Drug Metabolism Letters - Volume 9, Issue 1, 2015

Recent guidance/guidelines for industry recommend that cytochrome P450 induction can be assessed using human hepatocyte enzyme activity and/or mRNA levels to evaluate potential drug– drug interactions. To evaluate time-dependent cytochrome P450 induction precisely, induction of CYP1A2, CYP2B6, and CYP3A4 mRNA was confirmed (> 2-fold) by the treatment with omeprazole, phenobarbital, and rifampicin, respectively, for 24 or 48 h on day 3 from the start of culture. After 24 h, the fold induction of CYP1A2 with 3.6 and 1.8 x 104 HepaRG cells per well was lower than that for 7.2 x 104 cells. CYP1A2 induction levels at 24 h were higher than those after 48 h. In contrast, higher CYP2B6 inductions were confirmed after 48 h exposure than after 24 h, independent of the number of cells per well. To help reduce the use of human cryopreserved hepatocytes, typical P450-dependent enzyme activities were investigated in human HepaRG cells cultured in commercial hanging-drop plates. Newly designed 96-well hanging-drop plates were capable of maintaining human CYP3A-dependent midazolam hydroxylation activities for up to 4 days using only 10% of the recommended initial 7.2 x 104 cells per well. Favorable HepaRG function using hanging-drop plates was confirmed by detecting 1′- hydroxymidazolam O-glucuronide on day 3, suggesting an improvement over traditional control plates in which this metabolite can be detected for 24-well plates. These results suggest that the catalytic function and/or induction of CYP1A2, CYP2B6, and CYP3A4 can be readily assessed with reduced numbers of starting HepaRG cells cultured in three-dimensional cultures in drops prepared with hanging-drop plates.

This work presents an in vitro investigation of the effect of protein restriction on the metabolism of albendazole (ABZ). This study was conducted using liver microsomal fractions obtained from Wistar rats. For the quantitative analysis, a multidimensional High Performance Liquid Chromatography (2D HPLC) method was fully validated for the determination of the ABZ metabolites: albendazole sulfoxide, albendazole sulfone and albendazole 2-aminesulfone. The target compounds were directly extracted using a C8-RAM-BSA column (5.0 x 0.46 cm i.d.) and analyzed on a chromatographic chiral column containing amylose tris(3,5-dimethylphenylcarbamate) (150 x 4.6 mm i.d.). The in vitro biotransformation results showed that the protein restriction influenced the oxidative metabolism of ABZ. The production of R-(+)-ABZ-SO (1309 nmol/L) and S-(-)-ABZ-SO (1456 nmol/L) was higher in the control animals than in the animals fed with a diet containing 6% protein, which produced 778.7 nmol/L and 709.5 nmol/L for R-(+) and S-(-)-ABZ-SO enantiomers, respectively. These results were statistically inspected by Student´s t test and the results showed a significant difference between the two means (p < 0.05). Moreover, the production of ABZ-SO enantiomers was enantioselective where the S-(-)-ABZ-SO was formed in greater amounts than the R-(+)-ABZ-SO in control animals (p = 0.0231). However, the enantioselectivity was not observed when the in vitro biotransformation of ABZ was conducted using the microsomal fractions obtained from protein restriction animals (p > 0.05). Furthermore, animal nutritional condition could affect the pattern of ABZ sulphoxidation indicating that the protein nutrition affect primarily the formation of R-(+)-ABZSO and S-(-)-ABZ-SO enantiomers.

Sandwich culture of hepatocytes is commonly applied for the prediction of in vivo biliary clearance (CLbil). In this paper, we present a modified procedure for the determination of in vitro CLbil in sandwich culture of rat hepatocytes, which allows the estimation of the impact of uptake processes on the overall CLbil. The main point of this modification is the separation of uptake and efflux processes. Ten drugs from four biopharmaceutics drug disposition classification system classes were chosen in order to demonstrate the advantages of this method: 1) the uptake is performed identically before the canaliculi are opened, thus the efflux starts at the same intracellular concentration of the drugs and the effect of Ca2+/Mg2+ depletion on the uptake is excluded; 2) exact intracellular concentrations can be measured at the start and at the end of the efflux; 3) the biliary clearance can be determined irrespective of the uptake; 4) the canalicular and the sinusoidal transport can be measured simultaneously; 5) drug-drug interactions concerning uptake and efflux transporters can be estimated independently. Depending on the degree of uptake, CLbil,app (calculated using the concentration of drugs in the medium) was significantly higher (sulfasalazine, fluvastatin, rosuvastatin, atorvastatin) or lower (pravastatin, procainamide) than CLbil,int (calculated using the intracellular concentration of drugs). When the uptake had no impact on the CLbil, the apparent and intrinsic CLbil did not differ significantly (lovastatin, rifampicin, quetiapine). Our results confirm that transporters may play a significant role in the uptake of drugs both with high and poor permeability and solubility.

The ADME of Pacritinib (SB1518), an orally active JAK 2 inhibitor, was investigated in vitro and in vivo in preclinical species and humans. Pacritinib showed ~ 5 fold higher affinity to human plasma proteins relative to mouse in vitro. It was metabolized by human CYP3A4 in vitro, and did not significantly induce CYP3A and 1A2 in human hepatocytes. In vitro metabolism studies with mouse and human liver microsomes showed the presence of four major metabolites of Pacritinib -M1 (oxidation), M2 (dealkylation), M3 (oxidation), M4 (reduction). The in vitro and in vivo metabolic patterns observed in mice and humans were in good agreement. Qualitatively and quantitatively, none of the metabolites formed in vivo was > 10% of Pacritinib in mouse, dog and humans. Pacritinib showed systemic clearance of 8.0, 1.6, 1.6 l/h/kg, volume of distribution of 14.2, 7.9, 8.5 l/kg, t1/2 of 5.6, 6.0, 4.6 h, and oral bioavailability of 39, 10, and 24% in mouse, rat and dog, respectively. In radiolabeled mass balance and QWBA studies in mice, ~ 91% of the dose was recovered in feces, suggesting biliary clearance, and maximum radioactivity was seen in the gastrointestinal tract followed by the kidney, heart and low activity in the brain. The relatively high exposures of Pacritinib in humans might be attributed to its very high plasma protein binding, low metabolic and/or biliary clearance.

Kalanchoe crenata popularly known as “dog’s liver” is used in most African countries for the treatment of chronic diseases such as diabetes, asthma and HIV/AIDS related infections. The evaluation of K. crenata for herb-drug interactions has not been reported. This study therefore aims to evaluate the risk of K. crenata for herb-drug interaction in vitro. Crude methanol and fractions of K. crenata were incubated and preincubated with recombinant human CYP2C19 and CYP3A4. Comparative studies were conducted in both human liver microsomes and recombinant human CYP to ascertain the inhibition profile of the crude extract and the various fractions. The cocktail approach of recombinant human CYPs was conducted to confirm the inhibition potential of the fractions in the presence of other CYPs. The results showed significant time-dependent inhibition of tested samples on CYP3A4 with crude methanol (39KC), fractions 45A, 45B and 45D given IC50 fold decrease of 3.29, 2.26, 1.91 and 1.49, respective. Time dependent kinetic assessment of 39KC and 45D showed KI and kinact values for 39KC as 1.77μg/mL and 0.091 min-1 while that of 45D were 6.45 μg/mL and 0.024 min-1, respectively. Determination of kinact based on IC50 calculations yielded 0.015 and 0.04 min-1 for 39KC and 45D, respectively. Cocktail approach exhibited fold decreases in IC50 for all test fractions on CYP3A4 within the ranges of 2.10 – 4.10. At least one phytoconstituent in the crude methanol extract of Kalanchoe crenata is a reversible and time-dependent inhibitor of CYP3A4.