During the last decade, cDNA cloning has identified various gene families of drug transporters, and pharmacokinetic studies of drugs based on the molecular characteristics of transporters have advanced. We cloned and characterized two organic anion transporters OAT-K1 and OAT-K2 from the rat kidney. The expression of both transporters was limited to the kidney, especially the brush-border membranes of proximal tubules, with an apparent molecular mass of 40 kDa. Using MDCK or LLC-PK1 cells stably expressing OAT-K1, posttranslational cleavage was suggested to affect the membrane localization and functional characteristics; 50 kDa with multispecificity in the apical membrane of MDCK cells and 70 kDa with methotrexate specific transport in the basolateral membrane of LLC-PK1 cells. A wide variety of anionic compounds including methotrexate are bidirectionally transported via OAT-K1 and OAT-K2 across the apical membrane in the MDCK-transfectants. The urinary secretion of methotrexate was depressed in 5/6 nephrectomized rats in association with the selective loss of OAT-K1 and OAT-K2 expression, and both transporters were suggested to be target molecules for methotrexate-folinic acid rescue. In this review, recent advances in the study of OAT-K1 and OAT-K2 were summarized in comparison with other transporters.

View full abstract

To clarify whether CYP2C19 is involved in the overall metabolism of clarithromycin (CAM) or not, in vitro studies using human liver microsomes and recombinant CYPs were performed by an approach based on the disappearance rate of parent compound from the incubation mixture. In addition, the results of disappearance rate were compared with those obtained from the formation rates of the major metabolites of CAM, 14-(R)-hydroxy-CAM and N-demethyl-CAM.
The intrinsic clearance (CL_int) values determined from the disappearance of CAM in nine different human liver microsomes were highly correlated with the testosterone 6β-hydroxylation activity (r=0.957, p<0.001). The CL_int of CAM was markedly reduced by selective inhibitors of CYP3A4 (ketoconazole and troleandomycin) and by polyclonal antibodies raised against CYP3A4/5 in human liver microsomes. Among the 11 isoforms of recombinant human CYP, only CYP3A4 revealed the metabolic activity for the disappearance of CAM. These results were fairly consistent with those obtained from the conventional approach based on the formation of major metabolites of CAM. Comparison of the kinetic parameters estimated from the disappearance rate of CAM and the formation rates of 14-(R)-hydroxy-CAM and N-demethyl-CAM indicates that N-demethylation and 14-(R)-hydroxylation account for 65% of CL_int derived from the disappearance of CAM in human liver microsomes.
The findings suggest that CYP3A4 plays a predominant role in the overall metabolic clearance of CAM as well as in the formation of 14-(R)-hydroxy-CAM and N-demethyl-CAM. CYP2C19 does not appear to be involved in the overall metabolism of CAM at least in human liver microsomes. A combination of the disappearance rate of a parent compound and the formation rate of metabolites appears to be a useful approach for estimating the percentage contribution of the formation of metabolites to the overall metabolic clearance of a parent compound in vitro.

View full abstract

With advances in antiretroviral therapy, many HIV+ individuals are living longer lives and some are developing end-stage renal and/or hepatic disease requiring transplantation. These patients require concomitant use of immunosuppressants (e.g., cyclosporine [CsA]) and antiretrovirals (e.g., protease inhibitors [PIs]), which exhibit narrow therapeutic windows and are substrates and inhibitors of cytochrome P450 3A enzymes and the cellular transporter P-glycoprotein. In this pilot study, HIV+ subjects on either oral nelfinavir (NFV) or indinavir (IND) with nondetectable viral loads and normal renal and hepatic function had 12 hour pharmacokinetic (PK) studies on 3 separate days: PIs alone, PIs+intravenous CsA, and PIs+oral CsA to determine the extent of PK interactions between these medications. PIs and CsA concentrations were measured by LC/MS in plasma and whole blood, respectively. Nine subjects (n=7 on NFV, n=2 on IND) completed the study. Only the results of those subjects taking NFV are reported. Oral co-administration of CsA increased NFV T_max from 2.6±0.9 to 3.2±0.8 h (p<0.05), and AUC_0-∞ from 27.9±15.2 to 43.2±27.1 mg^*h/mL (p=0.06). Intravenous CsA did not appreciably alter oral pharmacokinetics of NFV. Both CsA and NFV PK parameters exhibited a high degree of intersubject variability, underscoring the need for routine therapeutic drug monitoring of both CsA and PIs in HIV+ subjects undergoing transplantation.

View full abstract

The ratio of the inhibitor concentration to the inhibition constant (K_i) is used as the index for predicting drug-drug interactions involving metabolic inhibition. The maximum unbound concentration in the circulation (I_{p, max, u}) and the maximum unbound concentration at the inlet to the liver (I_{u, max}) have been used for the inhibitor concentration. In the present study, the methods for predicting drug-drug interactions using these concentrations were evaluated by Monte Carlo simulation. Information on the pharmacokinetic parameters of drugs and the K_i values for cytochrome P450(CYP) were obtained from the literature. It was assumed that the pharmacokinetic parameters (intrinsic metabolic clearance, renal clearance and distribution volume for unbound fraction), serum protein binding and K_i value for substrate and inhibitor are all log-normally distributed. Correlations among the parameters were assessed and were used for further simulations. A change in AUC of the substrate following co-administration of the inhibitor was simulated 1000 times using the physiologically based pharmacokinetic (PBPK) model. The percent of the drug combinations which exhibited a significant increase in the AUC (>125%) was 16.2% of the total combinations. The cases where the I/K_i using I_{u, max} and I_{p, max, u} overestimated compared with the actual increased ratio of AUC (false positive prediction) were 41.2% and 16.7%, respectively. The cases where the predicted ratios of AUC from I/K_i using I_{u, max} and I_{p, max, u} were comparable with the actual ratio were 3.2% and 8.7%, respectively. The prediction using I_{p, max, u} was, thus, more reliable than that using I_{u, max}. However, in the case of I_{u, max}, there was no case where the actual increased ratio of AUC was greater than that predicted from I/K_i (false negative prediction). On the other hand, for I_{p, max, u}, the rate of false negative prediction was 1.4%. The present study indicates that I_{u, max} is better than I_{p, max, u} for avoiding false negative predictions and I_{p, max, u} is better than I_{u, max} for increasing the probability of true positive and true negative predictions and avoiding false positive predictions.
In conclusion, it is necessary to use both predictions involving I_{u, max} and I_{p, max, u} and to use them early on during the development stage of drug candidates. In order to finally choose which compound(s) to take forward to clinical trials, when predicting an interaction, the more quantitative and reliable method based on the PBPK model needs to be used.

View full abstract

The CYP3A4 enzyme is known for its atypical inhibition kinetics; ligand inhibition can differ depending upon the probe drug used. A high throughput-LCMS/MS CYP3A4 inhibition assay with four substrate drugs was developed to minimize the potential oversight of CYP3A4 inhibition. The assay uses a 96-well format, human liver microsomes, and four CYP3A4 substrate drugs, midazolam, testosterone, nifedipine and terfenadine. After incubation of the individual substrate with human liver microsomes, the reaction is stopped by solid phase extraction and the four probe metabolites produced are pooled and measured by LCMS/MS with multiple-ion-monitoring mode. Using this assay, the IC₅₀ values of fourteen compounds recognized as substrates/inhibitors of CYP3A4, were measured for the CYP3A4 catalyzed-metabolism of probe drugs. IC₅₀ values were also obtained for the common set of compounds by the microtiter plate fluorescent assays with cDNA-expressed CYP3A4. Comparison of the results from the two methods suggests that decision making should be cautiously executed to predict drug interaction potential caused by inhibition of CYP3A4 considering the gap between the two assays and various other factors.

View full abstract

A simple and sensitive method for the quantitation of theophylline (TP) in tears and plasma developed using gas chromatography/electron-impact ionization/mass spectrometry. Tears were collected by non-invasive Schirmer method. Plasma was pipetted on a Schirmer tear test strip (cutting to 5 mm×5 mm). Then, TP was converted directly into its pentafluorobenzoyl amide derivative without the need to perform any extraction from the biological fluid absorbed on Schiemer test paper and was quantified by gas chromatography-selected ion recordings with electron ionization mode. The concentrations in tears [C]t correlated very well with those of the free form in the plasma [Cf]p and those of the total form in the plasma [Cb+f]p. The ratios between TP concentrations in tears and plasma (free and total form) were as follows: [C]t/[Cb+f]p=0.53±0.20; [C]t/[Cf]p=1.21±0.19; [Cf]p/[Cb+f]p=0.44±0.14. The ratios of [C]t/[Cb+f]p, [C]t/[Cf]p and [Cf]p/[Cb+f]p were in good agreement with the previously published data.

View full abstract

Three novel single nucleotide polymorphisms (SNPs) were found in the UDP-glucuronosyltransferase (UGT) 1A9 gene from 97 Japanese subjects (47 cancer patients and 50 cardiovascular disease patients). The detected SNPs were as follows:
1) SNP, MPJ6_U1A006; GENE NAME, UGT1A9; ACCESSION NUMBER, AF297093; LENGTH, 25 bases; 5'-AATTCTCTTAGGG/TTTCTCAGATGCC-3'
2) SNP, MPJ6_U1A007; GENE NAME, UGT1A9; ACCESSION NUMBER, AF297093; LENGTH, 25 bases; 5'-TGTTACGGAGTAT/GGATCTCTACAGC-3'
3) SNP, MPJ6_U1A031; GENE NAME, UGT1A9; ACCESSION NUMBER, AF297093; LENGTH, 25 bases; 5'-ACTCATTCTCAGG/AGGGCATGAGGTG-3'
All three SNPs were located in exon 1 with frequencies of 0.036 for MPJ6_U1A006, and 0.005 for MPJ6_U1A007 and MPJ6_U1A031. SNP MPJ6_U1A007 (726T>G) results in formation of a termination codon TAG (Y242X). The other two SNPs, MPJ6_U1A006 (588G>T) and MPJ6_U1A031 (153G>A), result in synonymous changes (G196G and R51R, respectively).

View full abstract

Five novel single nucleotide polymorphisms (SNPs) were found in the EPHX1 gene from 96 Japanese epileptic patients. The detected SNPs were as follows:
1) SNP, MPJ6_EX1009; GENE NAME, EPHX1 ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-CCTCACTTCAGTG/ACTGGGCTTTGCC-3'.
2) SNP, MPJ6_EX1013; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-TCCGCAGCCAGGG/CAGGACGACAGCA-3'.
3) SNP, MPJ6_EX1026; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-GTTCTCCCTGGAC/TGACCTGCTGACC-3'.
4) SNP, MPJ6_EX1028; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-AGGCAGGGGGACG/AGCCAGTCTTGGG-3'.
5) SNP, MPJ6_EX1030; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-TGAAAAGTGGGTG/AAGGTTCAAGTAC-3'.
The frequencies were 0.016 for MPJ6_EX1028 (IVS8+54G>A) and 0.005 for the other SNPs. The SNP MPJ6_EX1013 (130G>C) results in an amino acid alteration (E44Q). The other three SNPs in the coding region, MPJ6_EX1009 (30G>A), MPJ6_EX1026 (1056C>T), and MPJ6_EX1030 (1239G>A) result in synonymous changes (V10V, D352D, and V413V, respectively).

View full abstract