Discovery of DS-6930, a potent selective PPARγ modulator. Part II: Lead optimization.

Attempts were made to reduce the lipophilicity of previously synthesized compound (II) for the avoidance of hepatotoxicity. The replacement of the left-hand side benzene with 2-pyridine resulted in the substantial loss of potency. Because poor membrane permeability was responsible for poor potency in vitro, the adjustment of lipophilicity was examined, which resulted in the discovery of dimethyl pyridine derivative (I, DS-6930). In preclinical studies, DS-6930 demonstrated high PPARγ agonist potency with robust plasma glucose reduction. DS-6930 maintained diminished PPARγ-related adverse effects upon toxicological evaluation in vivo, and demonstrated no hepatotoxicity. Cofactor recruitment assay showed that several cofactors, such as RIP140 and PGC1, were significantly recruited, whereas several canonical factors was not affected. This selective cofactor recruitment was caused due to the distinct binding mode of DS-6930. The calcium salt, DS-6930b, which is expected to be an effective inducer of insulin sensitization without edema, could be evaluated clinically in T2DM patients.

Discovery of DS-6930, a potent selective PPARγ modulator. Part I: Lead identification.

The lead identification of a novel potent selective PPARγ agonist, DS-6930 is reported. To avoid PPARγ-related adverse effects, a partial agonist was designed to prevent the direct interaction with helix 12 of PPARγ-LBD. Because the TZD group is known to interact with helix 12, the TZD in efatutazone (CS-7017) was replaced to discover novel PPARγ intermediate partial agonist 8i. The optimization of 8i yielded 13ac with high potency in vitro. Compound 13ac exhibited robust plasma glucose lowering effects comparable to those of rosiglitazone (3 mg/kg) in Zucker diabetic fatty rats. Upon toxicological evaluation, compound 13ac (300 mg/kg) induced hemodilution to a lower extent than rosiglitazone; however, 13ac elevated liver enzyme activities. X-ray crystallography revealed no direct interaction of 13ac with helix 12, and the additional lipophilic interactions are also suggested to be related to the maximum transcriptional activity of 13ac.

Comparison of photopic negative response (PhNR) between focal macular and full-field electroretinograms in monkeys.

PURPOSE: To compare the characteristics of the photopic negative response (PhNR) between the focal macular and full-field electroretinograms (ERGs) in monkeys. METHODS: Both focal macular and full-field photopic ERGs were recorded in four cynomolgus monkeys under identical stimulus and recording conditions except for which area of the retina was illuminated. The luminance and duration of red flash stimuli were varied in the presence of steady blue background illumination. These ERGs were recorded before and after intravitreal injection of tetrodotoxin (TTX). RESULTS: Several differences were identified between the focal macular and full-field ERGs, including: (1) The PhNR/b-wave amplitude ratio was higher in the focal macular than in the full-field ERGs, and (2) the stimulus threshold of the focal macular PhNR was lower than that of the full-field PhNR. For both macular and full-field stimulation conditions, (1) PhNR amplitude generally increased with increasing stimulus luminance; (2) PhNR implicit time was independent of the stimulus luminance; (3) PhNR amplitude and implicit time increased with increasing stimulus duration up to 50 ms, while a further increase in stimulus duration produced no change in amplitude or implicit time; and (4) PhNR amplitude was selectively attenuated by TTX. CONCLUSIONS: Both the focal macular and full-field PhNRs reflect the functional properties of the inner retina including the retinal ganglion cells (RGCs). Relative to the b-wave, the contribution is weighted more heavily in the focal macular than in the full-field PhNR. Furthermore, these results support the idea that the focal macular PhNR can be an indicator of the function of the macular RGCs.

Response Characteristics and Retinal Origin of the Photopic Negative Response of the Electroretinogram in Dogs.

PURPOSE: To investigate the response characteristics and retinal origin of the photopic negative response (PhNR) of the electroretinograms (ERGs) in dogs. METHODS: Photopic ERGs were elicited by white flash stimuli of different intensities under a steady white background illumination in four anesthetized dogs. These ERGs were also recorded in the same manner after intravitreal injection of tetrodotoxin (TTX). Additionally, retinal localization of voltage-gated sodium channel Nav 1.6 was assessed by immunohistochemistry. RESULTS: The amplitude of the a-wave and the PhNR was increased as the stimulus intensity was raised, while the amplitude of the b-wave was peaked at the moderate stimulus intensity of 3.09 cd·s/m2. TTX greatly attenuated the PhNR, while the reduction in the b-waves and a-wave was mild or insignificant. Nav 1.6-expression was specifically detected on the retinal ganglion cells (RGCs). CONCLUSIONS: Our results are consistent with the PhNR primarily derived from the inner retina including RGCs in dogs, suggesting that the PhNR can be used to monitor function of these retinal components in dogs.

Effects of R-102444 and its active metabolite R-96544, selective 5-HT2A receptor antagonists, on experimental acute and chronic pancreatitis: Additional evidence for possible involvement of 5-HT2A receptors in the development of experimental pancreatitis.

The effects of R-102444 ((2R, 4R)-4-lauroyloxy-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]phenoxy]ethyl-1-methylpyrrolidine hydrochloride) and its active metabolite R-96544 ((2R, 4R)-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]phenoxy]ethyl-4-hydroxy-1-methylpyrrolidine hydrochloride), potent and selective 5-hydroxytryptamine 2A (5-HT2A) receptor antagonists, on development of pancreatitis were investigated in experimental models of acute and chronic pancreatitis. Rat acute pancreatitis was induced by caerulein (20 microg/kg) intraperitoneal injection and by pancreatic duct ligation. In both the models, serum amylase and lipase activities were markedly increased. R-102444 dose-dependently reduced these enzyme activities at a dose range of 10 to 100 mg/kg (p.o.) for the caerulein model and 0.3 to 10 mg/kg (p.o.) for the ligation model. In a mouse model of acute pancreatitis induced by a choline-deficient, ethionine (0.5%)-supplemented diet, subcutaneous administration of R-96544 (10-100 mg/kg, bid) reduced serum amylase activity. Histological analysis showed that R-96544 dose-dependently attenuated pancreatic necrosis, inflammation and vacuolization. The effect of R-102444 was further examined in male Wistar Bonn/Kobori rats (4-9 months of age) which spontaneously show pancreatic fibrosis and parenchymal destruction compatible with human chronic pancreatitis. In Wistar Bonn/Kobori rats (from 3 to 9 months of age) fed a diet containing 0.017% and 0.17% of R-102444, pancreatic weight, pancreatic protein and amylase content were higher compared to those in non-treated pancreatitis control rats. Histological analysis showed that R-102444 suppressed parenchymal destruction and replacement with adipose tissue, indicating inhibition of pancreatic atrophy. These results clearly indicate that R-102444 and R-96544 inhibit the progression of acute and chronic pancreatitis and support the contention of possible involvement of 5-HT2A receptors in the progression of experimental pancreatitis.

Mitochondrial damage revealed by morphometric and semiquantitative analysis of mouse pup cardiomyocytes following in utero and postnatal exposure to zidovudine and lamivudine.

Zidovudine (ZDV), an antiretroviral drug used alone or in combination with other antiretroviral agents to treat HIV-infected pregnant women and their newborn infants, effectively reduces mother-to-child transmission of the virus. That myopathy and cardiomyopathy, related to mitochondrial damage, develop in some adults chronically treated with ZDV has long been known; recently, reports have suggested that similar adverse effects may occur in some infants exposed perinatally. Using a mouse model of human neonatal exposure, we treated pregnant CD-1 mice twice daily with doses of 75 mg/kg ZDV plus 37.5 mg/kg lamivudine throughout gestation and lactation; pups were exposed by direct gavage beginning postnatal day (PND) 4 and sacrificed on PND 28. Hearts were removed rapidly, and ventricles were processed for electron microscopy. Morphometric and semiquantitative morphological analyses were performed on three micrographs from each of three blocks from each of three females and three males from the control and treated groups. Treated mice showed significant increases in the mean area and decreases in the mean number of cardiomyocytic mitochondria compared to controls. We observed clusters of damaged mitochondria more frequently in treated animals than in controls; damage included fragmentation and loss of cristae. These results, demonstrating alterations in cardiomyocytic mitochondria of mice exposed in utero and postnatally, may model cardiac damage reported in human infants similarly exposed to ZDV. Critical insights derived from animal-model data like these may be used to mitigate risks to thousands of human infants receiving essential lifesaving therapy with antiretroviral drugs.

Cerivastatin induces type-I fiber-, not type-II fiber-, predominant muscular toxicity in the young male F344 rats.

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are associated with adverse skeletal muscle toxicity, but the underlying mechanism remains unclear. To investigate the pathological mechanism of statin-induced myotoxicity, cerivastatin (20 ppm; corresponding to 2 mg/kg/day) was dietarily administered to young male F344 rats for 10 days, and time-course clinical observations, measurement of plasma creatine kinase activity, and light and electron microscopy of type I fiber-predominant skeletal muscle (soleus) or type II fiber-predominant skeletal muscles (extensor digitorum longus and tibialis anterior), were performed. Clinical symptoms including weakness of hind limbs, staggering gait and body weight loss, accompanied by marked plasma creatinine kinase elevation in rats fed cerivastatin at around Day 6 to 8. Interestingly, microscopic examination revealed that cerivastatin-induced muscle damages characterized by hypercontraction (opaque) and necrosis of the fibers were of particular abundance in the soleus muscle at Day 8, whereas these histological lesions in the extensor digitorum longus and tibialis anterior were negligible, even at Day 9. Prior to manifestation of muscle damage, swollen mitochondria and autophagic vacuoles in the soleus were observed as the earliest ultra structural changes at Day 6; then activated lysosomes, disarray of myofibril and dilated sarcoplasmic reticulum vesicles became ubiquitous at Day 8. These results demonstrate that cerivastatin induces type I fiber-predominant muscles injury, which is associated with mitochondrial damage, in young male F344 rats. Since the rat exhibiting type I fiber-targeted injury is a unique animal model for statin-induced myotoxicity, it will be useful for gaining insight into mechanisms of statin-induced myotoxicity.

Effects of prasugrel, a novel P2Y(12) inhibitor, in rat models of cerebral and peripheral artery occlusive diseases.

Prasugrel is an orally available thienopyridyl prodrug with more potent in vivo antiplatelet effects compared to clopidogrel. In the present study, we examined the effects of prasugrel in rat models of cerebral and peripheral arterial occlusive diseases. Cerebral arterial thrombosis was induced by photochemical irradiation of the middle cerebral artery. Prasugrel (3 and 10 mg/kg) dose-relatedly and significantly reduced thrombus-mediated cerebral infarction 24 h after the irradiation. The effect of prasugrel was further examined in an embolic infarction model. Four h after an oral administration of prasugrel, non-occlusive thrombus formation in the right common carotid artery was initiated. In this model, prasugrel (0.3-3 mg/kg) reduced incidence, total area, and total number of cerebral infarcts in a dose-related manner 24 h after the vascular injury. Clopidogrel (10 or 30 mg/kg) was less potent than prasugrel at the doses tested on these thrombotic and embolic infarctions. Finally, the effect of prasugrel on lauric acid-induced peripheral arterial occlusive diseases was evaluated. After injection of lauric acid into the femoral artery, the lesions were scored for the following 10 days as they gradually progressed from the toe throughout the leg. Prasugrel (0.03-3 mg/kg/day) administered from the day before the lauric acid injection for 11 successive days inhibited the progression of the disease in a dose-related manner. Clopidogrel (3-30 mg/kg/day) showed similar effect but its effect was less potent than prasugrel. These results suggest that prasugrel could be a useful drug for preventing thromboembolic diseases including cerebral infarction and peripheral arterial occlusive diseases.

Epididymal sperm granuloma induced by chronic administration of 2-methylimidazole in B6C3F1 mice.

Two-year mouse and rat bioassay studies of 2-methylimidazole (2-MI) conducted by the National Toxicology Program revealed that epididymal sperm granuloma(SG)s occurred only in male B6C3F1 mice in a dose-related manner. The present study characterized 2-MI-induced SGs in these epididymides. Groups of 50 male B6C3F1 mice were fed diets containing 0, 625, 1250, or 2500 ppm 2-MI for 105 weeks; the doses were equivalent to average daily doses of approximately 13, 40, or 130 mg/kg. Testes and epididymides were histopathologically reexamined. 2-Methylimidazole increased the incidence of epididymal SGs (0%, 0%, 6%, 12%, respectively). Histologically, most of the SGs exhibited rupture of epididymal ducts with focal aggregations of macrophages in interstitia. Lesions occurred in the proximal caput of the epididymis and/or efferent ducts, not in the corpus and cauda. In the testis, incidences of germinal epithelial atrophy (GEA) increased dose-relatedly (2%, 8%, 16%, 28%, respectively). All mice with epididymal SG developed testicular GEA. The grading scores of testicular GEA tended to be more severe in mice with SGs than those without. No epididymal SG or testicular GEA was observed in 6-month-interim-sacrificed mice. The results imply that 2-year treatment of B6C3F1 mice with 2-MI can induce epididymal SGs, primarily followed by more severe testicular GEA. The potential mechanism of SG induction by 2-MI is discussed.

Susceptibility of signal transducer and activator of transcription-1-deficient mice to pulmonary fibrogenesis.

The signal transducer and activator of transcription (Stat)-1 mediates growth arrest and apoptosis. We postulated that lung fibrosis characterized by excessive proliferation of lung fibroblasts would be enhanced in Stat1-deficient (Stat1-/-) mice. Two weeks after bleomycin aspiration (3 U/kg), Stat1-/- mice exhibited a more severe fibroproliferative response and significantly elevated total lung collagen compared to wild-type mice. Growth factors [epidermal growth factor (EGF) or platelet-derived growth factor (PDGF)] enhanced [3H]thymidine uptake in lung fibroblasts isolated from Stat1-/- mice compared to wild-type mice. Interferon (IFN)-gamma, which signals growth arrest via Stat1, inhibited EGF- or PDGF-stimulated mitogenesis in wild-type fibroblasts but enhanced [3H]thymidine uptake in Stat1-/- fibroblasts. Moreover, IFN-gamma treatment in the absence of growth factors induced a concentration-dependent increase in [3H]thymidine uptake in Stat1-/- but not wild-type fibroblasts. Mitogen-activated protein kinase (ERK-1/2) phosphorylation in response to PDGF or EGF did not differ among Stat1-/- and wild-type fibroblasts. However, Stat3 phosphorylation induced by PDGF, EGF, or IFN-gamma increased twofold in Stat1-/- fibroblasts compared to wild-type fibroblasts. Our findings indicate that Stat1-/- mice are more susceptible to bleomycin-induced lung fibrosis than wild-type mice due to 1) enhanced fibroblast proliferation in response to growth factors (EGF and PDGF), 2) stimulation of fibroblast growth by a Stat1-independent IFN-gamma signaling pathway, and 3) increased activation of Stat3.

Follicular epithelial cell hypertrophy induced by chronic oral administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin in female Harlan Sprague-Dawley rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) affects the thyroid morphologically and/or functionally in adult animals. Recently, the National Toxicology Program conducted a 2-year gavage study of TCDD in female Harlan Sprague-Dawley rats. The only treatment-related alterations found in thyroid follicles were decreased luminal size and increased height of the follicular epithelial cells, without prominent protrusion into the lumen. The present study elucidated the nature of these follicular lesions. Thyroid glands of 10 rats each from the control, high (100 ng/kg/day)-dose, and stop-study (100 ng/kg/day, 30 weeks; vehicle to study termination) groups in the 2-year study were evaluated microscopically. Twenty randomly selected follicles were measured morphometrically in each animal. TCDD treatment significantly decreased the mean ratio of luminal/epithelial areas and increased the mean sectional epithelial height of the high-dose group compared to controls. Thyroid sections were immunostained with antibody against minichromosome maintenance (MCM) proteins, a novel cell-cycle biomarker. The MCM labeling index of the high-dose group was significantly higher than that of the control; however, the TUNEL labeling index was also higher in the high-dose group than the control. All data from the stop group were comparable to those from controls. These results indicate that the follicular cell response was hypertrophic and reversible. This information should contribute to diagnosis of nonneoplastic thyroid follicular lesions in rats.