Improved seizure liability detection by combining rat hippocampal brain slice electrophysiology with in vivo behavior observation following intracerebroventricular drug administration.

An adverse effect of drug candidates, seizure is a serious issue in drug development. Improving evaluation systems for seizure liability is crucial for selecting good candidates. Firstly, in vitro electrophysiological measurement by a multielectrode array system in rat hippocampal brain slices was employed to confirm an increase in electrically evoked population spike (PS) area, the occurrence of multiple population spikes (MPSs), and thereby the seizure liability of five positive control chemicals: picrotoxin, 4-aminopyridine, pentylenetetrazole, penicillin G, and chlorpromazine. Aspirin, a negative control, did not affect PS area or generate MPSs. Furthermore, baclofen, an anticonvulsant drug, decreased PS area and inhibited the increase in PS area or occurrence of MPSs induced by picrotoxin. A comparative study of seizure liability among carbapenem antibiotics revealed that tienam > carbenin > omegacin and finibax. Despite leading to a strong decrease in PS area, physostigmine, cisplatin, and paroxetine still produced MPSs. Therefore, the increase in PS area or the occurrence of the MPS are considered significant evaluation parameters for seizure liability. In contrast, the in vitro electrophysiological measurement could not detect the seizure liability of diphenhydramine or fluvoxamine. A follow-up study of in vivo mouse behavioral change induced by intracerebroventricular administration of these drugs clearly detected convulsions. The in vitro electrophysiological study using hippocampal brain slices combined with in vivo behavior observation study of drug candidates administered by intracerebroventricular injection can implement to assess the seizure liability of even small amounts, especially in the early stages of drug development.

Non-invasive differentiation of hepatic steatosis and steatohepatitis in a mouse model using nitroxyl radical as an MRI-contrast agent.

Drug-induced steatohepatitis is considered more serious than drug-induced hepatic steatosis, so that differentiating between the two is crucial in drug development. In addition, early detection of drug-induced steatohepatitis is considered important since recovery is possible with drug withdrawal. However, no method has been established to differentiate between the two. In the development of drug-induced steatohepatitis, reactive oxygen species (ROS) is excessively generated in the liver. It has been reported that ROS can be monitored with electron spin resonance (ESR) and dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) by using nitroxyl radicals, which are known to participate in various in vivo redox reactions. The decay/reduction rate, which is an index for monitoring nitroxyl radicals, has been reported to be increased in tissues with excessive ROS levels other than liver, but decreased in methionine choline deficient (MCD) diet-induced steatohepatitis with excess ROS. Therefore, looking to differentiate between drug-induced hepatic steatosis and steatohepatitis, we examined whether the reduction rate decreases in steatohepatitis other than the MCD-diet induced disease and whether the decrease could be detected by MRI. We used STAM™ mice in which hepatic steatosis and steatohepatitis developed sequentially under diabetic conditions. 3-carbamoyl-PROXYL (CmP), one of the nitroxyl radicals, was injected intravenously during the MRI procedure and the reduction rate was calculated. The reduction rate was significantly higher in early steatohepatitis than in hepatic steatosis and the control. Excess ROS in early steatohepatitis was detected by an immunohistochemical marker for ROS. Therefore, it was indicated that the increase or decrease in the reduction rate in steatohepatitis differs depending on the model, and early steatohepatitis could be noninvasively differentiated from hepatic steatosis using CmP in MRI. Since the change in direction of the reduction rate in steatohepatitis in clinical studies could be predicted by confirming the reduction rate in preclinical studies, the present method, which can be used consistently in clinical and preclinical studies, warrants consideration as a candidate monitoring method for differentiating between early drug-induced steatohepatitis and hepatic steatosis in drug development.

Comparison of 1H-magnetic resonance spectroscopy and blood biochemistry as methods for monitoring non-diffuse hepatic steatosis in a rat model.

No method of monitoring drug-induced hepatic steatosis has been established, which is a concern in drug development. Hepatic steatosis is divided into diffuse and non-diffuse forms according to the pattern of fat deposition. Diffuse hepatic steatosis was reported as evaluable by 1H-magnetic resonance spectroscopy (1H-MRS), which is used as an adjunct to the MRI examination. Blood biomarkers for hepatic steatosis have been also actively investigated. However, there are few reports to conduct 1H-MRS or blood test in human or animal non-diffuse hepatic steatosis with reference to histopathology. Therefore, to investigate whether non-diffuse hepatic steatosis can be monitored by 1H-MRS and/or blood samples, we compared histopathology to 1H-MRS and blood biochemistry in a non-diffuse hepatic steatosis rat model. Non-diffuse hepatic steatosis was induced by feeding rats the methionine choline deficient diet (MCDD) for 15 days. The evaluation sites of 1H-MRS and histopathological examination were three hepatic lobes in each animal. The hepatic fat fraction (HFF) and the hepatic fat area ratio (HFAR) were calculated from 1H-MRS spectra and digital histopathological images, respectively. Blood biochemistry analyses included triglycerides, total cholesterol, alanine aminotransferase, and aspartate aminotransferase. A strong correlation was found between HFFs and HFARs in each hepatic lobe (r = 0.78, p < 0.0001) in rats fed the MCDD. On the other hand, no correlation was found between blood biochemistry values and HFARs. This study showed that 1H-MRS parameters correlated with histopathological changes but blood biochemistry parameters didn't, so that it is suggested that 1H-MRS has the potential to be a monitoring method for non-diffuse hepatic steatosis in rats fed the MCDD. Given that 1H-MRS is commonly used in preclinical and clinical studies, 1H-MRS should be considered a candidate method for monitoring drug-induced hepatic steatosis.

Evaluation of in vivo MRI for detecting midodrine-induced arteritis in rats.

There are no specific and sensitive biomarkers for arteritis, and the occurrence of arteritis in nonclinical toxicological studies of a candidate drug makes development of the drug very difficult. However, we showed in a previous study that the high signal intensity region around the artery on magnetic resonance imaging (MRI) could be a candidate biomarker for detection of arteritis. The present study was conducted to clarify the details of midodrine hydrochloride (MH)-induced arteritis lesions and whether arteritis induced by a mechanism other than the vasodilatory effect, which was evaluated in a previous study, could be detected by MRI. MH is a selective peripherally acting alpha-1 adrenergic receptor agonist, known to induce arteritis due to its vasoconstrictor action, but there is not enough information about MH-induced arteritis. Based on the data obtained under multiple dosing conditions, MH was administered subcutaneously to each rat once daily for 2 days at a dose level of 40 mg/kg/day for MRI assessment. The mesenteric arteries were examined using in vivo MRI at 1 day or 7 days after administration of the final dose and examined histopathologically. On the day after the final dose, high signal intensity region around the artery was observed in animals with minimal perivascular lesions confirmed by histopathology and not observed in an animal without histological changes. On the 7th day after the final dose, no abnormality was observed in histopathological examinations and no high signal intensity regions were observed by MRI in any animal. In conclusion, although further investigation is needed to confirm that high signal intensity is a reliable biomarker for humans, it is suggested that high signal intensity around the artery could be a versatile candidate biomarker with high specificity and sensitivity.

Detection of fenoldopam-induced arteritis in rats using ex vivo / in vivo MRI.

A method capable of identifying drug-induced arteritis is highly desirable because no specific and sensitive biomarkers have yet been defined. Although magnetic resonance imaging (MRI) may be used to find a biomarker candidate for drug-induced arteritis, there are no reports on the evaluation of drug-induced arteritis by MRI. The present study was conducted to clarify whether Fenoldopam mesylate (FM)-induced arteritis in rats can be detected by MRI. FM, a dopamine (D1 receptor) agonist, is known to induce arteritis in rats. FM was administered subcutaneously to each rat once daily for 2 days at a dose of 100 mg/kg/day. These arteries were examined with ex vivo high-resolution MRI or postmortem MRI after euthanasia. These arteries were also examined using in vivo MRI on the day after final dosing or 3 days after administration of the final dose. These arteries were examined histopathologically in all experiments. The ex vivo MRI showed low-intensity areas and a high signal intensity region around the artery, and these findings were considered to be erythrocytes infiltrating the arterial wall and perivascular edema, respectively. In the in vivo study, the MRI of the FM-administered group showed a high signal intensity region around the artery. The perivascular edema observed histopathologically was recognized as a high signal intensity region around the artery on the image of MRI. In conclusion, detection of the high signal intensity region around the artery by MRI is considered to be a useful method for identifying arteritis. Although further investigation is needed to be a reliable biomarker, it is suggested that it could be a biomarker candidate.

Effects of microsampling on toxicity assessment of hematotoxic compounds in a general toxicity study in rats.

Microsampling (MS) has been increasingly used in toxicity studies reducing animal use for toxicokinetic analysis. However, especially for drugs with hematotoxic properties, the potential effects of MS on hematological parameters and subsequent toxicity assessment should be considered, while such properties are frequently unknown at the discovery stage. Here, we conducted a rat 2-week study of hematotoxic compounds and evaluated the effects of MS on toxicity assessment. Six-week-old female SD rats were orally dosed with vehicle, methylene blue trihydrate (MB: 300 mg/kg/day), or azathioprine (AZP: 12 and 24 mg/kg/day) for 2 weeks. Each treatment group was divided into non-MS and MS subgroups, and in the MS subgroups, 50 µL/time point of blood was collected from the jugular vein at 7 time points each on Days 1 and 13 of dosing. The test items included clinical signs, body weight, urinalysis, hematology, blood chemistry, necropsy, organ weight, and histopathology. In the MB non-MS subgroup, there were low values in red blood cell parameters, high values in reticulocytes and bilirubin, and increased extramedullary hematopoiesis, reflecting hemolytic anemia. In the AZP non-MS subgroup, there were low values of red and white blood cell parameters and decreased cellularity in the bone marrow, reflecting myelosuppression. The effects of MB and AZP were similarly observed in the MS subgroups, and the effects of MS on the toxicological endpoints were generally small. Based on these results, the effects of MS on toxicity assessment were considered to be small in rat toxicity studies even for hematotoxic compounds.

Current activities between the DevTox Berlin workshops and the Japanese Teratology Society Terminology Committee in harmonizing the terminology for classifying anomalies in laboratory animals in developmental toxicity studies: Report from the Satellite Workshop of the 60th Annual Meeting of the Japanese Teratology Society.

In recent years, the Japanese Teratology Society has worked with the DevTox Berlin Workshops project to provide internationally consistent terminology for teratogenic effects. This paper summarizes a satellite workshop of the 60th Annual Meeting of the Japanese Teratology Society, which was entitled "Current activities between DevTox Berlin Workshops to develop a harmonized terminology for classifying anomalies in laboratory animals in developmental toxicity studies." The Japanese Teratology Society - Laboratory Animal Terminology Project (JTS-LATP) reviewed "gray zone" anomalies and focused on developing criteria for reclassifying a large number of gray zone anomalies to clarify them and to make it easier to judge fetal categories. This effort will lead to international agreement, based on shared conceptions. The present article aimed to provide the reader with a summary of the issues discussed at the 2020 satellite meeting, which included discussions on open issues from the DevTox Berlin Workshops, ongoing work by the JTS-LATP on gray zone (GZ) anomalies, current industrial concerns, and future challenges.

The in vitro pharmacology and non-clinical cardiovascular safety studies of a novel 5-HT4 receptor agonist, DSP-6952.

The pharmacological activity of DSP-6952, a novel compound was investigated, compared to that of clinically efficacious gastrointestinal (GI) prokinetic 5-hydroxytryptamine4 (5-HT4) receptor agonists. DSP-6952 had a strong affinity of Ki = 51.9 nM for 5-HT4(b) receptor, and produced contraction in the isolated guinea pig colon with EC50 of 271.6 nM and low intrinsic activity of 57%, similar to tegaserod and mosapride. In the development of the 5-HT4 receptor agonists, cardiovascular risk was deliberately evaluated, because some related prokinetics were reported to cause with cardiovascular adverse events, such as ventricular arrhythmias or ischemia. DSP-6952 showed minimal effects up to 100 µM in human ether-a-go-go-related gene (hERG) channels or guinea pig cardiomyocytes. In telemetered conscious monkeys, DSP-6952 did not affect blood pressure or any electrocardiogram (ECG) up to 180 mg/kg, p.o.; however, DSP-6952 transiently increased heart rate, as well as in anesthetized dogs. The positive chronotropic effects of DSP-6952 were completely antagonized by a 5-HT4 receptor antagonist, and another 5-HT4 receptor agonist, TD-5108 also increased heart rate. These effects are considered a class effect seen in clinically developing and marketed 5-HT4 receptor agonists, and have not been regarded as a critical issue in clinical use. DSP-6952 did not induce contraction in the rabbit coronary artery up to 100 µM, which differed from tegaserod or sumatriptan. These results show that DSP-6952 does not have cardiac ischemic risk via coronary vasoconstriction. In conclusion, DSP-6952 is a promising GI prokinetic compound with partial 5-HT4 receptor agonistic activity as well as a favorable cardiovascular safety profile.

Categorization of fetal external findings in developmental toxicology studies by the Terminology Committee of the Japanese Teratology Society.

Categorization of fetal external findings in common laboratory animals, intended to make the agreement at Berlin Workshop in 2014 more practical, was proposed by the Terminology Committee of the Japanese Teratology Society at the Workshop in the 55th Japanese Teratology Society Annual Meeting in 2015. In the Workshop, 73 external findings, which had been categorized as "Gray zone" anomalies but not as "Malformation" or "Variation" in the 2014 Berlin Workshop, were discussed and classified as Malformation, "Non-structural abnormality," Variation, and "Not applicable." The proposal was based on the results of a survey conducted in 2014, where 20 facilities (including pharmaceutical, chemical, and pesticide companies and contract laboratories) and 2 selected expert teratologists in Japan were asked for their opinions on the categorization of these findings. Based on the discussion, Japanese Teratology Society members have agreed that 42 out of the 73 findings can be classified as Malformations (38), Non-structural abnormalities (3), Malformations/Non-structural abnormalities (1), and Variations (0), while the remaining 31 findings were recommended to be categorized as Not applicable for fetuses. The details of the classification are shown on the website of the Japanese Teratology Society (http://www.umin.ac.jp/cadb/External.pdf).

Improvement of the evaluation method for teratogenicity using zebrafish embryos.

The zebrafish has been considered as a suitable animal model for drug discovery, especially for evaluation of the teratogenicity, due to their small size, rapid development, transparency, and developmental similarities to mammalian development. These features of zebrafish make it possible to maintain them in culture plates, evaluate the teratogenicity in short term, conduct morphological assessment of each organ without any autopsy operation. The purpose of the present study was to improve an evaluation method for the teratogenicity of test compounds with high throughput ability and prediction rateusing zebrafish embryos. In this study, we established a modified evaluation method as using non-dechorionated embryos and observation a limited number of parameters without grading. Zebrafish embryos were exposed to test compounds from 5-6 to 144 hr post-fertilization, (hpf) corresponding to the organogenesis period. Morphological changes or functional abnormalities induced by test compound treatments were assessed and scored at 11 endpoints, and the potential of teratogenicity was judged based on the score. As a validation assay of the system, the potentials of 59 known teratogenic or non-teratogenic test compounds were evaluated using the present standard zebrafish assay, and the teratogenicity was correctly predicted in 90% (53/59) of all compounds with low false negative and false positive rates. These results indicated that the evaluation method using zebrafish for the teratogenicity we have improved was a valuable tool for early stage screening in drug discovery.

Historical control data on prenatal developmental toxicity studies in rabbits.

Historical control data on rabbit prenatal developmental toxicity studies, performed between 1994-2010, were obtained from 20 laboratories, including 11 pharmaceutical and chemical companies and nine contract laboratories, in Japan. In this paper, data were incorporated from a laboratory if the information was based on 10 studies or more. Japanese White rabbits and New Zealand White rabbits were used for prenatal developmental toxicity studies. The data included maternal reproductive findings at terminal cesarean sections and fetal findings including spontaneous incidences of morphological alterations. No noticeable differences between strains or laboratories were observed in the maternal reproductive and fetal developmental data. The inter-laboratory variations in the incidences of fetal external, visceral, and skeletal alterations seem to be due to differences in the selection of observation parameters, observation criteria, and classification of the findings, and terminology of fetal alterations.

Evaluation of ovarian toxicity of mono-(2-ethylhexyl) phthalate (MEHP) using cultured rat ovarian follicles.

Mono-(2-ethylhexyl) phthalate (MEHP) is the most toxic metabolite of di-(2-ethylhexyl) phthalate (DEHP). It has been reported that DEHP causes abnormal reproductive development in women, and suppresses estradiol synthesis and ovulation in female rats with diminished size of preovulatory follicles. The present study was conducted to evaluate the ovarian toxicity of MEHP using cultured rat ovarian follicles. Secondary follicles were isolated from the ovaries of 14-day-old female rats and cultured for 48 hr with MEHP (0, 10, 30, and 100 µg/ml). At 0, 24, and 48 hr of MEHP treatment, follicular diameters were measured. After the culture, viability and apoptosis of follicles were assessed, and progesterone, androstenedione, testosterone, and estradiol levels in culture media were measured. At 100 µg/ml, suppression of follicular development was observed, which is associated with decreased viability of follicles and apoptosis of granulosa cells. At this concentration, progesterone level increased markedly, whereas androstenedione, testosterone, and estradiol levels decreased. At 10 and 30 µg/ml, follicular development was not suppressed, no apoptotic change was observed, and the levels of all measured steroid hormones tended to increase. The combined levels of all steroid hormones increased at all concentrations of MEHP, and the increase implies that MEHP activates the synthetic pathway from cholesterol to estradiol including de novo synthesis of cholesterol. However, the progesterone/androstenedione ratio increased extremely at 100 µg/ml, and the increase implies that MEHP inhibits the conversion of progesterone to androstenedione. In conclusion, MEHP induces ovarian toxicity via suppression of follicular development and abnormal steroid hormone synthesis in cultured rat ovarian follicles.

Evaluation of ovarian toxicity of sodium valproate (VPA) using cultured rat ovarian follicles.

Sodium valproate (VPA) is a major antiepileptic drug that is widely used for the treatment of epilepsy as well as other neuropsychiatric diseases. The present study was conducted to evaluate the ovarian toxicity of VPA using cultured rat ovarian follicles. Secondary follicles were isolated from the ovaries of 14-day-old female rats and cultured for 48 hr with VPA (0, 0.2, 1.0, and 5.0 mM). At 0, 24, and 48 hr of VPA treatment, follicular diameters were measured. After the culture, viability of follicles and expression of aromatase in the follicles were assessed, and progesterone, androstenedione, testosterone, and estradiol levels in culture media were measured. At all concentrations of VPA, follicular development was suppressed, and androstenedione, testosterone, estradiol, and combined levels of all steroid hormones tended to decrease in association with suppression of aromatase expression in granulosa cells. Additionally, the suppression of follicular development was associated with decreased viability of follicles and an increased progesterone level at 5.0 mM of VPA. The decrease in the combined levels of all steroid hormones implies that VPA suppresses the synthetic pathway from cholesterol to estradiol including de novo synthesis of cholesterol. In conclusion, VPA induces ovarian toxicity via suppression of development and abnormal steroid hormone synthesis in cultured rat ovarian follicles.