Implications for the Predictivity of Cell-Based Developmental Toxicity Assays Developed Two Decades Apart.

Many in vitro developmental toxicity assays have been proposed over several decades. Since the late 1980s, we have made intermittent attempts to introduce in vitro assays as screening tests for developmental toxicity of in-house candidate products. Two cell-based assays which were developed two decades apart were intensively studied. One was an assay of inhibitory effects on mouse ascites tumor cell attachment to a concanavalin A-coated plastic sheet surface (MOT assay), which we studied in the early days of assay development. The other was an assay of inhibitory effects on the differentiation of mouse embryonic stem cell to beating heart cells (EST assay), which we assessed more recently. We evaluated the suitability of the assays for screening in-house candidates. The concordance rates with in vivo developmental toxicity were at the 60% level. The EST assay classified chemicals that inhibited cell proliferation as embryo-toxic. Both assays had a significant false positive rate. The assays were generally considered unsuitable for screening the developmental toxicity of our candidate compounds. Recent test systems adopt advanced technologies. Despite such evolution of materials and methods, the concordance rates of the EST and MOT systems were similar. This may suggest that the fundamental predictivity of in vitro developmental toxicity assays has remained basically unchanged for decades. To improve their predictivity, in vitro developmental toxicity assays should be strictly based on elucidated pathogenetic mechanisms of developmental toxicity.

Translational research into species differences of endocrine toxicity via steroidogenesis inhibition by SMP-028--for human safety in clinical study.

SMP-028 is a drug candidate developed for the treatment of asthma. In a 13-week repeated dose toxicity study of SMP-028 in rats and monkeys, differences of endocrine toxicological events between rats and monkeys were observed. In rats, these toxicological events mainly consisted of pathological changes in the adrenal, testis, ovary, and the other endocrine-related organs. On the other hand, in monkeys, no toxicological events were observed. The goal of this study is to try to understand the reason why only rats, but not monkeys, showed toxicological events following treatment with SMP-028 and to eventually predict the possible toxicological effect of this compound on human endocrine organs. Our results show that SMP-028 inhibits neutral cholesterol esterase more strongly than other steroidogenic enzymes in rats. Although SMP-028 also inhibits monkeys and human neutral cholesterol esterase, this inhibition is much weaker than that of rat neutral cholesterol esterase. These results indicate (1) that the difference in endocrine toxicological events between rats and monkeys is mainly due to inhibition of steroidogenesis by SMP-028 in rats, not in monkeys, and (2) that SMP-028 may not affect steroidogenesis in humans and therefore might cause no endocrine toxicological events in clinical studies.

Effect of SMP-028 on steroidogenesis in rats; mechanism of toxicological events on endocrine organs of rats.

SMP-028 is a new compound for treatment of asthma. Oral administration of SMP-028 to rats was associated with toxicological events in endocrine organs. These events mainly consisted of pathological changes in the adrenal gland, testis, prostate, seminal vesicle, ovaries, and uterus. In this study, we set to clarify whether SMP-028 inhibits steroidogenesis in primary culture cells obtained from rat endocrine organs in vitro. Adrenal cells, testicular cells, and ovarian cells were treated with SMP-028 and the production of steroid hormones, i.e., progesterone, aldosterone, corticosterone, total testosterone, and estradiol from these cells was measured by radioimmunoassay. We found that the production of progesterone from these cells treated with SMP-028 at 1 µM decreased to 16-67% that of the control. These findings indicate that SMP-028 inhibits steroidogenesis in rat endocrine organs in vitro. Considering that free maximum concentration in rats treated with SMP-028 are higher than the IC50 values for the inhibition of steroidogenesis in vitro, it is therefore believed that the toxicological events seen in rats following treatment with SMP-028 are due to inhibition of steroidogenesis in vivo.

Relationship between arterial and fibrous cap remodeling: a serial three-vessel intravascular ultrasound and optical coherence tomography study.

BACKGROUND: Positive arterial remodeling and thin fibrous cap are characteristics of rupture-prone or vulnerable plaque. The natural course of the fibrous cap thickness and the relationship between serial arterial remodeling and changes in fibrous cap thickness are unknown. Therefore, the purpose of this study was to evaluate the relationship between changes in fibrous cap thickness and arterial remodeling by using optical coherence tomography (OCT) and intravascular ultrasound (IVUS) during 6-month follow-up. METHODS AND RESULTS: Both IVUS and OCT examinations were performed on 108 vessels from 36 patients with ischemic heart disease who underwent percutaneous coronary intervention. Fifty-eight fibroatheromas were selected from 82 nonsignificant, nonculprit lesions (angiographic diameter stenosis, 25% to 75%; plaque burden, >40% by IVUS). Fibroatheroma was defined by OCT as lipid-rich plaque in >1 quadrant that has lipid. Thickness of the fibrous cap was measured by OCT. IVUS and OCT examinations were repeated at 6-month follow-up. Serial changes and relationships between IVUS indices and fibrous cap thickness were investigated. Overall, fibrous cap thickness (98.1±38.9 to 96.9±44.5 µm) as well as IVUS indices did not change significantly within 6 months. The percent changes in fibrous cap thickness correlated negatively and significantly (r=-0.54; P<0.0001; generalized estimating equation adjusted, r=-0.42; P=0.001) with the percent changes in external elastic membrane cross-sectional area. CONCLUSIONS: Arterial remodeling is related to changes in fibrous cap thickness. Positive arterial remodeling is not only an adaptive process, but also related to thinning of the fibrous cap.

The predictivity of preliminary embryo-fetal development (EFD) studies: results of a retrospective survey in Japanese pharmaceutical companies.

To explore the predictivity of dose range-finding (DRF) studies, we conducted asurvey by sending out questionnaires to 72 Japanese pharmaceutical companies.The survey yielded data for 108 and 85 compounds for which any embryo-fetaldevelopment (EFD) toxicities were observed in the definitive studies in rodentsand non-rodents, respectively. As a result of the analysis, 83% of studies inrodents and 80% in non-rodents showed EFD effects in the DRF studies. Whenfocusing on teratogenicity, 91% of studies in rodents and 100% in non-rodentswere judged "positive" in the DRF studies when all EFD toxicities were used asmarkers. When the effects of both the rodent and non-rodent studies wereevaluated together, the combination predictive value in the DRF studies was 96%for EFD toxicants and 100% for teratogens. To evaluate the influence of theexamination items, the predictive value was analyzed using 54 compounds forwhich full examinations (external, visceral and skeletal examination) wereconducted in both rodent and non-rodent DRF studies. When the results werejudged by including or excluding skeletal and visceral examinations results,the predictive values were not significantly different. In conclusion, theresults of this survey showed that a pair of the DRF studies in the rodents andnon-rodents is useful to increase the predictivity of DRF studies. In additionthe inclusion of observations such as fetal survival, body weight and externalexamination into the DRF studies are important to predict effects in thedefinitive studies.

Portopulmonary hypertension associated with congenital absence of the portal vein treated with bosentan.

Portopulmonary hypertension (PPHTN) is pulmonary arterial hypertension (PAH) associated with portal hypertension. It is a common condition among liver transplantation candidates; however, its association with congenital absence of the portal vein (CAPV) has not yet been established. CAPV is a very rare developmental anomaly, which is usually accompanied by abnormal mesenteric drainage that bypasses the liver. Here, we report a rare case of severe PPHTN secondary to CAPV.

Three-dimensional quantitation of mitral valve coaptation by a novel software system with transthoracic real-time three-dimensional echocardiography.

We investigated the degree of mitral valve coaptation with a custom quantitation software system using transthoracic three-dimensional (3D) echocardiography. With real-time 3D echocardiography, we obtained transthoracic volumetric images in 20 healthy subjects and 20 patients with dilated cardiomyopathy. With our novel software system, the surface area of mitral valve tenting in the onset of mitral leaflet closure [O] and the timing of maximum closure of mitral leaflet [M] were reconstructed for quantitative measurement. The coaptation index was calculated by the following formula: [(3D tenting surface area in O-3D tenting surface area in M)/3D tenting surface area in O]. The coaptation index in patients with dilated cardiomyopathy was significantly smaller than that in healthy subjects (11% +/- 4.1% vs. 18% +/- 8.0%, P = .004). The custom quantitation software system with 3D echocardiography allowed us to assess the degree of mitral valve coaptation.

Local release of catecholamines from the hearts of patients with tako-tsubo-like left ventricular dysfunction.

BACKGROUND: The precise mechanism of tako-tsubo-like left ventricular (LV) dysfunction remains unclear, although recent studies have shown that activation of sympathetic tone might be involved. However, local release of catecholamines from cardiac sympathetic efferent neurons in patients with tako-tsubo-like LV dysfunction remains poorly understood. The purpose of this study was to investigate evidence of local release of catecholamines from the hearts of patients with tako-tsubo-like LV dysfunction. METHODS AND RESULTS: Five consecutive patients with tako-tsubo-like LV dysfunction were studied. After confirming LV apical ballooning and a normal coronary angiogram, sampling of blood for the measurement of plasma catecholamine levels was performed at the aortic root (Ao) and coronary sinus (CS). In all 5 cases, increased local release of norepinephrine from the heart was documented (597, 4,238, 2,121, 486, 371 pg/ml at the Ao; 836, 5,719, 3,386, 658, 472 pg/ml at the CS, respectively). CONCLUSIONS: Increased cardiac catecholamines might cause the transient LV apical ballooning in patients with tako-tsubo-like LV dysfunction.

Rapid progression of mild to moderate aortic stenosis in patients older than 80 years.

OBJECTIVE: The purpose of this study was to evaluate the rate of progression of mild and moderate aortic stenosis in patients aged 80 years and older. METHODS: In all, 41 patients with mild and moderate aortic stenosis were included and divided into two groups by age: 19 patients aged 80 years and older (mean 84 +/- 4 years), and 21 patients younger than 80 years (mean 66 +/- 6 years). RESULTS: The rate of degression of aortic valve area was more rapid in the 80 years and older age group than that in the younger than 80 years age group (-0.05 +/- 0.06 and -0.10 +/- 0.08 cm2/y, P = .014). Univariate and multivariate analysis of the rate of degression of aortic valve area were performed, and age was the only independent predictor of the rate of degression of aortic valve area. CONCLUSION: Progression of mild and moderate aortic stenosis in patients aged 80 years and older was more rapid than that in those aged younger than 80 years.

Rate of progression of valvular aortic stenosis in patients undergoing dialysis.

BACKGROUND: In patients undergoing dialysis, aortic valve calcification and aortic stenosis are frequently found. However, the rate of progression of aortic stenosis is still unclear. METHODS: In all, 55 consecutive patients undergoing dialysis were followed up by echocardiography for a period of 12 months. Patients were divided into two groups: noncalcification (no or mildly calcified aortic valve) and calcification (moderate or heavily calcified aortic valve). RESULTS: The rate of progression of maximum aortic jet velocity and degression of aortic valve area were more rapid in calcification group than in noncalcification group (0.37 +/- 0.36 m/s and 0.17 +/- 0.29 m/s, P = .027; 0.17 +/- 0.15 cm2 and 0.04 +/- 0.07 cm2, P < .001, respectively). CONCLUSIONS: The degree of aortic stenosis progressed more rapidly in patients undergoing dialysis with aortic valve calcification than without aortic valve calcification.

Lack of evidence for endocrine disrupting effects in rats exposed to fenitrothion in utero and from weaning to maturation.

Fenitrothion is a broad-spectrum organophosphate insecticide. Recently, it has been reported to exert androgenic or anti-androgenic activity in in vitro and in vivo screening assays, although the effects appear equivocal in vivo. To provide a conclusive and comprehensive evaluation of fenitrothion, especially regarding its anti-androgenic activity in the reproductive and endocrine systems, we conducted a one-generation reproductive toxicity study at appropriately toxic dose levels with a number of sensitive endpoints for endocrine disruption. Fenitrothion was administered to Crj:CD(SD)IGS parental animals (P) at concentrations of 10, 20, and 60 ppm in the diet for 10 weeks prior to mating, and throughout mating, gestation and lactation. Their offspring (F1) were exposed from weaning until maturation at the age of 10 weeks. In the P generation, brain cholinesterase activity was remarkably reduced in the 60 ppm males and in the 20 and 60 ppm females. Reproductive performance, organ weights, histopathology, and sperm analytical parameters were not affected. In the F1 generation, no general toxicity or effects on anogenital distance, retention of areolae/nipples, onset of puberty, organ weights, histopathological findings, and sperm parameters were observed. In conclusion, fenitrothion had no effects on the reproductive or endocrine systems of the P and F1 generations, even at toxic doses that markedly suppressed brain cholinesterase activity in P animals. The results suggest that fenitrothion at in-use levels in the environment is unlikely to cause disruption of human endocrine systems.

[Bilateral coronary ostial stenosis after aortic valve replacement with freestyle stentless bioprosthesis: a case report].

An 80-year-old woman underwent aortic valve replacement with Freestyle stentless prosthetic valve for the stenosis. Four months later, she was admitted with myocardial ischemia. Coronary angiography revealed severe stenosis in the ostium of both right and left coronary arteries. Coronary artery bypass grafting was performed. One year later, percutaneous coronary intervention was carried out for the bilateral coronary arteries because of unstable angina. Intravascular ultrasonography demonstrated localized, membranous, homogeneous, and severe stenoses in the ostium of the right and left coronary arteries. Histological examination of a specimen taken by directional coronary atherectomy showed intimal hypertrophy, mucinous degeneration, and hyaline degeneration without reactive change. There were no findings of atherosclerosis. These clinical, angiographical histological and intravascular ultrasonography findings suggest that the immunological reaction to the heterograft was the mechanism of the bilateral ostial coronary arteries stenoses in the present case. The possibility of immunological reaction after aortic valve replacement with heterograft should be considered. There have been no report on intravascular echocardiographic and histological findings.