Less invasive, simultaneous, and continuous measurements of locomotor activity and body temperature using the nano tag® small accelerometer device in cynomolgus monkeys.

Locomotor activity and body temperature evaluations of cynomolgus monkeys are useful to understand the effects of drugs on the central nervous system. Here, we describe a simple, inexpensive, and less invasive evaluation method using the nano tag® (KISSEI COMTEC Co., Ltd.), a small three-axis accelerometer device with a temperature sensor. Nano tags® were subcutaneously implanted in four cynomolgus monkeys that had been intraperitoneally implanted with a telemetry transmitter. Then, body temperature and locomotor activity counts were simultaneously and continuously measured by both the nano tag® and telemetry transmitter for 14 days after nano tag® implantation. The invasiveness of the implantation surgery was evaluated by recovery after surgery, and the validity of each nano tag® parameter was evaluated by comparison with the telemetry system data. Additionally, locomotor activity and body temperature changes induced by treatment with ketamine, a noncompetitive N-methyl-d-aspartate receptor antagonist, were evaluated by the nano tag®. Recovery from nano tag® implantation surgery was observed at 7 days postoperative, indicating that nano tag® was less invasive than a telemetry transmitter. Both of the parameter profiles measured by nano tag® were approximately comparable to those of the telemetry system. Moreover, the nano tag® could detect ketamine-induced pharmacological changes of decreases in both parameters. The present study demonstrates that nano tag® is an effective, simple, and less invasive tool for locomotor activity and body temperature evaluations in cynomolgus monkeys. This proposed easier method could help researchers evaluate central nervous system effects in cynomolgus monkeys.

Estradiol dominance induces hemodilution and mild hematological alterations in mifepristone-treated rats.

We examined that an estradiol-dominant state against progesterone could affect hematological parameters through hemodilution because estradiol is known to increase plasma volume via oncotic pressure. We performed a 2- and 3-week repeated oral dose study with mifepristone, a progesterone receptor antagonist, in female rats and examined erythrocyte counts, hemoglobin, hematocrit, plasma volume, levels of estradiol and progesterone, water intake, and water loss. Mifepristone treatment decreased some hematological parameters mildly and increased plasma volume. There were no remarkable changes in the balance of water intake and water loss through urination. Both estradiol and progesterone levels and the ratio of estradiol to progesterone increased. Therefore, our findings indicate that repeated mifepristone treatment increases estradiol levels and plasma volume, resulting in lower erythrocyte counts, hemoglobin, and hematocrit. The present study proved the possible contribution of estradiol to understanding the toxicological significance of mifepristone-induced hemodilution.

Detection of micronuclei in hepatocytes isolated from young adult rats repeatedly treated with N-nitrosodi-n-propylamine.

To assess the effectiveness of the multiple dose liver micronucleus (MN) assay, the induction of micronuclei by N-nitrosodi-n-propylamine (NDPA), a genotoxic rodent carcinogen, was compared in hepatocytes (HEPs) and bone marrow (BM) cells. Young adult male rats were treated orally with NDPA at 6 weeks of age for 14 days using daily doses of 10, 20 and 40mg/kg. Samples of the liver and BM tissues were harvested from each animal one day following the last treatment with NDPA and were evaluated for the frequencies of micronucleated cells. Repeated doses with 40mg/kg/day of NDPA caused systemic and hepatic toxicity, including suppressed body weight gains and histopathological hepatic lesions. The frequencies of micronucleated HEPs were significantly increased in all the NDPA-treated groups in a dose-dependent manner. In contrast, the induction of micronuclei in the BM was undetectable, even at the high dose level of 40mg/kg, for which the inhibition of hematopoiesis was observed. For the detection of micronucleated HEPs induced by NDPA treatment, a 14-day administration period is adequate. The liver MN assay using naive young adult rats may be integrated into general repeated-dose toxicity studies including histopathological examinations. Our results suggest that the liver MN assay using multiple doses is more efficient and sensitive than the BM MN assay in detecting the in vivo genotoxic potential of NDPA.

ß3-adrenoceptor-mediated increased circulating transaminase levels in mice treated with its agonist BRL 37344.

Treatment with the selective ß(3)-adrenoceptor agonist BRL 37344 increased circulating levels of alanine transaminase (ALT) and aspartate transaminase (AST) in mice without causing hepatocellular injury. To clarify whether this was a ß(3)-adrenoceptor-mediated effect, the inhibitory effect of the selective ß(3)-adrenoceptor antagonist SR 59230A on the increase in circulating transaminase levels induced by BRL 37344 was examined. A single intraperitoneal dose of BRL 37344 alone initially increased insulin and non-esterified fatty acid (NEFA) dose-proportionally at 0.5 hr post-dose, findings considered attributable to ß(3)-adrenoceptor-stimulating effects. Levels of the gluconeogenic precursors pyruvate (PA) and lactate (LA) were increased corresponding to the change in insulin. Thereafter, glucose (GLU) level was decreased at 4 and 8 hr post-dose, suggesting disruption of glucose homeostasis. In association with these changes in glucose metabolism, transaminase levels were increased maximally at 4 hr post-dose. The transaminase changes were not accompanied by increases in circulating levels of other hepatocellular enzymes, including guanine deaminase (GUA), glutamate dehydrogenase (GLDH), and lactate dehydrogenase (LDH), or any morphological hepatocellular injury. Intraperitoneal pre-treatment with SR 59230A partly inhibited the effects of BRL 37344 alone, indicating that the increase in levels of circulating ALT by BRL 37344 was attributable to a ß(3)-adrenoceptor-stimulating effect. In conclusion, the ß(3)-adrenoceptor agonist BRL 37344 was shown to increase circulating transaminase levels in mice accompanied with dynamic changes in glucose metabolism. These findings suggest the possibility that circulating transaminase levels are increased as pharmacological effects of drugs disrupting glucose metabolism, and that hepatotoxic markers should be selected considering these effects to distinguish between acceptable pharmacology and toxicity.

Salbutamol inhibits lipopolysaccharide-induced inflammatory responses in rat peritoneal macrophages.

Acute and chronic inflammatory diseases are associated with the induction of inducible nitric oxide synthase (iNOS) and inducible heme oxygenase (HO-1). These inducible enzymes are up-regulated in macrophages subjected to inflammatory stimuli and oxidative stress. beta(2)-Adrenoceptor (AR) agonists, which function as bronchial dilators, are widely used for the treatment of asthma and chronic obstructive pulmonary disease (COPD). We examined whether salbutamol, a classical beta(2)-AR agonist, inhibits the induction of proinflammatory cytokines and stress inducible proteins. Rat macrophages obtained from the abdominal cavity were incubated with lipopolysaccharide (LPS) with or without salbutamol. Induction by LPS of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 was significantly inhibited (P < 0.05) by salbutamol treatment. Induction by LPS of iNOS mRNA and protein was also significantly inhibited (P < 0.05) by salbutamol. LPS-mediated increases in HO-1 mRNA and protein were not appreciably affected by salbutamol. One of the anti-inflammatory mechanisms of salbutamol was thus found to be inhibition of induction by LPS of extracellular stimulus-responsive kinase (ERK) 1/2 in macrophages. These findings suggest that salbutamol has the potential for use as an anti-inflammatory agent due to its suppression of LPS-induced TNF-alpha, and IL-6 and iNOS via ERK pathway without affecting HO-1 expression.

Renoprotective effect of the L/N-type calcium channel antagonist cilnidipine on puromycin aminonucleoside-induced nephrosis in rats.

The renoprotective effect of cilnidipine ((+/-)-2-methoxyethyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate, CAS 132203-70-4), a L/N-type calcium channel antagonist, on puromycin aminonucleoside (PAN)-induced nephrosis was investigated in rats. In the Experiment I, rats were given an intravenous injection of PAN (70 mg/kg). Cilnidipine (3 mg/kg/day) and enalapril (CAS 75847-73-3, 5 mg/kg/day) were administered orally from 6 days after treatment with PAN (day 6) to day 26, and urinary analysis was performed on days 9, 15, 20 and 27. In the Experiment II, nephrosis was also induced by intravenous injection of PAN (70 or 100 mg/kg) in rats which were treated with cilnidipine and enalapril from days 6 to 10. Systolic blood pressure was measured on day 7 and urinary analysis was performed on day 10. On day 11, serum was collected and the kidneys were removed for immunofluorescence staining for nephrin and podocin proteins. In PAN-treated rats, the daily urinary protein excretion was dramatically elevated on day 5, reached a peak on day 9 and gradually returned to a normal level from days 15 to 27. Cilnidipine (3 mg/kg/ day) significantly suppressed the increase in proteinuria on day 9 and also improved the decrease in creatinine clearance without evident effect on the blood pressure. Furthermore, the elevations in serum total cholesterol and triglyceride tended to be suppressed by cilnidipine. The expression of nephrin and podocin proteins in PAN-treated rats showed the granular pattern in the glomeruli, while the intensity of staining seemed to be dependent on the urinary protein excretion level in the cilnidipine-treated rats. The results obtained in this study suggest a renoprotective effect of cilnidipine in PAN-induced nephrosis in rats.

Induction of hepatic cytochrome P450 isoforms by nicardipine at therapeutic doses in spontaneously hypertensive rats.

Nicardipine hydrochloride (Nic), a calcium channel antagonist, is used for the treatment of hypertension. In the present study, we estimated its effects on the levels and activities of hepatic cytochrome P450 isoforms in spontaneously hypertensive rats given p.o. with Nic at a dose of 0.5, 2.5, 5, or 12.5 mg/kg at 24-hr intervals for 14 days. Therapeutic effects on the development of hypertension were observed at doses of 5 and 12.5 mg/kg/day. Significant increases in the levels of mRNAs and enzyme activities of hepatic P450 isoforms, CYP1A1 and/or CYP1A2, by 14-day repetitive treatment with Nic were observed at lower therapeutic doses, whereas the increase in protein levels for CYP1A2 was observed at a higher therapeutic dose of 12.5 mg/kg/day. Likewise, the activities of hepatic CYP2B and CYP3A subfamily enzymes were increased by the 14-day-treatment of Nic only at a therapeutic dose (12.5 mg/kg/day), whereas their mRNA and protein levels were increased at lower therapeutic doses. To date, the dihydropyridine family, including Nic, has been believed to have inhibitory effects on the activity of various cytochrome P450 enzymes, especially human CYP3A4. However, the present findings demonstrate for the first time that Nic-repetitive treatments at a therapeutic dose result in significant increases in the expressions and activities of hepatic CYP1A, CYP2B, and CYP3A subfamily enzymes. Therefore, the effects of dihydropyridine family on cytochrome P450 enzymes have to be further validated to provide information on its safe and beneficial therapeutic application.

Species difference in the induction of hepatic CYP1A subfamily enzymes, especially CYP1A2, by 2-methoxy-4-nitroaniline among rats, mice, and guinea pigs.

Species difference in the induction of hepatic cytochrome P450 CYP1A subfamily enzymes by 2-methoxy-4-nitroaniline (2-MeO-4-NA) was investigated among male F344 rats, C57BL/6 Cr mice, and Hartley guinea pigs. All species of animals were treated with a single ip injection of 2-MeO-4-NA (0.44 mmol/kg body weight), and changes in levels of the mRNA and protein of hepatic cytochrome P4501A (CYP1A) subfamily enzymes were examined by the methods of RT-PCR and Western blot, respectively. In addition, hepatic microsomal enzyme activities were measured using methoxyresorufin and ethoxyresorufin as substrates of CYP1A2 and CYP1A1, respectively. The overall results of the RT-PCR, Western blot, and measurement of the enzyme activity indicated that 2-MeO-4-NA-mediated induction of hepatic CYP1A subfamily enzymes, especially CYP1A2, occurred only in rats but not any other species of animals examined and that the species difference in the CYP1A induction was not necessarily correlated with that in pharmacokinetics of 2-MeO-4-NA. Furthermore, a luciferase reporter gene assay for screening of the ligands of arylhydrocarbon receptor (AhR) using a rat hepatic cell line suggested that 2-MeO-4-NA is not an AhR ligand. The present findings demonstrate for the first time the species difference in the 2-MeO-4-NA-mediated induction of hepatic CYP1A subfamily enzymes between rats and other rodents, mice and guinea pigs, and further propose an AhR-independent pathway for 2-MeO-4-NA-mediated induction in rats.

[Toxicity profile of silodosin (KMD-3213)].

The toxicity profile of silodosin, a selective alpha(1A)-adrenoceptor antagonist, was evaluated. The lethal doses were 800 mg/kg in rats and 1500 mg/kg in dogs. Repeated-dose studies revealed fatty degeneration of hepatocytes and an induction of drug-metabolizing enzymes at 15 mg/kg/day or more in male rats, mammary gland hyperplasia at 60 mg/kg/day or more in female rats, and degeneration of the seminiferous tubular epithelium at 25 mg/kg/day or more only in young dogs. Silodosin was negative in all mutagenicity studies, except for a weak positive in a chromosomal aberration assay conducted without metabolic activation. In carcinogenicity studies, mammary gland tumors and pituitary adenomas were increased in female mice given 150 mg/kg/day or more and 400 mg/kg/day respectively, while thyroid follicular cell carcinoma was increased in male rats given 150 mg/kg/day. Reproductive studies in rats revealed a decreased male fertility at 20 mg/kg/day or more and a prolonged estrous cycle at 60 mg/kg/day or more. Silodosin did not exhibit any teratogenic potential in either rats or rabbits, and had no effects on the postnatal development of rat offspring. In safety pharmacology studies, silodosin produced no severe effects on the central nervous, cardiovascular, or respiratory systems. In conclusion, silodosin exhibited adequate safety margins between the clinically recommended dose and those at which toxic effects or safety pharmacological changes were detected. As a new therapeutic drug for the micturition difficulties caused by benign prostatic hyperplasia, silodosin should have few serious side effects in clinical use.