Since 1994, researchers have isolated various genes encoding transporter proteins involved in drug uptake into and efflux from tissues that play key roles in the absorption, distribution and secretion of drugs in animals and humans. The pharmacokinetic characteristics of drugs that are substrates for these transporters are expected to be influenced by coadministered drugs that work as inhibitors or enhancers of the transporter function. This review deals with recent progress in molecular and functional research on drug transporters, and then with transporter-mediated drug interactions in absorption and secretion from the intestine, secretion from the kidney and liver, and transport across the blood-brain barrier in humans. Although the participation of the particular transporters in observed drug-drug interactions can be difficult to confirm in humans, this review focuses mainly on pharmacokinetic interactions of clinically important drugs.
To estimate the influence of repeated administration of drug metabolism inhibitors on the extent of drug interaction, we investigated the effects of single intravenous or repeated oral administration of itraconazole on the pharmacokinetics of midazolam in rats. In the single administration study, the plasma concentration of itraconazole was maintained by intravenous infusion, and midazolam was administered into the portal vein to investigate its kinetics. In the repeated administration study, the kinetics of midazolam was investigated after seven-day oral treatment with itraconazole. The in vitro metabolism of midazolam and the contents of cytochrome P450 were investigated using liver microsomes from the itraconazole-treated rats. The area under the curve (AUC) of midazolam was increased by 1.45- or 1.44-fold after single or repeated itraconazole treatment, respectively. Meanwhile, the liver concentrations of itraconazole after single administration and repeated administration were 38.2 and 20.3 (nmol/g), respectively. In vitro maximum metabolic reaction velocity (Vmax) and Michaelis-Menten constant (Km) of midazolam were increased from 2.26 to 3.84 (nmol/min/mg protein) and from 8.28 to 13.0 (μM) by single itraconazole treatment, respectively, and decreased from 2.23 to 1.17 (nmol/min/mg protein) and from 7.86 to 4.47 (μM) by repeated treatment, respectively. Correspondingly, the content of CYP3A2 was significantly altered by single or repeated itraconazole administration. The increases in AUC could be predicted only when the changes in Vmax and Km were taken into consideration, in addition to the hepatic unbound concentration of itraconazole. In conclusion, changes in enzyme kinetics should be taken into account to predict the extent of drug interaction after repeated treatment with inhibitors.
Salivary caffeine excretion rate test has been proposed for the evaluation of gastrointestinal transit characteristics of an oral patch preparation after administration to human volunteers instead of measuring the plasma or serum concentration in the early stages of formulation development. Patches having a diameter of 3.0 mm and containing caffeine as a model drug were prepared. The patches consisted of 1) the backing layer made of a water-insoluble polymer, 2) the drug-carrying layer composed of caffeine and a gel-forming polymer, and 3) the enteric polymer membrane. These three layer patches were filled into enteric capsules. Caffeine solution in an enteric capsule was used as the control preparation. After oral administration of each preparation to human volunteers at a dose of 50 mg of caffeine in a cross-over study with a wash-out period of two weeks, saliva samples were collected over 1 min at every sampling time for 12 h and salivary caffeine concentration was determined by a HPLC assay method. Salivary caffeine excretion rate (ER) was used for pharmacokinetic analysis. Mean residence time (MRT) and first-appearance time of caffeine into the saliva (Ti) were determined. To characterize the pharmacokinetics of caffeine, MRT-Ti values of patch and solution preparations were compared. Patch preparations had a Ti value of 2.33±0.33 h and showed significantly longer MRT-Ti, 3.87±0.21 h, as compared to the control preparation (MRT-Ti=1.04±0.38 h) under fasting condition (p<0.05). Food intake prolonged the gastric emptying time (GET) of the preparations with Ti values of 5.00±1.15 h for control preparation and 4.67±1.20 h for patch preparation. The MRT-Ti values were 0.62±0.20 h (control) and 2.45±0.73 h (patch). The results of this study indicate that the parameter, MRT-Ti, was useful in characterizing the transit characteristics of oral patch preparations than MRT itself and the presence of food affects the performance of the patch system.
After intravenous bolus administration of aprindine (AP) to conscious guinea pigs, the semilogarithmic plasma concentration versus time curve was linear at a dose of 2 mg/kg, but convex at doses of 5 and 10 mg/kg. AP concentrations immediately after administration (Cp0) were almost identical, irrespective of the dose received. The areas under the plasma concentration-time curves (AUCs) were proportional to the AP doses. At 2 mg/kg, the plasma total clearance (CLtot) of AP was high (279±80 mL/h), and its volume of distribution (Vdss) was large (245±99 mL). Total blood clearance and time-averaged blood clearance (CLave) values for AP were similar to those for R(+) propranolol (PL) after intravenous coadministration of R(+) PL (0.25 mg/kg) and AP (2 or 10 mg/kg). An in vitro serum protein binding study showed that the unbound fraction of AP was concentration-dependent. In guinea pigs pretreated with turpentine oil (2 mL/kg/day), the elimination of AP after intravenous doses of 2 and 5 mg/kg closely followed first-order kinetics, while Cp0 and AUC increased in proportion to the AP doses. The bound fraction of AP in the serum was larger after turpentine oil pretreatment than in normal guinea pig serum in vitro. From these observations, the nonlinear pharmacokinetics of AP observed in guinea pigs can be attributed to nonlinear serum protein binding.
The goal of this study was to classify and identify the ligand binding sites on α1-acid glycoprotein (AGP) from 3 species, in order to understand species differences with respect to both ligand binding properties and ligand interaction on protein binding. These characteristics of human, dog and bovine AGP were examined using the basic ligands chlorpromazine and auramine O, the acidic ligand acenocoumarin, and the steroid hormone progesterone. Ultrafiltration and fluorescence techniques were used to characterize the nature of the interactions, and the data were analyzed according to the method of Kragh-Hansen. Using a model analysis of the interaction, the ligand binding site on human AGP consists of at least 3 partially overlapping subsites: a basic ligand binding site, an acidic ligand binding site and a steroid hormone binding site. Moreover, dog and bovine AGP each have a basic ligand binding site and a steroid hormone binding site, which significantly overlap and affect each other. However, dog and bovine AGPs do not contain an acidic ligand binding region. The results of the fluorescence experiments indicate that the hydrophobic nature of the ligand binding pockets on the 3 AGPs are similar, but that their microviscosities are markedly different.
Absorption behavior of theophylline, categorized into Class I of Biopharmaceutics Classification System, orally administered as powders in rats was analyzed and predicted by Gastrointestinal-Transit-Absorption (GITA) model, which was modified to describe GI-transit kinetics and dissolution of powders orally administered. First of all, GI-transit kinetics of glass beads was examined to describe the transit kinetics of powders through GI tract in rats. The results showed that the gastric emptying of glass beads was slower than that of solution, but that there was not much difference in the transit rate constants through the small intestine and cecum between glass beads and solution. Furthermore, to introduce the dissolution process of theophylline powders into GITA model, an in-vitro dissolution test was examined for theophylline powders according to the Japanese Pharmacopoeia paddle method. The dissolution rate constants calculated based on the mean dissolution time were not so different in the range of pH from 1.2 to 6.5. Using the parameters for GI transit, dissolution and absorption obtained, the plasma concentration-time profile of theophylline after oral administration as powders to rats was predicted based on GITA model. The profile calculated was significantly correlated with the observed time course of plasma concentration for theophylline, and the parameters such as Cmax and AUC based on the predicted curve coincided with those on the observed data, showing that GITA model is useful for the prediction of the absorption behavior of drugs administered as powders. The simulation studies showed that about 80% of orally administered theophylline powders dissolved in the stomach and that the remaining powders rapidly moved to the lower jejunum and ileum, where they dissolved. Furthermore, it was suggested that theophylline is absorbed mostly in the upper small intestine, duodenum, upper jejunum and lower jejunum, after its oral administration as powders.
TSQ ESI MS/MS and ion trap ESI MS2 cleave protonated molecules. MS2 at m/z 332 of zotepine cleaved m/z 245 (10%), m/z 287 (5%) and m/z 315 (100%) fragment ions at protonated positions. MS2 at m/z 356 of tiaramide cleaved m/z 338 (18%), 313 (10%), 226 (100%), 198 (78%) and 131 (60%) fragment ions at protonated positions. The ESI ion trap MS produced new internal protonated molecules in an ion trap, such as m/z 113 and m/z 88 from m/z 131 protonated piperazinonium, and m/z 245 protonated 8-chloro dibenzo[b,f]thiepin. ESI ion trap (MS)n (n≥3) cleaved new internal protonated molecules. It also causes carbocation cleavage, α cleavage, onium cleavage and McLafferty cleavage. We can easily elucidate the structure of metabolites from the difference in m/z of corresponding fragments between unchanged compound and its metabolite. Reactive oxygen diradicals involved in cytochrome P-450 cycles react with electron rich groups and reactive C-H bonds of zotepine and tiaramide to produce metabolites of 2-hydroxyzotepine, 3-hydroxyzotepine, norzotepine, zotepine-N-oxide, zotepine-S-oxide, Tiaramide carboxylic acid, dehydroxyethyltiaramide and tiaramide-N-oxide. The strategy for structure elucidation of drug metabolites was established on the basis of the reactivity of unchanged drug with reactive oxygen diradicals involved in cytochrome P-450 cycles and theory associated with protonated molecules and (MS)n fragmentation of drug metabolites.
In order to understand the insulinomimetic activity of zinc(II) complexes, we studied the metallokinetic features of zinc in the blood of normal rats given the zinc complexes, bis(maltolato)zinc(II) (Zn(mal)2) and bis(6-methylpicolinato)zinc(II) (Zn(6mpa)2) by comparing each of them with an ionic form of zinc chloride (ZnCl2). The bioavailability of the zinc(II) complexes following oral administration was enhanced to 1.4-1.5-fold that of ZnCl2 with respect to zinc level. Based on the results of a metallokinetic analysis and administration method in normal rats, we examined the antidiabetic ability of the zinc(II) complexes in GK rats, a model animal of type 2 diabetes mellitus. High blood glucose levels of GK rats were normalized following intraperitoneal injections and oral administration of the zinc(II) complexes, in which the Zn(6mpa)2 complex was found to be more effective than Zn(mal)2. The present results are noteworthy, not only due to their potential relevance for clinical application, but also for the development of new zinc(II) complexes.
Japanese monkey liver contains multiple forms of dihydrodiol dehydrogenase with 3(20)α-hydroxysteroid dehydrogenase activity. Here we have purified the major and minor forms (DD1 and DD4) of the enzyme from Cynomolgus monkey liver, and isolated cDNA species for the two enzyme forms by reverse transcription-PCR. The cDNAs encoded proteins comprising of 323 amino acids, in which the sequence identity between DD1 and DD4 was 83%. The sequences deduced from the cDNAs for DD1 and DD4 perfectly matched the partial sequences of peptides derived from the respective enzymes. We also isolated the cDNAs for DD1 and DD4 of Japanese monkey liver, which had almost identical amino acid sequences with those of the respective enzymes of Cynomolgus monkey liver. The monkey DD1s and DD4s showed the highest sequence identity (94%) with AKR1C1 and AKR1C4, respectively, of four isoenzymes of human 3(20)α-hydroxysteroid dehydrogenase, which belongs to the aldo-keto reductase family. The substrate specificity and inhibitor sensitivity of the purified recombinant Cynomolgu monkey DD1 and Japanese monkey DD4 were also essentially identical to those of the recombinant AKR1C1 and AKR1C4, respectively, indicating that DD1 and DD4 are homologues of human AKR1C1 and AKR1C4, respectively. The mRNA for DD1 was detected only in liver, kidney, intestine and adrenal gland among Japanese monkey tissues, and that for DD4 was expressed in liver and kidney. These tissue distribution patterns differ from those of human AKR1C1 and AKR1C4, which are expressed ubiquitously and liver-specific, respectively. In addition, no mRNA for an enzyme corresponding to another isoenzyme (AKR1C2) of the human enzyme was detected in livers of the two monkey strains. The results suggest a difference in the metabolism of steroids and xenobiotics mediated by 3(20)α-hydroxysteroid dehydrogenase isoenzymes between monkeys and humans.
To investigate the association between NAT2 genotypes and the incidence of isoniazid (INH)-induced adverse reactions, in the hope of identifying a pharmacogenetic approach that could be useful in the prediction and prevention of adverse reactions in Japanese patients, we retrospectively studied the genotypes of NAT2 in 102 Japanese patients treated with INH (without rifampicin co-administration). The subjects were classified into three groups according to their genotypes: rapid-type, intermediate-type, and slow-type. The clinical conditions of the patients were followed-up in order to evaluate the development of any adverse drug reactions (ADRs) and correlate them with patient genotypes. Six out of the 102 patients (5.9%) developed various ADRs following INH treatment. These reactions included nausea/vomiting, fever, visual impairment, and peripheral neuritis. We found a statistically significant difference between the incidence of ADRs and NAT2 genotype. The incidence of ADRs was significantly higher in the slow type than in the other two types, as 5 out of the 6 ADR patients were of the slow-type, and the other one was of the intermediate-type, while no patients of the rapid-type developed any ADRs. The results indicated that the genes coding for slow acetylation were associated with the incidence of serious ADRs following INH treatment. Our findings suggest that determination of NAT2 genotype might be clinically useful in the evaluation of patients at high risk of developing ADRs induced by INH.
The effect of calcium polycarbophil on the absorption of cefdinir, cephalosporin derivative, was evaluated in both in vitro and in vivo studies. In the in vitro study, the release of cefdinir from a cellulose membrane in the presence or absence of metal cations was measured using the dissolution test procedure. In the in vivo study, volunteers and a randomized crossover design with two phases were used. In the first phase, the volunteers received 200 mg of cefdinir alone (Study 1); in the other phase, they received 200 mg of cefdinir and 1200 mg of fine calcium polycarbophil granules concomitantly (Study 2). The cefdinir concentrations in the samples or serum were measured by an UV-VIS spectrophotometer or high-performance liquid chromatography. Release in the presence of iron ions was slower than that in the absence of metal ions, however no difference was observed between release in the presence of calcium ions and that in the absence of metal ions. No difference was observed in AUC(0-10), Cmax and tmax between Study 1 and Study 2. The absorption of cefdinir was not affected by co-administration of calcium polycarbophil. Moreover, the in vitro study on the release of drugs from a cellulose membrane may predict the absorption of a drug caused by the formation of chelate complexes between the drug and metal ions.
An in vitro cell culture system for estimating the human blood-brain barrier (BBB) permeability of drugs is required for the development of drugs with effects on the central nervous system. In this study, cultured human brain microvascular endothelial cells (hBME) were characterized. hBME cells exhibited concentration-dependent uptake of L-Leu, L-Glu and L-Lys with Km values of 51.1±23.1 μM, 163.3±79.8 μM and 72.4±56.6 μM, respectively. The cellular accumulation of rhodamine123 in hBME cells was unaffected by P-glycoprotein (P-gp) substrates (cyclosporin A, quinidine and verapamil), while the accumulation in human P-gp-overexpressing cells was significantly increased in the presence of these P-gp substrates. RT-PCR revealed that hBME cells expressed large neutral amino acid transporter 1 (LAT1) and its associated molecule (4F2hc), excitatory amino acid transporter 3 (EAAT3), cationic amino acid transporter 1 (CAT1), glucose transporter 1 (GLUT1), monocarboxylic acid transporter 1 (MCT1) and multidrug resistance-associated protein 1 (MRP1). However, no expression of multidrug resistance protein 1 (MDR1) was detected. The results suggest that these amino acid transporters are functionally expressed at the human BBB, and that hBME cells retain the in vivo BBB transport functions and expression characteristics. Consequently, hBME cells should be a useful tool for studies of the human BBB.
Five novel single nucleotide polymorphisms (SNPs) were found in exon 3 and introns 1, 3, 7, and 8 in cytochrome P450 (CYP) 2C8 gene from 54 Japanese individuals, who were administered the anti-arrhythmic drug amiodarone. The detected SNPs were as follows: 1) SNP, MPJ6_2C8014; GENE NAME, CYP2C8; ACCESSION NUMBER, NT_008769; LENGTH, 25 bases; 5′-ATTCAGAAATATC/TGAATCTATGTGT-3′ 2) SNP, MPJ6_2C8015; GENE NAME, CYP2C8; ACCESSION NUMBER, NM_000770 and NT_008769; LENGTH, 25 bases; 5′-GGAGGAGTTGAGA/-AAAACCAAGGGT-3′. 3) SNP, MPJ6_2C8016; GENE NAME, CYP2C8; ACCESSION NUMBER, NT_008769; LENGTH, 25 bases; 5′-ATTTGTAAGATAT/-TGTTTAAAATTT-3′ 4) SNP, MPJ6_2C8017; GENE NAME, CYP2C8; ACCESSION NUMBER, NT_008769; LENGTH, 25 bases; 5′-TTGGTTCCAACCC/TTCTAACAACACA-3′ 5) SNP, MPJ6_2C8018; GENE NAME, CYP2C8; ACCESSION NUMBER, NT_008769; LENGTH, 25 bases; 5′-GATAGCAAATATA/GTCTCTTTTTGTA-3′ Among these SNPs, MPJ6_2C8015 was expected to cause a frame-shift due to the deletion of adenine 471, resulting in amino acid alterations from codon 159 and an early stop codon at residue 177. Therefore, the variant enzyme is most likely to be inactive since it lacks 64% of the protein structure, including the heme-binding site and 5 out of 6 substrate recognition sites.