Dhiren R. Thakker

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

Abstract (±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (−)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (−)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (−)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (−)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ∼30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ∼5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.

Enhancing paracellular permeability by modulating epithelial tight junctions

The intestinal epithelium is a major barrier to the absorption of hydrophilic drugs. The presence of intercellular junctional complexes, particularly the tight junctions (zona occludens), renders the epithelium impervious to hydrophilic drugs, which cannot diffuse across the cells through the lipid bilayer of the cell membranes. There have been significant advances in understanding the structure and cellular regulation of tight junctions over the past decade. This article reviews current knowledge regarding the physiological regulation of tight junctions and paracellular permeability, and recent progress towards the rational design of agents that can effectively and safely increase paracellular permeability via modulation of tight junctions.

Applications of the Caco-2 model in the design and development of orally active drugs: elucidation of biochemical and physical barriers posed by the intestinal epithelium

Abstract Oral administration is the most important and preferred route of administration for small molecular weight conventional drugs. The overall bioavailability of an orally administered drug depends on many factors, including the physicochemical properties of the drug as well as the morphological and biochemical state of the intestinal epithelium. Our understanding of the factors governing oral delivery of drug molecules is far from complete. As a result, the approaches to solve the problems related to oral drug delivery are often empirical in nature. The multifaceted nature of the problems associated with poor oral bioavailability of drug molecules requires a systematic and reductionist approach to understand the underlying factors affecting the bioavailability and absorption of these molecules. Thus, use of appropriate in vitro models is extremely useful in elucidating the role of various physical and biochemical barriers (such as metabolic enzymes, drug transporters, and the multidrug resistance (MDR) P-glycoprotein) to drug absorption. In this chapter, we have discussed the use of one such in vitro model, i.e. the Caco-2 cells, in elucidating the roles of the physical and biochemical barriers to drug absorption posed by the intestinal epithelium. By using specific examples, we have illustrated how this improved understanding about the barriers to drug absorption can be used for the design and development of drug candidates with enhanced oral absorption.

Differences in mutagenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides

Summary Substantial differences in the mutagenic activities of the optically pure (+)- and (−)-enantiomers of the diastereomeric 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrenes were observed in strains TA98 and TA100 of Salmonella typhimurium and Chinese hamster V79 cells. In strains TA98 and TA100 (−)-7β,8α-dihydroxy-9β, 10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene was the most mutagenic compound, inducing from 1.3 to 9.5 times as many mutations as the three other optically active stereoisomers. In Chinese hamster cells (+)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene was the most mutagenic compound inducing from 6 to 18 times as many variant colonies as the three other isomers. These results with known metabolites of the environmental carcinogen benzo[a]pyrene represent the first report of differences in mutagenic activity among optical enantiomers.

Novel experimental parameters to quantify the modulation of absorptive and secretory transport of compounds by P-glycoprotein in cell culture models of intestinal epithelium

AbstractPurpose. The purpose of this work was to elucidate the asymmetric effect of P-gp on modulation of absorptive and secretory transport of compounds across polarized epithelium, to develop experimental parameters to quantify P-gp-mediated modulation of absorptive and secretory transport, and to elucidate how P-gp-mediated modulation of transport is affected by passive diffusion properties, interaction of the substrate with P-gp, and P-gp expression. Methods. The permeability of a set of P-gp substrates was determined in absorptive and secretory directions in Madine-Darby Canine kidney (MDCK), Caco-2, and MDR-MDCK monolayers. The transport was also determined in the presence of GW918, a non-competitive P-gp inhibitor, to quantify the permeability without the influence of P-gp. From these two experimental permeability values in each direction, two new parameters, absorptive quotient (AQ) and the secretory quotient (SQ), were defined to express the functional activity of P-gp during absorptive and secretory transport, respectively. Western blot analysis was used to quantify P-gp expression in these monolayers and in normal human intestinal. Results. P-gp expression in Caco-2 and MDR-MDCK monolayers was comparable to that in normal intestine, and much less in MDCK cells. For all models, the substrates encompassed a wide range of apparent permeability due to passive diffusion (PPD). The parameters AQ and SQ, calculated for all compounds, assessed the attenuation in absorptive and enhancement of secretory transport, respectively, normalized to the permeability due to passive diffusion. Analysis of these parameters showed that 1) P-gp affected absorptive and secretory transport differentially and 2) compounds could be stratified into distinct groups with respect to the modulation of their absorptive and secretory transport by P-gp. Compounds could be identified whose absorptive transport was either strongly affected or poorly affected by changes in P-gp expression. For certain compounds, AQ values showed parabolic relationship with respect to passive diffusivity, and for others AQ was unaffected by changes in passive diffusivity. Conclusions. The relationship between attenuation of absorptive transport and enhancement of secretory transport of compounds by P-gp is asymmetric, and different for different sets of compounds. The relationship between attenuation of absorption by P-gp and passive diffusivity of compounds, their interaction potential with P-gp, and levels of P-gp expression is complex; however, compounds can be classified into sets based on these relationships. A classification system that describes the functional activity of P-gp with respect to modulation of absorptive and secretory transport was developed from these results.

Efflux ratio cannot assess P-glycoprotein-mediated attenuation of absorptive transport: Asymmetric effect of P-glycoprotein on absorptive and secretory transport across Caco-2 cell monolayers

AbstractPurpose. The purpose of this work was to determine whether P-glycoprotein (P-gp) modulates absorptive and secretory transport equally across polarized epithelium (i.e., Caco-2 cell monolayers) for structurally diverse P-gp substrates, a requirement for the use of the efflux ratio to quantify P-gp-mediated attenuation of absorption across intestinal epithelium. Methods. Studies were performed in Caco-2 cell monolayers. Apparent permeability (Papp) in absorptive (Papp,AB) and secretory (Papp,BA) directions as well as efflux ratios (Papp,BA / Papp,AB) were determined for substrates as a function of concentration. Transport of these compounds (10 μM) was measured under normal conditions and in the presence of the P-gp inhibitor, GW918 (1 μM), to dissect the effect of P-gp on absorptive and secretory transport. Apparent biochemical constants of P-gp-mediated efflux activity were calculated for both transport directions. Results. Efflux ratios for rhodamine 123 and digoxin were comparable (approx. 10). However, transport studies in the presence of GW918 revealed that P-gp attenuated absorptive transport of digoxin by approx. 8-fold but had no effect on absorptive transport of rhodamine 123 (presumably because absorptive transport of rhodamine 123 occurs via paracellular route). The apparent Km for P-gp-mediated efflux of digoxin was >6-fold larger in absorptive vs. secretory direction. For structurally diverse P-gp substrates (acebutolol, colchicine, digoxin, etoposide, methylprednisolone, prednisolone, quinidine, and talinolol) apparent Km was approximately 3 to 8-fold greater in absorptive vs. secretory transport direction, whereas apparent Jmax was somewhat similar in both transport directions. Conclusions. P-gp-mediated efflux activity observed during absorptive and secretory transport was asymmetric for all substrates tested. For substrates that crossed polarized epithelium via transcellular pathway in both directions, this difference appears to be caused by greater apparent Km of P-gp-mediated efflux activity in absorptive vs. secretory direction. These results clearly suggest that use of efflux ratios could be misleading in predicting the extent to which P-gp attenuates the absorptive transport of substrates.

Rhodamine 123 requires carrier-mediated influx for its activity as a P-glycoprotein substrate in Caco-2 cells.

AbstractPurpose. The purpose of this work was to elucidate transport pathways of the P-glycoprotein (P-gp) substrates rhodamine 123 (R123) and doxorubicin across Caco-2 cells. Methods. Experiments were designed to identify saturable and nonsaturable transport processes and transport barriers for R123 and doxorubicin transport across Caco-2 cells. Confocal laser scanning microscopy (CLSM) imaged R123 transport under normal conditions and in the presence of the P-gp inhibitor, GW918 (used to abolish P-gp-mediated efflux activity). Results. R123 secretory Papp (Papp,BA) showed concentration dependence, whereas R123 absorptive Papp (Papp,AB) did not. Inhibition of P-gp efflux revealed that P-gp-mediated efflux had no effect on R123 or doxorubicin Papp,AB, but enhanced R123 and doxorubicin Papp,BA. In calcium-free medium, R123 Papp,AB increased 15-fold, indicating intercellular junctions are a barrier to R123 absorption. CLSM of R123 fluorescence during absorptive transport under normal conditions and in the presence of GW918 was identical, and was limited to paracellular space, confirming that P-gp is not a barrier to R123 absorption. CLSM revealed that R123 fluorescence during secretory transport under normal conditions and in the presence of GW918 was localized intracellularly and in paracellular space. R123 and doxorubicin uptake across Caco-2 cells basolateral membrane was saturable. Conclusions. R123 absorptive transport occurs primarily by paracellular route, whereas R123 secretory transport involves influx across BL membrane mediated solely by a saturable process followed by apically directed efflux via P-gp. Doxorubicin utilizes similar transport pathways to cross Caco-2 cells.

Prodrugs of anticancer agents

Abstract Anticancer drugs are primarily cytotoxic agents, and exert their antitumor activity by interfering with some aspects of DNA replication, repair, translation, or cell division. Hence, cancer chemotherapy is typically associated with severe side effects. An important approach to alleviate toxicity of the anticancer agents is to prepare covalent derivatives i.e., prodrugs, which lack the cytotoxic activity, but which can be converted enzymatically or non-enzymatically to the cytotoxic agents upon administration to patients. Prodrugs have been used to improve the solubility, transport properties, and pharmacokinetic properties of anticancer agents. More importantly, several prodrug strategies have been developed that enable selective delivery of the cytotoxic agents to the tumor tissue, thereby significantly reducing the toxic side effects of the anticancer agents. These strategies include design of prodrugs based on elevated enzymes in tumor tissues, hypoxic environment inside the core of solid tumors, and tumor-specific antigens expressed on the surface of tumor cells. The utility of various prodrug strategies in improving the therapeutic index of anticancer agents as well as the limitations of these prodrug strategies are reviewed in this chapter.

Mechanism of Intestinal Absorption of Ranitidine and Ondansetron: Transport Across Caco-2 Cell Monolayers

We have investigated the transport of ranitidine and ondansetron across the Caco-2 cell monolayers. The apparent permeability coefficients (Papp) were unchanged throughout the concentration range studied, indicating a passive diffusion pathway across intestinal mucosa. No metabolism was observed for ranitidine and ondansetron during the incubation with Caco-2 cell monolayers. Papp values for ranitidine and ondansetron (bioavailability of 50 and ∼100% in humans, respectively) were 1.03 ± 0.17 × 10−7 and 1.83 ± 0.055 × 10−5 cm/sec, respectively. The Papp value for ranitidine was increased by 15- to 20-fold in a calcium-free medium or in the transport medium containing EDTA, whereas no significant change occurred with ondansetron, indicating that paracellular passive diffusion is not rate determining for ondansetron. Uptake of ondansetron by Caco-2 cell monolayers was 20- and 5-fold higher than that of ranitidine when the uptake study was carried out under sink conditions and at steady state. These results suggest that ranitidine and ondansetron are transported across Caco-2 cell monolayers predominantly via paracellular and transcellular pathways, respectively.

Mechanisms Underlying Saturable Intestinal Absorption of Metformin

The purpose of the study was to elucidate mechanisms of metformin absorptive transport to explain the dose-dependent absorption observed in humans. Apical (AP) and basolateral (BL) uptake and efflux as well as AP to BL (absorptive) transport across Caco-2 cell monolayers were evaluated over a range of concentrations. Transport was concentration-dependent and consisted of saturable and nonsaturable components (Km ∼ 0.05 mM, Jmax ∼ 1.0 pmol min-1 cm-2, and Kd, transport ∼ 10 nl min-1 cm-2). AP uptake data also revealed the presence of saturable and nonsaturable components (Km ∼ 0.9 mM, Vmax ∼ 330 pmol min-1 mg of protein-1, and Kd, uptake ∼ 0.04 μl min-1 mg of protein-1). BL efflux was rate-limiting to transcellular transport of metformin; AP efflux was 7-fold greater than BL efflux and was not inhibited by N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GW918), a P-glycoprotein inhibitor. AP efflux was trans-stimulated by metformin and prototypical substrates of organic cation transporters, suggesting that a cation-specific bidirectional transport mechanism mediated the AP efflux of metformin. BL efflux of intracellular metformin was much less efficient in comparison with the overall transport, with BL efflux clearance accounting for ∼7 and ∼13% of the overall transport clearance at 0.05 and 10 mM metformin concentrations, respectively. Kinetic modeling of cellular accumulation and transport processes supports the finding that transport occurs almost exclusively via the paracellular route (∼90%) and that the paracellular transport is saturable. This report provides strong evidence for a saturable mechanism in the paracellular space and provides insight into possible mechanisms for the dose dependence of metformin absorption in vivo.