Genome-Wide Association Study Identifies Genetic Polymorphisms Associated with Estimated Minimum Effective Concentration of Fentanyl in Patients Undergoing Laparoscopic-Assisted Colectomy.

Sensitivity to opioids varies widely among individuals. To identify potential candidate single-nucleotide polymorphisms (SNPs) that may significantly contribute to individual differences in the minimum effective concentration (MEC) of an opioid, fentanyl, we conducted a three-stage genome-wide association study (GWAS) using whole-genome genotyping arrays in 350 patients who underwent laparoscopic-assisted colectomy. To estimate the MEC of fentanyl, plasma and effect-site concentrations of fentanyl over the 24 h postoperative period were estimated with a pharmacokinetic simulation model based on initial bolus doses and subsequent patient-controlled analgesia doses of fentanyl. Plasma and effect-site MECs of fentanyl were indicated by fentanyl concentrations, estimated immediately before each patient-controlled analgesia dose. The GWAS revealed that an intergenic SNP, rs966775, that mapped to 5p13 had significant associations with the plasma MEC averaged over the 6 h postoperative period and the effect-site MEC averaged over the 12 h postoperative period. The minor G allele of rs966775 was associated with increases in these MECs of fentanyl. The nearest protein-coding gene around this SNP was DRD1, encoding the dopamine D1 receptor. In the gene-based analysis, the association was significant for the SERP2 gene in the dominant model. Our findings provide valuable information for personalized pain treatment after laparoscopic-assisted colectomy.

Physiologically-based pharmacokinetic model-based translation of OATP1B-mediated drug-drug interactions from coproporphyrin I to probe drugs.

The accurate prediction of OATP1B-mediated drug-drug interactions (DDIs) is challenging for drug development. Here, we report a physiologically-based pharmacokinetic (PBPK) model analysis for clinical DDI data generated in heathy subjects who received oral doses of cyclosporin A (CysA; 20 and 75 mg) as an OATP1B inhibitor, and the probe drugs (pitavastatin, rosuvastatin, and valsartan). PBPK models of CysA and probe compounds were combined assuming inhibition of hepatic uptake of endogenous coproporphyrin I (CP-I) by CysA. In vivo Ki of unbound CysA for OATP1B (Ki,OATP1B ), and the overall intrinsic hepatic clearance per body weight of CP-I (CLint,all,unit ) were optimized to account for the CP-I data (Ki,OATP1B , 0.536 ± 0.041 nM; CLint,all,unit , 41.9 ± 4.3 L/h/kg). DDI simulation using Ki,OATP1B reproduced the dose-dependent effect of CysA (20 and 75 mg) and the dosing interval (1 and 3 h) on the time profiles of blood concentrations of pitavastatin and rosuvastatin, but DDI simulation using in vitro Ki,OATP1B failed. The Cluster Gauss-Newton method was used to conduct parameter optimization using 1000 initial parameter sets for the seven pharmacokinetic parameters of CP-I (ß, CLint, all , Fa Fg , Rdif , fbile , fsyn , and vsyn ), and Ki,OATP1B and Ki,MRP2 of CysA. Based on the accepted 546 parameter sets, the range of CLint, all and Ki,OATP1B was narrowed, with coefficients of variation of 12.4% and 11.5%, respectively, indicating that these parameters were practically identifiable. These results suggest that PBPK model analysis of CP-I is a promising translational approach to predict OATP1B-mediated DDIs in drug development.

Effect of Cyclosporin A and Impact of Dose Staggering on OATP1B1/1B3 Endogenous Substrates and Drug Probes for Assessing Clinical Drug Interactions.

This study was designed to assess the quantitative performance of endogenous biomarkers for organic anion transporting polypeptide (OATP) 1B1/1B3-mediated drug-drug interactions (DDIs). Ten healthy volunteers orally received OATP1B1/1B3 probe cocktail (0.2 mg pitavastatin, 1 mg rosuvastatin, and 2 mg valsartan) and an oral dose of cyclosporin A (CysA, 20 mg and 75 mg) separated by a 1-hour interval (20 mg (-1 hour), and 75 mg (-1 hour)). CysA 75 mg was also given with a 3-hour interval (75 mg (-3 hours)) to examine the persistence of OATP1B1/1B3 inhibition. The area under the plasma concentration-time curve ratios (AUCRs) were 1.63, 3.46, and 2.38 (pitavastatin), 1.39, 2.16, and 1.81 (rosuvastatin), and 1.42, 1.77, and 1.85 (valsartan), at 20 mg, 75 mg (-1 hour) and 75 mg (-3 hours) of CysA, respectively. CysA effect on OATP1B1/1B3 was unlikely to persist at the dose examined. Among 26 putative OATP1B1/1B3 biomarkers evaluated, AUCR and maximum concentration ratio (Cmax R) of CP-I showed the highest Pearson's correlation coefficient with CysA AUC (0.94 and 0.93, respectively). Correlation between AUCR of pitavastatin, and Cmax R or AUCR of CP-I were consistent between this study and our previous study using rifampicin as an OATP1B1/1B3 inhibitor. Nonlinear regression analysis of AUCR-1 of pitavastatin and CP-I against CysA Cmax yielded Ki,OATP1B1/1B3,app (109 ± 35 and 176 ± 42 nM, respectively), similar to the Ki ,OATP1B1/1B3 estimated by our physiologically-based pharmacokinetic model analysis described previously (107 nM). The endogenous OATP1B1/1B3 biomarkers, particularly Cmax R and AUCR of CP-I, corroborates OATP1B1/1B3 inhibition and yields valuable information that improve accurate DDI predictions in drug development, and enhance our understanding of interindividual variability in the magnitude of DDIs.

Comparison of pharmacokinetics of newly discovered aromatase inhibitors by a cassette microdosing approach in healthy Japanese subjects.

The aim of the present study is to investigate the pharmacokinetics of our newly developed aromatase inhibitors (cetrozole and TMD-322) in healthy subjects by a cassette microdose strategy. A cocktail of cetrozole and TMD-322 was administered intravenously or orally (1.98 µg for each drug) to six healthy volunteers in a crossover fashion. Anastrozole (1.98 µg) was also included in the oral cocktail. Total body clearance and bioavailability were 12.1 ± 7.1 mL/min/kg and 34.9 ± 32.3% for cetrozole, and 16.8 ± 3.5 mL/min/kg and 18.4 ± 12.2% for TMD-322, respectively. The area under the plasma concentration-time curves of cetrozole and TMD-322 after oral administration was markedly lower than that of anastrozole because of their high hepatic clearance. Two subjects out of six exhibited 4- and 17-fold larger exposure of cetrozole than the others following intravenous and oral administration, respectively. Such variation was not observed for TMD-322 and anastrozole. Extensive metabolism of cetrozole and TMD-322 was observed in the CYP2C19 expression system among the test CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). We report the first clinical investigation of our aromatase inhibitors by a cassette microdose strategy in healthy Japanese subjects. This strategy offers an optional approach for candidate selection as a phase zero study in drug development.

Evaluation of Oatp and Mrp2 activities in hepatobiliary excretion using newly developed positron emission tomography tracer [11C]dehydropravastatin in rats.

We developed a pravastatin derivative, sodium (3R,5R)-3,5-dihydroxy-7-((1S,2S,6S,8S)-6-hydroxy-2-methyl-8-((1-[(11)C]-(E)-2-methyl-but-2-enoyl)oxy)-1,2,6,7,8,8a-hexahydronaphthalen-1-yl)heptanoate ([(11)C]DPV), as a positron emission tomography (PET) probe for noninvasive measurement of hepatobiliary transport, and conducted pharmacokinetic analysis in rats as a feasibility study for future clinical study. Transport activities of DPV in freshly isolated rat hepatocytes and rodent multidrug resistance-associated protein 2 (rMrp2; human, MRP2)-expressing membrane vesicles were similar to those of pravastatin. Rifampicin diminished the uptake of DPV and pravastatin by the hepatocytes, with similar inhibition potency. [(11)C]DPV underwent biotransformation to produce at least two metabolites in rat, but metabolism of [(11)C]DPV occurred negligibly in human hepatocytes during a 90-minute incubation. After intravenous injection, [(11)C]DPV was mainly distributed to the liver and kidneys, where the tissue uptake clearances (CLuptake,liver and CLuptake,kidney) were blood-flow-limited (73.6 ± 4.8 and 24.6 ± 0.6 ml/min per kilogram, respectively). Systemic elimination of [(11)C]DPV was delayed in rifampicin-treated rat and an Mrp2-deficient mutant rat, Eisai hyperbilirubinemic mutant rat (EHBR). Rifampicin treatment decreased both CLuptake,liver and CLuptake,kidney of [(11)C]DPV by 30% (P < 0.05), whereas these parameters were unchanged in EHBR. Meanwhile, the canalicular efflux clearance (CLint,bile) of [(11)C]DPV, which was 12.2 ± 1.5 ml/min per kilogram in the control rat, decreased by 60% and 89% in rifampicin-treated rat and EHBR (P < 0.05), respectively. These results indicate that [(11)C]DPV is taken up into the liver by organic anion-transporting polypeptides (rodent, Oatps; human, OATP) and excreted into bile by Mrp2 in rat, and that rifampicin may inhibit Mrp2 as well as Oatps, and consequently increase systemic exposure of [(11)C]DPV. PET using [(11)C]DPV is feasible for studies prior to the future clinical investigation of OATP and MRP2 functionality, especially for personalized medicine.

Investigation of the effect of active efflux at the blood-brain barrier on the distribution of nonsteroidal aromatase inhibitors in the central nervous system.

The brain distribution of nonsteroidal aromatase inhibitors was investigated in mice to understand their interactions with brain aromatase. The brain-to-plasma ratio (Kp,brain , mL/g brain) of anastrozole was 0.0299 ± 0.0068, which was lower than that of letrozole (0.383 ± 0.048) and vorozole (0.185 ± 0.031) despite their similar physicochemical properties. The brain-to-plasma unbound concentration ratio of anastrozole, measured using microdialysis, was 0.118 ± 0.037 mL/g brain. In situ mouse brain perfusion also demonstrated that the uptake clearance [mL/(min·g brain)] of anastrozole by the brain (0.108 ± 0.018) was lower than that for letrozole and vorozole (0.422 ± 0.068 and 0.910 ± 0.152, respectively). Anastrozole and vorozole were transported by P-glycoprotein (P-gp) in vitro, whereas none of the compounds were transported by breast cancer resistance protein (BCRP). The Kp,brain of anastrozole and vorozole were increased by 12- and 3.3-fold, respectively, in Mdr1a/b/Bcrp(-/-) mice. IC50 (nM) of anastrozole and letrozole against human aromatase was 12.9 ± 0.7 and 3.59 ± 0.75, respectively. Taken together, these results suggest that active efflux mediated by P-gp at the blood-brain barrier limits the effect of anastrozole in the central nervous system, whereas vorozole and letrozole easily traverse the barrier.

Dynamic analysis of fluid distribution in the gastrointestinal tract in rats: positron emission tomography imaging after oral administration of nonabsorbable marker, [(18)F]Deoxyfluoropoly(ethylene glycol).

To develop potent drugs for oral use, information on their pharmacokinetic (PK) properties after oral administration is of great importance. We have recently reported the utility of positron emission tomography (PET) for the analysis of gastrointestinal (GI) absorption of radiolabeled compounds. In this study, PET image analysis was performed in rats using a novel PET probe, [(18)F]deoxyfluoropoly(ethylene glycol)s, with an average molecular weight of 2 kDa ([(18)F]FPEG), as a nonabsorbable marker to elaborate the GI physiology in more detail, such as segmental transition of the administered water, and fluid volume and distribution in the intestine. After oral administration of [(18)F]FPEG solution to rats, a 90 min PET scan with continuous blood sampling was performed, and then the disposition of radioactivity in each part of GI tract was investigated. From blood PK analysis, it was confirmed that the bioavailability of [(18)F]FPEG was quite low in rats. PET image analysis showed that the radioactivity after oral administration of [(18)F]FPEG solution rapidly passed through the stomach, spread into the proximal small intestine, and then transited toward the distal region of the small intestine without decreasing the radioactivity during GI transition. Radiometabolite analysis revealed that the radioactivity in intestinal mucosal tissues, blood, and urine was mainly derived from unchanged [(18)F]FPEG. It was also found that the volume of interest (VOI) after oral administration of the radiotracer enables an understanding of the time-dependent manner of effective fluid volume changes in the stomach and the small intestine. In addition, the rate constant of the intestinal transition of radioactivity in each intestinal segment was calculated by kinetic model analysis, which revealed that PET analysis enables us to determine the GI transit from the same individuals and that it is applicable to determine site-specific intestinal absorption. In conclusion, we demonstrated the high potency of PET imaging technique to elucidate the distribution of orally administered solution in the GI tract in vivo.

Evaluation of breast cancer resistance protein function in hepatobiliary and renal excretion using PET with 11C-SC-62807.

UNLABELLED: A quantitative PET imaging method was used to assess the in vivo kinetics of hepatobiliary and renal excretion of the breast cancer resistance protein (Bcrp) substrate (11)C-SC-62807 in mice. METHODS: Serial abdominal PET scans were collected in wild-type and Bcrp knockout (Bcrp(-/-)) mice after intravenous injection of (11)C-SC-62807. Venous blood samples and PET images were obtained at frequent intervals up to 30 min after radiotracer administration. Dynamic PET data were analyzed to determine the canalicular and brush-border efflux clearances in the liver and kidney (CL(int,bile,liver) and CL(int,urine,kidney), respectively). RESULTS: SC-62807 is an in vitro substrate of mouse Bcrp and human BCRP. Radioactivity associated with (11)C-SC-62807 was predominantly found in the blood, liver, bile, and urine 30 min after administration. Both biliary and urinary excretion of radioactivity was markedly lower in Bcrp(-/-) mice than in wild-type mice, suggesting greater systemic exposure in Bcrp(-/-) mice. Both the CL(int,bile,liver) and the CL(int,urine,kidney) were significantly lower in Bcrp(-/-) mice (74% ± 10% and 99% ± 1% lower than controls, respectively). We also found that (11)C-SC-62807 is a substrate of the organic anion-transporting polypeptides OATP1B1 and OATP1B3 in vitro. CONCLUSION: The present study demonstrated that Bcrp plays a significant role in the efflux of (11)C-SC-62807 in mouse liver and kidney. We also demonstrated the feasibility of PET using (11)C-SC-62807 to study the activity of BCRP in humans.

Synthesis of [(11)C]dehydropravastatin, a PET probe potentially useful for studying OATP1B1 and MRP2 transporters in the liver.

Drug transporters mediate the uptake and elimination of drugs in various organs; therefore, having knowledge of how a transporter functions in the body would play a key role in ensuring drug efficacy in in vivo systems. In this context, we designed and synthesized [(11)C]dehydropravastatin, a novel PET probe that would be potentially useful for evaluation of the functions of the OATP1B1 and MRP2 transporters, based on the use of palladium(0)-mediated rapid C-[(11)C]methylation (viz., the rapid cross-coupling between [(11)C]methyl iodide and a boron intermediate).

PET imaging-based evaluation of hepatobiliary transport in humans with (15R)-11C-TIC-Me.

UNLABELLED: It is well accepted that drug transporters play a pivotal role in hepatobiliary excretion of anionic drugs, in which drug-drug interactions and genetic polymorphisms are known to cause variations. However, PET probes for in vivo functional characterization of these transporters have not been established yet. We used PET to investigate hepatic uptake and subsequent canalicular efflux of (11)C-labeled (15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin methyl ester [(15R)-(11)C-TIC-Me)] in healthy subjects. METHODS: Serial PET scans of the abdominal region in healthy male subjects were obtained with or without the organic anion-transporting polypeptide (OATP) inhibitor rifampicin after intravenous injection of (15R)-(11)C-TIC-Me as a radiotracer. Venous blood samples and PET images were obtained at frequent intervals up to 30 min after administration of the PET tracer. Dynamic imaging data were evaluated by integration plots of data collected for 2-10 min and for 10-30 min after tracer administration for the determination of tissue uptake clearance and biliary efflux clearance, respectively. RESULTS: After rapid hydrolysis in blood, the acid form-(11)C-labeled (15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin [(15R)-(11)C-TIC]-accumulated in the liver (37% of the dose by 17 min), and the radioactivity was then excreted into the bile (6.2% by 30 min). Rifampicin (600 mg by mouth), a potent OATP inhibitor, significantly reduced the radioactivity excreted into the bile (by 44%) by inhibiting both uptake (by 45%) and subsequent canalicular efflux (by 62%). (15R)-(11)C-TIC is an in vitro substrate of OATP1B1 and OATP1B3, and clinically relevant concentrations of rifampicin inhibited uptake by OATP1B1 and OATP1B3. These results demonstrated that in humans, (15R)-(11)C-TIC-associated radioactivity is excreted into the bile by organic anion transport systems. CONCLUSION: We demonstrated that PET image analysis with (15R)-(11)C-TIC-Me is useful for investigating variations in OATP function in the human hepatobiliary transport system.

Whole-body distribution and radiation dosimetry of [11C]telmisartan as a biomarker for hepatic organic anion transporting polypeptide (OATP) 1B3.

INTRODUCTION: Telmisartan, a nonpeptide angiotensin II AT1 receptor antagonist used as an antihypertensive drug, is specifically taken up by the liver through the OATP1B3. PET imaging with [(11)C]telmisartan is expected to provide information about the whole body pharmacokinetics of telmisartan as well as its transport property by OATP1B3. The purpose of the study was to determine the biodistribution and radiation dosimetry of [(11)C]telmisartan in humans. METHODS: Biodistribution of [(11)C]telmisartan was measured in three rats and six healthy male human volunteers. In the rat study, a dynamic emission scan was performed for 90 min. In the human study, dynamic whole-body PET images were acquired after intravenous injection of [(11)C]telmisartan. ROIs were defined for source organs on the PET images to measure time-course of [(11)C]telmisartan uptake as percentage injected dose and the number of disintegration for each organ. Radiation dosimetry was calculated with OLINDA/EXM. RESULTS: In the rat study, most radioactivity was rapidly taken up by the liver and part of it was excreted into the biliary tract and intestine. Extrapolating from the rat data, the effective dose for the adult human being was estimated to be 3.65±0.01 microSv/MBq (n=3). In the human study, most of the tracer was taken up by the liver as well, although not as rapidly as in the rat. The activity in the gall bladder and intestine increased gradually. The effective dose for the adult human being was 4.24±0.09 microSv/MBq (n=6). CONCLUSIONS: [(11)C]Telmisartan is a safe PET tracer with a dosimetry profile comparable to other common (11)C PET tracers.

The involvement of organic anion transporting polypeptide in the hepatic uptake of telmisartan in rats: PET studies with [¹¹C]telmisartan.

Telmisartan, a selective angiotensin II receptor antagonist, is primarily excreted via hepatobiliary transport. The predominant contribution of organic anion transporting polypeptide (OATP) 1B3 in its hepatic uptake of telmisartan has been demonstrated by in vitro transport studies. In the present study, a quantitative positron emission tomography (PET) methodology was developed for in vivo kinetic assessment of hepatobiliary transport of telmisartan. Serial abdominal PET scans were performed in rats following intravenous administration of [(11)C]telmisartan as a radiotracer. PET scans revealed that [(11)C]telmisartan was localized primarily in the liver and some of the radioactivity moved to the intestine, which corresponds to biliary excretion. Radiometabolite analysis by radiometric HPLC showed that [(11)C]telmisartan was converted to its acylglucuronide, which was mainly detected in bile, but little in plasma and liver. Integration plot analysis revealed that [(11)C]telmisartan was taken up into the liver as rapidly as the hepatic blood flow rate, and the radiometabolite was subsequently excreted into the bile. When rifampicin, a typical Oatp inhibitor, was coadministered with [(11)C]telmisartan in rats, hepatic uptake clearance of [(11)C]telmisartan was significantly decreased, whereas biliary efflux clearance was not changed. Coinjection with unlabeled telmisartan (4 and 10 mg/kg) also decreased hepatic uptake clearance of [(11)C]telmisartan. On the other hand, PET imaging analysis revealed a significant increase of biliary efflux when telmisartan dose was increased to more than 4 mg/kg. These results suggested that the hepatic uptake of [(11)C]telmisartan mainly consists of a saturable process mediated by Oatps in rats, according to noninvasive real-time measurement of tissue radioactivity with the use of PET. The present study with rats is expected to provide the feasibility of PET imaging study to quantitatively estimate OATP1B3 function in humans.

In vivo expression of cyclooxygenase-1 in activated microglia and macrophages during neuroinflammation visualized by PET with 11C-ketoprofen methyl ester.

UNLABELLED: Cyclooxygenase (COX)-1 and -2 are prostanoid-synthesizing enzymes that play important roles in the regulation of neuroinflammation and in the development of neurodegenerative disorders. However, the specific functions of these isoforms are still unclear. We recently developed (11)C-labeled ketoprofen methyl ester as a PET probe that targets the COXs for imaging neuroinflammation, though its responsible isoform is yet to be determined. In the present study, we performed ex vivo and in vivo imaging studies with (11)C-ketoprofen methyl ester and determined the contributions of the COX isoforms during the neuroinflammatory process. METHODS: To identify the COX isoform responsible for (11)C-ketoprofen methyl ester in the brain, we examined the ex vivo autoradiography of (11)C-ketoprofen methyl ester using COX-deficient mice. Time-dependent changes in accumulation of (11)C-ketoprofen methyl ester during the neuroinflammatory process were evaluated by PET in rats with hemispheric neuroinflammation induced by intrastriatal injection of lipopolysaccharide or quinolinic acid. In both rat models, cell-type specificity of COX isoform expression during neuroinflammation was identified immunohistochemically. RESULTS: Ex vivo autoradiographic analysis of COX-deficient mice revealed a significant reduction of (11)C-ketoprofen methyl ester accumulation only in COX-1-deficient mice, not COX-2-deficient mice. PET of rats after intrastriatal injection of lipopolysaccharide showed a significant increase in accumulation of (11)C-ketoprofen methyl ester in the inflamed area. This increase was evident at the early phase of 6 h, peaked at day 1, and then returned to basal levels by day 7. In addition, immunohistochemical analysis revealed that the population of activated microglia and macrophages was elevated at the early phase with COX-1 expression but not COX-2. A significant increase in (11)C-ketoprofen methyl ester accumulation was also observed at day 1 after intrastriatal injection of quinolinic acid, with increased COX-1-expressing activated microglia and macrophages. CONCLUSION: We have identified (11)C-ketoprofen methyl ester as a COX-1-selective PET probe, and using this, we have also demonstrated a time-dependent expression of COX-1 in activated microglia and macrophages during the neuroinflammatory process in the living brain. Thus, COX-1 may play a crucial role in the pathology of neuroinflammation and might be a critical target for the diagnosis and therapy of neurodegenerative disorders.

Increase in hypothalamic aromatase in macaque monkeys treated with anabolic-androgenic steroids: PET study with [11C]vorozole.

In an earlier study in rodents, we showed that the aromatase that converts androgens to estrogens in the preoptic area and bed nucleus of stria terminalis was significantly increased in concentration after exposure to anabolic-androgenic steroids. To confirm whether this occurs in primates, we conducted a positron emission tomographic study using macaque monkeys. Male rhesus monkeys were treated with nandrolone decanoate for 3 weeks. To measure aromatase concentrations, we performed positron emission tomographic imaging using a 11C-labeled specific aromatase inhibitor, [11C]vorozole. After treatment with nandrolone, significant increase in [11C]vorozole binding was observed in the hypothalamus but not other areas including the amygdala, which is also aromatase enriched. These findings in monkeys are consistent with those we obtained earlier in rats. These findings strongly suggest that aromatase in the hypothalamus may play a crucial role in the emotional instability of anabolic-androgenic steroids abusers.

Structure-activity relationship study of novel NR2B-selective antagonists with arylamides to avoid reactive metabolites formation.

A novel potent NMDA-NR2B selective antagonist without the reactive metabolites formation issue was identified. Through this study, a close correlation between reactive metabolites formation and calculated HOMO energies of parent compounds was found.