Strategies to improve the prediction accuracy of hepatic intrinsic clearance of three antidiabetic drugs: Application of the extended clearance concept and consideration of the effect of albumin on CYP2C metabolism and OATP1B-mediated hepatic uptake.

The antidiabetic drugs glibenclamide, repaglinide, and nateglinide are well-known substrates for hepatic uptake transporters of the organic anion transporting polypeptide (OATP) family and metabolizing enzymes of the cytochrome P450 (CYP) 2C subfamily. The systemic exposure of these drugs varies substantially among individuals, impacted by genetic polymorphisms of transporters and metabolizing enzymes as well as drug-drug interactions. The use of the conventional in vitro-in vivo extrapolation (IVIVE) method was found to underestimate their hepatic intrinsic clearance (CLint,all); the clinically observed CLint,all values were ≥10-fold higher than the predicted values from in vitro data. In order to improve the accuracy in predicting CLint,all of these drugs, the following modifications were implemented; i) the extended clearance concept was applied during IVIVE processes, ii) albumin was added to metabolic assays using human liver microsomes (to minimize the impact of intrinsic inhibitors on kinetic parameters for CYP2C-mediated metabolism) and to hepatic uptake assays (to accommodate the enhanced hepatic uptake observed with albumin-bound drugs), and iii) differing rates of efflux and influx via diffusion were used. The IVIVE method with these modifications yielded the predicted CLint,all values from in vitro data in closer agreement with the CLint,all values observed in vivo; the fold differences between the predicted and observed CLint,all values reduced from 13-15 to 5.9-6.7. Our current approach offers an improvement in the prediction of CLint,all and further investigations are warranted to enhance the prediction accuracy of IVIVE.

Simulation of human plasma concentration-time profiles of the partial glucokinase activator PF-04937319 and its disproportionate N-demethylated metabolite using humanized chimeric mice and semi-physiological pharmacokinetic modeling.

1. The partial glucokinase activator N,N-dimethyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (PF-04937319) is biotransformed in humans to N-methyl-5-((2-methyl-6-((5-methylpyrazin-2-yl)carbamoyl)benzofuran-4-yl)oxy)pyrimidine-2-carboxamide (M1), accounting for ∼65% of total exposure at steady state. 2. As the disproportionately abundant nature of M1 could not be reliably predicted from in vitro metabolism studies, we evaluated a chimeric mouse model with humanized liver on TK-NOG background for its ability to retrospectively predict human disposition of PF-04937319. Since livers of chimeric mice were enlarged by hyperplasia and contained remnant mouse hepatocytes, hepatic intrinsic clearances normalized for liver weight, metabolite formation and liver to plasma concentration ratios were plotted against the replacement index by human hepatocytes and extrapolated to those in the virtual chimeric mouse with 100% humanized liver. 3. Semi-physiological pharmacokinetic analyses using the above parameters revealed that simulated concentration curves of PF-04937319 and M1 were approximately superimposed with the observed clinical data in humans. 4. Finally, qualitative profiling of circulating metabolites in humanized chimeric mice dosed with PF-04937319 or M1 also revealed the presence of a carbinolamide metabolite, identified in the clinical study as a human-specific metabolite. The case study demonstrates that humanized chimeric mice may be potentially useful in preclinical discovery towards studying disproportionate or human-specific metabolism of drug candidates.

Formation of the accumulative human metabolite and human-specific glutathione conjugate of diclofenac in TK-NOG chimeric mice with humanized livers.

3'-Hydroxy-4'-methoxydiclofenac (VI) is a human-specific metabolite known to accumulate in the plasma of patients after repeated administration of diclofenac sodium. Diclofenac also produces glutathione-conjugated metabolites, some of which are human-specific. In the present study, we investigated whether these metabolites could be generated in humanized chimeric mice produced from TK-NOG mice. After a single oral administration of diclofenac to humanized mice, the unchanged drug in plasma peaked at 0.25 hour and then declined with a half-life (t1/2) of 2.4 hours. 4'-Hydroxydiclofenac (II) and 3'-hydroxydiclofenac also peaked at 0.25 hour and were undetectable within 24 hours. However, VI peaked at 8 hours and declined with a t1/2 of 13 hours. When diclofenac was given once per day, peak and trough levels of VI reached plateau within 3 days. Studies with administration of II suggested VI was generated via II as an intermediate. Among six reported glutathione-conjugated metabolites of diclofenac, M1 (5-hydroxy-4-(glutathion-S-yl)diclofenac) to M6 (2'-(glutathion-S-yl)monoclofenac), we found three dichlorinated conjugates [M1, M2 (4'-hydroxy-3'-(glutathion-S-yl)diclofenac), and M3 (5-hydroxy-6-(glutathion-S-yl)diclofenac)], and a single monochlorinated conjugate [M4 (2'-hydroxy-3'-(glutathion-S-yl)monoclofenac) or M5 (4'-hydroxy-2'-(glutathion-S-yl)monoclofenac)], in the bile of humanized chimeric mice. M4 and M5 are positional isomers and have been previously reported as human-specific in vitro metabolites likely generated via arene oxide and quinone imine-type intermediates, respectively. The biliary monochlorinated metabolite exhibited the same mass spectrum as those of M4 and M5, and we discuss whether this conjugate corresponded to M4 or M5. Overall, humanized TK-NOG chimeric mice were considered to be a functional tool for the study of drug metabolism of diclofenac in humans.

Effect of intestinal first-pass hydrolysis on the oral bioavailability of an ester prodrug of fexofenadine.

The contribution of intestinal first-pass hydrolysis to oral bioavailability was evaluated in rats using a model prodrug of fexofenadine (FXD), which has poor oral bioavailability. The prodrug, ethyl-FXD, has high membrane permeability but the oral bioavailability of FXD derived from ethyl-FXD was only 6.2%. Ethyl-FXD was not detected in the plasma, whereas FXD was detected, indicating complete first-pass hydrolysis. In in vitro experiments, hydrolase activity for ethyl-FXD was higher in the liver and blood than that in the intestine. However, the high blood protein binding of ethyl-FXD resulted in a high hepatic availability (F(h) = 88%). The complete bioconversion of ethyl-FXD in the in vivo oral administration is difficult to explain by first-pass hydrolysis in the liver and blood. Interestingly, in an in situ rat jejunal single-pass perfusion experiment, 84% of the ethyl-FXD taken up into enterocytes was hydrolyzed. Furthermore, only one-fifth of the FXD formed in mucosa reached the mesenteric vein because of its P-glycoprotein-mediated efflux into the intestinal lumen. These findings indicate that the intestinal bioconversion of ester prodrugs to their parent drugs is a key factor in determining their oral bioavailability.

Comprehensive quantitative and qualitative liquid chromatography-radioisotope-mass spectrometry analysis for safety testing of tolbutamide metabolites without standard samples.

Liquid chromatography-radioisotope-mass spectrometry (LC-RI-MS) analysis was used to determine the structures of 12 (four previously unknown) (14) C-tolbutamide (TB) metabolites in rat biological samples (plasma, urine, bile, feces, and microsomes). The four novel metabolites are ω-carboxy TB, hydroxyl TB (HTB)-O-glucuronide, TB-ortho or meta-glutathion, and tolylsulphoaminocarbo-glutathion. In rat plasma, after oral administration of (14) C-TB at therapeutic dose (1 mg/kg) and microdose (1.67 µg/kg), the total RI and six metabolites [HTB, carboxy TB (CTB), M1: desbutyl TB, M2: ω-hydroxyl TB, M3: α-hydroxyl TB, and M4: ω-1-hydroxyl TB] were quantified by LC-RI-MS. The plasma concentrations were calculated using their response factors (MS-RI intensity ratio) without standard samples, and the area under the curve (AUC) of plasma concentration per time for evaluation of Safety Testing of Drug Metabolites (MIST) was calculated using the ratio of TB metabolites AUC/total RI AUC. The ratios were as follows: TB 94.5% and HTB 2.5% for the microdose (1.67 µg/kg) and TB 95.6%, HTB 0.96%, CTB 0.065%, M1 0.62%, M2 0.0035%, M3 0.077%, and M4 0.015% for the therapeutic dose (1 mg/kg). These values were less than 10% of the MIST criteria.

In vitro P-glycoprotein interactions and steady-state pharmacokinetic interactions between tolvaptan and digoxin in healthy subjects.

Interactions between tolvaptan and digoxin were determined in an open-label, sequential study where 14 healthy subjects received tolvaptan 60 mg once daily (QD) on days 1 and 12 to 16 and digoxin 0.25 mg QD on days 5 to 16. Mean maximal concentrations (C(max)) and area under the curve during the dosing interval (AUC(τ)) for digoxin with tolvaptan (day 16) were increased 1.27- and 1.18-fold compared with digoxin alone (day 11); mean renal clearance of digoxin was decreased by 59% (P < .05). Tolvaptan C(max) and AUC(0-24h) for a single dose with digoxin (day 12) were each increased about 10% compared with tolvaptan alone (day 1). Tolvaptan did not accumulate upon multiple dosing. After a single dose of tolvaptan (day 1, day 12), 24-hour urine volume was about 7.5 L. As expected, after 5 days of tolvaptan, 24-hour urine volume decreased about 20%. In vitro studies in control and MDR1-expressing LLC-PK1 cells indicate that tolvaptan is a substrate of P-glycoprotein. Tolvaptan (50 µM) inhibited basolateral to apical digoxin secretion to the same extent as 30 µM verapamil; the IC50 of tolvaptan was determined to be 15.9 µM. The increase in steady-state digoxin concentrations is likely mediated by tolvaptan inhibition of digoxin secretion.

In vitro evaluation of cytochrome P450 and glucuronidation activities in hepatocytes isolated from liver-humanized mice.

Cryopreserved human (h-) hepatocytes are currently regarded as the best in vitro model for predicting human intrinsic clearance of xenobiotics. Although fresh h-hepatocytes have greater plating efficiency on dishes and greater metabolic activities than cryopreserved cells, performing reproducible studies using fresh hepatocytes from the same donor and having an "on demand" supply of fresh hepatocytes are not possible. In this study, cryopreserved h-hepatocytes were transplanted into albumin enhancer/promoter-driven, urokinase-type plasminogen activator, transgenic/severe combined immunodeficient (uPA/SCID) mice to produce chimeric mice, the livers of which were largely replaced with h-hepatocytes. We determined whether the chimeric mouse could serve as a novel source of fresh h-hepatocytes for in vitro studies. h-Hepatocytes were isolated from chimeric mice (chimeric hepatocytes), and cytochrome P450 (P450) activities were determined. Compared with cryopreserved cells, the P450 (1A2, 2C9, 2C19, 2D6, 2E1, 3A) activities of fresh chimeric hepatocytes were similar or greater. Moreover, ketoprofen was more actively metabolized through glucuronide conjugates by fresh chimeric hepatocytes than by cryopreserved cells. We conclude that chimeric mice may be a useful tool for supplying fresh h-hepatocytes on demand that provide high and stable phase I enzyme and glucuronidation activities.

Possible protective role of pregnenolone-16 alpha-carbonitrile in lithocholic acid-induced hepatotoxicity through enhanced hepatic lipogenesis.

Lithocholic acid (LCA) feeding causes both liver parenchymal and cholestatic damages in experimental animals. Although pregnenolone-16 alpha-carbonitrile (PCN)-mediated protection against LCA-induced hepatocyte injury may be explained by induction of drug metabolizing enzymes, the protection from the delayed cholestasis remains incompletely understood. Thus, the PCN-mediated protective mechanism has been studied from the point of modification of lipid metabolism. At an early stage of LCA feeding, an imbalance of biliary bile acid and phospholipid excretion was observed. Co-treatment with PCN reversed the increase in serum alanine aminotransferase (ALT) as well as alkaline phosphatase (ALP) activities and hepatic hydrophobic bile acid levels. LCA feeding decreased hepatic mRNA levels of several fatty acid- and phospholipid-related genes before elevation of serum ALT and ALP activities. On the other hand, PCN co-treatment reversed the decrease in the mRNA levels and hepatic levels of phospholipids, triglycerides and free fatty acids. PCN co-treatment also reversed the decrease in biliary phospholipid output in LCA-fed mice. Treatment with PCN alone increased hepatic phospholipid, triglyceride and free fatty acid concentrations. Hepatic fatty acid and phosphatidylcholine synthetic activities increased in mice treated with PCN alone or PCN and LCA, compared to control mice, whereas these activities decreased in LCA-fed mice. These results suggest the possibility that PCN-mediated stimulation of lipogenesis contributes to the protection from lithocholic acid-induced hepatotoxicity.

Development of a novel system for estimating human intestinal absorption using Caco-2 cells in the absence of esterase activity.

Both mRNA and protein levels of the carboxylesterase (CES) isozymes, hCE1 and hCE2, in Caco-2 cells increase in a time-dependent manner, but hCE1 levels are always higher than those of hCE2. In human small intestine, however, the picture is reversed, with hCE2 being the predominant isozyme. Drugs hydrolyzed by hCE1 but not by hCE2 can be hydrolyzed in Caco-2 cells, but they are barely hydrolyzed in human small intestine. The results in Caco-2 cells can be misleading as a predictor of what will happen in human small intestine. In the present study, we proposed a novel method for predicting the absorption of prodrugs in the absence of CES-mediated hydrolysis in Caco-2 cells. The specific inhibition against CES was achieved using bis-p-nitrophenyl phosphate (BNPP). The optimal concentration of BNPP was determined at 200 microM by measuring the transport and hydrolysis of O-butyryl-propranolol (butyryl-PL) as a probe. BNPP concentrations of more than 200 microM inhibited 86% of hydrolysis of butyryl-PL, resulting in an increase in its apparent permeability. Treatment with 200 microM BNPP did not affect paracellular transport, passive diffusion, or carrier-mediated transport. Furthermore, the proposed evaluation system was tested for ethyl fexofenadine (ethyl-FXD), which is a superior substrate for hCE1 but a poor one for hCE2. CES-mediated hydrolysis of ethyl-FXD was 94% inhibited by 200 microM BNPP, and ethyl-FXD was passively transported as an intact prodrug. From the above observations, the novel evaluation system is effective for the prediction of human intestinal absorption of ester-type prodrugs.

Absorption, distribution, and excretion of 14C-labeled tacrolimus (FK506) after a single or repeated ocular instillation in rabbits.

PURPOSE: The aim of this study was to investigate the absorption, distribution, and excretion of radioactivity in male rabbits after a single or repeated instillation of (14)C-labeled tacrolimus (FK506) ophthalmic suspension or an intravenous (i.v.) administration of (14)C-FK506. METHODS: The 0.3% (14)C-FK506 suspension was administered in single and repeated (three times, 5-min intervals) instillation studies, and 1 mg/kg of (14)C-FK506 was administered in the i.v. dose study. RESULTS: Results for single and repeated instillation studies were similar. In eyeball microautoradiograms, 15 min after dosing, the level of radioactivity in the cornea was the highest, followed by conjunctiva. After 1 h, little specific distribution was detected in the corneal epithelium, stroma, or Descemet's membrane. At 24 h, the level of radioactivity in the cornea decreased. Whole-body autoradiograms showed that the radioactivity was distributed to the digestive tract through the nasal meatus and esophagus and then was excreted into the feces. In the i.v. dose study, the distribution of radioactivity in whole-body autoradiographs was similar to that in quantitative tissue distribution measurements. The excretion of radioactivity in the urine and feces up to 168 h were 4.5 and 94.9%, respectively. CONCLUSIONS: After the ocular instillation, FK506 is first absorbed in the cornea, conjunctiva, and nasolacrimal duct, and then the rest is distributed to digestive tract through the nasal meatus and esophagus, after which it is excreted mainly into the feces.

Cardiac sympathetic rejuvenation: a link between nerve function and cardiac hypertrophy.

Neuronal function and innervation density is regulated by target organ-derived neurotrophic factors. Although cardiac hypertrophy drastically alternates the expression of various growth factors such as endothelin-1, angiotensin II, and leukemia inhibitory factor, little is known about nerve growth factor expression and its effect on the cardiac sympathetic nerves. This study investigated the impact of pressure overload-induced cardiac hypertrophy on the innervation density and cellular function of cardiac sympathetic nerves, including kinetics of norepinephrine synthesis and reuptake, and neuronal gene expression. Right ventricular hypertrophy was induced by monocrotaline treatment in Wistar rats. Newly developed cardiac sympathetic nerves expressing beta(3)-tubulin (axonal marker), GAP43 (growth-associated cone marker), and tyrosine hydroxylase were markedly increased only in the right ventricle, in parallel with nerve growth factor upregulation. However, norepinephrine and dopamine content was paradoxically attenuated, and the protein and kinase activity of tyrosine hydroxylase were markedly downregulated in the right ventricle. The reuptake of [(125)I]-metaiodobenzylguanidine and [(3)H]-norepinephrine were also significantly diminished in the right ventricle, indicating functional downregulation in cardiac sympathetic nerves. Interestingly, we found cardiac sympathetic nerves in hypertrophic right ventricles strongly expressed highly polysialylated neural cell adhesion molecule (PSA-NCAM) (an immature neuron marker) as well as neonatal heart. Taken together, pressure overload induced anatomical sympathetic hyperinnervation but simultaneously caused deterioration of neuronal cellular function. This phenomenon was explained by the rejuvenation of cardiac sympathetic nerves as well as the hypertrophic cardiomyocytes, which also showed the fetal form gene expression.

Cytochrome p450 expression of cultured rat small hepatocytes after long-term cryopreservation.

Small hepatocytes (SHs) are hepatic progenitor cells that can be cryopreserved for a long time. After thawing, the cells can proliferate and, when treated with Matrigel, they can differentiate into mature hepatocytes (MHs). In this study, we investigated whether cryopreserved SHs could express cytochromes P450 (P450s), whether P450 expression was induced by appropriate inducers, and whether P450 activities were measurable. 3-Methylcholanthrene (3-MC), phenobarbital (PB), pregnenolone-16alpha-carbonitrile (PCN), and ethanol were used as inducers for CYP1A, 2B, 3A, and 2E, respectively. Immunoblot analysis indicated that cryopreserved SHs constitutively expressed CYP1A1/2, CYP2E1, and CYP3A2 as much as 26 days after plating. Significant expression of CYP1A1/2 and 3A2 in the cells treated with Matrigel was induced by 3-MC and PCN, respectively. Although Matrigel did not up-regulate the enzymatic activity of CYP1A, CYP3A and CYP2E activities increased. Induction of CYP1A and CYP3A activities by each inducer was observed in cryopreserved cells treated with Matrigel. Although the expression of CYP2B1 could be detected in subcultured SHs treated with PB, it was not detected in cryopreserved SHs. The activity of NADPH-cytochrome P450 reductase was measured in both subcultured and cryopreserved SHs, although the activities in both were approximately 30% of that of MHs. Profiles of (14)C-testosterone metabolites were examined in cultured MHs and in cryopreserved SHs by high-performance liquid chromatography. Similar peaks for testosterone metabolites in MHs and SHs were observed in the same elution time. These results indicate that, although induction of CYP3A and 2B in cryopreserved SHs is inferior to that in subcultured ones, SHs can maintain the expression and activities of P450s after long-term cryopreservation.

Thiol-based SAHA analogues as potent histone deacetylase inhibitors.

In order to find novel nonhydroxamate histone deacetylase (HDAC) inhibitors, a series of thiol-based compounds modeled after suberoylanilide hydroxamic acid (SAHA) was synthesized, and their inhibitory effect on HDACs was evaluated. Compound 6, in which the hydroxamic acid of SAHA was replaced by a thiol, was found to be as potent as SAHA, and optimization of this series led to the identification of HDAC inhibitors more potent than SAHA.

Novel histone deacetylase inhibitors: design, synthesis, enzyme inhibition, and binding mode study of SAHA-based non-hydroxamates.

In order to find novel non-hydroxamate histone deacetylase (HDAC) inhibitors, a series of compounds modeled after suberoylanilide hydroxamic acid (SAHA) were designed and synthesized as (i). substrate (acetyl lysine) analogues (compounds 3-7), (ii). analogues bearing various functional groups expected to chelate zinc ion (compounds 8-15), and (iii). analogues bearing nucleophilic functional groups which could bind covalently to HDACs (compounds 16-18). In this series, semicarbazide 8b and bromoacetamides 18b,c were found to be potent HDAC inhibitors for non-hydroxamates.

Use of ADME studies to confirm the safety of epsilon-polylysine as a preservative in food.

Epsilon-polylysine is a homopolymer of L-lysine, containing approximately 30 L-lysine subunits, as synthesized in aerobic bacterial fermentation by Streptomyces albulus. epsilon -Polylysine is approved for food use in Japan as an antimicrobial preservative. A series of pharmacokinetic and metabolic profile studies on epsilon -polylysine have been conducted in rats in order to provide a better understanding of the reason for its lack of toxicological effects in subchronic and chronic feeding bioassays using relatively high concentrations in the diet up to 50000 ppm. As reported in this article, epsilon -polylysine was practically non-toxic in an acute oral toxicity study in rats, with no mortality up to 5 g/kg and was not mutagenic in bacterial reversion assays. Absorption, distribution, metabolism and excretion (ADME) studies on 14C-radiolabeled epsilon -polylysine, given in a single dose to fasted male rats at 100mg/kg, revealed low absorption from the gastrointestinal tract. All but trace amounts of the dosed radioactivity was eliminated by excretion within 168 h and over 97% was accounted for in urine (1.2%), feces (92.9%), or expired air (3%) by 48 h. The sum of the cumulative excretion with routes associated with absorption in urine, expired air and carcass was 6.4% of total recovered radioactivity; approximately 94% of the dose of epsilon -polylysine passed unabsorbed through the gastrointestinal tract in the feces. Whole body autoradiography did not show concentration of absorbed epsilon -polylysine in any tissue or organ. Excretion half-lives of epsilon -polylysine equivalents in blood and plasma were 20 and 3.9 days, likely prolonged by the incorporation into protein of cleaved L-lysine. Metabolic profiles by HPLC analysis of plasma samples suggest that L-lysine is the predominant early metabolic by-product, likely from protease activity in the upper GI tract; only 0.2% of the administered parent compound was found in plasma. At 8-72 h, HPLC profiles show diminishing levels of epsilon -polylysine and L-lysine in plasma, accompanied by a shift to larger peaks of homopolymer fragments of varying subunit length, presumably from microbial degradation of epsilon -polylysine in the lower gut. HPLC profiles of urine and feces collected from 0 to 24 h post-dosing revealed three distinct peaks in urine, the first peak likely to be epsilon -polylysine and epsilon -polylysine less a few amino acid subunits, and the second, L-lysine and the third, a metabolite of L-lysine. Radiolabeled L-lysine was reduced from 67.2% of the radioactivity in plasma at 30 min to 7.5% at 4 h, indicating that L-lysine is readily removed from plasma from essential amino acid incorporation into protein. Based on the findings of the ADME studies and lack of toxicity in safety studies, the proposed use of epsilon -polylysine as a preservative in foods is considered to be safe.

New formulation of chemical peeling agent: 30% salicylic acid in polyethylene glycol. Absorption and distribution of 14C-salicylic acid in polyethylene glycol applied topically to skin of hairless mice.

Salicylic acid is used in chemical peeling procedures. However, they have caused many side effects, even salicylism. To achieve a salicylic acid peeling that would be safer for topical use, we recently developed a new formulation consisting of 30% salicylic acid in polyethylene glycol (PEG) vehicle. In an extension of our previous research, we studied the absorption of 30% salicylic acid labeled with 14C in PEG vehicle applied topically to the intact and damaged skin of male hairless mice. An ointment containing 3 mg salicylic acid in 10 mg vehicle was applied to both groups. In animals with intact skin, 1 h after application the plasma concentration of radioactivity was 1665.1 ng eq/ml, significantly lower than the 21437.6 ng eq/ml observed in mice with damaged skin. Microautoradiograms of intact skin showed that the level of radioactivity in the cornified cell layer was similar at 6 h after application. However, in damaged skin, the overall level of radioactivity showed a decrease by 3 h after application. In the carcasses remaining after the treated intact and damaged skin had been removed, 0.09 and 11.38% of the applied radioactivity remained, respectively. These findings confirm that 30% salicylic acid in PEG vehicle is little absorbed through the intact skin of hairless mice, and suggest that salicylism related to absorption through the skin of quantities of topically applied salicylic acid is not likely to occur in humans with intact skin during chemical peeling with this preparation. This new preparation of 30% salicylic acid in PEG vehicle is believed to be safe for application as a chemical peeling agent.