Calcineurin inhibitors, tacrolimus and cyclosporine, have been widely used to prevent the rejection or graft-versus-host disease after transplantations. Since these drugs have a narrow therapeutic range and show large inter- and intraindividual pharmacokinetic variability, frequent therapeutic drug monitoring is required to control their blood concentrations. Even with blood concentrations within the therapeutic range, some patients still experience acute rejection or infections. Tacrolimus and cyclosporine form a complex with their respective binding proteins, immunophilins, which in turn inhibit the phosphatase activity of calcineurin, a key enzyme in the activation of T lymphocytes. Pharmacodynamic assessment of calcineurin phosphatase activity in combination with the monitoring of blood concentrations has been studied. The inhibitory effects on calcineurin activity in peripheral blood mononuclear cells differed between tacrolimus and cyclosporine in transplant patients. The pharmacodynamics of both drugs shows great inter- as well as intraindividual variation, and acute rejection was associated with calcineurin activity. Calcineurin activity at trough time points was suggested as a single surrogate predictor for overall calcineurin activity throughout dosing periods. Monitoring of calcineurin phosphatase activity might be useful to determine the therapeutic range of tacrolimus and cyclosporine concentrations for an individual patient treated with a calcineurin inhibitor.
The pharmacokinetics of dihydroartemisinin (DHA) in a 5-day oral monotherapy regimen was investigated in ten adult Vietnamese patients with uncomplicated falciparum malaria. The patients were treated with a total dose of 900 mg DHA divided as single daily doses of 300, 300, 100, 100, and 100 mg from day 0 through day 4. There were no differences in the concentrations of DHA within the first two days of treatment. The pharmacokinetics of DHA in the acute phase, however, was significantly different from that in the convalescent phase of malaria. Reduced half-life (T1/2z) and lower area under concentration curve (AUC∞) values were observed on the final day of treatment in comparison to those obtained on the first day. These decreases in T1/2z and AUC∞ were observed in concordance with increased drug clearance (CL/F). Furthermore, the time required to reach maximum plasma DHA concentration (Tmax) on day 4 was shorter than that on day 0. Together, these findings suggest that the change in pharmacokinetics of DHA is related to the physiological change in malaria patients between the acute and convalescent phases of the disease.
The work described in this study aimed to express CYP2C8 wild-type and mutant proteins in bacterial expression system and to use the expressed proteins to investigate the structural and functional consequences of a reported allele CYP2C8*4 (carrying Ile264Met substitution) on protein activity. Ile264 was replaced by three different amino acids resulting in three mutant constructs, 2C8I264M, 2C8I264R and 2C8I264D. The presence of isoleucine at position 264 in CYP2C8 was found to be important for proper haem insertion and protein folding; whereas bulkier or charged residues were highly disruptive resulting in inactive proteins with minimum spectral and catalytic activities. This was evidenced from the low levels of Soret peak at 450 nm and negligible levels of tolbutamide methylhydroxylase activity. Kinetic study using paclitaxel indicated that all three mutants exhibited only 9.7 to 35.4% of the activity level observed in the wild-type. In addition, the mutants were more sensitive to proteinase K digestion, indicating a possible alteration of conformation. The combined effects of protein instability and compromised catalytic activity resulted in defective CYP2C8 protein which may have clinical implications in carriers of CYP2C8*4, particularly in terms of their capacity to clear potent drugs and their susceptibility to adverse drug reactions.
To study the metabolites of glycyrrhetic acid (GA) in rat bile, an analytical method was developed to identify GA and its metabolites by liquid chromatography mass spectrometry (LC-MS). Rat bile was collected after i.v. injection of GA. Three major GA-related peaks were detectable in rat bile by high-performance liquid chromatography (HPLC) analysis at 254 nm. LC-MS spectra showed their protonated molecular ions at m/z 727, 647, and 551. Furthermore, the three metabolites were also confirmed to exist in rat bile on LC-MS total ion chromatogram (TIC). Taken together with the susceptible nature to β-glucuronidase digestion and alkaline conditions, they were identified to be a novel sulfate-glucuronide diconjugate and the known monoglucuronide and sulfate conjugate, respectively.
We recently showed that arginine transport via cationic amino acid transporter 1 (CAT1), which transports arginine, lysine, ornithine, and histidine, is essential for erythropoiesis. In the present study, to confirm the importance of both arginine and CAT1 in erythropoiesis, we investigated the relationship of arginine uptake activity and differentiation and proliferation of blood cells by knockdown of the CAT1 gene using shRNA. Five short hairpin RNA (shRNA)-transfected K562 cell clones, in which the CAT1 mRNA expression level is decreased, and a vector-transfected clone were obtained. The differentiation to erythrocytes and proliferation of K562 cells were decreased by knockdown of CAT1. In addition, the initial uptake rate of [3H]arginine was decreased in the shRNA-transfected cell clones. The ratio of differentiation of CAT1-knockdown K562 cells was well correlated with the uptake activity for arginine by the cells (R2=0.59). These findings indicate that CAT1 is directly involved in erythropoiesis through supplying arginine to the blood cells.
Hepatocyte differentiation markers were expressed in the cells differentiated from mouse embryonic stem (ES) cells. In the differentiating ES cells, Cyp1a1 mRNA was highly expressed during the early to middle stage; Cyp2c29, Cyp2e1, Cyp3a11 and Cyp7a1 mRNAs were expressed only at the late stage; Cyp7b1 mRNA was expressed throughout all stages. Alpha-fetoprotein and albumin were co-expressed with Cyp3a and Cyp1a, respectively. Aryl hydrocarbon receptor, aryl hydrocarbon receptor nuclear translocator and glucocorticoid receptor mRNAs were detected in differentiating ES cells throughout the culture period. Pregnane X receptor mRNA was detected only in cells cultured for more than 24 days. The expression levels of Cyp2c29, Cyp3a11 and Cyp7a1 and G6p mRNAs were increased in embryoid bodies that were cultured with culture medium containing acid fibroblast growth factor, hepatocyte growth factor (HGF) and oncostatin M for 12 or 18 days, then the medium was replaced by that without HGF. These findings suggested that the expression levels of Cyp genes in hepatocytes differentiated from ES cells were markedly changed in individual enzymes during the course of differentiation, and that the duration of incubation with the addition of HGF affected the expression of Cyps and hepatocytes marker proteins.
This study aimed to clarify the differences in mRNA levels of cytochrome P450 (CYP) isoforms and nuclear receptors between Dark Agouti (DA) and Sprague-Dawley (SD) rats which are animal models for poor metabolizers and extensive metabolizers for CYP2D6, respectively. Using liver and small intestine tissues of both rat strains, we investigated the mRNA levels of CYP1A, 2A, 2B, 2C, 2D, 2E, and 3A subfamilies and nuclear receptors which regulate the transcription of CYP isoforms. In the liver, male DA rats showed a low CYP2D2 mRNA level but high mRNA levels of CYP3A1, 3A2, and 1A1 compared to SD rats. No significant difference was noted in other CYP isoforms. The mRNA levels of CAR were higher in DA rats than those in SD rats. In small intestine, the mRNA levels of CYP isoforms and nuclear receptors exhibited no significant strain differences. In addition, the activity of CYP3A in small intestinal microsome did not differ between SD and DA rats. Female DA rats exhibited higher mRNA levels of CYP3A1, 3A2, and 2B1 in the liver than female SD rats. In conclusion, the mRNA levels of CYP3A1 and 3A2 isoforms and CAR in the liver but not in the small intestines were different between DA and SD rats in both sexes.
This study was designed to clarify the pharmacological role of carnitine/organic cation transporter (Octn) family members in mouse heart. Immunohistochemical analysis revealed that Octn1 was exclusively expressed on endothelial cells in blood vessels. Octn2 was detected on the plasma membrane of cardiac muscle cells by immunoelectron microscopy. Octn3 was not detected in the heart. Integration plot analysis showed that coadministration of unlabeled L-carnitine reduced distribution of L-[3H]carnitine to the heart. L-[3H]Carnitine uptake in heart slices was reduced by carnitine analogs and various Octn2 substrates. L-[3H]Carnitine uptake by heart slices from juvenile visceral steatosis (jvs) mice, which have a hereditary octn2 gene deficiency, was negligible. Distribution of [3H]quinidine, another Octn2 substrate, to the heart was not reduced by L-carnitine, and [3H]quinidine uptake in heart slices was Na--independent and inhibited by cationic drugs, but not carnitine analogs. [3H]Quinidine uptake by heart slices from jvs mice was similar to that of wild-type mice. These results demonstrate that OCTN2 is functionally expressed on the plasma membrane of muscle cells and is involved in distribution of carnitine to the heart. Some mechanism(s) other than OCTN2 is involved in the distribution of quinidine to the heart.
The effect of several compounds, which are structurally analogous to glycerol, on carrier-mediated glycerol uptake was examined in HCT-15 cells to help clarifying the functional characteristics of the glycerol transport system. The carrier-mediated uptake of glycerol conformed to the Michaelis-Menten kinetics with a Michaelis constant of 21.1 μM and the tested compounds were all suggested to inhibit it competitively with the values of the inhibition constant (Ki) in the increasing order as follows: monobutyrin (41.0 μM)≤monoacetin (54.6 μM)<diglycerol (154 μM)<1,2-propanediol (1650 μM). Therefore, they all may possibly be substrates of the carrier-mediated glycerol transport system, for which the glycerol esters (monoacetin and monobutyrin) have the highest affinities among them. It was also found that S-(+)-enantiomer of 1,2-propanediol (Ki=484 μM) has a higher affinity than its R-(-)-enantiomer (Ki=19100 μM), indicating enantioselective recognition. These results support the suggestion that a specific carrier protein is involved in glycerol uptake in HCT-15 cells. It would be of interest to identify the carrier, which may be present also in some organs, and further investigate the possibility that glycerol ester derivatives of drugs might be delivered via the carrier.