A new safety index based on intrapulse monitoring of ultra-harmonic cavitation during ultrasound-induced blood-brain barrier opening procedures.

Ultrasound-induced blood-brain barrier (BBB) opening using microbubbles is a promising technique for local delivery of therapeutic molecules into the brain. The real-time control of the ultrasound dose delivered through the skull is necessary as the range of pressure for efficient and safe BBB opening is very narrow. Passive cavitation detection (PCD) is a method proposed to monitor the microbubble activity during ultrasound exposure. However, there is still no consensus on a reliable safety indicator able to predict potential damage in the brain. Current approaches for the control of the beam intensity based on PCD employ a full-pulse analysis and may suffer from a lack of sensitivity and poor reaction time. To overcome these limitations, we propose an intra-pulse analysis to monitor the evolution of the frequency content during ultrasound bursts. We hypothesized that the destabilization of microbubbles exposed to a critical level of ultrasound would result in the instantaneous generation of subharmonic and ultra-harmonic components. This specific signature was exploited to define a new sensitive indicator of the safety of the ultrasound protocol. The approach was validated in vivo in rats and non-human primates using a retrospective analysis. Our results demonstrate that intra-pulse monitoring was able to exhibit a sudden appearance of ultra-harmonics during the ultrasound excitation pulse. The repeated detection of such a signature within the excitation pulse was highly correlated with the occurrence of side effects such as hemorrhage and edema. Keeping the acoustic pressure at levels where no such sign of microbubble destabilization occurred resulted in safe BBB openings, as shown by MR images and gross pathology. This new indicator should be more sensitive than conventional full-pulse analysis and can be used to distinguish between potentially harmful and safe ultrasound conditions in the brain with very short reaction time.

The impact of earlier intervention by an antimicrobial stewardship team for specific antimicrobials in a single weekly intervention.

OBJECTIVE: The objective of this study was to evaluate the effects of earlier intervention by an antimicrobial stewardship team (AST) on antimicrobial use, antimicrobial resistance rates, and the clinical outcomes, without changing the weekly intervention schedule. METHODS: A retrospective study was conducted at Fukuoka University Hospital between April 2013 and March 2016. The effects were compared among three study periods (SP): SP1 (patients receiving anti-methicillin-resistant Staphylococcus aureus agents and carbapenems for ≥14 days), SP2 (patients receiving specific antimicrobials for ≥14 days), and SP3 (patients receiving specific antimicrobials regardless of the duration of treatment). RESULTS: The timing of AST intervention was shortened from an average of 15.5days after administration in SP1 to 4.2 days in SP3. The antimicrobial use density (AUD) of carbapenems and piperacillin-tazobactam decreased significantly (SP2 vs. SP3, p<0.05), and the costs of specific antimicrobials decreased (SP1, US$ 1080000; SP2, US$ 944000; SP3, US$ 763000). The rates of carbapenem resistance among Pseudomonas aeruginosa isolates showed a significant reduction from 16.2% in SP2 to 8.7% in SP3 (p<0.05). The mortality rate and length of stay did not change during the study period. CONCLUSIONS: Earlier intervention by an AST could contribute to the proper use of antimicrobials without adversely affecting patient outcomes.

Optical and transport properties correlation driven by amorphous/crystalline disorder in InP nanowires.

Indium phosphide nanowires with a single crystalline zinc-blend core and polycrystalline/amorphous shell were grown from a reliable route without the use of hazardous precursors. The nanowires are composed by a crystalline core covered by a polycrystalline shell, presenting typical lengths larger than 10 µm and diameters of 80-90 nm. Raman spectra taken from as-grown nanowires exhibited asymmetric line shapes with broadening towards higher wave numbers which can be attributed to phonon localization effects. It was found that optical phonons in the nanowires are localized in regions with average size of 3 nm, which seems to have the same order of magnitude of grain sizes in the polycrystalline shell. Regardless of the fact that the nanowires exhibit a crystalline core, any considerable degree of disorder can lead to a localized behaviour of carriers. In consequence, the variable range hopping was observed as the main transport instead of the usual thermal excitation mechanisms. Furthermore the hopping length was ten times smaller than nanowire cross-sections, confirming that the nanostructures do behave as a 3D system. Accordingly, the V-shape observed in PL spectra clearly demonstrates a very strong influence of the potential fluctuations on the exciton optical recombination. Such fluctuations can still be observed at low temperature regime, confirming that the amorphous/polycrystalline shell of the nanowires affects the exciton recombination in every laser power regime tested.

Chirp- and random-based coded ultrasonic excitation for localized blood-brain barrier opening.

Chirp- and random-based coded excitation methods have been proposed to reduce standing wave formation and improve focusing of transcranial ultrasound. However, no clear evidence has been shown to support the benefits of these ultrasonic excitation sequences in vivo. This study evaluates the chirp and periodic selection of random frequency (PSRF) coded-excitation methods for opening the blood-brain barrier (BBB) in mice. Three groups of mice (n = 15) were injected with polydisperse microbubbles and sonicated in the caudate putamen using the chirp/PSRF coded (bandwidth: 1.5­1.9 MHz, peak negative pressure: 0.52 MPa, duration: 30 s) or standard ultrasound (frequency: 1.5 MHz, pressure: 0.52 MPa, burst duration: 20 ms, duration: 5 min) sequences. T1-weighted contrast-enhanced MRI scans were performed to quantitatively analyze focused ultrasound induced BBB opening. The mean opening volumes evaluated from the MRI were mm3, mm3and mm3 for the chirp, random and regular sonications, respectively. The mean cavitation levels were V.s, V.s and V.s for the chirp, random and regular sonications, respectively. The chirp and PSRF coded pulsing sequences improved the BBB opening localization by inducing lower cavitation levels and smaller opening volumes compared to results of the regular sonication technique. Larger bandwidths were associated with more focused targeting but were limited by the frequency response of the transducer, the skull attenuation and the microbubbles optimal frequency range. The coded methods could therefore facilitate highly localized drug delivery as well as benefit other transcranial ultrasound techniques that use higher pressure levels and higher precision to induce the necessary bioeffects in a brain region while avoiding damage to the surrounding healthy tissue.

Population pharmacokinetic analysis of meropenem in Japanese adult patients.

WHAT IS KNOWN AND OBJECTIVE: Meropenem is frequently employed as an empirical treatment for serious infections, but there has been no report on its population pharmacokinetic parameters for Japanese patients. Our aim is to undertake a population pharmacokinetic analysis of meropenem using non-linear mixed effects model (NONMEM). METHODS: Data from 68 patients were analysed via NONMEM with the first-order method. The participants' covariates, including gender, age, actual body weight, serum creatinine, serum albumin, serum total protein and creatinine clearance, were analyzed by the forward inclusion and backward elimination method to identify their potential influence on meropenem pharmacokinetics. The adequacy of the constructed model was assessed by goodness-of-fit plots and the precision of the parameter estimated at each step of the model development. To assess the robustness of the estimated parameter, bootstrap analysis was performed. RESULTS AND DISCUSSION: The data were best described by a one-compartment model. The serum creatinine values modified by the below normal limit in our hospital (mSCR) were an influential covariate for clearance (CL): CL (L/h) = 11·1 × (mSCR/0·7)(-1). The volume of distribution was estimated as 33·6 L. The coefficient of variation of the inter-individual variability of CL and the residual variability were 52·1% and 0·827% µg/mL, respectively. A comparison of the population pharmacokinetic parameters of meropenem in the final model estimated in NONMEM with original data, and 1000 bootstrap samples shows that both sets of estimates were comparable, thereby indicating the robustness of the proposed model. WHAT IS NEW AND CONCLUSION: A population pharmacokinetic model that satisfactorily described the disposition and variability of meropenem in our Japanese population is described. NONMEM analysis showed that the clearance of meropenem depended on modified serum creatinine. The results of this study should help Japanese patients on meropenem by improving the prediction accuracy of dosing using the Bayesian method.

Effect of various estimates of renal function on prediction of vancomycin concentration by the population mean and Bayesian methods.

OBJECTIVE: Renal function was estimated in 129 elderly patients with methicillin-resistant Staphylococcus aureus (MRSA) who were treated with vancomycin (VCM). The estimation was performed by substituting serum creatinine (SCR) measured enzymatically and a value converted using the Jaffe method into the Cockcroft-Gault and Modification of Diet in Renal Disease (MDRD) equations. The serum trough level was predicted from three estimates of renal function by the population mean (PM) and Bayesian methods and the predictability was assessed. METHODS: Two-compartment model-based Japanese population parameters for VCM were used, and the mean prediction error (ME) and root mean squared error (RMSE) were calculated as indices of bias and accuracy, respectively, for predictions by the PM and Bayesian methods. RESULTS: The PM method gave the highest correlation with the measured value using the estimate of renal function obtained by substituting the Jaffe-converted SCR into the Cockcroft-Gault equation. There was no positive or negative bias in the ME and the value was significantly smaller than for other predicted data (P < 0.05). RMSE was also the smallest, indicating that this method increases the predictability of the serum VCM trough level. While, ME showed a negative bias for all values predicted by the Bayesian method, both the ME and RMSE were very small. CONCLUSION: In the application of the PM method for VCM treatment of elderly patients with MRSA, substitution of SCR based on the Jaffe method into the Cockcroft-Gault equation increases the predictability of the serum VCM trough level. The Bayesian method predicted the serum VCM trough level with high accuracy using any of the estimates of renal function.

Pharmacokinetics of YK754, a novel If channel inhibitor in rats, dogs and humans.

The pharmacokinetics of YM758, a novel funny If current channel (If channel) inhibitor, was investigated after single intravenous (i.v.) and oral dosing to rats and dogs, and partially compared with the results in humans by using liver microsomes. After i.v. administration, the plasma YM758 concentrations decreased, with an elimination half-life (t(1/2)) of 1.14-1.16 h in rats and 1.10-1.30 h in dogs. Total body clearance (CL(tot)) was 5.71-7.27 and 1.75-1.90 L/h/kg in rats and dogs, respectively which was comparable to the hepatic blood flow rate. In dogs, the pharmacokinetic profiles for i.v. bolus administration and continuous infusion did not differ. After oral administration, the levels of YM758 in rat plasma increased more than dose-proportionally, whereas almost linear pharmacokinetics were observed in dogs. Absolute bioavailability was 7.5%-16.6% in rats and 16.1%-22.0% in dogs. The plasma protein binding saturation of YM758 was observed in dogs and humans; this finding is consistent with the result that the major binding protein of YM758 in plasma is alpha1-acid glycoprotein (AGP), in particular in humans. The blood-to-plasma partition coefficient values were 1.36-1.42 in rats, 0.95-1.15 in dogs and 0.71-0.85 in humans. The results of the metabolic study on liver microsomes indicated that the non-linear pharmacokinetics of YM758 observed in rats may be partially due to a first-pass effect in the gastrointestinal tract and the liver.

Tissue distribution of YM758, a novel If channel inhibitor, in pregnant and lactating rats.

In this study the tissue distribution of radioactivity in pregnant and lactating rats was investigated by quantitatively determining radioactivity concentrations and by whole-body autoradioluminograms after a single oral administration of 14C-YM758. In addition, the transfer of radioactivity into the reproductive tissues, foetus, and milk is discussed in terms of the localization of transporters in syncytiotrophoblast and mammary gland. The radioactivity concentrations in the liver were the highest of all the tissues and organs tested at all the sampling times. The radioactivity in main tissues (liver and kidney), including reproductive tissues (amniotic fluid, placenta, ovary, and uterus), was not retained for a long time, as in the plasma. The tissue/plasma (T/P) ratio of radioactivity in the foetus was below 1.0, which might be due to Mdr1-mediated export of YM758 into blood via the blood-placenta barrier since YM758 is a substrate for hMDR1, not for hBCRP/rBcrp. The T/P ratio of radioactivity in the maternal milk 1 and 4 h after oral administration of 14C-YM758 was 7.2 and 11.0, respectively. To understand better the distribution of new drugs into the reproductive tissues/milk, and to interpret further the results of reproductive safety studies for drug development, the contribution of transporters expressed in the blood-placenta barrier and mammary gland to the drug-transfer into placenta and milk should be considered.

Effect of cationic drugs on the transporting activity of human and rat OCT/Oct 1-3 in vitro and implications for drug-drug interactions.

The inhibitory effects of cationic drugs (beta-adrenoreceptor antagonists, calcium (Ca)-channel blocker, I(f) channel inhibitor, antiarrhythmic drugs, and antibacterial drugs) that inhibit 1-methyl-4-phenylpyridinium (MPP) and/or metformin uptake into hOCT1-3/rOct1-3-expressing cells and human/rat hepatocytes were investigated in this study. The drug-drug interaction (DDI) potential of these drugs for the hOCT/rOct-mediated hepatic/renal uptake process was also assessed. The IC(50) values of cardiovascular drugs, including an I(f) channel inhibitor with a new mechanism of action, were greater for hOCT2/rOct2 than those for hOCT1/rOct1 or hOCT3/rOct3. No species differences in these values were observed between hOCTs and rOcts. As for hOCT2-mediated uptake, the IC(50) values of quinidine and the I(f) channel inhibitor for metformin uptake were lower than those for MPP uptake. However, previous clinical studies found that the IC(50) values of these drugs for hOCT1/rOct1 and hOCT2/rOct2 were much greater than their unbound plasma concentrations, which suggests that the DDIs of these cationic compounds may not be related to hOCT/rOct-mediated hepatic/renal uptake pathways. In addition, investigation of the luminal transporters of cationic compounds in the kidney, as well as the in vitro DDI potential of their inhibitors, is important for the clarification of cationic compound DDIs in humans.

Characterization of renal tubular apical efflux of zonampanel, an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist, in humans.

1. Zonampanel, a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist, is mainly excreted unchanged via renal tubular secretion. The renal apical transport transport of zonampanel was examined in this study using HEK293 cells expressing human organic anion transporter 4 (OAT4/SLC22A11), and membrane vesicles prepared from Sf-9 insect cells expressing human multidrug resistance-associated protein 2 (MRP2/ABCC2), MRP4 (ABCC4), and breast cancer resistance protein (BCRP/ABCG2). 2. Glutaric acid, a model dicarboxylate, trans-stimulated the uptake of [(14)C]zonampanel by OAT4, suggesting that zonampanel was transported by OAT4 via an exchange with dicarboxylate. Considering the endogenous dicarboxylate gradient, OAT4 seems to transport zonampanel in the direction of reabsorption rather than secretion. For MRP2, MRP4, and BCRP, zonampanel selectively inhibited the activity of MRP4 (K(i) = 41.3 microM). Marked transport of [(14)C]zonampanel was observed only for MRP4 (K(m) = 33.7 microM). 3. In conclusion, the data indicate that MRP4 was the apical efflux transporter that contributed to the active renal tubular secretion of zonampanel in humans, in concert with the apical reabsorption transporter OAT4 and basolateral uptake transporters.

Comparative evaluation of absorption, distribution, and excretion of YM758, a novel If channel inhibitor, between albino and non-albino rats.

1. YM758 is a novel If channel inhibitor for the treatment of stable angina and atrial fibrillation. The absorption, distribution, and excretion of YM758 have been investigated in albino and non-albino rats after a single oral administration of (14)C-YM758 monophosphate. 2. YM758 was well absorbed from all segments of the gastrointestinal tract except for the stomach. After oral administration, the ratio of AUC(0-1 h) between the plasma concentrations of radioactivity and the unchanged drug was estimated to be 17.7%, which suggests metabolism. 3. The distribution of the radioactivity derived from (14)C-YM758 in tissues was evaluated both in albino and non-albino rats. The radioactivity concentrations in most tissues were higher than those in plasma, which indicates that the radioactivity is well distributed to tissues. Extensive accumulation and slower elimination of radioactivity were noted in the thoracic aorta of albino and non-albino rats as well as in the eyeballs of non-albino rats. The recovery rates of radioactivity in urine and bile after oral dosing to bile duct-cannulated albino rats were 17.8% and 57.3%, respectively. 4. These results suggest that YM758 was extensively absorbed, subjected to metabolism, and excreted mainly into the bile after oral administration to rats, and extensive accumulation of the unchanged drug and/or metabolites into tissues such as the thoracic aorta and eyeballs was observed.

Functional involvement of the organic cation transporter 2 (rOct2) in the renal uptake of organic cations in rats.

This study examined the contribution made by organic cation transporters (hOCT/rOct) to the saturable component of the renal uptake of 1-methyl-4-phenylpyridinium, tetraethylammonium (TEA), cimetidine and metformin into rOct2-expressing HEK293 cells and rat kidney slices. All the test compounds accumulated in the rat kidney slices in a carrier-mediated manner. The Michaelis- Menten constant (K(m)) values for saturable uptake of TEA, cimetidine and metformin into rat kidney slices were relatively comparable with those for the rOct2-expressing HEK293 cells. In addition, the relative uptake activity values of TEA, cimetidine and metformin in rat kidney slices were similar to those in rOct2-expressing HEK293 cells. This suggests that the saturable components involved in the renal uptake of TEA, cimetidine and metformin are mediated mainly by rOct2. The saturable uptake profile of cationic compounds into rat kidney can be evaluated in both cDNA-expressing cells and rat kidney slices, as well as the transporter expression pattern. This approach can also be used to estimate the saturable uptake mechanism of cationic compounds into the human kidney when human kidney slices and hOCT2-expressing cells are used.

Evaluation of the inhibitory and induction potential of YM758, a novel If channel inhibitor, for human P450-mediated metabolism.

This study was designed to examine the in vitro metabolism of YM758, a novel cardiovascular agent, and to evaluate its potential to cause drug interactions and induction of CYP isozymes. After incubation with pooled human liver microsomes, YM758 was converted to two major metabolites (AS2036313-00, and YM-394111 or YM-394112). The formation of AS2036313-00, and YM-394111 or YM-394112 were mediated by CYP2D6 and CYP3A4, respectively, which was elucidated by using a bank of human liver microsomes and recombinant CYP enzymes in combination with the utilization of typical substrates and inhibitors. The Ki values of YM758 for midazolam, nifedipine, and metoprolol metabolism ranged from 59 to 340 microM, being much higher than the YM758 concentration in human plasma. The formation of AS2036313-00, and YM-394111 or YM-394112 was inhibited by quinidine and ketoconazole with Ki values of 140 and 0.24 microM, respectively, which indicates that YM758 metabolism may be affected by coadministration of strong CYP2D6 and 3A4 inhibitors in vivo, given the clinical plasma concentrations of quinidine and ketoconazole. After human hepatocytes were exposed to 10 microM YM758, microsomal activity and mRNA level for CYP1A2 were not induced while those for CYP3A4 were slightly induced. The tested concentration was much higher than that in human plasma, which suggests that the induction potential of YM758 is also negligible.

Functional involvement of organic cation transporter1 (OCT1/Oct1) in the hepatic uptake of organic cations in humans and rats.

The contribution of organic cation transporters to the saturable component in the hepatic uptake of 1-methyl-4-phenylpyridinium (MPP), tetraethylammonium (TEA), cimetidine, and metformin was examined by the use of human/rat organic cation transporter (hOCT1/rOct1)-expressing cells and human/rat hepatocytes. Transfection of rOct1 resulted in a considerable increase in the uptake of metformin, whereas that of hOCT1 resulted in only a slight increase. All test compounds (MPP, TEA, cimetidine, and metformin) accumulated in human and rat hepatocytes in a carrier-mediated manner. The Km values for the uptake of MPP, TEA, cimetidine, and metformin into human and rat hepatocytes were comparable with those into hOCT1 and rOct1-expressing cells, respectively. In addition, the relative uptake activities, which were obtained by normalizing the intrinsic uptake clearances of TEA, cimetidine, and metformin against those values of MPP in human and rat hepatocytes, were similar with the uptake activities in hOCT1 and rOct1, respectively. These results suggest that the saturable component in the hepatic uptake of these cationic compounds may be mediated mainly by hOCT1/rOct1; therefore, it is meaningful to evaluate the saturable uptake profile of cationic compounds by the liver using both hOCT1/rOct1-expressing cells and human/rat hepatocytes.

Comparison of the kinetic characteristics of inhibitory effects exerted by biguanides and H2-blockers on human and rat organic cation transporter-mediated transport: Insight into the development of drug candidates.

In this study, the comparison of the transport of substrates (1-methyl-4-phenylpydinium (MPP) and tetraethyl ammonium (TEA)) and the inhibition potency of the inhibitors (biguanides and H(2)-blockers) for human and rat organic cation transporters (hOCTs and rOcts), and the inhibition type of inhibitors for these transporters were investigated using HEK293 cells that stably express hOCT/rOct. The concentration-dependent uptake of [(3)H]-MPP and [(14)C]-TEA by hOCT1-3/rOct1-3 had K(m) values similar to those in the literature. It was also deduced that MPP and TEA are competitive inhibitors for hOCT1-2/rOct1-2. The K(i) values for phenformin inhibition of [(3)H]-MPP and [(14)C]-TEA uptake by hOCT1-3/rOct1-3 were lower than that for metformin. The [(3)H]-MPP uptake by hOCT1/rOct1 and hOCT3/rOct3 was inhibited by famotidine and ranitidine whereas that by hOCT2/rOct2 was not. The inhibitory potency of cimetidine for hOCT1-2 was very weak. In most cases, the differences in the V(max)/K(m) values of substrates and the K(i) values of inhibitors between hOCT and rOct were minor. The acquisition of information on OCT/Oct mediated-transport and/or inhibition such as that presented in this report is very useful for further understanding of certain aspects of uptake, distribution, and excretion for drug candidates.

Effects of tetrabromobisphenol A, brominated flame retardant, in ICR mice after prenatal and postnatal exposure.

Tetrabromobisphenol A (TBBPA), brominated flame retardant, is produced in the largest amounts globally for use in plastics or building materials. TBBPA has been detected in sediment, air at the dismantling plant or human serum samples. In the present study, we examined the effects of prenatal and postnatal exposure to TBBPA in mice. TBBPA (99.1% pure) in diet was administered to pregnant ICR mice at doses of 0% (control), 0.01%, 0.1% or 1.0% from gestational day 0 to weaning at postnatal day 27. The average daily food intake and body weight of dams showed no significant differences between the control and treated groups. There were no dose-related effects on reproductive data. Serum concentrations of total-cholesterol and liver weights of treated dams and offspring were higher than those of the control mice. Histological findings in treated dams or offspring showed the increase of focal necrosis of hepatocytes and inflammatory cell infiltration in the liver, and increase of dilation or atrophy of renal tubules and cyst in the kidney. TBBPA was developed as a new, safe class of flame retardant and was not highly toxic. However, the present data suggested that TBBPA caused a lipid metabolic disorder and hepatic or kidney lesion, under these conditions.

Genetic polymorphism of cytochrome P450s in beagles: possible influence of CYP1A2 deficiency on toxicological evaluations.

A number of human cytochrome P450 (CYP) isozymes have been shown to be genetically polymorphic, and extensive pharmaceutical studies have been conducted to characterize the clinical relevance of the polymorphism. Although the beagle is extensively used in the safety assessment studies of new drug candidates and agricultural chemicals, only a limited number of studies have been reported on the significance of the CYP isozyme polymorphism in dogs. Recently, a single nucleotide polymorphism that results in a deficiency of canine CYP1A2 was discovered. This deficiency was shown to significantly alter the pharmacokinetic behavior of two drugs, and can be associated with a large inter-individual difference in the kinetic behavior of a third. In this article, the five genetically polymorphic canine CYP isozymes that have been reported so far are reviewed, and the altered pharmacokinetics of the drugs concerned are described. Although little information on toxicological relevance has been reported, it is possible that the modified pharmacokinetics may also cause altered toxic responses as well. This phenomenon may occur only with the types of chemicals that are eliminated mainly through polymorphic-enzyme mediated metabolism. However, it is recommended that genetically pure beagles are used for the toxicity studies and safety assessment of new chemical entities in order to reduce the potential inter-individual differences.

Identification of metabolites of [14C]zonampanel, an a-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist, following intravenous infusion in healthy volunteers.

This study determined the pharmacokinetics, metabolism and excretion of an a-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist zonampanel monohydrate (YM872) after intravenous infusion of [14C]YM872 at 1 mg kg-1 h-1 for 2 h to four healthy male volunteers. Mean pharmacokinetic parameters of unchanged YM872 were 0.78 h for terminal half-life, 25.9 l h-1 for total clearance, 22.9 l h-1 for renal clearance, and 15.6 l for volume of distribution at steady-state. Urinary excretion of radioactivity accounted for 94.9% of the dose, and faecal excretion for only 0.5% of the dose. Measurement of YM872 concentrations by a high-performance liquid chromatography (HPLC)-ultraviolet method and radiometric HPLC metabolite profiling revealed that almost all of [14C]YM872 was excreted unchanged in the urine and that unchanged [14C]YM872 was the major circulating [14C] component in the plasma. Two minor metabolites, H1 and H2, detected in the urine and identified as the same chemical structures as those of the rat urinary metabolites, have a hydroxyamino group and an amino group, respectively, which were probably formed by reduction of the nitro group of YM872. These results show that virtually all of the administered YM872 remains unchanged, with urinary excretion representing the major elimination pathway. The high renal clearance implies tubular secretion of this drug.

Identification of the novel canine CYP1A2 1117 C > T SNP causing protein deletion.

The pharmacokinetics of YM-64227 (4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]-pyrimidine-2-(1H)-one), a novel and selective phosphodiesterase type 4 inhibitor, was characterized in beagle dogs. Based on the plasma parent drug to major hydroxylated metabolite ratio, 21 dogs were phenotyped as 16 extensive metabolizers (EM) and five poor metabolizers (PM). Nucleotide sequences of CYPs 1A2, 2B11, 2C21, 2D15, 2E1 and 3A12 were investigated in the EM and PM dogs. A CYP1A2 1117 C>T single nucleotide polymorphism was found, which resulted in an amino acid change from an Arg codon to a stop codon at position 373. All dogs phenotyped as PM were T/T homozygous, whereas EMs were C/C homozygous and C/T heterozygous. In Western blotting of liver microsomes, CYP1A protein expression was detected in the C/C and C/T types, but not in the T/T type. Of 65 dogs genotyped using genome DNA, the frequencies of the C and T alleles were 0.61 and 0.39, respectively, suggesting approximately 15% of the dogs would not express the CYP1A2 protein. The findings provide a coherent explanation for the inter-individual variability in the pharmacokinetics of CYP1A2 substrate drugs in dogs.