Effects of organic solvents on the time-dependent inhibition of CYP3A4 by diazepam.

The effects of organic solvents, acetonitrile, dimethyl sulfoxide (DMSO), and methanol, which are used to dissolve lipophilic test compounds and cytochrome P450 (P450) substrates, and carried into pre-incubation at 1% (v/v), on time-dependent inhibition of CYP3A4 by diazepam, were evaluated using human liver microsomes (HLM) and recombinant human P450 expressed microsomes (rCYPs). The inactivation kinetics of CYP3A4 by diazepam dissolved in acetonitrile and methanol were almost equal with k(inact)/K(I) values, 0.095 and 0.15 min(-1) mM(-1) for HLM and 1.1 and 1.4 min(-1) mM(-1) for rCYP3A4, respectively. In contrast, the inactivation by diazepam dissolved in 1% DMSO significantly decreased and the kinetic parameter could not be calculated. The formation rate of nordiazepam and temazepam metabolized from diazepam dissolved in DMSO were approximately half of those using substrate dissolved in acetonitrile and methanol in both HLM and rCYP3A4. Dixon plots revealed that the metabolism of diazepam in rCYP3A4 were inhibited by DMSO in a competitive or mixed-type manner with K(i) (inhibition constant) values of 6 and 24 mM for nordiazepam and temazepam, respectively. In conclusion, the time-dependent inhibition of CYP3A4 by diazepam was attenuated by DMSO, while acetonitrile and methanol had no effect. The metabolite formation profile under the conditions tested suggested that DMSO competitively inhibit the formation of the reactive metabolites of diazepam by CYP3A4. The effect of organic solvents should be taken into consideration when evaluating the in vitro time-dependent inhibition of new chemical entities.

Comparison of mechanism-based inhibition of human cytochrome P450 2C19 by ticlopidine, clopidogrel, and prasugrel.

Mechanism-based inhibition of CYP2C19 in human liver microsomes by the thienopyridine antiplatelet agents clopidogrel, prasugrel and their thiolactone metabolites was investigated by determining the time- and concentration-dependent inhibition of the activity of S-mephenytoin 4'-hydroxylase as typical CYP2C19 activity and compared with ticlopidine and its metabolite. Clopidogrel was shown to be a mechanism-based inhibitor of CYP2C19 with the inactivation kinetic parameters, k(inact) and K(I), equal to 0.0557 min(-1) and 14.3 microM, respectively, as well as ticlopidine (0.0739 min(-1) and 3.32 microM, respectively). The thiolactone metabolite of ticlopidine and clopidogrel inhibited CYP2C19 only in a concentration-dependent manner. In contrast, neither prasugrel nor its thiolactone metabolite inhibited CYP2C19 at concentrations up to 100 microM. The oxidation of the thiophene moiety of clopidogrel to form their respective thiolactones was found to be the critical reaction that produces the chemically reactive metabolites which cause the mechanism-based inhibition of CYP2C19. Estimation of in vivo drug-drug interaction using in vitro parameters predicted clinically observed data. For clopidogrel, there was no increase in the area under the curve (AUC) at its clinical dose level as predicted by the in vitro parameters, and for ticlopidine the prediction agreed with the clinically observed AUC increase. In conclusion, clopidogrel is potent mechanism-based inhibitors of CYP2C19 as well as ticlopidine, whereas prasugrel did not inactivate CYP2C19. Administration of prasugrel would not cause a clinically relevant interaction with CYP2C19.

Effect of procainamide on renal tubular transport of cimetidine in the isolated perfused rat kidney.

The effect of procainamide on renal tubular transport of cimetidine was studied in isolated perfused rat kidney based on the multiple indicator dilution (MID) technique. T-1824-labeled albumin (a vascular reference), [14C]creatine (an extracellular reference), and [3H]cimetidine were rapidly injected into the renal artery of isolated perfused rat kidney in the presence or absence of procainamide (100 microM) in the perfusate, and normalized outflow-time patterns were secured from rapidly sampled renal perfusate. A distributed two-compartmental model was fitted to the dilution data by non-linear least-squares regression, and the influx, efflux and sequestration rate constants were estimated. Net transport and influx processes of cimetidine were competitively inhibited by procainamide (PA), while the efflux and sequestration processes were increased. The increase in the values of the efflux and sequestration rate constants by addition of procainamide may be explained by the increase in the tissue binding of cimetidine. However, these three processes were not significantly affected by p-aminohippurate (PAH). These results suggest that both cimetidine and procainamide are secreted into the lumen by an organic base transport mechanism in the perfused kidney, in which the spatial organization and cell polarity of the kidney are maintained.

Sleep apnea and nocturnal myoclonus in the elderly.

Insomnia, daytime sleepiness, and nocturnal wandering, so common in the elderly, are caused largely by two specific pathophysiologic processes. Sleep apnea is a condition where respiration pauses during sleep, leading to arousal. Sleep apnea is due either to obstruction in the throat or failure of the central respiratory center. Periodic movements in sleep are characterized by frequent ankle and leg flexions, leading to arousal. Sleep apnea and periodic movements in sleep require specific diagnoses and treatments. Each process occurs in 20%-30% of people over 65, and perhaps the majority of older people have one or the other condition or both. Because of possible interactions with these sleep disorders, the widespread prescribing of sleeping pills to elderly patients is irrational and often dangerous. In the future, large-scale clinical trials will be needed to define effective long-term treatments for these conditions and to define when treatment is worthwhile.

Enhancing effect of bromovinyldeoxyuridine on antitumor activity of 5'-deoxy-5-fluorouridine against adenocarcinoma 755 in mice. Correlation with pharmacokinetics of plasma 5-fluorouracil levels.

5'-Deoxy-5-fluorouridine (DFUR), whether or not combined with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was pursued in BDF1 mice from both a pharmacokinetic viewpoint, following a single oral dose administration, and an anticancer viewpoint, following 5 daily oral doses in mice inoculated subcutaneously with adenocarcinoma 755 tumor cells. Half-life (t1/2) values for the elimination of DFUR and 5-fluorouracil (5-FU) from plasma following DFUR (100 mg/kg) administration were about 0.80 and 0.39 hr, respectively. Plasma 5-FU AUC (area under the curve) values following oral DFUR (100 mg/kg) was 0.224 micrograms.hr/ml. If DFUR (100 mg/kg) was combined with BVDU (10 mg/kg) the t1/2 and AUC values for 5-FU increased from 0.39 to 1.24 hr, and from 0.224 to 1.699 micrograms.hr/ml, respectively. Thus, BVDU significantly increased the plasma levels of 5-FU. It had no effect on the plasma levels of DFUR. At 100 mg/kg, DFUR did not show a significant antitumor activity. At 500 mg/kg it effected a 90% inhibition in tumor growth. When combined with BVDU (10 mg/kg), DFUR at 100, 200 and 300 mg/kg reduced tumor growth by 96, 100 and 100%, respectively. The antitumor activity achieved by DFUR, in the presence or absence of BVDU, correlated highly significantly with the AUC values for plasma 5-FU.

The effect of butoctamide hydrogen succinate on nocturnal sleep: all-night polygraphical studies.

Butoctamide hydrogen succinate (BAHS), related to an organic compound naturally occurring in the central nervous system (CNS), has been shown to increase REM sleep in chronically prepared cats. In the present study, we confirmed that BAHS increases REM sleep in healthy humans, the subjects were six males whose mean age was 21 years and the experiment covered eight consecutive nights. Identical capsules containing either a placebo (linolenic acid) or 600 mg BAHS were administered 1 h prior to recording, which was started at 11 p.m. There was little change in total sleep time, sleep efficiency index, sleep latency, REM sleep latency, or the number of REM sleep periods during the drug- as compared to the baseline periods. There were, however, significant increases in REM sleep and decreases in sleep stages 1 and 2. The night's sleep was divided into three equal portions and analysis of the percentage of sleep stages in each showed that REM sleep markedly increased in the middle third while stages 3 and 4 increased in the last third. A carryover effect of BAHS was recognized during the withdrawal period. The maximum percentage of BAHS-induced REM sleep was 34%. REM density during the drug periods tended to decrease. These results suggest that BAHS may be an efficacious hypnotic in that it increases REM sleep which is suppressed by other clinically used hypnotics.

Drug-increased REM sleep in aged subjects: butoctamide hydrogen succinate (BAHS).

Butoctamide hydrogen succinate (BAHS), which is related to an organic compound naturally occurring in CSF, has been demonstrated to increase REM sleep in cats and yound adults. In the present study, BAHS was confirmed also to increase REM sleep in healthy aged subjects. The subjects were six females (68-77 years of age). The experiment covered 8 consecutive nights. Identical capsules containing either a placebo (linoleic acid) or 600 mg BAHS were administered 1 h prior to recording, which was started at 9 PM. BAHS tended to stabilize sleep. The average number and percentage of REM periods increased significantly during the drug nights compared with the baseline nights (P less than 0.05 and P less than 0.02, respectively). The maximum percentage of BAHS-induced REM sleep was approximately 20%. REM sleep did not exceed the upper limit of the physiological range. A carry-over effect of BAHS occurred during the withdrawal nights. During the drug nights, and the average length of REM periods increased in each sleep cycle. The length especially increased significantly in cycle 3 (P less than 0.05). The interruptions of REM sleep decreased in number. The histogram of REM sleep showed that REM sleep increased in the middle and the latter part of the night with two apparent peaks. Though REM sleep increased, REM density decreased. The mechanisms by which BAHS increases REM density decreased. The mechanisms by which BAHS increases REM sleep suggests that BAHS increases serotonin in the brain, and that serotonin increases REM sleep secondarily. BAHS seems to be a unique drug which increases REM sleep, while other clinically used drugs suppress it.

Flunitrazepam effects on human sleep EEG spectra: Differences in NREM, REM and individual responses.

Flunitrazepam (FNZ) (4mg), an intermediate type benzodiazepine (BDZ) hypnotic, was administered orally to five healthy male subjects (Ss) for seven consecutive nights. Sleep EEG from the baseline night (BLN), the initial drug night (IDN), the fourth and the seventh drug nights (4DN, 7DN) was subjected to fast Fourier transform (FFT) analysis. During NREM sleep of 4DN and 7DN the sigma band (11.0-12.5 Hz) activity was similarly enhanced in every S. In REM of 4DN and 7DN beta band (23.0-29.0 Hz) was enhanced, but with larger variations among Ss. High intra-individual consistency of the relative EEG power patterns on 4DN and 7DN was observed. These results suggest that 1) EEG responses to FNZ are different in sleep states; explorations of these differences may provide better understandings of sleep mechanisms, and 2) individual variations in EEG responses may reflect individual variations of the BDZ receptor system. These methods may be useful for exploring receptor changes in neuropsychiatric disorders.

Theophylline delays human sleep phase.

Twelve healthy male volunteers were given theophylline 250 mg in order to test effects on 24-hr rhythms. Rhythms of sleep/wake and subjective sleepiness were delayed. Ingestion of xanthines such as theophylline in coffee, tea, colas and chocolate may contribute to some sleep disorders. Theophylline might likewise be useful in treating disorders of circadian oscillators.

Flunitrazepam effects on human sleep EEG spectra. II: Sigma and beta alterations during NREM sleep.

Flunitrazepam (FNZ) is known to enhance the higher EEG frequencies, including sigma (10-15 Hz) and beta (20-28 Hz). Both sigma and beta frequency bands show an inverse relationship with delta (0.3-3 Hz) during NREM periods, as we have previously reported. It is not known whether generation of these two EEG frequencies is mediated by the same or different neuronal mechanisms. In this report, we compare alterations of delta, sigma and beta EEG induced by FNZ (4 mg) orally administered to five healthy male subjects for seven consecutive nights. Sleep EEG on the baseline night (BLN), and the fourth and seventh drug nights (4DN, 7DN) was subjected to fast Fourier transform (FFT) analysis. On drug nights, sigma was enhanced without regard to delta amount, but beta was enhanced only during epochs containing low delta. Thus, sigma and beta EEG were altered differently by the same pharmacological agent. These results suggest that sigma and beta EEG are mediated by different neuronal mechanisms.

No basic rest-activity cycle in head, wrist or ankle.

Kleitman's theory of a Basic Rest-Activity Cycle (BRAC) was tested by recording activity of the head, wrists, and left ankle from 10 healthy subjects. No 90-100 min rhythms in activity were found.

Sleep and respiration under acute hypobaric hypoxia.

The purpose of this study was to elucidate the effects of acute hypobaric hypoxia on nocturnal sleep architecture and respiratory responses in a hypobaric simulator. Five healthy young males (19-23 years old) were recruited to sleep for 8 h at sea level and at simulated altitudes of 1,500, 3,000, and 4,000 m in the simulator (61.2 m3, 20 degrees C, and 60% RH). Each experimental run was separated by at least 3 d. Standard polysomnograph, respiration, and arterial oxygen saturation (SaO2) during sleep were observed. 1) SaO2 decreased significantly with increasing altitude. At 4,000 m, SaO2 showed its lowest value during 1 to 3 h after sleep onset. 2) Sleep architecture below 3,000 m showed almost the same pattern. However, reduction in REM sleep and increased wakefulness were observed at 4,000 m, though such sleep disturbance was not observed in the first one-third of the night spent in bed. 3) Periodic breathing (PB) with apnea and/or hypopnea developed in all subjects above 3,000 m. PB tended to appear in light sleep, though sleep was not always disturbed by PB. It might be concluded that there was no sleep disturbance up to 3,000 m altitude. Nocturnal sleep at 4,000 m, however, was disturbed after a few hours from sleep onset by severe hypoxemia induced by multiplicative effects of hypoxia and hypoventilation during deep sleep. At high altitude, PB seems to not induce arousals consistently, which was different from sleep apnea syndrome at sea level.

Long interspersed element-1 is differentially regulated by food-borne carcinogens via the aryl hydrocarbon receptor.

A single human cell contains more than 5.0 × 10(5) copies of long interspersed element-1 (L1), 80-100 of which are competent for retrotransposition (L1-RTP). Recent observations have revealed the presence of de novo L1 insertions in various tumors, but little is known about its mechanism. Here, we found that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), food-borne carcinogens that are present in broiled meats, induced L1-RTP. This induction was dependent on a cellular cascade comprising the aryl hydrocarbon receptor (AhR), a mitogen-activated protein kinase, and CCAAT/enhancer-binding protein ß. Notably, these compounds exhibited differential induction of L1-RTP. MeIQx-induced L1-RTP was dependent on AhR nuclear translocator 1 (ARNT1), a counterpart of AhR required for gene expression in response to environmental pollutants. By contrast, PhIP-induced L1-RTP did not require ARNT1 but was dependent on estrogen receptor α (ERα) and AhR repressor. In vivo studies using transgenic mice harboring the human L1 gene indicated that PhIP-induced L1-RTP was reproducibly detected in the mammary gland, which is a target organ of PhIP-induced carcinoma. Moreover, picomolar levels of each compound induced L1-RTP, which is comparable to the PhIP concentration detected in human breast milk. Data suggest that somatic cells possess machineries that induce L1-RTP in response to the carcinogenic compounds. Together with data showing that micromolar levels of heterocyclic amines (HCAs) were non-genotoxic, our observations indicate that L1-RTP by environmental compounds is a novel type of genomic instability, further suggesting that analysis of L1-RTP by HCAs is a novel approach to clarification of modes of carcinogenesis.

Prediction of fluoroquinolone-induced elevation in serum creatinine levels: a case of drug-endogenous substance interaction involving the inhibition of renal secretion.

The aim of this study was to examine the mechanism underlying the elevation in serum creatinine levels caused by a novel des-fluoro(6)-quinolone antibacterial agent, DX-619, in healthy subjects. hOCT2 showed a prominent uptake of creatinine (K(m) = 56.4 mmol/l) among renal organic ion transporters. DX-619 is a potent inhibitor of hOCT2 (K(i) = 0.94 micromol/l), hMATE1 (0.82 µmol/l), and hMATE2-K (0.10 micromol/l). The pharmacokinetic model involving the inhibition of hOCT2 (model 1), hOCT2, and MATE1 or MATE2-K (model 2) could predict the elevation in serum creatinine levels in individual subjects receiving DX-619. This assumes that a significant contribution of tubular secretion (59, 38, and 31%) and reabsorption ranged from 3-50, 4-30, and 5-21% in model 1, -2a (hOCT2/hMATE1), and -2b (hOCT2/hMATE2-K), respectively, for creatinine. In conclusion, DX-619, at its therapeutic dose, is able to inhibit hOCT2, hMATE1, and hMATE2-K, leading to a significant inhibition of tubular secretion of creatinine and consequently to elevation of serum creatinine levels.

The effects of air mattress upon sleep and bed climate.

The purpose of this study was to investigate the effects of an air mattress upon sleep and bed climate. This air mattress, which employs a pump and timer to increase or decrease the inflation pressure in order to cure and prevent decubitus was tested. Six healthy female volunteers, aged 18 to 23, served as subjects. The experiments were carried out under three conditions: using regular Futon (Futon), the air mattress with pump and timer activated (Air+) and the same mattress without pump and timer activated (Air-). Room temperature and relative humidity were controlled at 22-23 degrees C and RH 50-60% respectively. Subjects' sleep was monitored by using EEG machine throughout the night, and subject's body temperature and bed climate were also continuously checked. Subjective estimation of bed and sleep were obtained before and after the recording sessions. Sleep onset latency and wake after sleep onset tended to be reduced in Air+ compared to Futon and Air-. The time and percentage of Stage 3 was increased significantly in the middle one third of the night in Air+. A significant difference was observed in bed climate of the waist area. Temperature tended to be higher in Futon than in Air+ and Air-, while relative and absolute humidity were significantly higher in Air+ and Air-. Significant difference between Air+ and Air- was observed only during one hour after sleep recordings started. Thermal sensation in the morning was cooler and comfort sensation tended to be better in Air+ and Air-. Subjective sleep estimation was somewhat good under all conditions. These results suggest that although these air mattresses do not affect sleep, we have to be cautious in using these mattresses as relative and absolute humidity were kept higher than with Futon. Further study on materials and construction of these air mattresses to decrease the humidity is needed.

The effects of a newly designed air mattress upon sleep and bed climate.

In our previous study, an air mattress with three series of air cells with inflation pressure, that was increased/decreased in time interval of 20 min, did not affect sleep quality and quantity, although the relative and absolute humidity inside the bedding was kept significantly higher than that of a Futon. The purpose of this study was to confirm the effects of a newly designed air mattress upon sleep and bed climate. In this newly designed air mattress, the cell series and time interval was reduced. Six healthy female volunteers, aged 18-22, served as subjects. The experiment was carried out under two conditions: using a regular Futon (Futon), and a newly-designed air mattress with the timer and pump activated (Airmat). The room temperature and relative humidity were controlled at 22-23 degrees C and RH 50-60%, respectively. The subjects' sleep was monitored by using an EEG machine and their skin temperatures and bed climates were also measured continuously. Subjective evaluations of bed comfort and sleep were obtained before and after the recording sessions. Sleep onset latency, wake after sleep onset and the sleep efficiency index showed no significant differences between the two conditions. A significant difference was observed in the bed climate of the waist area. The temperature of the waist was lower overall under the Airmat than the Futon, while relative humidity was higher under the Airmat. Absolute humidity also tended to be higher in the Airmat. Sleep evaluation and comfort sensation were good under both conditions. Although sleep was not disturbed and subjective sleep evaluation tended to be better in Airmat, our results indicate that changing the time intervals and cell series until this air mattress level is not effective in decreasing the bed climate humidity.

Polarized efflux of mono- and diacid metabolites of ME3229, an ester-type prodrug of a glycoprotein IIb/IIIa receptor antagonist, in rat small intestine.

ME3229 is an ester-type prodrug of a glycoprotein IIb/IIIa receptor antagonist ME3277. In our previous study, it was shown that only a small part of the drug taken up into the enterocytes reached the mesenteric vein, mainly due to transporter-mediated efflux of its hydrolyzed metabolites formed in the cells. To characterize the efflux transport system for the metabolites, the transport of the diacid metabolite ME3277 and the monoacid metabolites PM-10 and PM-11 were studied. ME3277 and PM-10 were preferentially transported in the serosal-to-mucosal direction across the rat small intestine in the presence of glucose. Permeability of ME3277 across monolayer of Caco-2 cells with P-glycoprotein (P-gp) and indomethacin-sensitive efflux pump expression did not show any directionality and verapamil, an inhibitor of P-gp, and indomethacin did not affect the permeability of ME3277 across rat intestinal tissue. Directional transport was not site specific and was observed in the Eisai hyperbilirubinemic rat whose canalicular multispecific organic anion transporter/multidrug resistance-associated protein (cMOAT/MRP2) is hereditarily defective as well as in normal rats. The efflux transport of ME3277 was inhibited by 1-naphthol, 1-choloro-2,4-dinitrobenzene, and sulfobromophthalein, and efflux of ME3277 and monoacid metabolites from intestinal tissue preloaded with ME3229 fell in the presence of 1-naphthol and sulfobromophthalein. These results demonstrate that mono- and diacid metabolites of ME3229 were pumped out into the gut lumen by an energy-dependent transport system located on the mucosal membrane of intestinal tissue and distinct from either P-gp, indomethacin-sensitive efflux pump or canalicular multispecific organic anion transporter/MRP2. An inhibition study suggested that this unknown transporter has a substrate specificity similar to that of MRP transporter families.