Simplifying the Extended Clearance Concept Classification System (EC3S) to Guide Clearance Prediction in Drug Discovery.

PURPOSE: The Extended Clearance Concept Classification System was established as a development-stage tool to provide a framework for identifying fundamental mechanism(s) governing drug disposition in humans. In the present study, the applicability of the EC3S in drug discovery has been investigated. In its current format, the EC3S relies on low-throughput hepatocyte uptake data, which are not frequently generated in a discovery setting. METHODS: A relationship between hepatocyte uptake clearance and MDCK permeability was first established along with intrinsic clearance from human liver microsomes. The performance of this approach was examined by categorizing 64 drugs into EC3S classes and comparing the predicted major elimination pathway(s) to that observed in humans. As an extension of the work, the ability of the simplified EC3S to predict human systemic clearance based on intrinsic clearance generated using in-vitro metabolic systems was evaluated. RESULTS: The assessment enabled the use of MDCK permeability and unscaled unbound intrinsic clearance to generate cut-off criteria to categorize compounds into four EC3S classes: Class 12ab, 2cd, 34ab, and 34cd, with major elimination mechanism(s) assigned to each class. The predictivity analysis suggested that systemic clearance could generally be predicted within threefold for EC3S class 12ab and 34ab compounds. For classes 2cd and 34cd, systemic clearance was poorly predicted using in-vitro systems explored in this study. CONCLUSION: Collectively, our simplified classification approach is expected to facilitate the identification of mechanism(s) involved in drug elimination, faster resolution of in-vitro to in-vivo disconnects, and better design of mechanistic pharmacokinetic studies in drug discovery.

Quantification of Accurate Composition and Total Abundance of Homologous Proteins by Conserved-Plus-Surrogate Peptide Approach: Quantification of UDP Glucuronosyltransferases in Human Tissues.

Characterization of accurate compositions and total abundance of homologous drug-metabolizing enzymes, such as UDP glucuronosyltransferases (UGTs), is important for predicting the fractional contribution of individual isoforms involved in the metabolism of a drug for applications in physiologically based pharmacokinetic (PBPK) modeling. Conventional targeted proteomics utilizes surrogate peptides, which often results in high technical and interlaboratory variability due to peptide-specific digestion leading to data inconsistencies. To address this problem, we developed a novel conserved-plus-surrogate peptide (CPSP) approach for determining the accurate compositions and total or cumulative abundance of homologous UGTs in commercially available pooled human liver microsomes (HLM), human intestinal microsomes (HIM), human kidney microsomes (HKM), and human liver S9 (HLS9) fraction. The relative percent composition of UGT1A and UGT2B isoforms in the human liver was 35:5:36:11:13 for UGT1A1:1A3:1A4:1A6:1A9 and 20:32:22:21:5 for UGT2B4:2B7:2B10:2B15:2B17. The human kidney and intestine also showed unique compositions of UGT1As and UGT2Bs. The reproducibility of the approach was validated by assessing correlations of UGT compositions between HLM and HLS9 (R2> 0.91). The analysis of the conserved peptides also provided the abundance for individual UGT isoforms included in this investigation as well as the total abundance (pmol/mg protein) of UGT1As and UGT2Bs across tissues, i.e., 268 and 342 (HLM), 21 and 92 (HIM), and 138 and 99 (HKM), respectively. The CPSP approach could be used for applications in the in-vitro-to-in-vivo extrapolation of drug metabolism and PBPK modeling. SIGNIFICANCE STATEMENT: We quantified the absolute compositions and total abundance of UDP glucuronosyltransferases (UGTs) in pooled human liver, intestine, and kidney microsomes using a novel conserved-plus-surrogate peptide (CPSP) approach. The CPSP approach addresses the surrogate peptide-specific variability in the determination of the absolute composition of UGTs. The data presented in this manuscript are applicable for the estimation of the fraction metabolized by individual UGTs towards better in vitro-to-in vivo extrapolation of UGT-mediated drug metabolism.

The Discovery of 3-((4-Chloro-3-methoxyphenyl)amino)-1-((3R,4S)-4-cyanotetrahydro-2H-pyran-3-yl)-1H-pyrazole-4-carboxamide, a Highly Ligand Efficient and Efficacious Janus Kinase 1 Selective Inhibitor with Favorable Pharmacokinetic Properties.

The discovery of a potent selective low dose Janus kinase 1 (JAK1) inhibitor suitable for clinical evaluation is described. As part of an overall goal to minimize dose, we pursued a medicinal chemistry strategy focused on optimization of key parameters that influence dose size, including lowering human Clint and increasing intrinsic potency, bioavailability, and solubility. To impact these multiple parameters simultaneously, we used lipophilic ligand efficiency as a key metric to track changes in the physicochemical properties of our analogs, which led to improvements in overall compound quality. In parallel, structural information guided advancements in JAK1 selectivity by informing on new vector space, which enabled the discovery of a unique key amino acid difference between JAK1 (Glu966) and JAK2 (Asp939). This difference was exploited to consistently produce analogs with the best balance of JAK1 selectivity, efficacy, and projected human dose, ultimately culminating in the discovery of compound 28.

Evaluation of JAK3 Biology in Autoimmune Disease Using a Highly Selective, Irreversible JAK3 Inhibitor.

Reversible janus associated kinase (JAK) inhibitors such as tofacitinib and decernotinib block cytokine signaling and are efficacious in treating autoimmune diseases. However, therapeutic doses are limited due to inhibition of other JAK/signal transducer and activator of transcription pathways associated with hematopoiesis, lipid biogenesis, infection, and immune responses. A selective JAK3 inhibitor may have a better therapeutic index; however, until recently, no compounds have been described that maintain JAK3 selectivity in cells, as well as against the kinome, with good physicochemical properties to test the JAK3 hypothesis in vivo. To quantify the biochemical basis for JAK isozyme selectivity, we determined that the apparent Km value for each JAK isozyme ranged from 31.8 to 2.9 µM for JAK1 and JAK3, respectively. To confirm compound activity in cells, we developed a novel enzyme complementation assay that read activity of single JAK isozymes in a cellular context. Reversible JAK3 inhibitors cannot achieve sufficient selectivity against other isozymes in the cellular context due to inherent differences in enzyme ATP Km values. Therefore, we developed irreversible JAK3 compounds that are potent and highly selective in vitro in cells and against the kinome. Compound 2, a potent inhibitor of JAK3 (0.15 nM) was 4300-fold selective for JAK3 over JAK1 in enzyme assays, 67-fold [interleukin (IL)-2 versus IL-6] or 140-fold [IL-2 versus erythropoietin or granulocyte-macrophage colony-stimulating factor (GMCSF)] selective in cellular reporter assays and >35-fold selective in human peripheral blood mononuclear cell assays (IL-7 versus IL-6 or GMCSF). In vivo, selective JAK3 inhibition was sufficient to block the development of inflammation in a rat model of rheumatoid arthritis, while sparing hematopoiesis.

A Pharmacokinetic Modeling Approach to Predict the Contribution of Active Metabolites to Human Efficacious Dose.

A preclinical drug candidate, MRK-1 (Merck candidate drug parent compound), was found to elicit tumor regression in a mouse xenograft model. Analysis of samples from these studies revealed significant levels of two circulating metabolites, whose identities were confirmed by comparison with authentic standards using liquid chromatography-tandem mass spectrometry. These metabolites were found to have an in vitro potency similar to that of MRK-1 against the pharmacological target and were therefore thought to contribute to the observed efficacy. To predict this contribution in humans, a pharmacokinetic (PK) modeling approach was developed. At the mouse efficacious dose, the areas under the plasma concentration time curves (AUCs) of the active metabolites were normalized by their in vitro potency compared with MRK-1. These normalized metabolite AUCs were added to that of MRK-1 to yield a composite efficacious unbound AUC, expressed as "parent drug equivalents," which was used as the target AUC for predictions of the human efficacious dose. In vitro and preclinical PK studies afforded predictions of the PK of MRK-1 and the two active metabolites in human as well as the relative pathway flux to each metabolite. These were used to construct a PK model (Berkeley Madonna, version 8.3.18; Berkeley Madonna Inc., University of California, Berkeley, CA) and to predict the human dose required to achieve the target parent equivalent exposure. These predictions were used to inform on the feasibility of the human dose in terms of size, frequency, formulation, and likely safety margins, as well as to aid in the design of preclinical safety studies.

Structure-based design and development of (benz)imidazole pyridones as JAK1-selective kinase inhibitors.

The mammalian Janus Kinases (JAK1, JAK2, JAK3 and TYK2) are intracellular, non-receptor tyrosine kinases whose activities have been associated in the literature and the clinic with a variety of hyperproliferative diseases and immunological disorders. At the onset of the program, it was hypothesized that a JAK1 selective compound over JAK2 could lead to an improved therapeutic index relative to marketed non-selective JAK inhibitors by avoiding the clinical AEs, such as anemia, presumably associated with JAK2 inhibition. During the course of the JAK1 program, a number of diverse chemical scaffolds were identified from both uHTS campaigns and de novo scaffold design. As part of this effort, a (benz)imidazole scaffold evolved via a scaffold-hopping exercise from a mature chemical series. Concurrent crystallography-driven exploration of the ribose pocket and the solvent front led to analogs with optimized kinome and JAK1 selectivities over the JAK2 isoform by targeting several residues unique to JAK1, such as Arg-879 and Glu-966.

Potent benzoazepinone γ-secretase modulators with improved bioavailability.

The triazolyl amide γ-secretase modulators are potent alternatives to the cinnamyl amides that have entered the clinic for the treatment of Alzheimer's disease. Herein we build on the lead benzoazepinones described in our prior communication with imidazomethoxyarene moiety alternatives that offer opportunities to fine tune physical properties as well as address hERG binding and PK. Both half-life and bioavailability were significantly improved, especially in dog, with robust brain Aß42 lowering maintained in both transgenic mouse and rat.

Discovery of novel triazolobenzazepinones as γ-secretase modulators with central Aß42 lowering in rodents and rhesus monkeys.

Synthesis and SAR studies of novel triazolobenzazepinones as gamma secretase modulators (GSMs) are presented in this communication. Starting from our azepinone leads, optimization studies toward improving central lowering of Aß42 led to the discovery of novel benzo-fused azepinones. Several benzazepinones were profiled in vivo and found to lower brain Aß42 levels in Sprague Dawley rats and transgenic APP-YAC mice in a dose-dependent manner after a single oral dose. Compound 34 was further progressed into a pilot study in our cisterna-magna-ported rhesus monkey model, where we observed robust lowering of CSF Aß42 levels.

siRNA-mediated knockdown of P450 oxidoreductase in rats: a tool to reduce metabolism by CYPs and increase exposure of high clearance compounds.

PURPOSE: To develop a tool based on siRNA-mediated knockdown of hepatic P450 oxidoreductase (POR) to decrease the CYP-mediated metabolism of small molecule drugs that suffer from rapid metabolism in vivo, with the aim of improving plasma exposure of these drugs. METHODS: siRNA against the POR gene was delivered using lipid nanoparticles (LNPs) into rats. The time course of POR mRNA knockdown, POR protein knockdown, and loss of POR enzyme activity was monitored. The rat livers were harvested to produce microsomes to determine the impact of POR knockdown on the metabolism of several probe substrates. Midazolam (a CYP3A substrate with high intrinsic clearance) was administered into LNP-treated rats to determine the impact of POR knockdown on midazolam pharmacokinetics. RESULTS: Hepatic POR mRNA and protein levels were significantly reduced by administering siRNA and the maximum POR enzyme activity reduction (~85%) occurred 2 weeks post-dose. In vitro analysis showed significant reductions in metabolism of probe substrates due to POR knockdown in liver, and in vivo POR knockdown resulted in greater than 10-fold increases in midazolam plasma concentrations following oral dosing. CONCLUSIONS: Anti-POR siRNA can be used to significantly reduce hepatic metabolism by various CYPs as well as greatly increase the bioavailability of high clearance compounds following an oral dose, thus enabling it to be used as a tool to increase drug exposure in vivo.

Discovery of 1-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl]-N-(pyridin-2-ylmethyl)methanesulfonamide (MK-8033): A Specific c-Met/Ron dual kinase inhibitor with preferential affinity for the activated state of c-Met.

This report documents the first example of a specific inhibitor of protein kinases with preferential binding to the activated kinase conformation: 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one 11r (MK-8033), a dual c-Met/Ron inhibitor under investigation as a treatment for cancer. The design of 11r was based on the desire to reduce time-dependent inhibition of CYP3A4 (TDI) by members of this structural class. A novel two-step protocol for the synthesis of benzylic sulfonamides was developed to access 11r and analogues. We provide a rationale for the observed selectivity based on X-ray crystallographic evidence and discuss selectivity trends with additional examples. Importantly, 11r provides full inhibition of tumor growth in a c-Met amplified (GTL-16) subcutaneous tumor xenograft model and may have an advantage over inactive form kinase inhibitors due to equal potency against a panel of oncogenic activating mutations of c-Met in contrast to c-Met inhibitors without preferential binding to the active kinase conformation.

Discovery of a 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one (MK-2461) inhibitor of c-Met kinase for the treatment of cancer.

c-Met is a transmembrane tyrosine kinase that mediates activation of several signaling pathways implicated in aggressive cancer phenotypes. In recent years, research into this area has highlighted c-Met as an attractive cancer drug target, triggering a number of approaches to disrupt aberrant c-Met signaling. Screening efforts identified a unique class of 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one kinase inhibitors, exemplified by 1. Subsequent SAR studies led to the development of 81 (MK-2461), a potent inhibitor of c-Met that was efficacious in preclinical animal models of tumor suppression. In addition, biochemical studies and X-ray analysis have revealed that this unique class of kinase inhibitors binds preferentially to the activated (phosphorylated) form of the kinase. This report details the development of 81 and provides a description of its unique biochemical properties.

Direct determination of the ratio of unbound fraction in plasma to unbound fraction in microsomal system (fu p/fu mic) for refined prediction of phase I mediated metabolic hepatic clearance.

At the drug discovery stage, in vivo metabolic hepatic clearance (CL(hep)) is commonly predicted using in vitro parent compound disappearance data generated in liver microsomes or hepatocytes. Correction for the unbound fraction of a compound in the in vitro system and in plasma/serum is known to be critical for the accuracy of metabolic clearance predictions. Discrete generation of these required experimental parameters can be laborious. Herein, we describe a straightforward and direct approach to obtain the ratio of unbound fraction in plasma (fu(p)) to unbound fraction in the microsomal system (fu(mic)) of a small molecule compound using equilibrium dialysis. Experimental conditions were optimized with respect to incubation time, temperature, and plate shaking speed. Results obtained from this system were validated for a set of test compounds by comparison to individually measured fu(p) and fu(mic) data using ultracentrifugation. The correlation for fu(p)/fu(mic) between the two methods for a set of 23 data points was very good with R(2) of 0.94, slope of 1.05 and an intercept of 0.007. The impact of microsomal binding on predicted CL(hep) was illustrated for a tightly bound compound using a series of incubations with increasing concentration of monkey liver microsomal protein. Alteration of this experimental parameter profoundly affected calculated CL(hep) using the well-stirred model. Significant differences were observed in the prediction when the model was corrected for fu(p) only; in contrast, the model corrected for plasma protein and microsomal protein binding predicted clearance values independent of the microsomal protein concentration.

MK-2461, a novel multitargeted kinase inhibitor, preferentially inhibits the activated c-Met receptor.

The receptor tyrosine kinase c-Met is an attractive target for therapeutic blockade in cancer. Here, we describe MK-2461, a novel ATP-competitive multitargeted inhibitor of activated c-Met. MK-2461 inhibited in vitro phosphorylation of a peptide substrate recognized by wild-type or oncogenic c-Met kinases (N1100Y, Y1230C, Y1230H, Y1235D, and M1250T) with IC(50) values of 0.4 to 2.5 nmol/L. In contrast, MK-2461 was several hundredfold less potent as an inhibitor of c-Met autophosphorylation at the kinase activation loop. In tumor cells, MK-2461 effectively suppressed constitutive or ligand-induced phosphorylation of the juxtamembrane domain and COOH-terminal docking site of c-Met, and its downstream signaling to the phosphoinositide 3-kinase-AKT and Ras-extracellular signal-regulated kinase pathways, without inhibiting autophosphorylation of the c-Met activation loop. BIAcore studies indicated 6-fold tighter binding to c-Met when it was phosphorylated, suggesting that MK-2461 binds preferentially to activated c-Met. MK-2461 displayed significant inhibitory activities against fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor, and other receptor tyrosine kinases. In cell culture, MK-2461 inhibited hepatocyte growth factor/c-Met-dependent mitogenesis, migration, cell scatter, and tubulogenesis. Seven of 10 MK-2461-sensitive tumor cell lines identified from a large panel harbored genomic amplification of MET or FGFR2. In a murine xenograft model of c-Met-dependent gastric cancer, a well-tolerated oral regimen of MK-2461 administered at 100 mg/kg twice daily effectively suppressed c-Met signaling and tumor growth. Similarly, MK-2461 inhibited the growth of tumors formed by s.c. injection of mouse NIH-3T3 cells expressing oncogenic c-Met mutants. Taken together, our findings support further preclinical development of MK-2461 for cancer therapy.

Validation of the use of nonnaive surgically catheterized rats for pharmacokinetics studies.

Although large animals, such as dogs and nonhuman primates, often are used for more than 1 pharmacokinetics study, common practice is to use only naive rodents for pharmacokinetics studies. We undertook a series of studies to validate whether surgically cannulated nonnaive rats could be used again after a 7-d washout. When vascular catheters are cared for appropriately, we find that they remain patent for more than 2 wk, with negligible drug carryover. Hematocrit decreased approximately 11% after pharmacokinetics studies but rebounded to prestudy levels after a 7-d washout. We empirically tested whether drugs known to alter drug disposition (1-aminobenzotriazole and quinidine) had residual effects on drug disposition after a 7-d washout and found that they did not. This finding suggests that after a 7-d washout, nonnaive rats likely would produce pharmacokinetics data similar to those of naive rats. We also tested reference compounds in naive and nonnaive rats and found no difference in pharmacokinetics parameters. Using surgically cannulated rats for a second study was feasible because of the relatively noninvasive nature of pharmacokinetics sampling (unrestrained rats attached to automated blood samplers). In addition, reusing surgically altered animals yields considerable cost savings. Our studies indicate that pharmacokinetics parameters did not differ significantly between naive and nonnaive rats. Cost-benefit analysis, monetary considerations, and validation studies support using rats for a second study after a 7-d washout period.

The discovery of 6-amino nicotinamides as potent and selective histone deacetylase inhibitors.

This communication highlights the development of a nicotinamide series of histone deacetylase inhibitors within the benzamide structural class. Extensive exploration around the nicotinamide core led to the discovery of a class I selective HDAC inhibitor that possesses excellent intrinsic and cell-based potency, acceptable ancillary pharmacology, favorable pharmacokinetics, sustained pharmacodynamics in vitro, and achieves in vivo efficacy in an HCT116 xenograft model.

Design of novel histone deacetylase inhibitors.

Histone deacetylase (HDAC) inhibitors that target Class I and Class II HDACs are of synthetic and therapeutic interest and ongoing clinical studies indicate that they show great promise for the treatment of cancer. Moreover, Zolinza (vorinostat) was recently approved by the FDA for the treatment of the cutaneous manifestations of cutaneous T-cell lymphoma [Nat. Rev. Drug Disc. 2007, 6, 21]. As part of a broader effort to more fully explore the structure-activity relationships (SAR) of HDAC inhibitors, we sought to identify novel HDAC inhibitor structures through iterative design by utilizing low affinity ligands as synthetic starting points for SAR development. Novel and potent HDAC inhibitors have been identified using this approach and herein we report the optimization of the recognition elements of a novel series of malonyl-derived HDAC inhibitors.

The effect of opiates on the activity of human placental aromatase/CYP19.

Aromatase, cytochrome P450 19, is a key enzyme in the biosynthesis of estrogens by the human placenta. It is also the major placental enzyme that metabolizes the opiates L-acetylmethadol (LAAM), methadone, and buprenorphine (BUP). Methadone and BUP are used in treatment of the opiate addict and are competitive inhibitors of testosterone conversion to estradiol (E(2)) and 16alpha-hydroxytestosterone (16-OHT) to estriol (E(3)) by aromatase. The aim of this investigation is to determine the effect of 20 opiates, which can be administered to pregnant patients for therapeutic indications or abused, on E(2) and E(3) formation by placental aromatase. Data obtained indicated that the opiates increased, inhibited, or had no effect on aromatase activity. Their effect on E(3) formation was more pronounced than that on E(2) due to the lower affinity of 16-OHT than testosterone to aromatase. The K(i) values for the opiates that inhibited E(3) formation were sufentanil, 7 +/- 1 microM; LAAM, 13 +/- 8 microM; fentanyl, 25 +/- 5 microM; oxycodone, 92 +/- 22 microM; codeine, 218 +/- 69 microM; (+)-pentazocine, 225 +/- 73 microM. The agonists morphine, heroin, hydromorphone, oxymorphone, hydrocodone, propoxyphene, meperidine, levorphanol, dextrorphan, and (-)-pentazocine and the antagonists naloxone and naltrexone caused an increase in E(3) formation by 124-160% of control but had no effect on E(2) formation. Moreover, oxycodone and codeine did not inhibit E(2) formation and the IC(50) values for fentanyl, sufentanil, and (+)-pentazocine were >1000 microM. It is unlikely that the acute administration of the opiates that inhibit estrogen formation would affect maternal and/or neonatal outcome. However, the effects of abusing any of them during the entire pregnancy are unclear at this time.

Methadone metabolism by early gestational age placentas.

The objective of this study was to identify the enzyme that metabolizes methadone in preterm placentas. Microsomal fractions were obtained from preterm (17 to 34 weeks) placentas (36 total; 12 per each gestational age group) and their activity in metabolizing methadone to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was determined. The enzyme catalyzing the reaction was identified by using chemical inhibitors selective for various cytochrome P450 isozymes and monoclonal antibodies raised against them. The metabolism of methadone by microsomes revealed saturation kinetics. Methadone was N-demethylated to EDDP by aromatase. The affinity of methadone to aromatase (apparent Km) did not change with gestation, but the activity of the enzyme (Vmax) increased and varied widely between individual placentas. Aromatase/CYP19 is the placental enzyme metabolizing methadone during pregnancy. The variability in enzyme activity among individuals should be reflected by the concentration of methadone in the fetal circulation and might be one of the factors affecting the incidence and intensity of neonatal abstinence syndrome.

The effect of methadone and buprenorphine on human placental aromatase.

Methadone and buprenorphine (BUP) are used for treatment of the pregnant opiate addict. CYP19/aromatase is the major placental enzyme responsible for the metabolism of methadone to 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and BUP to norbuprenorphine (norBUP). The aim of this investigation was to determine the effects of methadone and BUP on the activity of placental microsomal aromatase in the conversion of its endogenous substrates testosterone to 17beta-estradiol (E(2)) and 16alpha-hydroxytestosterone (16-OHT) to estriol (E(3)). The conversion of testosterone and 16-OHT by human placental microsomes exhibited saturation kinetics, and the apparent K(m) values were 0.2 +/- 1 and 6 +/- 3 microM, respectively. V(max) values for E(2) and E(3) formation were 70 +/- 16 and 28 +/- 10 pmol/mg proteinmin, respectively. Also, data obtained revealed that methadone and BUP are competitive inhibitors of testosterone conversion to E(2) and 16-OHT to E(3). The K(i) for methadone inhibition of E(2) and E(3) formation were 393 +/-144 and 53 +/- 28 microM, respectively, and for BUP the K(i) was 36 +/- 9 and 6 +/- 1 microM. The higher potency of the two opiates and their metabolites in inhibiting E(3) formation is in agreement with the lower affinity of 16-OHT than testosterone to aromatase. Moreover, the metabolites EDDP and norBUP were weaker inhibitors of aromatase than their parent compounds. The determined inhibition constants of methadone and BUP for E(3) formation by a cDNA-expressed CYP19 preparation were similar to those for placental microsomes. Therefore, data reported here suggest that methadone, BUP, and their metabolites are inhibitors of androgen aromatization in the placental biosynthesis of estrogens.

Bidirectional transfer of methadone across human placenta.

Methadone maintenance programs are considered the standard of care for the pregnant opiate addict. However, data on changes in methadone pharmacokinetics (PK) during pregnancy are limited and do not include its disposition by the placenta due to obvious ethical and safety considerations. Accordingly, investigations in our laboratory are focusing on human placental disposition of opiates including methadone. Recently, we reported on methadone metabolism by placental aromatase and provide here data on its bidirectional transfer across the tissue utilizing the technique of dual perfusion of placental lobule. The concentrations of the opiate transfused into the term placental tissue were those reported for its in vivo levels in the maternal serum of women under treatment with the drug. Data obtained indicated that the opiate has no adverse effects on placental viability and functional parameters and that it is retained by the tissue. Also, methadone transfer and its clearance index in the fetal to maternal direction (0.97+/-0.05) was significantly higher (P<0.05) than in the maternal to fetal (0.83+/-0.09). The observed asymmetry in methadone transfer could be explained by the unidirectional activity of the efflux transporter P glycoprotein (P-gp) that is highly expressed in variable amounts in trophoblast tissue. Therefore, placental disposition of methadone might be an important contributor to the regulation of its concentration in the fetal circulation and consequently may affect the incidence and intensity of neonatal abstinence syndrome for women treated with the drug during pregnancy.