A method combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem-mass spectrometry to detect circulating immune complexes between therapeutic monoclonal antibodies and anti-drug antibodies in animals.

Therapeutic monoclonal antibodies can potentially induce unwanted immune responses, resulting in the production of anti-drug antibodies (ADAs). The binding of ADAs to drugs and subsequent formation of immune complexes (ICs) can trigger various responses, dependent on the size, concentration, and subclass of ADAs. To better understand the impact of ADAs on pharmacokinetics, pharmacodynamics, and toxicological profiles, a bioanalytical method was developed for the detection of ICs between human monoclonal immunoglobulin G (IgG) and ADAs in biological samples. Regarding the experimental procedure, in brief, the human antibody-specific ICs and unbound human antibody in biological samples are separated through blue native polyacrylamide gel electrophoresis (BN-PAGE). The target fractions are then cut from the gel, followed by in-gel trypsin digestion and subsequent liquid chromatography tandem-mass spectrometry (LC-MS/MS) to monitor the human IgG-specific peptide. This method was able to detect various types of human antibodies with a lower limit of detection of 10 µg/mL in monkey serum. The assay performance for the detection of ICs was demonstrated using spiked samples, and pre-incubated ICs in monkey serum were clearly detected. Taken together, these findings indicate that our method enables a semi-quantitative analysis for estimating the ratio of human antibody included ICs in comparison to the total antibody. This method was successfully applied to an in vivo study using mice, and the data helped explain the unexpectedly rapid clearance of a humanized antibody due to the formation of large ICs. The combination of the separation of ICs by BN-PAGE and the detection of the human IgG-specific peptide by LC-MS/MS is a useful general bioanalytical approach for the detection of ICs in animals.

A comparative study for detecting CYP3A induction by CYP3A probe drugs and endogenous markers in cynomolgus monkeys.

CYP3A probe drugs such as midazolam and endogenous markers, and plasma 4ß-hydroxycholesterol (4ß-OHC) and urinary 6ß-hydroxycortisol-to-cortisol ratios (6ß-OHC/C) have been used as markers of CYP3A induction in cynomolgus monkeys, as with humans. However, there is limited information on their sensitivity and ability to detect CYP3A induction, as most studies were evaluated only at a high dose of the inducer, rifampicin (RIF; 20 mg/kg). In the present study, the CYP3A induction by RIF over a range doses of 0.2, 2 and 20 mg/kg (n = 4) was examined using CYP3A probe drugs (midazolam, triazolam and alprazolam) and the plasma and urinary endogenous CYP3A markers (4ß-OHC and 6ß-OHC/C). The sensitivity and relationship for detecting CYP3A induction was compared among the markers. Four days repeated oral administration of rifampicin to cynomolgus monkeys reduced the area under the plasma concentration-time curve of all CYP3A probe drugs in a rifampicin dose-dependent manner. Although the endogenous CYP3A markers (4ß-OHC and 6ß-OHC/C) were also changed for the middle (2 mg/kg) and high (20 mg/kg) doses of rifampicin, the fold-changes were relatively small, and CYP3A induction could not be detected at the lowest dose of rifampicin (0.2 mg/kg). In conclusion, CYP3A probe drugs are more sensitive for detecting CYP3A induction than endogenous CYP3A markers in cynomolgus monkeys, even for a short experimental period.

Reduced renal clearance of cefotaxime in asians with a low-frequency polymorphism of OAT3 (SLC22A8).

Organic anion transporter 3 (OAT3, SLC22A8), a transporter expressed on the basolateral membrane of the proximal tubule, plays a critical role in the renal excretion of organic anions including many therapeutic drugs. The goal of this study was to evaluate the in vivo effects of the OAT3-Ile305Phe variant (rs11568482), present at 3.5% allele frequency in Asians, on drug disposition with a focus on cefotaxime, a cephalosporin antibiotic. In HEK293-Flp-In cells, the OAT3-Ile305Phe variant had a lower maximum cefotaxime transport activity, Vmax , [159 ± 3 nmol*(mg protein)(-1) /min (mean ± SD)] compared with the reference OAT3 [305 ± 28 nmol*(mg protein)(-1) /min, (mean ± SD), p < 0.01], whereas the Michaelis-Menten constant values (Km ) did not differ. In healthy volunteers, we found volunteers that were heterozygous for the Ile305Phe variant and had a significantly lower cefotaxime renal clearance (CLR ; mean ± SD: 84.8 ± 32.1 mL/min, n = 5) compared with volunteers that were homozygous for the reference allele (158 ± 44.1 mL/min, n = 10; p = 0.006). Furthermore, the net secretory component of cefotaxime renal clearance (CLsec ) was reduced in volunteers heterozygous for the variant allele [33.3 ± 31.8 mL/min (mean ± SD)] compared with volunteers homozygous for the OAT3 reference allele [97.0 ± 42.2 mL/min (mean ± SD), p = 0.01]. In summary, our study suggests that a low-frequency reduced-function polymorphism of OAT3 associates with reduced cefotaxime CLR and CL(sec) .

Investigation of drug-drug interactions caused by human pregnane X receptor-mediated induction of CYP3A4 and CYP2C subfamilies in chimeric mice with a humanized liver.

The induction of cytochrome P450 (P450) enzymes is one of the risk factors for drug-drug interactions (DDIs). To date, the human pregnane X receptor (PXR)-mediated CYP3A4 induction has been well studied. In addition to CYP3A4, the expression of CYP2C subfamily is also regulated by PXR, and the DDIs caused by the induction of CYP2C enzymes have been reported to have a major clinical impact. The purpose of the present study was to investigate whether chimeric mice with a humanized liver (PXB mice) can be a suitable animal model for investigating the PXR-mediated induction of CYP2C subfamily, together with CYP3A4. We evaluated the inductive effect of rifampicin (RIF), a typical human PXR ligand, on the plasma exposure to the four P450 substrate drugs (triazolam/CYP3A4, pioglitazone/CYP2C8, (S)-warfarin/CYP2C9, and (S)-(-)-mephenytoin/CYP2C19) by cassette dosing in PXB mice. The induction of several drug-metabolizing enzymes and transporters in the liver was also examined by measuring the enzyme activity and mRNA expression levels. Significant reductions in the exposure to triazolam, pioglitazone, and (S)-(-)-mephenytoin, but not to (S)-warfarin, were observed. In contrast to the in vivo results, all the four P450 isoforms, including CYP2C9, were elevated by RIF treatment. The discrepancy in the (S)-warfarin results between in vivo and in vitro studies may be attributed to the relatively small contribution of CYP2C9 to (S)-warfarin elimination in the PXB mice used in this study. In summary, PXB mice are a useful animal model to examine DDIs caused by PXR-mediated induction of CYP2C and CYP3A4.

Quantitative prediction of human pregnane X receptor and cytochrome P450 3A4 mediated drug-drug interaction in a novel multiple humanized mouse line.

Cytochrome P450 (P450) 3A4 is the predominant P450 enzyme expressed in human liver and intestine, and it is involved in the metabolism of approximately 50% of clinically used drugs. Because of the differences in the multiplicity of CYP3A genes and the poor correlation of substrate specificity of CYP3A proteins between species, the extrapolation of CYP3A-mediated metabolism of a drug from animals to man is difficult. This situation is further complicated by the fact that the predictability of the clinically common drug-drug interaction of pregnane X receptor (PXR)-mediated CYP3A4 induction by animal studies is limited as a result of marked species differences in the interaction of many drugs with this receptor. Here we describe a novel multiple humanized mouse line that combines a humanization for PXR, the closely related constitutive androstane receptor, and a replacement of the mouse Cyp3a cluster with a large human genomic region carrying CYP3A4 and CYP3A7. We provide evidence that this model shows a human-like CYP3A4 induction response to different PXR activators, that it allows the ranking of these activators according to their potency to induce CYP3A4 expression in the human liver, and that it provides an experimental approach to quantitatively predict PXR/CYP3A4-mediated drug-drug interactions in humans.

Genetic variation in the proximal promoter of ABC and SLC superfamilies: liver and kidney specific expression and promoter activity predict variation.

Membrane transporters play crucial roles in the cellular uptake and efflux of an array of small molecules including nutrients, environmental toxins, and many clinically used drugs. We hypothesized that common genetic variation in the proximal promoter regions of transporter genes contribute to observed variation in drug response. A total of 579 polymorphisms were identified in the proximal promoters (-250 to +50 bp) and flanking 5' sequence of 107 transporters in the ATP Binding Cassette (ABC) and Solute Carrier (SLC) superfamilies in 272 DNA samples from ethnically diverse populations. Many transporter promoters contained multiple common polymorphisms. Using a sliding window analysis, we observed that, on average, nucleotide diversity (pi) was lowest at approximately 300 bp upstream of the transcription start site, suggesting that this region may harbor important functional elements. The proximal promoters of transporters that were highly expressed in the liver had greater nucleotide diversity than those that were highly expressed in the kidney consistent with greater negative selective pressure on the promoters of kidney transporters. Twenty-one promoters were evaluated for activity using reporter assays. Greater nucleotide diversity was observed in promoters with strong activity compared to promoters with weak activity, suggesting that weak promoters are under more negative selective pressure than promoters with high activity. Collectively, these results suggest that the proximal promoter region of membrane transporters is rich in variation and that variants in these regions may play a role in interindividual variation in drug disposition and response.

Inhibitory effect of the new orally active CCR4 antagonist K327 on CCR4+CD4+ T cell migration into the lung of mice with ovalbumin-induced lung allergic inflammation.

CC chemokine receptor 4 (CCR4) is expressed on Th2 cells, found in inflamed tissues of allergic diseases, and is therefore suspected to be involved in the pathogenesis of allergic diseases by controlling Th2 cell migration into inflamed tissues. The aim of the present study was to investigate the inhibitory effect of a selective CCR4 antagonist, K327 [6-cyclopropancarbonyl-4-(2,4-dichlorobenzylamino)-2-(4-[2-(piperidin-1-yl)ethyl] piperazin-1-yl)-7,8-dihydro-5H-pyrido (4,3-d)pyrimidine], on the recruitment of CCR4+CD4+ T cells to the airway of mice with ovalbumin-induced allergic airway inflammation. K327 was administered to mice in which CCR4+CD4+ T cell accumulation was elicited by multiple inhalations of aerosolized ovalbumin. K327 significantly and dose-dependently inhibited the recruitment of CCR4+CD4+ T cells with an ID(50 )value of 44 mg/kg, p.o. twice daily. The antiasthmatic potential of K327 was also demonstrated by the fact that K327 suppressed the elevation of Th2 cytokines and airway eosinophilia. These results indicate that CCR4 antagonists can control in vivo migration of Th2 cells which express CCR4 and, presumably, serve as a new class of therapeutic agent for allergy.

Functional genetic variation in the basal promoter of the organic cation/carnitine transporters OCTN1 (SLC22A4) and OCTN2 (SLC22A5).

The organic cation/ergothioneine transporter OCTN1 (SLC22A4) and the high-affinity carnitine transporter OCTN2 (SLC22A5), play an important role in the disposition of xenobiotics and endogenous compounds. Here, we analyzed the sequence of the proximal promoter regions of OCTN1 and OCTN2 in four ethnic groups and determined the effects of the identified genetic variants on transcriptional activities and mRNA expression. Six variants were found in the proximal promoter of OCTN1, one of which showed high allele frequency ranging from 13 to 34% in samples from individuals with ancestries in Africa, Europe, China, and Mexico. OCTN1 haplotypes had similar activities as the reference in luciferase reporter assays. For OCTN2, three of the seven variants identified in the proximal promoter showed allele frequencies greater than 29.5% in all populations, with the exception of -207C>G (rs2631367) that was monomorphic in Asian Americans. OCTN2 haplotypes containing -207G, present in all populations, were associated with a gain of function in luciferase reporter assays. Consistent with reporter assays, OCTN2 mRNA expression levels in lymphoblastoid cell lines (LCLs) from gene expression analysis were greater in samples carrying a marker for -207G. This SNP seems to contribute to racial differences in OCTN2 mRNA expression levels in LCLs. Our study with healthy subjects (n = 16) homozygous for either -207C or -207G, showed no appreciable effect of this SNP on carnitine disposition. However, there were significant effects of gender on carnitine plasma levels (p < 0.01). Further in vivo studies of OCTN2 promoter variants on carnitine disposition and variation in drug response are warranted.

Inhibition of oat3-mediated renal uptake as a mechanism for drug-drug interaction between fexofenadine and probenecid.

Fexofenadine, a nonsedating antihistamine drug, is effective for the treatment of seasonal allergic rhinitis and chronic urticaria. Simultaneous administration of probenecid increases the plasma concentration of fexofenadine due to an inhibition of its renal elimination in healthy volunteers (Clin Pharmacol Ther 77:17-23, 2005). The purpose of the present study is to investigate the possibility that the drug-drug interaction between fexofenadine and probenecid involves the renal basolateral uptake process. The uptake of fexofenadine was determined in HEK293 cells expressing human organic anion transporter 1 (OAT1/SLC22A6), OAT2 (SLC22A7), OAT3 (SLC22A8), and organic cation transporter 2 (OCT2/SLC22A2). Only hOAT3-HEK showed a significantly greater accumulation of fexofenadine than that in vector-HEK, which was saturable with K(m) and V(max) values of 70.2 microM and 120 pmol/min/mg protein, respectively. Inhibition potency of probenecid for the uptake of fexofenadine was compared between hOAT3 and organic anion-transporting peptide 1B3 (hOATP1B3), a transporter responsible for the hepatic uptake of fexofenadine (Drug Metab Dispos 33:1477-1481, 2005). The K(i) values were determined to be 1.30 and 130 microM for hOAT3 and hOATP1B3, respectively, with Hill coefficients of 0.76 and 0.64, respectively. The K(i) value of probenecid for hOAT3, but not for hOATP1B3, was significantly lower than the maximum unbound plasma concentration of probenecid at clinical dosages. These results suggest that the renal drug-drug interaction between fexofenadine and probenecid is probably explained by an inhibition of the renal uptake of fexofenadine via hOAT3, at least in part.

Is the monkey an appropriate animal model to examine drug-drug interactions involving renal clearance? Effect of probenecid on the renal elimination of H2 receptor antagonists.

The renal drug-drug interaction between famotidine (an H(2) receptor antagonist) and probenecid has not been reproduced in rats. We have proposed that this is caused by a species difference in the transport activity by human/rat organic anion transporter (OAT) 3 and the expression of organic cation transporter (OCT) 1 in the rodent kidney. Since monkey OATs (mkOATs) exhibit similar transport activities to human orthologs, it is hypothesized that in vivo studies in monkeys will allow a more precise prediction of renal drug-drug interactions in humans. Famotidine and cimetidine were efficiently taken up by mkOAT3-expressing human embryonic kidney cells (Km, 154 and 71 microM, respectively), and their uptake was strongly inhibited by probenecid (Ki, 3.0-5.7 microM). Quantification of mkOCT1 and mkOCT2 mRNAs in the monkey kidney using real-time reverse transcription-polymerase chain reaction revealed their predominant expression in the liver and kidney, respectively. Crossover studies were conducted in cynomolgus monkeys. Famotidine was given by i.v. administration, with or without probenecid. Probenecid treatment caused a 65% reduction in the renal clearance (0.426 +/- 0.079 versus 0.165 +/- 0.027 l/h/kg) and a 90% reduction in the tubular secretion clearance (0.275 +/- 0.075 versus 0.0230 +/- 0.0217 l/h/kg), whereas it had no effect on the renal clearance of cimetidine. In contrast to the species-dependent effect of probenecid, allometric scaling using animal data (rat, dog, and monkey) successfully predicted the renal and tubular secretion clearance of famotidine in humans. These results suggest that monkeys are more appropriate animal species for predicting the renal drug-drug interactions in humans.

A species difference in the transport activities of H2 receptor antagonists by rat and human renal organic anion and cation transporters.

A clinical drug-drug interaction between famotidine (a H2 receptor antagonist) and probenecid has not been reproduced in rats. The present study hypothesized that the species-dependent probenecid sensitivity is due to a species difference in the contribution of renal organic anion and cation transporters. The transport activities of the H2 receptor antagonists (cimetidine, famotidine, and ranitidine) by rat and human basolateral organic anion and cation transporters [human organic anion transporter (hOAT) 1, hOAT2, r/hOAT3, rat organic cation transporter (rOct) 1, and r/hOCT2] were compared using their cDNA transfectants. The transport activities (Vmax/Km) of famotidine (Km, 345 microM) by rOat3 were 8- and 15-fold lower than those of cimetidine (Km, 91 microM) and ranitidine (Km, 155 microM), respectively, whereas the activity by hOAT3 (Km, 124 microM) was 3-fold lower than that of cimetidine (Km, 149 microM) but similar to that of ranitidine (Km, 234 microM). Comparison of the relative transport activity with regard to that of cimetidine suggests that famotidine was more efficiently transported by hOAT3 than rOat3, and vice versa, for ranitidine. Only ranitidine was efficiently transported by hOAT2 (Km, 396 microM). rOct1 accepts all of the H2 receptor antagonists with a similar activity, whereas the transport activities of ranitidine and famotidine (Km, 61/56 microM) by r/hOCT2 were markedly lower than that of cimetidine (Km, 69/73 microM). Probenecid was a potent inhibitor of r/OAT3 (Ki, 2.6-5.8 microM), whereas it did not interact with OCTs. These results suggest that, in addition to the absence of OCT1 in human kidney, a species difference in the transport activity by hOAT3 and rOat3 accounts, at least in part, for the species difference in the drug-drug interaction between famotidine and probenecid.

Molecular cloning and functional analyses of OAT1 and OAT3 from cynomolgus monkey kidney.

PURPOSE: The functional characterization of monkey OAT1 (SLC22A6) and OAT3 (SLC22A8) was carried out to elucidate species differences in the OAT1- and OAT3-mediated transport between monkey and human. METHODS: The cDNAs of monkey OAT1 and OAT3 were isolated from monkey kidney, and their stable transfectants were established in HEK293 cells (mkOAT1- and mkOAT3-HEK). Transport studies were performed using cDNA transfectants, and kinetic parameters were compared among rat, monkey and human. RESULTS: The amino acid sequences of mkOAT1 and mkOAT3 exhibit 97% and 96% identity to their corresponding human orthologues. For OAT1, there was no obvious species difference in the K(m) values and the relative transport activities of 11 substrates with regard to p-aminohippurate transport. For OAT3, there was no species difference in the K(m) values and in the relative transport activities of nine substrates with regard to benzylpenicillin transport between monkey and human. However, the relative transport activities of indoxyl sulfate, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, and estrone-3-sulfate showed a difference between primates and rat and gave a poor correlation. CONCLUSIONS: These results suggest that monkey is a good predictor of the renal uptake of organic anions in the human.

P-glycoprotein plays a major role in the efflux of fexofenadine in the small intestine and blood-brain barrier, but only a limited role in its biliary excretion.

Fexofenadine is a selective, nonsedating H(1)-receptor antagonist approved for symptoms of allergic conditions, which is mainly excreted into feces via biliary excretion. The purpose of this study is to investigate its pharmacokinetics in mice and rats to determine the role of P-glycoprotein (P-gp) in its biliary excretion. In mice, biliary excretion clearance (17 ml/min/kg) accounted for almost 60% of the total body clearance (30 ml/min/kg). Comparing the pharmacokinetics after intravenous and oral administration indicated that the bioavailability of fexofenadine was at most 2% in mice. Knockout of Mdr1a/1b P-gp did not affect the biliary excretion clearance with regard to both plasma and liver concentrations, whereas the absence of P-gp caused a 6-fold increase in the plasma concentration after oral administration. In addition, the steady-state brain-to-plasma concentration ratio of fexofenadine was approximately 3-fold higher in Mdr1a/1b P-gp knockout mice than in wild-type mice. Together, these results show that P-glycoprotein plays an important role in efflux transport in the brain and small intestine but only a limited role in biliary excretion in mice. In addition, there was no difference in the biliary excretion between normal and hereditarily multidrug resistance-associated protein 2 (Mrp2)-deficient mutant rats (Eisai hyperbilirubinemic rats) and between wild-type and breast cancer resistance protein (Bcrp) knockout mice. These results suggest that the biliary excretion of fexofenadine is mediated by unknown transporters distinct from P-gp, Mrp2, and Bcrp.