Collaborative study using common samples to evaluate the performance of anti-drug antibody assays constructed by different companies.

This study was undertaken to evaluate the performance of anti-drug antibody (ADA) assays constructed by each participating company using common samples including ADA, drug and human serum. The ADA assays constructed by each company showed good sensitivity and precision for evaluation of ADA. Cut points for screening and confirmatory assays and assay selectivity were determined by various calculation methods. In evaluations of blind ADA samples, nearly similar results were obtained by the study companies in determinations of whether samples were positive or negative except at the lowest sample concentration (5 ng/mL). In measurement of drug tolerance, for almost samples containing ADA and drugs, more positive results were obtained in assays using acid dissociation compared to those without acid dissociation. Overall, the performance of ADA assays constructed by the 10 companies participating in this study was acceptable in terms of sensitivity and reproducibility for detection and evaluation of immunogenicity in both patients and healthy subjects. On the other hand, based on results for samples containing ADA and drugs, validity of results for ADA assays conducted without acid dissociation was less meaningful and more difficult to evaluate. Thus, acid dissociation was confirmed to be useful for improving drug tolerance.

Pharmacokinetics of miriplatin in experimental dog models of hepatic or renal impairment.

Miriplatin is an anticancer platinum complex for treatment of hepatocellular carcinomas by intra-hepatic arterial injection suspended in an iodinated ethyl ester of fatty acids from poppy seed oil as a carrier. Effects of liver and kidney function on( 14)C-miriplatin pharmacokinetics were assessed using dog models of hepatic and renal impairment introduced by thioacetamide exposure and 7/8 nephrectomy, respectively. Miriplatin was selectively delivered to the liver; platinum and radioactive component were gradually released into systemic circulation and excreted into urine. Microautoradiographic analysis of liver specimens showed( 14)C-miriplatin to be localized in blood vessels and/or macrophage-like cells. These features of miriplatin disposition were not affected by hepatic impairment. Thus, in clinical settings, hepatic impairment would not be expected to affect the intra-hepatic distribution and systemic pharmacokinetics of miriplatin. In dogs with renal impairment, although inconclusive, plasma concentrations of ultrafilterable platinum and radioactivity increased due to reduction in renal clearance.

Targeted disruption of the multidrug and toxin extrusion 1 (mate1) gene in mice reduces renal secretion of metformin.

Multidrug and toxin extrusion 1 (MATE1/SLC47A1) is important for excretion of organic cations in the kidney and liver, where it is located on the luminal side. Although its functional and regulatory characteristics have been clarified, its pharmacokinetic roles in vivo have yet to be elucidated. In the present study, to clarify the relevance of MATE1 in vivo, targeted disruption of the murine Mate1 gene was carried out. The lack of Mate1 expression in the kidney and liver was confirmed by reverse transcription-polymerase chain reaction and Western blot analysis. The mRNA levels of other organic cation transporters such as Octs did not differ significantly between wild-type [Mate1(+/+)] and Mate1 knockout [Mate1(-/-)] mice. It is noteworthy that the Mate1(-/-) mice were viable and fertile. Pharmacokinetic characterization was carried out using metformin, a typical substrate of MATE1. After a single intravenous administration of metformin (5 mg/kg), a 2-fold increase in the area under the blood concentration-time curve for 60 min (AUC(0-60)) of metformin in Mate1(-/-) mice was observed. Urinary excretion of metformin for 60 min after the intravenous administration was significantly decreased in Mate1(-/-) mice compared with Mate1(+/+) mice. The renal clearance (CL(ren)) and renal secretory clearance (CL(sec)) of metformin in Mate1(-/-) mice were approximately 18 and 14% of those in Mate1(+/+) mice, respectively. This is the first report to demonstrate an essential role of MATE1 in systemic clearance of metformin.

Regulatory mechanism governing the diurnal rhythm of intestinal H+/peptide cotransporter 1 (PEPT1).

The intestinal H(+)/peptide cotransporter 1 (PEPT1) plays important roles as a nutrient and drug transporter. Previously, we reported that rat intestinal PEPT1 showed a diurnal rhythm and that this rhythm is closely related to the feeding schedule. Furthermore, we also demonstrated that transcription factors, Sp1, Cdx2, and peroxisome proliferator-activated receptor-alpha (PPAR-alpha) contribute to the basal, intestine-specific, and fasting-induced expression of PEPT1, respectively. In this study, to clarify the molecular mechanism governing the diurnal rhythm of PEPT1 expression, we compared expression profiles of these transcription factors under two kinds of feeding schedules. The intestinal Sp1 and Cdx2 did not show a circadian accumulation of mRNA or response to the daytime feeding regimen. Plasma free fatty acids, endogenous PPAR-alpha ligands, exhibited a robust circadian fluctuation in phase with that of PEPT1. However, subsequent experiments using PPAR-alpha-null mice revealed the absence of any association between the circadian rhythm of PEPT1 and PPAR-alpha. We then focused on the clock genes (Clock, Bmal1, Per1-2, and Cry1) and clock-controlled gene, albumin D site-binding protein (DBP). A robust and coordinated circadian expression of the clock genes was observed, and daytime feeding entirely inverted the phase except for Clock. The expression of DBP was in phase with that of PEPT1 in both groups. Electrophoretic mobility shift assays and reporter assays revealed that DBP has the ability to bind the DBP binding site located in the distal promoter region of the rat PEPT1 gene and induce the transcriptional activity. These findings indicate that DBP plays pivotal roles in the circadian oscillation of PEPT1.

Induction of intestinal peptide transporter 1 expression during fasting is mediated via peroxisome proliferator-activated receptor alpha.

We previously demonstrated that starvation markedly increased the amount of mRNA and protein levels of the intestinal H+/peptide cotransporter (PEPT1) in rats, leading to altered pharmacokinetics of the PEPT1 substrates. In the present study, the mechanism underlying this augmentation was investigated. We focused on peroxisome proliferator-activated receptor alpha (PPARalpha), which plays a pivotal role in the adaptive response to fasting in the liver and other tissues. In 48-h fasted rats, the expression level of PPARalpha mRNA in the small intestine markedly increased, accompanied by the elevation of serum free fatty acids, which are endogenous PPARalpha ligands. Oral administration of the synthetic PPARalpha ligand WY-14643 to fed rats increased the mRNA level of intestinal PEPT1. Furthermore, treatment of the human intestinal model, Caco-2 cells, with WY-14643 resulted in enhanced PEPT1 mRNA expression and uptake activity of glycylsarcosine. In the small intestine of PPARalpha-null mice, augmentation of PEPT1 mRNA during fasting was completely abolished. In the kidney, fasting did not induce PEPT1 expression in either PPARalpha-null or wild-type mice. Together, these results indicate that PPARalpha plays critical roles in fasting-induced intestinal PEPT1 expression. In addition to the well-established roles of PPARalpha, we propose a novel function of PPARalpha in the small intestine, that is, the regulation of nitrogen absorption through PEPT1 during fasting.

The transcription factor Cdx2 regulates the intestine-specific expression of human peptide transporter 1 through functional interaction with Sp1.

H(+)/peptide cotransporter 1 (PEPT1, SLC15A1) localized at the brush-border membranes of intestinal epithelial cells plays important roles in the intestinal absorption of small peptides and a variety of peptidemimetic drugs. We previously demonstrated that transcription factor Sp1 functions as a basal transcriptional regulator of human PEPT1. However, the factor responsible for the intestine-specific expression of PEPT1 remains unknown. In the present study, we investigated the effect of the intestinal transcription factors on the transcription of the PEPT1 gene and found that only Cdx2 markedly trans-activated the PEPT1 promoter. However, the promoter region responsible for this effect lacked a typical Cdx2-binding sequence, but instead, possessed some Sp1-binding sites. In vitro experiments using Caco-2 cells showed that (1) mutation of the Sp1-binding site diminished the effect of Cdx2, (2) co-expression of Cdx2 and Sp1 synergistically trans-activated the PEPT1 promoter and (3) Sp1 protein was immunoprecipitated with Cdx2 protein. These results raise the possibility that Cdx2 modulates the PEPT1 promoter by interaction with Sp1. The significance of Cdx2 in vivo for PEPT1 regulation was shown by the determination of mRNA levels of Cdx2 and PEPT1 in human tissue. In gastric samples, some with intestinal metaplasia, the levels of PEPT1 and Cdx2 mRNA were highly correlated. Taken together, the present study suggests that Cdx2 plays a key role in the transcriptional regulation of the intestine-specific expression of PEPT1, possibly through interaction with Sp1.

Characterization of the human peptide transporter PEPT1 promoter: Sp1 functions as a basal transcriptional regulator of human PEPT1.

H+-coupled peptide transporter 1 (PEPT1, SLC15A1) localized at the brush-border membranes of intestinal epithelial cells plays an important role in the intestinal absorption of small peptides and a variety of peptidemimetic drugs. PEPT1 is regulated by various factors, including hormones, dietary conditions, some pharmaceutics, and diurnal rhythm. But there is little information about the transcriptional regulation of PEPT1. In the present study, therefore, we cloned the human (h)PEPT1 promoter region and examined its promoter activity using a human intestinal cell line, Caco-2. Deletion analysis of the hPEPT1 promoter suggested that the region spanning -172 to -35 bp was essential for basal transcriptional activity. This region lacked a TATA-box but contained some GC-rich sites that supposedly bind with the transcription factor Sp1. Mutational analysis revealed that three of these putative Sp1 sites contributed to the transcriptional activity. EMSA showed that Sp1 bound to two GC-rich sites. Furthermore, inhibition of Sp1 binding by mithramycin A treatment significantly reduced the transcriptional activity. Finally, overexpression of Sp1 increased the transcriptional activity in a dose-dependent manner. This study reports the first characterization of the hPEPT1 promoter and shows the significant role of Sp1 in the basal transcriptional regulation of hPEPT1.

In vitro drug-drug interactions with perospirone and concomitantly administered drugs in human liver microsomes.

In vitro metabolism studies were conducted to assess drug-drug interactions between perospirone, an antipsychotic agent, and concomitantly administered drugs--biperiden, flunitrazepam, haloperidol, and diazepam--using human liver microsomes. The metabolism of perospirone in the presence of 100 microg/ml drugs was decreased to 45-73% of that in their absence, whereas no effects were observed with any of the drugs at 1 microg/ml or lower. The effects of perospirone on the metabolism of concomitantly administered drugs were also assessed, and no inhibitory effect was observed. Thus, the metabolism of perospirone and concomitantly administered drugs did not demonstrate any marked mutual inhibition in the human liver microsomes. On the other hand, the perospirone metabolism was markedly reduced by ketoconazole indicating a major role for CYP 3A4. Based on the inhibition constant (Ki) for perospirone metabolism and the plasma unbound concentration of ketoconazole, in vivo perospirone clearance was estimated to be reduced to 64-90% of the control level. Thus careful attention should be paid to the possibility of increase in unchanged perospirone concentration when perospirone is co-administered with drugs that are known as CYP3A4 inhibitors, including macrolide antibiotics and other imidazole antifungals.