Tomoe Fujita

Functional analysis of polymorphisms in the organic anion transporter, SLC22A6 (OAT1)

Objectives The organic anion transporter, OAT1 (SLC22A6), plays a role in the renal elimination of many drugs and environmental toxins. The goal of this study was to identify and functionally characterize OAT1 variants as a first step towards understanding whether genetic variation in OAT1 may contribute to interindividual differences in renal elimination of xenobiotics. Methods As part of a larger study, 276 DNA samples from an ethnically diverse population were screened and 12 coding region variants of OAT1 were identified. The non-synonymous variants were then constructed and characterized in Xenopus laevis oocytes. A small family-based clinical study was conducted to determine the renal elimination of a model OAT1 substrate, adefovir (an antiviral agent) in human subjects who possessed a non-functional variant, OAT1-R454Q. Results Six non-synonymous variants were identified; two (OAT1-R50 H and OAT1-R293W) were present at ≥1% in at least one ethnic population. These two variants exhibited normal uptake of p-aminohippurate, ochratoxin A and methotrexate assayed in X. laevis oocytes. One variant, OAT1-R454Q, was non-functional with respect to the above substrates. In the clinical study, there was no significant decrease in the renal secretory clearance of adefovir in family members heterozygous for OAT1-454Q in comparison to those with the reference transporter, OAT1-454R. Conclusions These data indicate that the coding region of OAT1 has low genetic and functional diversity and suggest that coding region variants of OAT1 may not contribute substantially to interindividual differences in renal elimination of xenobiotics.

Suppressed angiogenesis in kininogen-deficiencies.

We investigated whether the kinin-generating system enhanced angiogenesis in chronic and proliferative granuloma and in tumor-surrounding stroma. In rat sponge implants, angiogenesis was gradually developed in normal Brown Norway Kitasato rats (BN-Ki). The development of angiogenesis was significantly suppressed in kininogen-deficient Brown Norway Katholiek rats (BN-Ka). The angiogenesis enhanced by basic fibroblast growth factor was also significantly less marked in BN-Ka than in BN-Ki. Naturally occurring angiogenesis was significantly suppressed by B1 or B2 antagonist. mRNA of vascular endothelial growth factor was more highly expressed in the granulation tissues in BN-Ki than in BN-Ka. Daily topical injections of aprotinin, but not of soy bean trypsin inhibitor, suppressed angiogenesis. Daily topical injections of low-molecular weight kininogen enhanced angiogenesis in BN-Ka. Topical injections of serum from BN-Ki, but not from BN-Ka, also facilitated angiogenesis in BN-Ka. FR190997, a nonpeptide mimic of bradykinin, promoted angiogenesis markedly, with concomitant increases in vascular endothelial growth factor mRNA. Angiogenesis in the granulation tissues around the implanted Millipore chambers containing Walker-256 cells was markedly more suppressed in BN-Ka than in BN-Ki. Our results suggest that endogenous kinin generated from the tissue kallikrein-kinin system enhances angiogenesis in chronic and proliferative granuloma and in the stroma surrounding a tumor. Thus, the agents for the kinin-generating system and/or kinin receptor signaling may become useful tools for controlling angiogenesis.

Pharmacodynamics and Pharmacokinetics of AMG 531, a Thrombopoiesis-Stimulating Peptibody, in Healthy Japanese Subjects: A Randomized, Placebo-Controlled Study

AMG 531 is a novel thrombopoiesis‐stimulating peptibody being investigated for the treatment of chronic immune thrombocytopenic purpura. This double‐blind, phase I study evaluated the safety, pharmacodynamics, and pharmacokinetics of AMG 531 in healthy Japanese men. Thirty subjects were randomly assigned 4:1 (AMG 531/placebo) to receive 1 dose of AMG 531 (0.3, 1, or 2 μg/kg) or placebo by subcutaneous injection; subjects were evaluated for 6 weeks. AMG 531 was generally well tolerated, with adverse events similar to placebo. Treatment‐related adverse events (headache, “feeling hot,” malaise) were reported for 5 of 24 AMG 531‐treated subjects. Platelets generated after exposure to AMG 531 functioned normally. Four of 8 subjects receiving 1 μg/kg and 7 of 8 receiving 2 μ/kg had platelet count increases ≥1.5‐fold over baseline, an effect similar to that seen in non‐Japanese subjects. Serum AMG 531 concentrations were below the lower limit of quantification in all but 2 subjects receiving 2 μg/kg.

Allergy medication in Japanese volunteers: treatment effect of single doses on nocturnal sleep architecture and next day residual effects.

ABSTRACT Objectives: To evaluate the acute effects of two histamine H1-receptor antagonists on nocturnal sleep architecture and on next day cognitive function and psychomotor performance. Methods: This was a single-site, randomized, double-blind, 3-way crossover study, comparing the effects of a single dose of chlorpheniramine (6 mg), fexofenadine (120 mg) and placebo in 18 healthy (male and female) Japanese volunteers aged 20–55 years. Volunteers were resident for 3 days and each period was separated by a minimum 5‐day washout period. The three treatments were administered at 23.00 h. Overnight sleep was measured from 23.00 h to 07.00 h using polysomnography. Residual effects were studied at 07.00 h and 9.00 h the next morning, with the latency to sleep (sleep latency test) measured at 09.30 h. Results: Compared with placebo, chlorpheniramine increased the latencies to sleep onset and rapid eye movement (REM) sleep ( p ≤ 0.05 for both), and reduced the duration of REM sleep ( p ≤ 0.01), but this was not observed with fexofenadine. As far as residual effects the next morning were concerned there were decrements in performance with chlorpheniramine, but not with fexofenadine. Chlorpheniramine 6 mg impaired divided attention ( p < 0.001), vigilance ( p < 0.05), working memory ( p < 0.0001) and sensori-motor performance ( p < 0.01), and the latency to daytime sleep was reduced ( p < 0.0001). Six adverse events possibly related to study medication were reported during the study, three of which were related to placebo, two to fexofenadine and one to chlorpheniramine. Conclusion: These findings suggest that a single nocturnal dose of fexofenadine has advantages over the first-generation antihistamine chlorpheniramine, being free of disruption of night-time sleep and detrimental effects on cognitive performance the next day. It is likely that this advantage will remain with chronic ingestion, but this would need to be confirmed.

Nonlinear Pharmacokinetics of Oral Quinidine and Verapamil in Healthy Subjects: A Clinical Microdosing Study

Microdosing studies are effective in enabling the early identification of the pharmacokinetic properties of compounds administered to humans. However, the nonlinearity of the pharmacokinetics between microdose and therapeutic dose, attributable to the saturation of metabolic enzymes and transporters, is a major concern. Verapamil and quinidine are good substrates of both the multidrug resistance 1 transporter (MDR1) and the cytochrome P450 (CYP) 3A4 enzyme (CYP3A4). We investigated their dose‐dependent pharmacokinetics in healthy subjects. Four different doses of verapamil or quinidine were administered orally to each subject, and the plasma concentrations of the parent drugs and their major metabolites were measured. The dose‐normalized area under the plasma concentration–time curve (AUC) values of quinidine and verapamil increased in a dose‐dependent manner and were 2.6‐ and 2.3‐fold higher, respectively, at the therapeutic dose than at microdose. These results suggest that the nonlinear pharmacokinetics of these drugs is caused mainly by the saturation of MDR1 and/or CYP3A4 in the small intestine.

Inhibitory effects of p-aminohippurate and probenecid on the renal clearance of adefovir and benzylpenicillin as probe drugs for organic anion transporter (OAT) 1 and OAT3 in humans

Probe substrates for, and inhibitors of, specific transporters are desired to evaluate quantitatively the in vivo functions of transporters in humans. Based on published data, adefovir and benzylpenicillin were selected as organic anion transporter (OAT) 1- and OAT3-selective probe substrates, respectively. In human kidney slices, probenecid potently inhibited the uptake of both adefovir and benzylpenicillin with inhibition constant (Ki) values of 18.6±5.1 and 12.6±4.2μM, respectively, whereas p-aminohippurate (PAH) preferentially inhibited adefovir uptake. A clinical drug-interaction study involving healthy subjects was performed to investigate the dose-dependent inhibition potencies of probenecid and PAH on the renal clearance of the probe substrates. Adefovir or benzylpenicillin was coadministered with different oral doses of probenecid (500, 750, or 1500mg) or intravenous PAH infusion rates (70, 120, or 210mg/min/person) to the same subject using a crossover design. The renal clearance of adefovir was reduced by 45% and 46% in the subjects treated with the maximum dose of probenecid and PAH, respectively, which was in accordance with the results of in vitro inhibition study. On the other hand, renal clearance of benzylpenicillin was reduced by 78% in the subjects treated with the maximum dose of probenecid (1500mg), which could be explained by its in vitro Ki values. However, PAH unexpectedly increased the renal clearance of benzylpenicillin by 47%. These results suggest that adefovir and benzylpenicillin can be used as probe drugs for OAT1 and OAT3, respectively, and that PAH can be used to investigate the role of OAT1 in the urinary excretion of drugs in humans, whereas it may modulate other transport processes in the kidney.

Effect of MKC‐733, a 5‐HT3 receptor partial agonist, on bowel motility and symptoms in subjects with constipation: an exploratory study

Background:  MKC‐733, a 5‐HT3 receptor partial agonist, is a novel enteroprokinetic compound.

In vivo transfer of antisense oligonucleotide against urinary kininase blunts deoxycorticosterone acetate-salt hypertension in rats

We have previously reported that the renal kallikrein‐kinin system suppressed the development of deoxycorticosterone acetate (DOCA)‐salt hypertension. Kinins were degraded in the kidney mainly by carboxypeptidase Y (CPY)‐like kininase. Blockade of renal kinin degradation may reduce hypertension in the developmental stage. We constructed an antisense oligonucleotide against rat CPY homologue (5′‐CAT‐CTC‐TGC‐TTC‐CTT‐GTG‐TC‐3′, AS) and its randomized control oligonucleotide (5′‐TCC‐TTC‐CTG‐CTT‐GAG‐TTC‐CT‐3′, RC), and prepared an HVJ‐liposome complex that prolongs and increases the effectiveness of the antisense oligonucleotide. Antisense oligonucleotide was transfected (25 nmole rat−1, in terms of nucleotide) into the kidney from the renal artery. Blood pressure was measured through a catheter inserted into the abdominal aorta. Mean blood pressure (MBP) in DOCA‐salt treated (for 2 weeks) Sprague Dawley strain rats was 130±3 mmHg (n=11), and was reduced significantly (P<0.05) more by AS transfection (122±4 mmHg, n=6) than by RC treatment (137±6 mmHg, n=5) 4 days after the transfection. This reduction in MBP was accompanied by increased urinary sodium excretion (AS, 8.4±1.5 mmole day−1; RC, 4.6±0.5 mmole day−1, P<0.05) and a reduction in urinary CPY‐like kininase activity. Ebelactone B (5 mg kg−1, twice a day, p.o.), an inhibitor for urinary CPY‐like kininase, also reduced MBP and induced natriuresis to the same degree as AS. Lisinopril, an inhibitor for angiotensin converting enzyme (ACE) failed to reduce the elevated MBP. These results suggest that CPY‐like kininase may have more contribution than ACE to degrade kinin in the kidney, and that knockdown of CPY‐like kininase in the kidney may partly prevent rat DOCA‐salt hypertension.

Microdose study of a P‐glycoprotein substrate, fexofenadine, using a non‐radioisotope‐labelled drug and LC/MS/MS

Objective:  Fexofenadine is a P‐glycoprotein substrate of low bioavailability. It is primarily excreted into faeces as a parent drug via biliary excretion. The predictability from microdose data for the drug absorbed via transporters such as P‐glycoprotein is not known. Therefore, this study assessed the predictability of therapeutic‐dose pharmacokinetics of fexofenadine from microdosing data using non‐radioisotope‐labelled drug and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS).

A Nonpeptide Mimic of Bradykinin Blunts the Development of Hypertension in Young Spontaneously Hypertensive Rats

We tested whether FR190997, a nonpeptide B(2) agonist, prevented the development of hypertension in young spontaneously hypertensive rats (SHR), which secrete less kallikrein into the urine than do Wistar-Kyoto rats. An intra-arterial (IA) injection of FR190997 (0.3 to 30 nmol/kg) caused dose-dependent hypotension in conscious Sprague-Dawley rats. Although the maximum hypotensive potency of FR190997 equaled that of bradykinin, its action lasted approximately 10 times as long. Hoe140 (100 nmol/kg IA) significantly blocked the hypotensive response induced by FR190997 (10 nmol/kg). Atropine (100 nmol/kg IA) did not affect this response. A selective infusion of FR190997 into the renal artery induced natriuresis and diuresis in anesthetized rabbits. A continuous infusion (2 nmol. 10 mL(-1). h(-1) per rat) of FR190997 into the abdominal aorta of young SHR (6 weeks old, n=6) for 6 days significantly (P<0.05) reduced mean blood pressure to 114+/-6 (day 2) and 110+/-6 (day 5) mm Hg, from 149+/-7 and 162+/-6 mm Hg, respectively, in vehicle-infused rats (n=6). At 8 days after continuous infusion (day 14), mean blood pressure (148+/-5 mm Hg) in FR190997-infused rats remained significantly (P<0. 05) lower than that in vehicle-infused rats (190+/-6 mm Hg), almost the peak value. The mesenteric artery isolated from FR190997-treated rats (day 14) had lower contractile sensitivity to norepinephrine than that from vehicle-treated rats. These results suggested that the continuous infusion of a nonpeptide B(2) agonist may prevent hypertension if performed in the critical phase.