Drug transporters expressed in various tissues play a significant role in drug disposition. By regulating the function of such transporters, it may be possible to eventually develop drugs with ideal pharmacokinetic profiles. In this article, we summarize the significant role played by drug transporters in drug disposition, focusing particularly on their potential use during the drug development process. The ability to manipulate transporter function offers the opportunity of being able to deliver a drug to the target organ, avoiding distribution to other organs (thereby reducing the chance of toxic side-effects), controlling the elimination process, and/or improving oral bioavailability. During drug development, it would be very useful to be able to select a lead compound that may or may not interact with transporters, depending on whether such an interaction is desirable. The use of specific inhibitors of transporters is also an attractive approach to controlling drug disposition, leading to improved efficacy. Currently, optimizing the pharmacokinetic properties of a drug during the early stages of its development is widely accepted as being of great importance. High-throughput screening systems using transporter gene transfected cells or computational (in silico) approaches are efficient tools for assessing transport activity during the early stage of drug development. In addition, drug-drug interactions involving drug transporters and functional genetic polymorphisms of drug transporters are also described. It would also be extremely valuable to be able to quantitatively predict inter-individual pharmacokinetic differences caused by transporter polymorphisms or pharmacokinetic changes caused by drug-drug interactions involving transporters during drug development.
20-Hydroxyeicosatetraenoic acid (20-HETE) has been shown to be an arachidonic acid metabolite of the cytochrome P450 (CYP) enzymes belonging to the CYP4A subfamily and is a predominant regulator of renal vascular tone and tubular ion reabsorption in rat kidney. CYP4A8 is one of the CYP4A enzymes expressed in rat kidney, but its contribution to 20-HETE formation has not been assessed. In order to clarify that the role of CYP4A8, we have developed bacterial expression systems for the expression of recombinant CYP4A8 (rCYP4A8). We also produced an antibody against rCYP4A8 which was used for immunoinhibition and immunohistochemical studies. In a reconstituted system, rCYP4A8 sufficiently catalyzed 20-HETE formation as well as prostaglandin A1 ω-hydroxylation, a marker activity for CYP4A8. In addition, anti-rCYP4A8 sera significantly inhibited prostaglandin A1 ω-hydroxylation and strongly inhibited arachidonic acid ω-hydroxylation in rat kidney microsomes. These observations suggested for the first time that CYP4A8 also contributed to 20-HETE formation in rat kidney. Furthermore, immunohistochemstry suggested that CYP4A8 is present in preglomerular arteries, where 20-HETE has been established to be a vasoconstrictor.
A cDNA clone designated as CYP2C43 was isolated from the rhesus monkey liver cDNA library. The first 16 amino acid residues at the N-terminal region of this cDNA product were identical with those of P450 CMLd which have been purified and characterized as S-mephenytoin 4′-hydroxylase in monkey liver. The respective nucleotide and deduced amino acid sequences of CYP2C43 were 83% and 77%, identical to those of monkey CYP2C20. Antibody against CYP2C9 detected a protein in the microsomes of yeast transformed CYP2C43 expression plasmid. The specific content of recombinant CYP2C43 was 78.0 pmol/mg protein and the yield was 4.23 nmol/l of the culture. CYP2C43 was able to metabolize S-mephenytoin stereo-selectively. The activity for S-mephenytoin in the microsomes reconstituted with or without cytochrome b5 was found to be 96.2 or 23.7 pmol/min/nmol P450, respectively. CYP2C43, however, did not show any oxidative activity for tolbutamide. These results indicate that CYP2C43 is the second identified member of the monkey CYP2C subfamily and a cDNA clone encoding P450 CMLd in monkey.
Most cephalosporin antibiotics are excreted into urine via glomerular filtration and active tubular secretion by renal organic anion transporters. In this study, we investigated the interaction of cephalosporins with rat organic anion transporter rOAT1, mainly expressed at the basolateral membrane of the renal proximal tubules, using Xenopus laevis oocytes, to assess the roles of rOAT1 in renal excretion of cephalosporin antibiotics. The expression of rOAT1 significantly stimulated the uptake of cefazolin, cefotiam and cephalexin into oocytes, but not of cefoperazone. The inhibition constants of these cephalosporins to rOAT1-mediated p-aminohippurate (PAH) uptake were 72 μM for cefazolin, 298 μM for cefoperazone, 718 μM for cefotiam and 6 mM for cephalexin. Eadie-Hofstee plot analysis revealed that cefoperazone as well as cefotiam inhibited rOAT1-mediated PAH uptake competitively. These results suggest that rOAT1 mediates basolateral uptake of cephalosporin antibiotics in the renal tubules. Furthermore, it is suggested that a minor contribution of the kidney to cefoperazone excretion could be related to the finding that cefoperazone is a poor substrate of rOAT1.
Accumulating evidence suggests that several ATP-binding cassette (ABC) transporters mediate the elimination of anticancer drugs from cancer cells and thereby confer drug resistance. SN-38-selected PC-6/SN2-5H human lung carcinoma cells were shown to overexpress ABCG2 with the reduced intracellular accumulation of SN-38, the active metabolite of irinotecan. We have recently demonstrated that plasma membrane vesicles prepared from those cells transported SN-38 in an ATP-dependent manner, and it was suggested that ABCG2 is involved in the active extrusion of SN-38 from cancer cells. In the present study, we have cloned the cDNA of ABCG2 from PC-6/SN2-5H human lung carcinoma cells, expressed ABCG2 in Sf9 insect cells, and characterized its function. Sequence analysis has revealed that the cloned ABCG2 has an arginine at the amino acid position 482, as does the wild type. Expression of the cloned ABCG2 in Sf9 cell membranes was detected by immunoblotting with the BXP-21 antibody. Contrary to our expectation, however, ATPase activity in the cell membranes expressing ABCG2 was stimulated by neither SN-38 nor rhodamine 123. It is suggested that there is a partner protein of ABCG2 required for heterodimer formation to exhibit transport activity toward SN-38.
The effects of co-administration of cationic proteins on the in vivo disposition characteristics of recombinant human interleukin-11 (rhIL-11) in mice and on the renal disposition in the perfused rat kidney were investigated. Following a bolus intravenous injection of 10 μg/kg 111In-labeled rhIL-11, along with cationic proteins at high dose (50 mg/kg), the plasma clearance of 111In-labeled rhIL-11 was significantly decreased mainly due to a reduction in the hepatic clearance of 111In-labeled rhIL-11. The effect on the renal clearance was relatively small, suggesting that the kidney has a high clearance capacity. The urinary excretion ratio increased by a factor of 2 or 4 with co-administration, suggesting that the cationic character of rhIL-11is involved in tubular re-absorption. An in situ renal disposition study supports these postulations. Thus, the renal and hepatic disposition of rhIL-11 is based on nonspecific cationic interaction. These data suggest that an efficient delivery system for this cytokine would require the reduction of electrostatic interaction of this molecule with these tissues in order to reduce the plasma clearance rate. These findings provide useful information for the construction of an rhIL-11 delivery system.
Endogenous and exogenous compounds having a carboxyl group, such as α-arylpropionic acid derivatives, undergo a phase II metabolic reaction to produce an amino acid conjugate through the acyl CoA thioester as well as the acyl glucuronide. It was previously shown that flurbiprofen, one of the nonsteroidal anti-inflammatory drugs, is not subjected to activation of the carboxyl group by the CoA thioester ligase, suggesting that acyl glucuronidation is the main phase II metabolic pathway. Recent observations, however, have demonstrated that the nonenzymatic formation of a covalently protein-bound drug, which is produced by the action of the acyl glucuronide, may cause hypersensitive reactions. Accordingly, a reliable method to measure diastereomeric flurbiprofen glucuronides in human biological fluids is required. In this study, we describe a liquid chromatographic/mass spectrometric method with a simple column switching technique to determine diastereomeric flurbiprofen acyl glucuronides in human urine specimens. The optimal conditions for the electrospray ionization were established based on the effects of orifice and ring lens voltages as well as mobile phase additives. The proposed method applied to urine specimens demonstrates high accuracy and reproducibility for the determination of flurbiprofen glucuronides in a quantitative range from 0.74 to 146.5 μg/mL, with a detection limit of 7.4 pg (17.6 fmol)/injection of S-flurbiprofen glucuronide, at a signal-to-noise ratio of 10 under the selected ion-monitoring mode. The urinary concentration of R-flurbiprofen glucuronides in healthy subjects determined by the proposed method were 6.8-29.4 μg/mL, and those values were slightly higher than that of S-flurbiprofen glucuronides (3.9-18.0 μg/mL).
Three non-synonymous single nucleotide polymorphisms (SNPs) in the CYP3A4 gene were found in 34 cell lines derived from Japanese individuals. These three SNPs (T185S, L293P, and T363M)1 have been previously reported, but little is known about the effect that these polymorphisms, especially T185S, have on catalytic activity. We measured testosterone hydroxylation in wild-type CYP3A4 and these three variants using a mammalian expression system. Testosterone 6β-, 2β-, and 15β-hydroxylations by the variant CYP3A4 forms T363M (<40%) and T185S (<60%) were reduced as compared with the wild-type in transient expression assays. L293P was similar to the wild-type in testosterone 6β- and 2β-hydroxylase activities. Western blot analysis confirmed lower amounts of CYP3A4 protein in the T363M and T185S variants than in the wild-type. Interestingly, Northern blot analysis showed no significant difference among mRNA levels between the wild-type and variants. These results suggest that the T363M and T185S substitutions in CYP3A4 affect either protein expression or stability. These established cell lines provided useful CYP3A4 SNP information in the Japanese.
We determined the CYP2D6 alleles in cell lines derived from 81 Japanese individuals with the CYP450 probe arrays using an Affymetrix GeneChip apparatus. Sequencing of the CYP2D6 exons from these same cell lines was performed to determine the accuracy of the allele calls by the Affymetrix probe array. Comparison of the results showed differences in the data from three cell lines for the CYP2D6*10 alleles between these two methods. These results indicated that the CYP450 probe array must be utilized cautiously for typing CYP2D6*10 alleles, which are frequently observed in the Japanese population.
We explored genetic polymorphisms in a Thai population which exhibited a low capacity to metabolize coumarin. The following two silent single nucleotide polymorphisms (SNPs) were found: 1) SNP, 020228Kiyotani001; GENE NAME, CYP2A6; ACCESSION NUMBER, NT_011139; LENGTH, 25 base; 5′-AAACTACCTGCAG/TCTGAACACAGAG-3′. 2) SNP, 020228Kiyotani002; GENE NAME, CYP2A6; ACCESSION NUMBER, NT_011139; LENGTH, 25 base; 5′-AATCCCCAGCAC/TTTCCTGAATGAG-3′. These two mutations (G144A and C1245T), which were located in exon 1 and exon 8 of the CYP2A6 gene, were found in two subjects among nine poor metabolizers for coumarin.
A novel single nucleotide polymorphism (SNP) was found in exon 6 of the UDP-glucuronosyltransferase (UGT) 2B15 gene from healthy Japanese populations. The SNP was as follows: SNP, 020228Toide001; GENE NAME, UGT2B15; ACCESSION NUMBER, U08854, AF180322, and NM_001078; LENGTH, 25 base; 5′-AGCTTGCCAAAAC/AAGGAAAGAAGAA-3′. This SNP was expected to cause a change of an amino acid residue at the position 523 (Thr to Lys) located in a putative co-factor binding region. The allele frequency of this SNP was 79% in Japanese, suggesting this polymorphism to be a major genotype in Japanese people.