Increased plasma drug concentration and decreased additional insulin secretion following oral administration of glimepiride in Spontaneously Diabetic Torii rats.

We aimed to evaluate the pharmacokinetics and pharmacological effects of glimepiride in the Spontaneously Diabetic Torii (SDT) rat, which is a non-obese model of type 2 diabetes. After oral administration of glimepiride (10 mg/kg), the maximum plasma concentrations and the area under the curve from 0 to 6 h of glimepiride in SDT rats were significantly higher than those in age-matched Sprague-Dawley rats. Whereas, additional insulin secretion following glimepiride treatment was markedly reduced in SDT rats. Thus, the SDT rat can be regarded as a model that reflects type 2 diabetes with reduced insulin secretory capacity. Our findings suggested that glimepiride could be ineffective in sever type 2 diabetic patients.

Myrcene and terpene regulation of TRPV1.

Nociceptive Transient Receptor Potential channels such as TRPV1 are targets for treating pain. Both antagonism and agonism of TRP channels can promote analgesia, through inactivation and chronic desensitization. Since plant-derived mixtures of cannabinoids and the Cannabis component myrcene have been suggested as pain therapeutics, we screened terpenes found in Cannabis for activity at TRPV1. We used inducible expression of TRPV1 to examine TRPV1-dependency of terpene-induced calcium flux responses. Terpenes contribute differentially to calcium fluxes via TRPV1 induced by Cannabis-mimetic cannabinoid/terpenoid mixtures. Myrcene dominates the TRPV1-mediated calcium responses seen with terpenoid mixtures. Myrcene-induced calcium influx is inhibited by the TRPV1 inhibitor capsazepine and Myrcene elicits TRPV1 currents in the whole-cell patch-clamp configuration. TRPV1 currents are highly sensitive to internal calcium. When Myrcene currents are evoked, they are distinct from capsaicin responses on the basis of Imax and their lack of shift to a pore-dilated state. Myrcene pre-application and residency at TRPV1 appears to negatively impact subsequent responses to TRPV1 ligands such as Cannabidiol, indicating allosteric modulation and possible competition by Myrcene. Molecular docking studies suggest a non-covalent interaction site for Myrcene in TRPV1 and identifies key residues that form partially overlapping Myrcene and Cannabidiol binding sites. We identify several non-Cannabis plant-derived sources of Myrcene and other compounds targeting nociceptive TRPs using a data mining approach focused on analgesics suggested by non-Western Traditional Medical Systems. These data establish TRPV1 as a target of Myrcene and suggest the therapeutic potential of analgesic formulations containing Myrcene.

The relationship between the glenoid track and the range of shoulder motion: A cadaver study.

INTRODUCTION: The concept of the glenoid track has been proposed to evaluate the risk of dislocation. The glenoid track width was demonstrated to be 84% of the glenoid width in cadaveric shoulders and 83% in live shoulders. HYPOTHESIS: The glenoid track width seems to be affected by the range of motion. PURPOSE: The purpose of this study was to determine the relationship between the glenoid track and the range of shoulder motion. METHODS: Ten fresh-frozen cadaveric shoulders were used. The specimen was fixed to a shoulder-positioning device. The anterior rim of the glenoid was marked on the humeral head using a Kirschner wire with the arm in 60° of abduction. This marking was repeated with the arm in (1) horizontal flexion/extension and (2) internal/external rotations (0° to max). The distances from the Kirschner wire markings to the footprint of the rotator cuff tendon were measured. RESULTS: The greater the angle of the horizontal extension or external rotation, the smaller the glenoid track width, whereas the greater the angle of the horizontal flexion or internal rotation, the greater the glenoid track width. There was a negative relationship between them. The horizontal flexion/extension motion was demonstrated to affect the glenoid track width more than the internal/external rotation motion. CONCLUSION: The glenoid track width decreased with the increase of horizontal extension. We should consider the range of horizontal extension angle when applying the glenoid track concept in clinical practice. TYPE OF STUDY: Laboratory study.

Development of a novel algorithm for detecting glucocorticoid-induced diabetes mellitus using a medical information database.

WHAT IS KNOWN AND OBJECTIVE: Glucocorticoid-induced diabetes mellitus (GIDM) increases the risk of diabetes mellitus (DM)-related complications but is generally difficult to detect in clinical settings. The criteria for diagnosing GIDM have not been established. Recently, medical information databases (MIDs) have been used in post-marketing surveillance (PMS) studies. We conducted a pharmacoepidemiological study to develop an algorithm for detecting GIDM using MID. METHODS: We selected 1214 inpatients who were newly prescribed with a typical glucocorticoid, prednisolone, during hospitalization from 2008 to 2014 from an MID of Hamamatsu University Hospital in Japan. GIDM was screened based on fasting blood glucose (FBG) and haemoglobin A1c (HbA1c) levels according to the current Japan Diabetes Society (JDS) DM criteria, and its predictability was evaluated by an expert's review of medical records. We investigated further candidate screening factors using receiver operating characteristics analysis. RESULTS: Sixty-three inpatients were identified by the JDS DM criteria. Of these, 33 patients were definitely diagnosed as having GIDM by expert's review (positive predictive value = 52·4%). To develop a highly predictive algorithm, we compared the characteristics of inpatients diagnosed with definite GIDM and those diagnosed as non-GIDM. The maximum levels of HbA1c in patients with GIDM were significantly higher than those of patients with non-GIDM (66·9 mmol/mol vs. 58·7 mmol/mol, P < 0·001). The patients with GIDM had significantly higher relative increase in maximum level of HbA1c (RIM-HbA1c) than those with non-GIDM (0·3 vs. 0·03, P < 0·001). However, we did not observe a significant difference in those of fasting blood glucose (FBG) levels. We applied the RIM-HbA1c as a second screening factor to improve the detection of GIDM. It showed that a 13% increase in RIM-HbA1c separated patients with from patients without GIDM. WHAT IS NEW AND CONCLUSIONS: Patients with GIDM had significantly higher RIM-HbA1c than patients with non-GIDM. There was a 13% increase in RIM-HbA1c in patients with GIDM compared to the others. Our detection algorithm for GIDM using an MID achieved high sensitivity and specificity, and was superior to one based only on the current JDS DM criteria. Our results suggest that monitoring changes in HbA1c levels is important for detecting GIDM and adds to current diagnostic criteria for type 2 DM.

Evaluation of two Japanese regulatory actions using medical information databases: a 'Dear Doctor' letter to restrict oseltamivir use in teenagers, and label change caution against co-administration of omeprazole with clopidogrel.

WHAT IS KNOWN AND OBJECTIVE: The implementation of appropriate epidemiological methodology using medical information databases (MIDs) to evaluate the effects of regulatory actions has been highly anticipated. To assess scientific methods for active pharmacovigilance using MIDs, we conducted a quantitative assessment of the impact of two regulatory actions by the Japanese government: (i) restriction of use of oseltamivir in teenagers in March 2007 and (ii) caution against the co-administration of omeprazole (OPZ) with clopidogrel (CPG) in April 2010. METHODS: Data were obtained from four hub hospitals in Japan. We measured the seasonal proportion of patients prescribed oseltamivir to those prescribed neuraminidase inhibitors for the 2002/2003 to 2010/2011 seasons. The monthly proportion of patients co-administered OPZ and CPG (OPZ+CPG) to those prescribed CPG was measured from May 2009 to April 2011. We evaluated the changes observed with implementation of the regulatory actions. To estimate the impact of the actions, we conducted segmented regression analysis using interrupted time series data. The impact was assessed by two parameter estimates of the regression model: the change in level for short-term effects and change in trend for long-term effects. RESULTS AND DISCUSSION: The use of oseltamivir in the target 10-19 years age group showed a significant and large decline (63·16%) immediately after the intervention (P = 0·0008). No change was observed in OPZ+CPG, although there was a relative inhibitory trend for OPZ+CPG compared with co-administration of lansoprazole or rabeprazole with CPG as the control group. When restricted to new users of CPG, the stratified results were consistent with the overall results. WHAT IS NEW AND CONCLUSION: The current analysis demonstrates the effectiveness of two regulatory actions. The results of the current study indicate that MID research can contribute to assessing and improving pharmacovigilance activities.

An algorithm for the identification of heparin-induced thrombocytopenia using a medical information database.

WHAT IS KNOWN AND OBJECTIVE: Using effective algorithms for extracting relevant drug and patient information from electronic medical information data systems is likely to form an increasingly important aspect of pharmacovigilance. To this end, we aimed to develop and validate a novel algorithm for detecting heparin-induced thrombocytopenia (HIT) using a medical information database (MID) and for identifying possible risk factors for HIT. METHODS: We developed a new algorithm for detecting HIT based on platelet count at distinct time-points and diagnostic information from patients treated with unfractionated heparin (UFH) from April 2008 through March 2012 at Hospital of Hamamatsu University School of Medicine, Japan. Definitive diagnoses of HIT were made through reviews of the medical records by a skilled haematologist. The performance of the algorithm was assessed using the positive predictive value (PPV). Multivariate logistic regression analysis was used to identify possible risk factors for HIT. RESULTS AND DISCUSSION: This algorithm detected 47 patients with suspected HIT in the source population (n = 2875). Of these, 41 were identified as patients with definitive HIT after review of the medical records. The PPV for the algorithm was 87·2%, and the frequency of definitive HIT was 1·4%. Longer-term treatment (≥4 days) with UFH was identified as a risk factor for HIT, with an odds ratio of 5·38 (95% CI: 2·35-12·32) for definitive HIT. WHAT IS NEW AND CONCLUSION: We developed a novel, high-PPV detection algorithm for HIT and identified longer-term treatment with UFH as a risk factor for HIT. Our results support the utility of MIDs for improving pharmacovigilance.

Mitochondrial swelling and incipient outer membrane rupture in preapoptotic and apoptotic cells.

Outer mitochondrial membrane (OMM) rupture was first noted in isolated mitochondria in which the inner mitochondrial membrane (IMM) had lost its selective permeability. This phenomenon referred to as mitochondrial permeability transition (MPT) refers to a permeabilized inner membrane that originates a large swelling in the mitochondrial matrix, which distends the outer membrane until it ruptures. Here, we have expanded previous electron microscopic observations that in apoptotic cells, OMM rupture is not caused by a membrane stretching promoted by a markedly swollen matrix. It is shown that the widths of the ruptured regions of the OMM vary from 6 to 250 nm. Independent of the perforation size, herniation of the mitochondrial matrix appeared to have resulted in pushing the IMM through the perforation. A large, long focal herniation of the mitochondrial matrix, covered with the IMM, was associated with a rupture of the OMM that was as small as 6 nm. Contextually, the collapse of the selective permeability of the IMM may precede or follow the release of the mitochondrial proteins of the intermembrane space into the cytoplasm. When the MPT is a late event, exit of the intermembrane space proteins to the cytoplasm is unimpeded and occurs through channels that transverse the outer membrane, because so far, the inner membrane is impermeable. No channel within the outer membrane can expose to the cytoplasm a permeable inner membrane, because it would serve as a conduit for local herniation of the mitochondrial matrix.

Rapid and validated fluorometric HPLC method for determination of gabapentin in human plasma and urine for clinical application.

WHAT IS KNOWN AND OBJECTIVE: A rapid derivatization and validated HPLC method for gabapentin in human plasma and urine is needed for clinical use. The objective of this study was to establish a rapid and validated analytical method for the determination of gabapentin in human plasma and urine using isocratic fluorometric HPLC for clinical application. METHODS: This analytical method is based on precolumn fluorescent derivatization using 4-fluoro-7-nitro-benzofurazan. The derivatization was coupled to fast HPLC separation using a 2·3 µm-particle size ODS column (100 × 4·6 mm i.d.). RESULTS AND DISCUSSION: The derivatization of gabapentin was optimized and HPLC separation was achieved over an ODS column with a run time of 3·5 min. Calibration curves in human plasma and urine were linear over the concentration ranges of 0·05-10 and 10-1000 µg/mL, respectively. Intra- and inter-assay precision and accuracy values of plasma were within 8·0% and 101-109% and within 8·3% and 94-108%, respectively. Those of urine were within 8·5% and 97-106% and within 9·5% and 97-105%, respectively. This validated method was applied to a pharmacokinetic study in healthy subjects. Interindividual variations in plasma disposition and urinary excretion of gabapentin were observed. WHAT IS NEW AND CONCLUSION: A rapid and validated isocratic fluorometric HPLC method for the determination of gabapentin in human plasma and urine for clinical application has been established. This method can be utilized to evaluate the pharmacokinetic disposition of gabapentin in humans.

Effective plasma concentrations of mycophenolic acid and its glucuronide in systemic lupus erythematosus patients in the remission-maintenance phase.

WHAT IS KNOWN AND OBJECTIVE: Mycophenolate mofetil (MMF) has been reported recently to be effective in the treatment of systemic lupus erythematosus (SLE). The therapeutic range of mycophenolic acid (MPA) in SLE in the remission-maintenance phase remains to be clarified. The aim of this study was to evaluate the therapeutic efficacy of MMF and predose plasma concentrations of MPA and its phenolic glucuronide (MPAG) in patients with SLE in the remission-maintenance phase. METHODS: Thirty-one patients with SLE receiving a fixed dosage regimen of MMF (median and interquartile range, 1500 and 1000-2000mg/day) for at least 1month and who had not experienced any adverse drug reactions for more than 3months were enrolled. RESULTS: Significant improvement was observed after MMF administration in total haemolytic complement CH(50) and its fractions C3 and C4, immunoglobulins IgG, IgA and IgM, anti-dsDNA antibody, serum concentration of albumin and red blood cell count, even though the mean daily dose of prednisolone was significantly reduced (P=0·02). Median predose plasma concentrations of MPA and MPAG were 1·95 and 26·2µg/mL (interquartile ranges, 0·94-2·96 and 18·6-53·7 µg/mL). Predose plasma concentrations of MPA and MPAG correlated significantly with MMF dose (r=0·64, P<0·01 and r=0·39, P=0·03). WHAT IS NEW AND CONCLUSIONS: MMF improved clinical laboratory markers and reduced prednisolone dosage in SLE patients with predose plasma concentration of MPA and MPAG in the interquartile ranges of 0·94-2·96 and 18·6-53·7µg/mL, respectively.

Cyclosporine alters correlation between free and total mycophenolic acid in kidney transplant recipients in the initial phase.

WHAT IS KNOWN AND OBJECTIVE: The factors affecting the pharmacokinetics of free mycophenolic acid (MPA) and its phenolic glucuronide (MPAG) are still unclear. The aim of this study was to evaluate the influence of cyclosporine on the pharmacokinetics of free MPA and MPAG. METHODS: Seventy-seven kidney transplant recipients (23 were in an initial phase and 54 in a stable phase; 41 were treated with cyclosporine and 36 with tacrolimus) were enrolled. Free and total MPA and MPAG were determined using HPLC. The correlations between free and total predose concentrations (C(0) ) of MPA or MPAG were evaluated separately in patients receiving calcineurin inhibitor medications. RESULTS AND DISCUSSION: Serum concentration of albumin was lower in the initial phase than in the stable phase. A higher ratio of free MPAG C(0) to free MPA C(0) was observed in cyclosporine-treated than tacrolimus-treated kidney transplant recipients. Free MPA C(0) correlated weakly with total MPA C(0) in kidney transplant recipients treated with cyclosporine in the initial phase (ρ= 0·53, P = 0·06). WHAT IS NEW AND CONCLUSION: Cyclosporine increased the ratio of free MPAG C(0) to free MPA C(0) and varied the free fraction of MPA in the hypoalbuminaemic kidney transplant recipients in the initial phase.

Prescription trends for treatment of paediatric gastroenteritis at a Japanese hospital between 1997 and 2007.

OBJECTIVE: We aimed to investigate recent trends in prescriptions for the treatment of paediatric gastroenteritis in Japan over a 10-year period (1997-2007). METHODS: In this retrospective cohort study, we collected data for 2295 prescriptions for 1241 putative cases of paediatric gastroenteritis, which were treated between 1997 and 2007 at Hamamatsu University Hospital, Hamamatsu, Japan. RESULTS: The most frequently prescribed drugs were probiotics (n = 621), followed by anti-emetics (n = 474). In most years between 1997 and 2007, more cases were treated with probiotics than with any other drug type (30.6-63.3% of cases), with the percentage increasing between 2005 and 2007. In contrast, the frequencies of anti-emetic and antipyretic prescriptions remained fairly stable, and prescriptions for antibiotics decreased slightly over the study period. Anti-emetics were commonly used in this hospital. CONCLUSION: Although experimental evidence upon which to base recommendations is lacking, Japanese evidence-based guidelines are critical for improving the quality of treatment of paediatric gastroenteritis.

Assisting the diagnosis of Graves' hyperthyroidism with Bayesian-type and SOM-type neural networks by making use of a set of three routine tests and their correlation with free T4.

In our previous paper, we proposed a novel screening method that assists the diagnosis of Graves' hyperthyroidism via two types of neural networks by making use of routine test data. This method can be applied by non-specialists during physical check-ups at a low cost and is expected to lead to rapid referrals for examination and treatment by thyroid specialists, that is, to improve patients' QOL. In this report, the amount of female sample data was increased and routine test data (14 parameters) from 120 subjects with a known diagnosis (35 patients with Graves' hyperthyroidism and 85 healthy volunteers) were adopted as training data, before 171 individuals who had also undergone the same routine tests at the Tohoku University Hospital were screened by the network for Graves' hyperthyroidism. The present re-examination of the screening method showed its high screening ability with the set of parameters used (low serum creatinine was added to the established measures of elevated alkaline phosphatase and low total cholesterol that appear in the Graves' hyperthyroidism guidelines) and robustness due to the increase of the training sample data. It was also found that there is a strong correlation between the three parameters and serum free thyroxine (FT4) in Graves' hyperthyroidism, which supports the usefulness of our screening method.

Validated LC coupled to ESI-MS/MS analysis for fentanyl in human plasma and UV analysis in applied reservoir transdermal patches using a simple and rapid procedure.

BACKGROUND AND OBJECTIVES: Fentanyl has been used for cancer pain in transdermal formulation. The aim of the present study was to establish an analytical method for fentanyl in human plasma and in an applied transdermal reservoir patch (Reservoir-TTS), as well as for therapeutic monitoring of fentanyl in cancer patients. METHOD: Electro-spray ionization mass spectrometric (ESI-MS/MS) analysis followed solid phase extraction (SPE) from human plasma and drug reservoir extraction from an applied Reservoir-TTS. Each separation was completed within 9 min using an ODS column (particle size, 3 microm, 2.0 mm i.d. x 75 mm) with 25% acetonitrile containing 5 mm ammonium acetate at pH 3.5. In the ESI-MS/MS analysis, the calibration curve for fentanyl was linear over a concentration range of 0.05-7.2 ng/mL in human plasma. The extraction efficiency of fentanyl in the human plasma was more than 95%. The intra- and interassay precision and accuracy were within 7% and 97.3-101.2%, respectively. The lower LOQ for fentanyl was 0.05 ng/mL in the human plasma. The extraction of the 25 microg/h and 50 microg/h Reservoir-TTS gave reproducible recoveries of 88.3% and 90.9%, respectively. The plasma concentration of fentanyl showed large interindividual variation in 31 patients with cancer pain. CONCLUSION: The method described is simple, accurate, and reproducible, and should be helpful for the therapeutic monitoring of fentanyl in cancer patients.

Relationship between the pharyngeal airway space and craniofacial morphology, taking into account head posture.

This study assessed the relationship between craniofacial characteristics and the size of the pharyngeal airway space (PAS), taking into account head posture. Sixty dental students 25-30 years of age (30 men and 30 women) were examined by lateral cephalometry. The data were corrected with the use of appropriate regression equations for the PAS. The PAS significantly correlated with hyoid position, maxillary and mandibular size, maxillary and mandibular prognathism, and mandibular inclination. A large, anteriorly positioned mandible was associated with a large PAS-TP (the most proximal distance between the posterior pharyngeal wall and the tongue base). Uvula length and PNS-Ba (the distance between the most posterior point of the hard palate and the most inferior point of the anterior foramen magnum) correlated with PAS-UP (the most proximal distance between the posterior pharyngeal wall and uvula). Our results suggest that the anteroposterior dimension of the PAS is substantially affected by the size of the enclosure surrounding the PAS, including the maxilla, mandible and soft palate.

Computed tomographic analysis of the position and course of the mandibular canal: relevance to the sagittal split ramus osteotomy.

The aim of this study was to investigate the position and course of the mandibular canal through the mandibular ramus using computed tomographic (CT) imaging and to relate the findings to performing sagittal split ramus osteotomies. The mandibles of 35 patients with skeletal Class III prognathism with symmetry (12 males and 23 females) were observed on transaxial computed tomograms acquired with a slice thickness of 2 mm. The position and course of the mandibular canal from the mandibular foramen to the mandibular body at the level of the second molar were measured at four specific locations in the same plane. Among the 70 rami examined, lack of a bone marrow space on the buccal side, including a fusion type anatomy with no buccal side cortical bone of the mandibular canal, were observed at the CT location between the mandibular foramen and mandibular angle. Our results suggest that special care must be taken when sagittal splitting is performed, and the safest location for the buccal corticotomy is anterior to the mandibular angle.

Thermodynamic stability of base pairs between 2-hydroxyadenine and incoming nucleotides as a determinant of nucleotide incorporation specificity during replication.

We investigated the thermodynamic stability of double-stranded DNAs with an oxidative DNA lesion, 2-hydroxyadenine (2-OH-Ade), in two different sequence contexts (5'-GA*C-3' and 5'-TA*A-3', A* represents 2-OH-Ade). When an A*-N pair (N, any nucleotide base) was located in the center of a duplex, the thermodynamic stabilities of the duplexes were similar for all the natural bases except A (N = T, C and G). On the other hand, for the duplexes with the A*-N pair at the end, which mimic the nucleotide incorporation step, the stabilities of the duplexes were dependent on their sequence. The order of stability is T > G > C >> A in the 5'-GA*C-3' sequences and T > A > C > G in the 5'-TA*A-3' sequences. Because T/G/C and T/A are nucleotides incorporated opposite to 2-OH-Ade in the 5'-GA*C-3' and 5'-TA*A-3' sequences, respectively, these results agree with the tendency of mutagenic misincorporation of the nucleotides opposite to 2-OH-Ade in vitro. Thus, the thermodynamic stability of the A*-N base pair may be an important factor for the mutation spectra of 2-OH-Ade.

Age difference in simultaneous permeation and metabolism of ethyl nicotinate in rat skin.

The age-dependent characteristics of transdermal permeation of ethyl nicotinate (EN) and its metabolism to nicotinic acid (NA) were examined in rats at the ages of a fetus at 21 d, 3, 10, 50, 270 and 360 d. Skin transport in vitro was investigated using mounted skin in side-by-side diffusion chambers, and flux of EN and NA was determined. With developing and aging in rats, EN flux from EN-saturated solution declined gradually, however, NA flux increased drastically at 10 and 50 d. To elucidate the mechanism of this age-dependent escalation of NA flux, a hydrolysis study was performed using skin homogenate, and the Michaelis-Menten parameters (Vmax and Km) of EN were evaluated. Vmax and Vmax/Km ratio showed the same tendency with NA flux/total (EN+NA) flux ratio, suggesting that skin esterases in rats are developed gradually after birth, then increase markedly and become steady in the adult period. On the other hand, the affinity parameter, Km, was almost the same among all ages. Moreover, metabolic saturation of esterase during the transdermal process occurred in all ages, and maximal NA flux and EN concentration in the donor compartment for the maximal NA flux were also affected by age. These findings indicated that the discrepancy in transdermal profiles of EN among the ages tested was dominantly due to the difference in the development of esterase in the growth process.

Inhibitory effect of azole antifungal agents on the glucuronidation of lorazepam using rabbit liver microsomes in vitro.

Azole antifungal agents (azoles) have inhibitory effects on the cytochrome P450. However, the effect of azoles on conjugative metabolism has not been given much attention. Lorazepam (LZP), a benzodiazepine sedative agent, is known to be metabolized by uridine 5'-diphosphate (UDP)-glucuronyltransferase. Herein we report investigation of the effect of azoles on the enzyme-kinetics of glucuronidation of lorazepam using rabbit liver microsomes in vitro. The Km and Vmax for LZP glucuronidation were determined to be 0.26+/-0.08 mM and 1.25+/-0.21 nmol/min/mg protein, respectively, when evaluated in the presence of a detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) (0.8 mg/mg protein). Azoles fluconazole, miconazole, and ketoconazole competitively inhibited the glucuronidation of LZP, with Ki values of 7.17+/-4.78 mM, 0.17+/-0.08 mM, and 0.092+/-0.026 mM, respectively. These results are comparable to the previously reported Ki values of azoles with zidovudine (AZT) glucuronidation (1.4, 0.18, and 0.08 mM for fluconazole, miconazole, and ketoconazole, respectively) [Sampol et al., Br. J. Clin. Pharmacol., 40, 83-86, 1995]. Therefore, in order to avoid possible side effects of LZP, the concomitant administration of LZP and azoles should be carefully evaluated.

In vitro selection of aptamers that act with Zn2+.

An in vitro selection was carried out with Zn2+ to isolate novel RNA molecules, zinc-dependent aptamers, that bind to HIV-1 Tat protein. RNAs bound to Tat were collected by using a nitrocellulose filter from a library of random RNAs and regenerated to the next generation of the RNA library by subsequent reverse transcription, polymerase chain reaction, and transcription. Sequences of the selected RNAs were determined after 6 and 12 rounds of the selection. The control clones after normal selection procedure with Mg2+ had a consensus UUG that resembled essential sequences of TAR or Arg aptamers. On the other hand, many unique sequences were revealed from a library selected with Zn2+ and the RNA with most abundant sequence (clone 31) bound to Tat tightly only when Zn2+ existed. The secondary structure of clone 31 RNA was predicted by using a computational prediction with our thermodynamic parameters and enzymatic scission of the RNA. Several model RNAs were prepared and the binding property of these RNAs to Tat were investigated. As a result, all the model RNAs did not reproduce the binding property of clone 31. Therefore, the Tat aptamer that acts with Zn2+ should require a relatively longer region of the sequence which is able to offer tertiary cooperation of several motifs for the binding.

In vivo and in vitro toxicodynamic analyses of new quinolone-and nonsteroidal anti-inflammatory drug-induced effects on the central nervous system.

We investigated the correlation between an in vivo isobologram based on the concentrations of new quinolones (NQs) in brain tissue and the administration of nonsteroidal anti-inflammatory drugs (NSAIDs) for the occurrence of convulsions in mice and an in vitro isobologram based on the concentrations of both drugs for changes in the gamma-aminobutyric acid (GABA)-induced current response in Xenopus oocytes injected with mRNA from mouse brains in the presence of NQs and/or NSAIDs. After the administration of enoxacin (ENX) in the presence or absence of felbinac (FLB), ketoprofen (KTP), or flurbiprofen (FRP), a synergistic effect was observed in the isobologram based on the threshold concentration in brain tissue between mice with convulsions and those without convulsions. The three NSAIDs did not affect the pharmacokinetic behavior of ENX in the brain. However, the ENX-induced inhibition of the GABA response in the GABAA receptor expressed in Xenopus oocytes was enhanced in the presence of the three NSAIDs. The inhibition ratio profiles of the GABA responses for both drugs were analyzed with a newly developed toxicodynamic model. The inhibitory profiles for ENX in the presence of NSAIDs followed the order KTP (1.2 microM) > FRP (0. 3 microM) > FLB (0.2 microM). These were 50- to 280-fold smaller than those observed in the absence of NSAIDs. The inhibition ratio (0.01 to 0.02) of the GABAA receptor in the presence of both drugs was well-fitted to the isobologram based on threshold concentrations of both drugs in brain tissue between mice with convulsions and those without convulsions, despite the presence of NSAIDs. In mice with convulsions, the inhibitory profiles of the threshold concentrations of both drugs in brain tissue of mice with convulsions and those without convulsions can be predicted quantitatively by using in vitro GABA response data and toxicodynamic model.