Genetic variations in cytochrome P450 2D6 (CYP2D6) contribute to interindividual variability in the metabolism of clinically used drugs, e.g., tamoxifen. CYP2D6 is genetically polymorphic and is associated with large interindividual variations in therapeutic efficacy and drug toxicity. In this study, we performed an in vitro analysis of 50 allelic variants of CYP2D6 proteins. Wild-type CYP2D6.1 and 49 variants were transiently expressed in COS-7 cells, and the enzymatic activities of the CYP2D6 variants were characterized using N-desmethyltamoxifen as a substrate. The kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of N-desmethyltamoxifen 4-hydroxylation were determined. Among the 50 CYP2D6 variants, the kinetic parameters for N-desmethyltamoxifen 4-hydroxylation were determined for 20 CYP2D6 variants. On the other hand, the kinetic parameters of 30 CYP2D6 variants could not be determined because the amount of metabolite produced was at or below the detection limit at the lower substrate concentrations. Among them, 8 variants, i.e., CYP2D6.2, .9, .26, .28, .32, .43, .45, and .70, showed decreased intrinsic clearance at <50% of CYP2D6.1. The comprehensive in vitro assessment of CYP2D6 variants provides novel insights into allele-specific activity towards tamoxifen and may be valuable when interpreting in vivo studies.
Folic acid (FA) is a water-soluble vitamin, and orally ingested FA is absorbed from the small intestine by the proton-coupled folate transporter (PCFT). In the present study, we investigated whether epigallocatechin gallate (EGCG), one of the tea catechins, affects the transport of FA by PCFT. EGCG inhibited the uptake of FA into Caco-2 cells and human PCFT-expressing HEK293 cells (PCFT-HEK293 cells). The initial rate of uptake of FA into PCFT-HEK293 cells followed Michaelis–Menten kinetics (Km = 1.9 µM). Dixon plots revealed that PCFT-mediated FA uptake was competitively inhibited by EGCG (Ki ≒ 9 µM). The uptake of the PCFT substrate methotrexate (MTX) was competitively inhibited by EGCG as well (Ki ≒ 15 µM). In conclusion, it is suggested that when FA or MTX is ingested with tea, it is likely that the intestinal absorption of these compounds by PCFT is inhibited, which could result in insufficient efficacy.
We investigated the utility of three-dimensional (3D) spheroid cultures of human hepatocytes in discovering drug metabolites. Metabolites of acetaminophen, diclofenac, lamotrigine, midazolam, propranolol and salbutamol were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) to measure enzyme activities in this system cultured for 2 and 7 days. Sequential metabolic reactions by Phase I and then Phase II enzymes were found in diclofenac [CYP2C9 and UDP-glucuronyltransferases (UGTs)], midazolam (CYP3A4 and UGTs) and propranolol (CYP1A2/2D6 and UGTs). Moreover, lamotrigine and salbutamol were metabolized to lamotrigine-N-glucuronide and salbutamol 4-O-sulfate, respectively. These metabolites, which are human specific, could be observed in clinical studies, but not in conventional hepatic culture systems as in previous reports. Acetaminophen was metabolized to glucuronide and sulfate conjugates, and N-acetyl-p-benzo-quinoneimine (NAPQI) and its metabolites were not observed. In addition, mRNA of drug-metabolism enzymes [CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, UGT1A1, UGT2B7, sulfotransferase 1A1 (SULT1A1) and glutathione S-transferase pi 1 (GSTP1)], which were measured by qRT-PCR, were expressed in the human hepatocyte spheroids. In conclusion, these results suggest that human hepatocyte spheroids are useful in discovering drug metabolites.
Significant inter-individual variability of exposure for CYP2C19 substrates may be only partly due to genetic polymorphism. Therefore, the in vivo inter-individual variability in hepatic intrinsic clearance (CLint,h) of CYP2C19 substrates was estimated from reported AUC values using Monte Carlo simulations. The coefficient of variation (CV) for CLint,h in poor metabolizers (PM) expected from genotypes CYP2C19*2/*2, CYP2C19*3/*3 or CYP2C19*2/*3 was estimated as 25.8% from the CV for AUC of omeprazole in PMs. With this, CVs of CLint,h in extensive metabolizers (EM: CYP2C19*1/*1), intermediate metabolizers (IM: CYP2C19*1/*2 or *3) and ultra-rapid metabolizers (UM), CYP2C19*17/*17 and *1/*17, were estimated as 66.0%, 55.8%, 6.8% and 48.0%, respectively. To validate these CVs, variability in the AUC of CYP2C19 substrates lansoprazole and rabeprazole, partially metabolized by CYP3A4 in EMs and IMs, were simulated using the CV in CLint,h for CYP2C19 EMs and IMs and 33% of the CV previously reported for CYP3A4. Published values were within 2.5–97.5 percentile range of simulated CVs for the AUC. Furthermore, simulated CVs for the AUC of omeprazole and lansoprazole in ungenotyped populations were comparable with published values. Thus, estimated CLint,h variability can predict variability in the AUC of drugs metabolized not only by CYP2C19 but also by multiple enzymes.
Prostaglandin (PG) E2 is involved in neuroinflammation and neurotoxicity, and the cerebral PGE2 concentration is increased in neurodegenerative diseases. Because the intracerebral concentration of l-glutamate (L-Glu) is reported to be also elevated in neurodegenerative diseases, it has been proposed that L-Glu affects PGE2 dynamics in the brain, and thus exacerbates neural excitotoxicity. The purpose of this study was to investigate the effect of intracerebral L-Glu on PGE2 elimination across the blood-brain barrier (BBB) in rats by using the intracerebral microinjection technique. [3H]PGE2 injected into the cerebral cortex was eliminated from the brain in rats, and the apparent brain-to-blood [3H]PGE2 efflux clearance was found to be 60.1 µL/(min·g brain). Intracerebral pre-administration of 50 mM L-Glu significantly inhibited [3H]PGE2 elimination across the BBB and this L-Glu-induced inhibition was abolished by co-administration of an intracellular Ca2+ chelator. The intracellular Ca2+ concentration is reported to be increased via N-methyl-d-aspartate (NMDA)-type L-Glu receptors (NMDAR) and [3H]PGE2 elimination was attenuated by intracerebral pre-administration of a mixture of NMDA and d-serine. Moreover, the co-administration of antagonists of NMDAR with L-Glu abolished the attenuation of PGE2 elimination induced by intracerebral L-Glu administration. These results suggest that L-Glu attenuates BBB-mediated PGE2 elimination via NMDAR-mediated processes.
Brivaracetam is a high-affinity synaptic vesicle protein 2A ligand, in phase 3 clinical development for epilepsy. A phase 1, single-center, randomized, double-blind, placebo-controlled, single (2.5–100 mg) and multiple (2.5–50 mg twice daily) rising oral dose study (N01209) was conducted to assess the adverse event profile and pharmacokinetics of brivaracetam in healthy Japanese men, and the influence of the cytochrome P450 (CYP) 2C19 genotype. Plasma and urine were collected serially for analysis of brivaracetam and its three main metabolites: acid, hydroxy and hydroxy acid. Overall, 79/80 randomized participants completed the study. Brivaracetam was generally well tolerated. After single- and multiple-dose administration, brivaracetam was rapidly absorbed, with dose-proportional pharmacokinetics over the dose ranges tested. Steady state was reached after 2 days of repeated dosing. Brivaracetam clearance (averaged across the five single dose levels) was reduced from 0.99 mL/min/kg in homozygous extensive metabolizers (EM; n = 10) to 0.81 mL/min/kg (−18%) in heterozygous EM (n = 17) and 0.70 mL/min/kg (−29%) in poor metabolizers (PM; n = 9). Exposure and urinary excretion of hydroxy metabolite were reduced 10-fold in PM participants, compared with EM participants. Results suggest that brivaracetam is hydroxylated by CYP2C19, but this pathway is minor compared with hydrolysis to the acid metabolite.
The effect of carrageenan-induced acute peripheral inflammation (API) on the pharmacokinetics of the hepatically metabolizing compound midazolam (MDZ) was investigated in rats. Rats were subcutaneously treated with λ-carrageenan in the hind paw to induce API. When MDZ was intravenously administered in male rats, it was demonstrated that the plasma concentration profile of MDZ slightly alters in API rats compared with that in normal rats, while the plasma concentrations of its metabolites, 4-hydroxy and 1′-hydroxy MDZ, are markedly reduced with delayed appearances in API rats. In the incubation study with rat liver microsomes, it was clearly indicated that the generation rates of the two metabolites decrease in API rats. Western blot analysis revealed that hepatic CYP3A1 expression increases, while CYP3A2 expression decreases in API rats. In female rats, in which CYP3A2 is barely expressed in the liver, MDZ metabolism is little affected by API. These findings indicate that the hepatic handling of a therapeutic compound varies with API, largely due to altered hepatic expression of the drug-metabolizing enzyme.
This study aimed to establish culture conditions which are able to give the differentiation of induced pluripotent (iPS) cells to hepatocytes. To this end, we examined the usefulness of a culture medium containing the components involved in the intermediary metabolism in the liver. More specifically, we examined the effect of the “modified L-15 medium” containing galactose, phenylalanine and ornitine, but deprived of glucose, tyrosine, arginine and pyruvic acid. The medium was altered according to changes in the expression of enzymes that participate in liver-specific pathways. After 25 days of differentiation, the differentiated cells expressed hepatocyte markers and drug-metabolizing enzymes. These expression levels were increased using modified L-15 medium. The survival of human fetal liver cells and the death of human fibroblasts were observed during culture in modified L-15 medium. Most of the cells that differentiated from human iPS cells using modified L-15 medium were stained by anti-human albumin antibody. These results suggest that iPS cells can be converted to high purity-differentiated hepatocytes by cultivating them in modified L-15 medium.
The drug interaction between new quinolone antibiotics (NQs) and polyvalent metal cation products, leading to a significant decrease in the absorption of NQ, is considered to be attributable to the formation of poorly absorbable chelate and physicochemical adsorption of NQs to cation products. To clarify the mechanisms of this drug interaction in detail, we investigated the effects of Al3+ or Mg2+ on the membrane permeation profile of ciprofloxacin (CPFX) across human colon carcinoma cell lines (Caco-2) in monolayer culture, and characterized the adsorption nature of CPFX to polyvalent metal cation products under physiological conditions. As a result, Al3+ or Mg2+ partially but not fully impaired the permeation of CPFX across Caco-2 monolayer up to 30% or 60% of control, respectively. Physicochemical adsorption of CPFX to cation products was not observed under physiological pH. In conclusion, two possible mechanisms investigated, the decrease in the permeability of CPFX by chelate formation and adsorption of CPFX to polyvalent metal cation products, may partially but not fully explain the extent of the drug interaction clinically observed.
In central nervous system drug discovery, cerebrospinal fluid (CSF) drug concentration (CCSF) has been widely used as a surrogate for unbound brain concentrations (Cu,brain). However, previous rodent studies demonstrated that when drugs undergo active efflux by transporters, such as P-glycoprotein (P-gp), at the blood-brain barrier, the CCSF overestimates the corresponding Cu,brain. To investigate the utility of CCSF as a surrogate for interstitial fluid (ISF) concentration (CISF) in nonhuman primates, this study simultaneously determined the CCSF and CISF of 12 compounds, including P-gp substrates, under steady-state conditions in cynomolgus monkeys using intracerebral microdialysis coupled with cisternal CSF sampling. Unbound plasma concentrations of non- or weak P-gp substrates were within 2.2-fold of the CISF or CCSF, whereas typical P-gp substrates (risperidone, verapamil, desloratadine, and quinidine) showed ISF-to-plasma unbound (Kp,uu,ISF) and CSF-to-plasma unbound concentration ratios (Kp,uu,CSF) that were appreciably lower than unity. Although the Kp,uu,CSF of quinidine, verapamil, and desloratadine showed a trend of overestimating the Kp,uu,ISF, Kp,uu,CSF showed a good agreement with Kp,uu,ISF within 3-fold variations for all compounds examined. Cu,brain of some basic compounds, as determined using brain homogenates, overestimated the CISF and CCSF. Therefore, CCSF could be used as a surrogate for CISF in nonhuman primates.
Neonatal Fc receptor (FcRn), a heterodimer of MHC class I-like protein and β2-microglobulin, encoded by FCGRT and B2M, respectively, is important for recycling immunoglobulin G (IgG) antibodies by binding with the Fc region of IgG. Cynomolgus macaques are important animal species used in the evaluation of therapeutic antibodies, largely due to sequence similarities of target proteins to those of humans. Because the function of FcRn could be modified by mutations in FCGRT or B2M, 71 cynomolgus and 24 rhesus macaques were analyzed in the present study. A total of 21 variants were identified, of which 4 were non-synonymous in FCGRT. Fifteen variants were unique to cynomolgus macaques, of which 3, 2, and 5 were unique to cynomolgus macaques bred in China (MacfaCHN), Cambodia (MacfaCAM), and Indonesia (MacfaIDN), respectively. Five variants were shared by MacfaCHN and MacfaCAM, but not by MacfaIDN. In B2M, only 5 variants were found, including 2 non-synonymous variants. Tissue expression analysis showed that cynomolgus FCGRT and B2M were widely expressed in the 10 tissue types analyzed. None of the non-synonymous variants of FCGRT or B2M found changes in the amino acid residues known to be important for FcRn function, suggesting that substantial inter-animal variability of FcRn is not expected for the cynomolgus macaques analyzed.