extracted rat serum. The LC/MS/MS method was further validated by comparing serum concentrations of METH and AMP to serum concentrations previously determined using an LC/[ (3)H]-METH assay with radiochemical detection. Finally, the LC/MS/MS method was used to study the pharmacokinetics of METH and AMP after a 1mg/kg intravenous bolus dose of METH to female Sprague-Dawley rats. more...

These studies examined the effects of a high-affinity anti-(+)-methamphetamine monoclonal antibody (mAb; KD = 11 nM) on (+)-methamphetamine [(+)-METH] and (+)-amphetamine [(+)-AMP] serum and tissue disposition and serum protein binding following i.v. (+)-METH administration. Male Sprague-Dawley rats were pretreated with a buffer solution (control rats) or with anti-(+)-METH mAb [equimolar in binding sites to the (+)-METH dose]. The next day, both groups received a 1 mg/kg i.v. (+)-METH dose. At various time points after (+)-METH administration, rats were sacrificed (n = 3 per time point), and serum and tissues were collected. (+)-METH serum protein binding was increased from approximately 5% in controls to approximately 88 to 99% in the mAb-treated rats. The (+)-METH area under the concentration versus time curves from 0 to 4.5 h (AUC0-4.5 h) in mAb-treated rats showed an increase of >6600% for serum and a decrease of >60% for brain, compared with buffer-treated controls. Differential effects of anti-METH mAb on (+)-METH concentrations were observed in other tissues. For example, in the liver, anti-(+)-METH mAb caused significant increases in (+)-METH concentrations. The AUC0-4.5 h for (+)-AMP, a pharmacologically active metabolite, was decreased by approximately 50% in all tissues examined. These data show that pretreatment with an anti-(+)-METH mAb can significantly alter the disposition of (+)-METH and (+)-AMP in rats. Since the mAb has no significant cross-reactivity with (+)-AMP, the data suggest that the mAb reduced (+)-METH metabolic clearance through high-affinity binding to (+)-METH. Finally, rapidly equilibrating tissues, like the brain, appear to be preferentially protected by the mAb. more...

The range of medical effects and complications resulting from excessive use of drugs of abuse like phencyclidine (PCP) has hindered the development of effective medications. Drug-specific monoclonal antibodies (mAbs) provide an appealing medication approach since they can be selective for the drug, without concern for the sites of action of the drug. The use of mAb medications has been considered impractical because it is commonly believed that very large doses of mAb would be required to treat the adverse medical effects resulting from excessive drug use. In this study, a single dose of an anti-PCP mAb was found to significantly reduce the negative health impact of excessive, prolonged PCP treatment in rats (18 mg/kg/day for 2 weeks). The protective effects were mAb dose-dependent, and mAb doses as low as 1/100th the molar equivalent amount of the PCP body burden were effective at preventing PCP-induced deaths, reducing PCP-induced behaviors, reducing PCP brain concentrations, and improving the general health status of the animals. They also show that treatment with monoclonal antibody medications can have medically important outcomes without the need to neutralize the entire dose of the offending drug. These results could help establish the feasibility of using carefully designed monoclonal antibody medications to treat drug abuse and addiction, a chronic and re-occurring illness of the central nervous system. more...

Our studies examined pharmacokinetic mechanisms involved in high-affinity (K(d) approximately 11 nM) monoclonal antibody-based antagonism of (+)-methamphetamine-induced locomotor effects. Male rats received (+)-methamphetamine (0.3, 1, or 3 mg/kg i.v.) followed 30 min later by saline or anti-(+)-methamphetamine monoclonal antibody. All groups received a constant dose of monoclonal antibody that was equimolar in binding sites to the body burden of a 1 mg/kg i.v. (+)-methamphetamine dose 30 min after administration. The monoclonal antibody antagonized locomotor effects due to 0.3 and 1 mg/kg (+)-methamphetamine. In contrast, monoclonal antibody treatment increased locomotor activity due to 3 mg/kg (+)-methamphetamine. We also investigated the serum and brain pharmacokinetics of (+)-methamphetamine without and with the monoclonal antibody. Rats received (+)-methamphetamine (1 mg/kg i.v.) followed by saline or monoclonal antibody treatment at 30 min. The monoclonal antibody significantly increased serum methamphetamine concentrations and significantly decreased brain methamphetamine concentrations. These data indicate that anti-(+)-methamphetamine monoclonal antibody-induced pharmacodynamics are complex, but are related to time-dependent changes in (+)-methamphetamine brain distribution., (Copyright 2003 Elsevier Science B.V.) more...

These studies tested the hypothesis that a single dose of high-affinity anti-phencyclidine monoclonal antibody (anti-PCP mAb) provides long-term protection against behavioral effects of repeated PCP administration in rats. Rats were treated with saline, nonspecific bovine IgG (NS-IgG), or anti-PCP mAb (1.0 g/kg). The next morning, the rats were challenged with escalating i.v. doses of PCP (0.32, 0.56, and 1.0 mg/kg) at 90-min intervals. This regimen was repeated every 3 days for 2 weeks. In the saline and NS-IgG control groups, PCP yielded reproducible and linear dose-dependent effects that remained constant during the experiment. In contrast, the anti-PCP mAb treatment blocked PCP effects on day 1, and sustained significant (P < 0.05) reductions in drug effects for the entire 2-week experiment. Brain PCP concentrations (determined at study termination) were reduced by ~55%, whereas serum concentrations were increased over 4000% compared with controls. Thus, a single dose of antibody medication provided long-term reductions in drug effects and brain concentrations, beyond the expected capacity of the drug-antibody interaction. These data challenge current concepts about in vivo dose dependence and unimolecular interaction between antibody binding sites and small molecules and establish that neuroprotection by mAbs may have an unique mechanism of action. more...

Previous dose range-finding studies with nonylphenol (NP) administered to rats in a soy- and alfalfa-free diet showed apparent feminization of several endpoints in male rats at doses of 25 ppm and above. One possible mechanism contributing to these effects is a reduction of testosterone at critical developmental periods. The present study was conducted as an adjunct to a multigeneration study and was designed to examine the effect of NP on testosterone production. Male rats in the F1 and F2 generations were exposed through their dams or directly to various dietary doses of NP (0, 25, 200 and 750 ppm) throughout gestation and until sacrifice at either postnatal day 2 (PND2), PND50, or PND140. Male pups in the F3 generation were examined only on PND2. At PND2, serum testosterone levels were significantly decreased in all groups exposed to NP in the F1 generation, but not in the F2 or F3 generations. The activity of 17alpha-hydroxylase/C17, 20 lyase (P450c17) in PND2 testicular homogenates was not affected by NP treatment. In F1 and F2 PND50 and PND140 rats, NP treatment did not affect serum testosterone levels. The absolute dorsolateral prostate weight was increased in the 200 and 750 ppm dose groups only in the F1 PND50 rats, however, no significant effects were observed in other male reproductive organs. NP treatment did not affect P450c17 activity in microsomes prepared from testes of F1 PND50 or PND140 rats. However, P450c17 activity was significantly decreased in testicular microsomes of F(2) PND50 (200 and 750 ppm dose groups) and PND140 (25, 200, and 750 ppm dose groups) rats. A decrease in testicular beta-nicotinamide adenine dinucleotide phosphate (NADPH) P450 reductase was also observed in all PND50 and PND140 NP-exposed rats of the F1 and F2 generations. The ability of NP to directly inhibit P450c17 activity in vitro at concentrations of 1-100 microM was also demonstrated. These results indicate that NP can inhibit the activity of enzymes involved in testosterone synthesis, but suggest minimal effects on testosterone or testosterone-dependent endpoints via this mechanism. more...

The objective of this study was to examine effects of estrogenic agents of varying potencies (genistein, p-nonylphenol, and ethinyl estradiol) on hepatic testosterone metabolism, cytochrome P-450 (CYP450) enzymes, and ERalpha expression. These endpoints were examined as potential biomarkers of, and contributors to, endocrine disruptive activity. Exposure occurred during critical developmental periods, from gestational day 7 through weaning via the mothers' diet. Thereafter, rats were exposed via their diet to the compounds until puberty (postnatal day 50). Testosterone hydroxylase and 5alpha-reductase activities, CYP2C and CYP3A levels were determined. In general, the compounds were more active in male rats than female rats. The only effect observed in female rats was at the 250 ppm genistein dose, in which an approximately 40% increase in 5alpha-reductase activity was observed. In male rats, genistein treatment had mixed effects on testosterone metabolism. The 1250 ppm dose decreased both CYP2C and CYP3A protein levels. Nonylphenol had the most profound effects on testosterone metabolism and CYP450 expression in male rats, with effects occurring at doses as low as 25 ppm. An increase in 5alpha-reductase activity and a decrease in the formation of 16alpha-OH-, 2alpha-OH-testosterone metabolites, CYP2C and CYP3A protein were observed. EE2 decreased the formation of several testosterone metabolites and CYP2C protein. All compounds had some effect on hepatic ERalpha expression, although a consistent effect was not observed. This study demonstrates that the test compounds can influence hepatic testosterone hydroxylase activity and CYP450 expression, as well as ERalpha expression, although these activities cannot be directly related to estrogenic activity. more...

Human microsomal epoxide hydrolase (mEH; EC 3.3.2.3) is an important biotransformation enzyme and potential risk determinant for pathologies such as cancer and teratogenesis. Currently, the effects of chemical exposures on human mEH gene expression are largely unknown, but they may constitute a unique modifier of disease susceptibility. To examine this issue, we exposed cultures of primary human hepatocytes isolated from seven donors to prototypic chemical inducers [such as phenobarbital (PB), polyaromatic hydrocarbons, dexamethasone, butylated hydroxyanisole, and ciprofibrate]. Basal levels of mEH RNA and protein were detected readily in untreated cells. Chemical treatment of cultured hepatocytes resulted in variable mEH RNA and protein expression, but, in general, only modest modulatory effects were detected following these exposures. The maximum increase in mEH RNA expression observed was approximately 3.5-fold following Arochlor 1254 exposure. Immunochemical levels of mEH protein were quantified for all treatment groups in three cultures and demonstrated less overall variation and, in general, a lack of concordance with corresponding mEH RNA levels. Cytochrome P450 (CYP) 1A2 and 3A mRNA levels were measured before and following exposure to beta-naphthaflavone and PB, respectively, to permit independent evaluation of hepatocyte inducer responsiveness. Substantial increases in RNA expression levels for both the CYP1A2 and CYP3A genes demonstrated that the hepatocyte cultures were robust and highly responsive to inducer treatment. These results indicate that the mEH gene in human hepatocytes is only modestly responsive to chemical exposures. more...

Microsomal epoxide hydrolase (mEH) is a key biotransformation enzyme that is variably expressed in humans. Genetic polymorphisms in the mEH gene have been identified that result in amino acid substitutions in the corresponding enzyme. Results of expression analyses of the mEH allelic variants in vitro suggest that the mutations do not affect the specific activity of the mEH enzyme, but may alter post-transcriptional regulation of mEH. To identify potential post-transcriptional mechanisms that influence mEH expression, the translational efficiency, mRNA half-life, and protein half-life of mEH allelic variants were determined. Constructs encoding each of the four mEH alleles were transcribed in vitro and translated. No differences were detected in the rate of protein synthesis among the variant transcripts, indicating that the previously characterized coding region polymorphisms do not appear to affect translational efficiency. mEH variant RNA half-lives were determined in transfected COS-1 cells, but no differences in decay rates were apparent among the polymorphic constructs. Half-lives of the polymorphic mEH proteins were determined in transiently transfected COS-1 cells treated with the protein synthesis inhibitor cycloheximide. Calculated protein half-lives were: Y113/H139, 15.2 h; H113/H139, 10.7 h, Y113/H139, 16.9 h and H113/R139, 16.0 h. The protein half-lives calculated for the polymorphic variants exhibited the same rank order as mEH protein and activity levels determined previously from expression experiments in vitro and therefore suggest that polymorphic amino acid substitution may result in altered protein stability. However, the differences noted were not statistically significant at the P < 0.05 level, and therefore additional study is required to firmly establish causative relationships. more...

These studies examined the microsomal brain metabolism of phencyclidine (PCP) in male and female Sprague-Dawley rats. Several monohydroxylated metabolites of PCP were detected including cis- and trans-1-(1-phenyl-4-hydroxycyclohexyl)piperidine (c-PPC and t-PPC) and 1-(1-phenylcyclohexyl)-4-hydroxypiperidine (PCHP). The in vitro formation of these metabolites required NADPH and was inhibited by carbon monoxide. c-PPC was formed in the male and female brain microsomes at rates of 7.1 +/- 1.3 and 5.7 +/- 1.1 fmol/min per mg, respectively, while t-PPC was formed at rates of 16.2 +/- 3.3 and 16.5 +/- 4.2 fmol/min per mg. PCHP had the highest formation rate at 50.7 +/- 8.9 and 48.2 +/- 8.8 fmol/min per mg, respectively. Although previous studies with rat liver microsomes find higher levels of PCP metabolism in male rats and the formation of an irreversibly bound metabolite in male rats, the present study of brain metabolism found no sex differences in brain metabolism. The formation of PCP metabolites in male rat livers is at least partially mediated by the male-specific isozyme CYP2C11, and possibly CYP2D1. Nevertheless, the formation of the major brain metabolite, PCHP, was not inhibited by an anti-CYP2C11 or an anti-CYP2D6 antibody. However, PCHP formation was inhibited by drug inhibitors of CYP2D1-mediated metabolism, suggesting the involvement of a CYP2D isoform. These data indicate brain metabolism of PCP is significant, but unlike the liver it is not sexually dimorphic. more...

These studies examined in vitro metabolism of phencyclidine (PCP) in a series of human liver microsomes (N = 10). Each sample was characterized for cytochrome P450 (CYP) content and for CYP1A, CYP2A, CYP2C, CYP2D, CYP2E, CYP3A, CYP4A, and lauric acid 11-hydroxylation metabolic activities. At least five PCP metabolites (c-PPC, t-PPC, PCHP, an unknown metabolite, and an irreversibly bound metabolite) were formed by the various human liver microsomes. Nevertheless, there was a large degree of inter-individual variation in the metabolite formation. For example, the irreversibly bound metabolite was formed in detectable amounts in only four of the ten samples. c-PPC, t-PPC and the irreversibly bound PCP metabolite formation rates significantly correlated with CYP3A activity. The CYP3A inhibitor troleandomycin was used to inhibit the formation of PCP metabolites. Troleandomycin inhibition was dose dependent with the highest dose producing complete inhibition of the formation of c-PPC, t-PPC, PCHP, and the irreversibly bound metabolite. In addition, PCP inhibited CYP3A-mediated testosterone 6 beta-hydroxylation by 50%. Furthermore, the relative intensity of CYP3A immunoreactive proteins significantly correlated with testosterone 6 beta-hydroxylation and with PCP metabolite formation (except for the unknown metabolite). PCHP formation also correlated with CYP1A activity, while the formation of the unknown PCP metabolite correlated with CYP2A activity. These studies suggest that several CYP isoforms contribute to PCP metabolism and that CYP3A plays a major role in PCP biotransformation in human liver microsomes. more...

Interindividual variation in the expression of human microsomal epoxide hydrolase (mEH) may be an important risk factor for chemically induced toxicities, including cancer and teratogenesis. In this study, phenotypic variability and mEH genetic polymorphisms were examined in a bank of 40 transplant-quality human liver samples. Immunochemically determined protein content, enzymatic activities, polymorphic amino acids, as well as mEH RNA levels were evaluated in parallel. Enzymatic activity was assessed using (+/-)-benzo[a]pyrene-4,5-epoxide at 2 substrate concentrations. The relative hydrolyzing activities obtained using saturating substrate levels were highly correlated (r = 0.85) with results derived from limiting substrate concentrations and exhibit approximately an 8-fold range in activity levels across the panel of 40 liver samples. mEH enzyme activity also demonstrated strong correlation (r > or = 0.74) with an 8.4-fold variation determined for mEH protein content within the same samples. However, these protein/activity measurements were poorly correlated (r < or = 0.23) with mEH RNA levels, which exhibited a 49-fold variation. Two common polymorphic amino acid loci in the mEH protein did not exclusively account for variation in enzymatic activity, although this conclusion is confounded by heterozygousity in the samples. These data demonstrate the extent of hepatic mEH functional variability in well-preserved human tissues and suggest that polymorphism of mEH protein expression is regulated in part by posttranscriptional controls, which may include nonstructural regulatory regions of the mEH transcript. more...

Four peptides (pep23-33, pep26-38, pep283-297, and pep409-419) corresponding to unique sequences in rat cytochrome P450 (CYP) 2D1 and/or human CYP2D6 were selected for production of antipeptide antibodies. Rat liver microsomal protein was recognized by anti-serum to all four peptides in ELISAs; however, antisera against pep23-33 and pep26-36 proved not usable for any other applications. Western blots of microsomal protein from a cell line specifically expressing human CYP2D6 revealed that antisera to pep283-297 and pep409-419 recognized a single protein band of the same molecular size as CYP2D6. Antisera to pep283-297 and pep409-419 recognized a rat microsomal protein presumed to be CYP2D1, because it comigrated with human CYP2D6 and had an apparent molecular size of 52 kDa. An unknown protein of approximately 85 kDa was also recognized by pep409-419. Recognition of microsomal protein(s) by antisera to pep283-297 and pep409-419 was blocked by pep283-297 or a bovine serum albumin-pep409-419 conjugate, respectively. Antiserum to pep283-297 was used to analyze sex and strain differences in liver microsomes prepared from Sprague-Dawley, Fischer 344, and Dark Agouti male and female rats. Sprague-Dawley and Fischer 344 rats expressed similar amounts of CYP2D1, but expression in Dark Agouti rats was significantly lower. The antiserum did not detect a sexual dimorphism in any of the strains. A significant correlation between antipeptide283-297 immunoreactivity and Vmax for dextromethorphan O-demethylation existed in female rat strains; however, this relationship did not exist in male rat strains.(ABSTRACT TRUNCATED AT 250 WORDS) more...

We determined whether chronic dosing with phencyclidine (PCP) could affect the in vitro function of liver microsomal enzymes in male Sprague-Dawley rats. PCP chronic dosing of rats (n = 3 per group) for 3 days with 2.5, 10 and 18 mg/kg/day caused a dose-dependent decrease (23, 36 and 53%, respectively) in the ability of the microsomal enzymes to bind covalently PCP metabolites. The 10- and 18-mg/kg/day dosing groups were significantly different from the 3-day saline-infused control group (P < .05). The results from time-dependent dosing studies indicated PCP covalent binding was significantly reduced (P < .05) in rats (n = 3 per group) infused with 18 mg/kg/day of PCP for 1, 2, 3, 4 and 10 days. Subsequently, it returned to near control values in rats infused for 20 days. In parallel with the time-dependent decreases in covalent binding, the concentrations of at least three phase I PCP mono- and dihydroxylated metabolites were also significantly reduced (P < .05) at the earlier time periods of dosing (3 and 10 days), but the rate of their formation returned to near normal values by 20 days of dosing. Total cytochrome P450 content did not differ from the control groups at any of the doses or time points. As dose- and time-dependent decreases in covalent binding suggested a specific metabolic pathway or isoenzyme was affected, we studied the affect on specific isoenzyme pathways. For these studies a series of cytochrome P450 inhibitors were used.(ABSTRACT TRUNCATED AT 250 WORDS) more...