Kinetics of diazepam metabolism in rat hepatic microsomes and hepatocytes and their use in predicting in vivo hepatic clearance

1. The rates of diazepam (DZ) metabolism to the primary metabolites 3-hydroxydi-azepam, 4'-hydroxydiazepam and nordiazepam were studied in vitro using rat hepatic microsomes and hepatocytes. 4'-hydroxydiazepam had the largest intrinsic clearance (V_max/K_m ratio, CL_int) in both microsomes and hepatocytes representing 49 and 70% of total metabolism respectively. Whereas the contribution of 3-hydroxydiazepam was similar in both systems (21–24%), the N-demethylation pathway was greater in microsomes (27%) than hepatocytes (9%).

2. The pharmacokinetics of DZ were determined in vivo using the intraportal route to avoid blood flow limitations due to the high clearance of DZ. No dose dependency was observed in either clearance or steady state volume of distribution, which were estimated to be 38 ml/min/SRW (where SRW is a standard rat weight of 250?g) and 1.3 L/SRW respectively. Blood binding of DZ was concentration independent, the unbound fraction being 0.22.

3. Scaling factors were used to relate the in vitro CL_int to the in vivo unbound clearance. Hepatocytes (123 ml/min/SRW) produced a more realistic prediction for the in vivo value (174 ml/min/SRW) than microsomes (41 ml/min/SRW). This situation is believed to arise from the quantitative differences in the three metabolic pathways in the two in vitro systems. It is speculated that end product inhibition is responsible for reduced total metabolism in microsomes whereas hepatocytes operate kinetically in a manner close to in vivo.     

A Novel Oligodeoxynucleotide Inhibitor of Thrombin. I. In VitroMetabolic Stability in Plasma and Serum

Purpose. To determine the degradation rates and pathways of GS-522, a potent oligodeoxynucleotide (GGTTGGTGTGGTTGG) inhibitor of thrombin, in serum and plasma. Methods. A stability-indicating, anion-exchange HPLC method was developed and used to determine concentrations of GS-522 and metabolites. Results. In monkey plasma at 2 µM or below, the degradation of GS-522 can be fit to a first-order exponential with a k^p _obs ~ 0.01 min^–1. At 3 µM and above the degradation process deviates from a monoexponential decay profile. An initial fast degradation process is followed by a slower phase with an observed rate constant equal to that observed at 2 µM and below. In monkey serum, the K_Mand V_maxare 8.4 µM and 0.87 µM min^–1, respectively. Conclusions. The kinetics are consistent with an equilibrium binding of GS-522 to prothrombin in plasma (K_d = 50 nM) which saturates at GS-522 concentrations >2 µM. Compared to a scrambled sequence (GGTGGTGGTTGTGGT), with no defined tertiary structure, GS-522 is 4-fold more stable in serum. The metabolic profile in plasma is consistent with a 3-exonuclease catalyzed hydrolysis of GS-522.     

In vitro inhibition and induction of human cytochrome P450 enzymes by SIPI5357, a potential antidepressant

This study investigated the in vitro drug–drug interaction potential of SIPI5357, an arylalkanol‐piperazine derivative used in the treatment of depression. Drug–drug interaction occurs via inhibition or induction of enzymes involved in their metabolism. In human liver microsomes, SIPI5357 showed the strongest inhibition of CYP2D6, followed by CYP3A4 (testosterone) and CYP2C8. Inhibition was observed in a concentration‐dependent manner, with IC₅₀ values of 18.45 µM, 36.63 µM (CYP3A4/testosterone), 89.23 µM, respectively. SIPI5357 was predicted not to cause significant metabolic drug–drug interaction via inhibition of CYP1A2, CYP2C9, CYP2C19, CYP2E1 or CYP3A4 (midazolam) because the IC₅₀ values for these enzymes were both >100 µM (200 times maximum plasma concentration [C_max]). SIPI5357 showed a mixed model inhibition of CYP2D6 (K_i = 11.12 µM). The value of [I]/K_i for CYP2D6 inhibition by SIPI5357 is below the FDA cut‐off value of 0.1; it is therefore reasonable to assume that SIPI5357 will not cause significant CYP2D6 inhibition. However, positive controls (50 µM omeprazole and 25 µM rifampin) caused the anticipated CYP induction, but the highest concentration of SIPI5357 (5 µM; 10 times plasma C_max) had a minimal effect on CYP1A2 and CYP3A4 mRNA levels in freshly isolated human hepatocytes, suggesting that SIPI5357 is not an inducer of these enzymes. However, significant induction of CYP2B6 was observed at 0.5 µM and 5 µM. In conclusion, SIPI5357 might cause drug–drug interaction via induction of CYP2B6. The in vivo drug–drug interaction potential deserves further investigation. Copyright © 2015 John Wiley & Sons, Ltd.     

A Functional Assay for Quantitation of the Apparent Affinities of Ligands of P-Glycoprotein in Caco-2 Cells

Purpose. To develop a facile functional assay for quantitative determination of the apparent affinities of compounds that interact with the taxol binding site of P-glcoprotein (P-gp) in Caco-2 cell monolayers. Methods. A transport inhibition approach was taken to determine the inhibitory effects of compounds on the active transport of [³H]-taxol, a known substrate of P-gp. The apparent affinities (K_I values) of the compounds were quantitatively determined based on the inhibitory effects of the compounds on the active transport of [³H]-taxol. Intact Caco-2 cell monolayers were utilized for transport inhibition studies. Samples were analyzed by liquid scintillation counting. Results. [³H]-Taxol (0.04 M) showed polarized transport with the basolateral (BL) to apical (AP) flux rate being about 10-20 times faster than the flux rate in the AP-to-BL direction. This difference in [³H]-taxol flux could be totally abolished by inclusion of (±)-verapamil (0.2 mM), a known inhibitor of P-gp, in the incubation medium. However, inclusion of probenecid (1.0 mM), a known inhibitor for the multidrug resistance associated protein (MRP), did not significantly affect the transport of [³H]-taxol under the same conditions. These results suggest that P-gp, not MRP, was involved in taxol transport. Quinidine, daunorubicin, verapamil, taxol, doxorubicin, vinblastine, etoposide, and celiprolol were examined as inhibitors of the BL-to-AP transport of [³H]-taxol with resulting K_I values of 1.5 ± 0.8, 2.5 ± 1.0, 3.0 ± 0.3, 7.3 ± 0.7, 8.5 ± 2.8, 36.5 ± 1.5, 276 ± 69, and 313 ± 112 M, respectively. With the exception of that of quinidine, these K_I values were comparable with literature values. Conclusions. This assay allows a facile quantitation of the apparent affinities of compounds to the taxol-binding site in P-gp; however, this assay does not permit the differentiation of substrates and inhibitors. The potential of drug-drug interactions involving the taxol binding site of P-gp can be conveniently estimated using the protocol described in this paper.     

Effect of mdr1a P-glycoprotein gene disruption, gender, and substrate concentration on brain uptake of selected compounds

Purpose. This study assessed the influence of mdr1a P-glycoprotein (P-gp) gene disruption, gender and concentration on initial brain uptake clearance (Cl _up) of morphine, quinidine and verapamil. Methods. Cl _up of radiolabeled substrates was determined in P-gp-competent and deficient [mdr1a(–/–)] mice by in situ brain perfusion. Brain:plasma distribution of substrates after i.v. administration was determined in both strains. Results. Genetic disruption of mdr1a P-gp resulted in 1.3-, 6.6- and 14-fold increases in Cl _up for morphine, verapamil and quinidine, respectively. With the exception of small differences for verapamil, gender did not affect Cl _up. Saturable transport of verapamil and quinidine was observed only in P-gp-competent mice, with apparent IC ₅₀ values for efflux of 8.6 ± 2.3 M and 36 ± 2 M, respectively. VerapamilCl _up was 50% higher in mdr1a(+/–) vs. mdr1a(+/+) mice; no such difference was observed for quinidine. In P-gp-competent mice, uptake of verapamil and quinidine was unaffected by organic vehicles. Plasma decreased VER Cl _up to a greater extent in the presence of P-gp. The influence of P-gp in situ was lower than, but correlated with, the effect in vivo. Conclusions. P-gp decreases Cl _up of morphine, verapamil and quinidine in situ with little or no influence of gender, but this effect cannot fully account for the effects of P-gp in vivo. P-gp is the only saturable transport mechanism for verapamil and quinidine at the murine blood-brain barrier. The influence of protein binding on Cl _up may be enhanced by P-gp-mediated efflux.     

Prediction of in Vivo Tissue Distribution from in Vitro Data. 2. Correlation Between in Vitro and in Vivo Tissue Distribution of a Homologous Series of Nine 5-n-Alkyl-5-Ethyl Barbituric Acids

Purpose. To evaluate the ability to determine accurate in vivo tissue-to-unbound plasma distribution coefficients (Kpu_e) from in vitro data. Methods. Fresh pieces of fifteen rat tissues/organs were incubated at 37°C with a homologous series of nine barbiturates covering a wide range of lipophilicity (Log P 0.02 to 4.13). Steady-state in vivo Kpu _e values were estimated from the tissue and plasma concentrations following simultaneous dosing by constant rate i.v. infusion of all nine barbiturates. Drug concentrations in the tissues and media were determined by HPLC with UV or mass spectrometric detection. Results. The pharmacokinetics of the barbiturate series following constant rate i.v. infusion indicated a range of clearance (0.49 to 30 ml.min^\-1.kg^\-1) and volume of distribution at steady state (0.51 to 1.9 l.kg^\-1) values. Good agreement was observed between the in vitro and in vivo Kpu values, although for the most lipophilic barbiturates the in vitro data underpredicted the in vivo tissue distribution for all tissues. Conclusions. The in vitro system for predicting the extent of in vivo tissue distribution works well for compounds of widely differing lipophilicity, although for the most lipophilic drugs it may result in an underprediction of in vivo values.     

P-Glycoprotein Inhibition Leads to Enhanced Disruptive Effects by Anti-Microtubule Cytostatics at the In Vitro Blood-Brain Barrier

Purpose. To investigate whether P–glycoprotein (Pgp) protects the in vitro BBB against the cytotoxic effects of anti–tumour drugs. Methods. In an in vitro BBB coculture model the influence of the anti–microtubule drugs vinblastine, colchicine, paclitaxel and the non–antimicrotubule drugs doxorubicin, fluorouracil and etoposide in the absence or presence of Pgp modulators on the trans–endothelial electrical resistance (TEER), which is an indicator for the integrity, was investigated. Results. In the absence of Pgp modulators vinblastine, colchicine and paclitaxel dose dependently decreased TEER values to less than 20% of control. Non–anti–microtubule drugs did not affect TEER values. Following competitive inhibition of Pgp by various Pgp modulators and substrates, even low concentrations of vinblastine, colchicine and paclitaxel substantially decreased TEER. IC₅₀ values of LY 335979, SDZ–PSC 833, cyclosporin A, and verapamil were 0.03, 0.25, 0.46, and 13.7 M, respectively. Conclusions. These results indicate that Pgp normally protects the in vitro BBB against the disruptive effects of anti–microtubule drugs, but its integrity is lost when anti–microtubule drugs are used in combination with potent Pgp modulators. In addition, this procedure offers the possibility to characterize Pgp modulators and substrates with respect to their efficacy and to elucidate drug interactions at the level of Pgp.     

Metabolism of bupropion by baboon hepatic and placental microsomes

The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics. Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by hepatic microsomes (K_m 36 ± 6 μM, V_max 258 ± 32 pmol mg protein⁻¹ min⁻¹), however the formation of OH-BUP by placental microsomes was below the limit of quantification. The apparent K_m values of bupropion for the formation of TB and EB by hepatic and placental microsomes were similar. The selective inhibitors of CYP2B6 (ticlopidine and phencyclidine) and monoclonal antibodies raised against human CYP2B6 isozyme caused 80% inhibition of OH-BUP formation by baboon hepatic microsomes. The chemical inhibitors of aldo-keto reductases (flufenamic acid), carbonyl reductases (menadione), and 11β-hydroxysteroid dehydrogenases (18β-glycyrrhetinic acid) significantly decreased the formation of TB and EB by hepatic and placental microsomes. Data indicate that CYP2B of baboon hepatic microsomes is responsible for biotransformation of bupropion to OH-BUP, while hepatic and placental short chain dehydrogenases/reductases and to a lesser extent aldo-keto reductases are responsible for the reduction of bupropion to TB and EB.     

Characterization of the Transport Properties of a Quinolone Antibiotic,Fleroxacin, in Rat Choroid Plexus

Purpose. It is reported that the cerebrospinal fluid (CSF) to plasma unbound concentration ratio of fleroxacin at steady-state is approximately 0.5 in experimental animals. These results can be accounted for by assuming the presence of an active transport system for the efflux of this compound across the choroid plexus. In the present study, the transport system for fleroxacin was characterized in isolated rat choroid plexus. Methods. Choroid plexus was isolated from the lateral ventricles of rats. The accumulation of [¹⁴C] fleroxacin or [³H] benzylpenicillin by the choroid plexus was examined by the centrifugal filtration method. Results. The accumulation of [¹⁴C] fleroxacin by the rat isolated choroid plexus was significantly inhibited by metabolic inhibitors (rotenone, 30 µM and carbonyl cyanide p-trifluorometh oxyphenylhydrazone (FCCP), 100 µM) and sulfhydryl reagent (p-chloromer-curibenzenesulfonic acid (PCMBS), 100 µM). This accumulation was composed of a saturable component (V_max = 240 pmol·min^–1·µl tissue^–1, K_m = 664 µM) and non-saturable one (P = 0.424 min^–1·µl tissue^–1). Accumulation of fleroxacin was competitively inhibited by benzylpenicillin and probenecid with K_i values of 29 µM and 51 µM, respectively. These values are comparable with the K_m of benzylpenicillin transport and the K_i of probenecid for the benzylpenicillin transport at the choroid plexus, respectively. Furthermore, fleroxacin inhibited competitively the accumulation of [³H] benzylpenicillin with a K_i of 384 µM, a value comparable with the K_m of [¹⁴C] fleroxacin transport. Conclusions. Fleroxacin and benzylpenicillin showed mutual competitive inhibition, suggesting that both are transported via a common transport system in the choroid plexus and are pumped out from CSF into the circulation.     

Inhibition of In Vitro Metabolism of Simvastatin by Itraconazole in Humans and Prediction of In Vivo Drug-Drug Interactions

Purpose. To evaluate an interaction between simvastatin and itraconazole in in vitro studies and to attempt a quantitative prediction of in vivo interaction in humans. Methods. The inhibitory effect of itraconazole on simvastatin metabolism was evaluated using human liver microsomes and the K_i values were calculated for the unbound drug in the reaction mixture. A physiologically-based pharmacokinetic model was used to predict the maximum in vivo drug-drug interaction. Results. Itraconazole competitively inhibited the metabolism of simvastatin to M-1 and M-2 with K_i values in the nM range. The area under the curve (AUC) of simvastatin after concomitant dosing with itraconazole was predicted to increase ca. 84-101-fold compared with that without administration of itraconazole. Taking into consideration the fact that this method predicts the maximum interaction, this agrees well with the clinical observation of a 19-fold increase. A similar prediction, based on the K_i value without taking into account the drug adsorption to microsomes, led to an underevaluation of the interaction. Conclusions. It was demonstrated that the competitive inhibition of CYP3A4-mediated simvastatin metabolism by itraconazole is the main cause of the drug interaction and that a K_i value corrected for drug adsorption to microsomes is the key factor in quantitatively predicting the maximum in vivo drug interactions.     

The metabolism of the 5HT3 antagonists ondansetron,alosetron and GR87442 I: A comparison of in vitro and in vivo metabolism and in vitro enzyme kinetics in rat,dog and human hepatocytes,microsomes and recombinant human enzymes

The metabolism of the structurally related 5HT₃ antagonists ondansetron, alosetron and GR87442 in the rat, dog and human was determined in hepatocytes, liver microsomes and human recombinant microsomes. The profiles of phase I metabolites were similar in human hepatocytes and microsomes. The metabolites of all three compounds produced in rat, dog and human microsomes and hepatocytes were similar to those seen in vivo, with the major routes of metabolism being N-dealkylation and/or hydroxylation. There was more extensive metabolic processing in hepatocytes than in microsomes; however, sequential metabolism was less extensive in vitro compared with in vivo. The pharmacokinetics of the three 5HT₃ antagonists investigated were dominated by CYP3A4 (and/or 2C9) compared with CYP1A2 in man, possibly determined by enzyme capacity rather than relative enzyme affinity. These data support the use of rat, dog and human hepatocytes for the prediction of in vivo metabolites of ondansetron, alosetron and GR87442.     

Effects of Selective Serotonin Reuptake Inhibitors on Timolol Metabolism in Human Liver Microsomes and Cryo‐Preserved Hepatocytes

Abstract: Timolol has been widely used in the treatment of glaucoma. Topically applied, timolol may cause adverse cardiovascular effects due to systemic absorption through the nasolacrimal duct. Timolol is mainly metabolized by cytochrome P450 2D6 (CYP2D6) in the liver. The aim of the present study was to characterize further the metabolism of timolol in vitro. Especially the effect of several drugs such as selective serotonin reuptake inhibitors on the metabolism of timolol was evaluated. In human liver microsomes, four timolol metabolites were identified, in cryo‐preserved hepatocytes nine. In both in vitro experiments, the hydroxy metabolite M1 was the main metabolite. The in vivo half‐life predicted for timolol was 3.7 hr. in cryo‐preserved hepatocytes, corresponding to the half‐life of timolol in humans in vivo. Fluoxetine, paroxetine, sertraline, citalopram and fluvoxamine inhibited the formation of M1 in microsomes with IC₅₀ values of 1.4, 2.0, 3.5, 21 and 20 μM, respectively. In human cryo‐preserved hepatocytes, the IC₅₀ values for fluoxetine, paroxetine and fluvoxamine were 0.7, 0.5 and 5.9 μM, respectively. In conclusion, compounds known to be potent CYP2D6 inhibitors inhibited timolol metabolism in in vitro experiments. The present results strongly suggest that fluoxetine and paroxetine may significantly affect the metabolism of timolol also in vivo and may thus potentiate the adverse cardiovascular effects of topically administered timolol.     

Comparison of CYP2D6 Content and Metoprolol Oxidation Between Microsomes Isolated from Human Livers and Small Intestines

Purpose. To assess the role of intestinal CYP2D6 in oral first-pass drug clearance by comparing the enzyme content and catalytic activity of a prototype CYP2D6 substrate, metoprolol, between microsomes prepared from human intestinal mucosa and from human livers. Methods. Microsomes were prepared from a panel of 31 human livers and 19 human intestinal jejunal mucosa. Microsomes were also obtained from the jejunum, duodenum and ileum of four other human intestines to assess regional distribution of intestinal CYP2D6. CYP2D6 content (pmole/mg microsomal protein) was determined by Western blot. CYP2D6 activity was measured by -hydroxylation and O-demethylation of metoprolol. Results. Kinetic studies with microsomes from select livers (n = 6) and jejunal mucosa (n = 5) yielded K_M estimates of 26 ± 9 M and 44 ± 17 M, respectively. The mean V_max (per mg protein) for total formation of -OH-M and ODM was 14-fold higher for the liver microsomes compared to the jejunal microsomes. Comparisons across intestinal regions showed that CYP2D6 protein content and catalytic activity were in the order of jejunum > duodenum > ileum. Excluding the poor metabolizer genotype donors, CYP2D6 content varied 13-and 100-fold across the panels of human livers (n = 31) and jejunal mucosa (n = 19), respectively. Metoprolol -hydroxylation activity and CYP2D6 content were highly correlated in the liver microsomes (r = 0.84, p < 0.001) and jejunal microsomes (r = 0.75, p < 0.05). Using the well-stirred model, the mean microsomal intrinsic clearance (i.e., V_max/K_M) for the livers and jejunum were scaled to predict their respective in vivo organ intrinsic clearance and first-pass extraction ratio. Hepatic and intestinal first-pass extractions of metoprolol were predicted to be 48% and 0.85%, respectively. Conclusions. A much lower abundance and activity of CYP2D6 are present in human intestinal mucosa than in human liver. Intestinal mucosal metabolism contributes minimally to the first-pass effect of orally administered CYP2D6 substrates, unless they have exceptionally high microsomal intrinsic clearances and/or long residence time in the intestinal epithelium.     

The Use of Human Hepatocytes to Select Compounds Based on Their Expected Hepatic Extraction Ratios in Humans

Purpose. The present investigation retrospectively evaluates the use of human hepatocytes to classify compounds into low, intermediate or high hepatic extraction ratio in man. Methods. A simple approach was used to correlate the in vivo hepatic extraction ratio of a number of compounds in man (literature and in-house data) with the corresponding in vitro clearance which was determined in human hepatocytes. The present approach assumes that, for compounds eliminated mainly through liver metabolism, intrinsic clearance is the major determinant for their in vivo hepatic extraction ratio and subsequently their bioavailability in man. The test compounds were selected to represent a broad range of extraction ratios and a variety of metabolic pathways. Results. The present data show that in vitro clearances in human hepatocytes are predictive for the hepatic extraction ratios in vivo in man. Most of the test compounds (n = 19) were successfully classified based upon human hepatocyte data into low, intermediate or high hepatic extraction compounds, i.e. compounds with potential for high, intermediate or low bioavailabilities in humans. Conclusions. The present approach, validated so far with 19 test compounds, appears to be a valuable tool to screen for compounds with respect to liver first-pass metabolism at an early phase of drug discovery.     

Characterization of in vitro metabolites of luotonin A in human liver microsomes using electrospray/tandem mass spectrometry

1. The pharmacological activity of luotonin A varies, depending on the type of functional group and the site of derivatization. To understand the in vivo efficacy of luotonin A, the in vitro metabolism of luotonin A was investigated in human liver microsomes and recombinant cDNA-expressed cytochromeP450 (CYP).

2. Incubation of luotonin A with pooled human liver microsomes in the presence of NADPH-generating system resulted in the formation of four metabolites and the structures of each metabolite were tentatively characterized on the basis of electrospray tandem mass spectra.

3. The main metabolic pathway of luotonin A in human liver microsomes was hydroxylation, resulting in the generation of two mono-hydroxyl metabolites (M1 and M2) and two di-hydroxyl metabolites (M3 and M4). CYP1A2 was primarily involved in hydroxylation of the quinolone moiety (M1 andM3), while CYP3A4 was mainly responsible for hydroxylation of the quinazoline moiety of luotonin A (M2 and M4) in human liver microsomes.     

Are MDCK Cells Transfected with the Human MDR1 Gene a Good Model of the Human Intestinal Mucosa?

Purpose. To investigate whether Madin-Darby canine kidney cells transfected with the human MDR1 gene (MDCK-MDR1) are a good model of the human intestinal mucosa. Methods. P-glycoprotein (P-gp) expression in Caco-2 cells was compared with P-gp expression in MDCK wild- type (MDCK-WT) and MDCK-MDR1 cells using Western blotting methods. The polarized efflux activities of P-gp(s) in MDCK-MDR1 cells, MDCK-WT cells, and Caco-2 cells were compared using digoxin as a substrate. Apparent Michaelis-Menten constants (K _M,V _max) for the efflux of vinblastine in these three cell lines were determined. Apparent inhibition constants (K _I) of known substrates/inhibitors of P-gp were determined by measuring their effects on the efflux of digoxin in Caco-2 or MDCK-MDR1 cell monolayers. Results. MDCK-MDR1 cells expressed higher levels of P-gp compared to Caco-2 and MDCK-WT cells, as estimated by Western blots. Two isoforms of P-gp were expressed in Caco-2 and MDCK cells migrating with molecular weights of 150 kDa and 170 kDa. In MDCK-MDR1 cells, the 150 kDa isoforms appeared to be overexpressed. The MDCK-MDR1 cells exhibited higher polarized efflux of [³H]-digoxin than did Caco-2 and MDCK-WT cells. K _M values of vinblastine in Caco-2, MDCK-WT, and MDCK-MDR1 cells were 89.2 ± 26.1, 24.5 ± 1.1, and 252.8 ± 134.7 M, respectively, whereas V _max values were 1.77 ± 0.22, 0.42 ± 0.01, and 2.43 ± 0.86 pmolcm^–2s^–1, respectively. Known P-gp substrates/inhibitors showed, in general, lower K _I values for inhibition of digoxin efflux in Caco-2 cells than in MDCK-MDR1 cells. Conclusions. These data suggest that the MDCK-MDR1 cells overexpress the 150 kDa isoform of P-gp. MDCK-MDR1 cells are a useful model for screening the P-gp substrate activity of drugs and drug candidates. However, the apparent kinetics constants and affinities of substrates determined in the MDCK-MDR1 cell model may be different than the values obtained in Caco-2 cells. These differences in substrate activity could result from differences in the relative expression levels of total P-gp in Caco-2 and MDCK-MDR1 cells and/or differences in the partitioning of substrates into these two cell membrane bilayers.     

Inhibition of human liver microsomal CYP by nateglinide

Objectives Nateglinide is metabolized by CYP2C9 and CYP3A4, therefore drug–drug interactions through cytochrome P450 (CYP) inhibition may occur. In this study, we examined the inhibitory effects of nateglinide and its major metabolite N‐[trans‐4‐(1‐hydroxy‐1‐methylethyl)‐cyclohexanecarbonyl]‐d ‐phenylalanine (M1) on various CYP isoforms in human liver microsomes. Methods We used typical substrates (7‐ethoxyresorufin for CYP1A1/2, tolbutamide for CYP2C9, S‐mephenytoin for CYP2C19, bufuralol for CYP2D6, chlorzoxazone for CYP2E1 and midazolam for CYP3A4) in the evaluation of the inhibitory effects, and examined the possibility of mechanism‐based inhibition (MBI) by evaluating the influence of pre‐incubation in the inhibition. Key findings The results showed that nateglinide inhibited CYP2C9 and CYP2C19 with an IC50_app (apparent value of the 50% inhibitory concentration) of 125 μmol/l and 946 μmol/l, respectively, while M1 did not inhibit any of the CYP isoforms. The inhibition constant (K_i) value of the inhibitory effect of nateglinide on CYP2C9 and the 1 + I_in,max,u/K_i value were estimated (where I_in,max,u= the maximum unbound concentration of nateglinide). The 1 + I_in,max,u/K_i value was 1.02 (close to 1), suggesting a low risk of drug–drug interactions. The influence of pre‐incubation on the inhibition by nateglinide of CYP3A4, CYP2C9 and CYP2C19 was examined. The results revealed that the inhibition of CYP by nateglinide was not influenced by pre‐incubation, and that the possibility of MBI is very low. Conclusions The possibility of drug–drug interactions involving nateglinide that might be attributable to CYP inhibition is low.     

Differential inhibitory effects of phenytoin,diclofenac, phenylbutazone and a series of sulfonam ides on hepatic cytochrom e P4502C activity in vitro,and correlation with som e m olecular descriptors in the dwarf goat (Caprus hircus aegagrus).

1. The aim of the present study was to investigate the potency of various sulfonamides to inhibit tolbutamide hydroxylation (a CYP2C activity) in hepatic microsomal fractions and hepatocytes of the dwarf goat. Also a number of suggested substrates for human CYP2C9 was investigated. 2. From Dixon plots (microsomal fractions) it was observed that all compounds were competitive inhibitors of tolbutamide hydroxylation. Phenytoin (PT) showed the lowest K_i. K_i for the sulfonamides ranged between 205 and 4546 μM, sulfadoxine having the lowest K_i followed by sulfadimethoxine, sulfamoxole, sulfadimidine and sulfaphenazole. 3. In hepatocytes sulfaphenazole and diclofenac were the most potent inhibitors. 4. Out data indicate that PT, diclofenac (DF) and phenylbutazone (PBZ) are relative strong competitive inhibitors of tolbutamide hydroxylation and they are probably also substrates for the same enzyme. Differential inhibition of tolbutamide hydroxylation by sulfonamides was observed. 5. Correlation of structural parameters with the inhibition constant or the inhibition in hepatocytes showed that molecular volume, polarisability and molecular surface area are important parameters in determining the rate of inhibition of tolbutamide hydroxylation by sulfonamides in both microsomes and hepatocytes. In addition, log P_oct are also involved oct in determining inhibition constants in microsomal fractions.     

Metabolite kinetics of ondansetron in rat. Comparison of hepatic microsomes,isolated hepatocytes and liver slices,with in vivo disposition

1. The kinetics of hydroxylation and N-demethylation of ondansetron have been determined in freshly isolated hepatocytes, hepatic microsomes and precision-cut liver slices from the male Sprague-Dawley rat. In vivo studies have also been carried out to characterize the pharmacokinetics of ondansetron and in vitro data have been assessed for their value as predictors of hepatic clearance.

2. In the three in vitro systems, the formation of hydroxylated and demethylated metabolites were characterized as a function of substrate concentration by a high-affinity, low-capacity site and a low-affinity, high-capacity site which was not saturated over the concentration range studied (2.5–500 μM). Slices gave consistently higher K_m's (20 and 30 μM for hydroxylation and demethylation respectively) than hepatocytes (3 and 13 μM respectively) and microsomes (2 and 5 μM respectively). The rank order of V_max and CL_int was the same for each system; hydroxylation rates exceeding demethylation rates. Although two hydroxylations (7- and 8-hydroxy metabolites) occurred exclusively in microsomes, these are believed to originate from a common precursor.

3. The high CL_int of ondansetron (150 ml/min/SRW, where SRW is a standard rat weight of 250 g) is well predicted by scaling either microsomal clearance for microsomal protein recovery or hepatocyte clearance for hepatocellularity (212 and 135 ml/min/SRW respectively). In contrast, the use of liver slice data scaled to a whole liver substantially underestimates CL_int (9 ml/min/SRW).     

Brain Penetration and In Vivo Recovery of NMDA Receptor Antagonists Amantadine and Memantine: A Quantitative Microdialysis Study

Purpose. To determine free brain concentrations of the clinically used uncompetitive NMDA antagonists memantine and amantadine using microdialysis corrected for in vivo recovery in relations to serum, CSF and brain tissue levels and their in vitro potency at NMDA receptors. Methods. Microdialysis corrected for in vivo recovery was used to determine brain ECF concentrations after steady-state administration of either memantine or amantadine. Additionally CSF, serum, and brain tissue were analyzed. Results. Following 7 days of infusion of memantine or amantadine (20 and 100 mg/kg/day respectively) whole brain concentrations were 44-and 16-fold higher than free concentrations in serum respectively. The free brain ECF concentration of memantine (0.83 ± 0.05 M) was comparable to free serum and CSF concentrations. In case of amantadine, it was lower. A higher in vivo than in vitro recovery was found for memantine. Conclusions. At clinically relevant doses memantine reaches a brain ECF concentration in range of its affinity for the NMDA receptor and close to its free serum concentration. This is not the case for amantadine and different mechanisms of action may be operational.