2-Deoxy-alpha-ecdysone from ovaries and eggs of the silkworm, Bombyx mori.

From ovaries dissected from developing and emerged adults of the silkworm, Bombyx mori, two substances having high molting hormone activity were isolated. One of these was identified as 2-deoxy-alpha-ecdysone by means of high-pressure liquid chromatography and mass spectrometry. Although this compound had previously been isolated from the fern Blechnum minus and postulated to be the precursor of alpha-ecdysone, it had not been obtained from insect material. The compound is also contained in the form of a conjugate in ovaries as well as in diapausing silkworm embryos.

Role of angiotensin II in renal injury of deoxycorticosterone acetate-salt hypertensive rats.

To investigate the role of angiotensin II (Ang II) in hypertension-induced tissue injury, we gave TCV-116 (1 mg/kg per day PO), a nonpeptide Ang II type I receptor antagonist, or enalapril (10 mg/kg per day PO) to deoxycorticosterone acetate (DOCA)-salt hypertensive rats for 3 weeks and examined the effects on tissue mRNA levels for transforming growth factor-beta 1 (TGF-beta 1) and extracellular matrix components. Tissue mRNA levels were measured by Northern blot analysis. Renal mRNA levels for TGF-beta 1; types I, III, and IV collagen; and fibronectin in DOCA-salt hypertensive rats were increased by severalfold (P < .01) compared with sham-operated rats. In the aorta of DOCA-salt hypertensive rats, TGF-beta 1 and fibronectin mRNA levels were increased, but types I, III, and IV collagen mRNAs did not increase. In the heart, increased mRNA was found only for fibronectin. Thus, these gene expressions are regulated in a tissue-specific manner. TCV-116 or enalapril did not lower blood pressure in DOCA-salt hypertensive rats. However, the increase in renal mRNAs for TGF-beta 1 and extracellular matrix components in DOCA-salt hypertensive rats was significantly inhibited by treatment with TCV-116 or enalapril, which was associated with a significant decrease in urinary protein and albumin excretions and histological improvement of renal lesions. In contrast, in the aorta and heart these gene expressions were not affected by TCV-116 or enalapril. Thus, local Ang II may contribute to renal injury of DOCA-salt hypertension by stimulating the gene expression of TGF-beta 1 and extracellular matrix components.

TCV-116 inhibits renal interstitial and glomerular injury in glomerulosclerotic rats.

TCV-116 and enalapril were given in two stages: (early phase) for 6 to 10 weeks to 5/6 nephrectomized (NX) rats two weeks after nephrectomy, 12-week-old Wistar fatty (WF) rats and 7-week-old spontaneously hypercholesterolemic (SHC) rats; and (late phase) for 6 to 16 weeks to 5/6 NX rats 11 weeks after nephrectomy, 27-week-old WF rats and 10-week-old SHC rats. Urinary albumin, blood pressure (BP), glomerular filtration rate (GFR) and renal histology were examined. In the early phase, both agents inhibited proteinuria and tended to inhibit glomerulosclerosis. TCV-116 also inhibited interstitial inflammation. The antiproteinuric effects did not necessarily correlate with the BP-lowering effects. In the late phase, both agents showed equal antiproteinuric and antihypertensive effects. In 5/6NX and WF rats, TCV-116 inhibited tubulointerstitial inflammation/fibrosis, glomerulosclerosis and renal dysfunction, but enalapril had little effect on these parameters. In the SHC rats, TCV-116 inhibited renal tubulointerstitial inflammation and glomerulosclerosis, but enalapril inhibited only glomerulosclerosis. After drug administration, there was a positive correlation between proteinuria and BP, and a negative correlation between the severity of tissue damage and GFR, but not BP. These findings suggest that initial BP-independent tubulointerstitial inflammation may enhance glomerulosclerosis in the late phase, and TCV-116 might prevent the development of glomerulosclerosis through inhibition of angiotensin II-mediated tubulointerstitial damage in these models.

Effect of pituitary transplants on the LH-RH concentrations in the medial basal hypothalamus and hypophyseal portal blood.

The effects of hyperprolactinemia on catecholamine turnover in the medial basal hypothalamus (MBH) and on the luteinizing hormone-releasing hormone (LH-RH) concentrations in MBH and hypophyseal portal blood were investigated in female Wistar rats. Chronic endogenous hyperprolactinemia was produced by implantation of anterior pituitary glands under the kidney capsule. Catecholamine turnover in the MBH was studied by inhibiting monoamine oxidase and then measuring the accumulation of catecholamines by high-performance liquid chromatography with electrochemical detection. Rats bearing pituitary transplants exhibited: (1) persistent vaginal diestrus within 3-6 days of the implantation; (2) increased serum concentrations of prolactin (PRL); (3) decreased serum concentrations of luteinizing hormone (LH); (4) increased pituitary concentrations of LH and follicle-stimulating hormone (FSH); (5) increased turnovers of dopamine in the MBH; and (6) decreased concentrations of LH-RH in the MBH and in plasma of hypophyseal portal blood. These findings suggest that chronic hyperprolactinemia may increase dopaminergic tone in the MBH that may inhibit LH-RH secretion from the MBH, and LH release from the pituitary. These processes may be responsible for disturbances of cyclic pituitary-ovarian activity.

Possible mechanism for the anemia induced by candesartan cilexetil (TCV-116), an angiotensin II receptor antagonist, in rats.

Candesartan cilexetil (TCV-116), an angiotensin II receptor antagonist, was administered orally to male F344/Jcl and Crj:CD (SD) rats at 1000 mg kg(-1) day(-1) for 1-28 days, and the possible mechanism for the anemia induced by TCV-116 was investigated. In the TCV-116 group, the erythrocyte count, hematocrit value and hemoglobin concentration were decreased by 7-8% as compared with the values in the control group after dosing for 28 days. The plasma and renal erythropoietin levels, the reticulocyte count in the peripheral blood and the erythroid cell count upon bone marrow examination were decreased on day 7, but there were no accompanying histopathological renal lesions. Renal blood flow was increased, and mean blood pressure was decreased after TCV-116. These results suggest that the primary cause of the anemia induced by TCV-116 treatment is the increase in renal blood flow followed by a decrease in erythropoietin production.

Candesartan cilexetil: a review of its preclinical pharmacology.

Candesartan is a highly potent, long-acting and selective angiotensin II type 1 (AT1) receptor blocker. It is administered orally as the inactive prodrug candesartan cilexetil which is rapidly and completely converted to candesartan during gastrointestinal absorption. In vitro studies have shown that candesartan acts as an insurmountable angiotensin II receptor antagonist, binding tightly to and dissociating slowly from the AT1 receptor. The above characteristics are thought to contribute to the marked and long-lasting antihypertensive effects of candesartan cilexetil in several animal models of hypertension. These included rodent models of renal hypertension in which candesartan cilexetil also demonstrated efficacy equivalent to or greater than enalapril. In other animal models, candesartan cilexetil reduced the incidence of stroke, renal dysfunction and renal disease while reducing cardiac and vascular hypertrophy. Furthermore, candesartan cilexetil conferred some protection against cerebral and renal damage at a dose that had no blood pressure-lowering effect. In toxicity and general pharmacology studies, candesartan cilexetil was shown to possess a 'clean' profile with a large safety margin. Also it did not potentiate chemical- or autocoid-induced cough or anaphylactoid reactions.

Improved method for determination of catecholamines in rat brain by isolation on boric acid gel and high-performance liquid chromatography with electrochemical detection.

An improved sensitive, simple and time-saving method for determining catecholamine (CA) in rat brain is described. The method involves isolation on boric acid gel and high-performance liquid chromatography with electrochemical detection. Boric acid gel effectively adsorbs CA at weakly alkaline pH and the over-all recoveries of 5 ng and 10 ng samples of authentic norepinephrine (NE) and dopamine (DA) added to a homogenate of rat brain were 98.9 +/- 9.2% and 103.4 %/- 9.3% for NE and 96.2 +/- 4.6% and 99.4 +/- 4.8% for DA, respectively. Intra-assay variation was 5.3% (5 ng) and 3.0% (10 ng) for NE and 4.4% (5 ng) and 3.8% (10 ng) for DA. Inter-assay variation was 7.7% (1 ng) for NE and 5.0% (1 ng) for DA. With this analytical system, the lowest amount of NE or DA detectable was 40 pg. Application of this method to determination of the DA and NE contents of rat hypothalamus during estrous cycle revealed significant increases in the turnovers of both in the proestrus stage. This method should be useful for routine determination of plasma NE and DA because it is sensitive and inexpensive.

Nonselective ETA/ETB receptor antagonist blocks proliferation of rat vascular smooth muscle cells after balloon angioplasty.

To elucidate the role of endothelin receptor subtypes in the abnormal proliferation of vascular smooth muscle cells (VSMC) associated with vascular injury, we have investigated the effects of a novel and potent nonselective ETA/ETB receptor antagonist (TAK-044) on the proliferation of rat VSMC in vitro and in vivo. TAK-044 dose-dependently inhibited DNA synthesis stimulated by 10(-7) M ET-1 in cultured rat VSMC from the late passage with the approximate IC50 of 6 x 10(-8) M. After balloon angioplasty, the neointimal lesion in the injured carotid arteries in the TAK-044-treated group (0.052 +/- 0.014 mm2) was significantly (p < 0.05) decreased compared to that in control group (0.26 +/- 0.045 mm2), while the medial surface area was not affected. The intima/media ratio in the TAK-044 group (31 +/- 6%) also significantly (p < 0.05) decreased from that of the control group (148 +/- 25%). Our data suggest that nonselective ETA/ETB receptor antagonists may be therapeutic potential for prevention against the intimal thickening associated with vascular injury.

Stimulatory effect of luteinizing hormone on the development and maintenance of 5 alpha-reduced steroid-producing testicular interstitial cell tumors in Fischer 344 rats.

The development and maintenance of testicular interstitial cell (IC) tumors under the conditions of low LH levels were examined in aged Fischer 344 rats, and the enzyme activities for androgen synthesis in the tumors were estimated. Sixty-week old rats received injections of an LH-RH agonist (LH-RHa) for 28 weeks or were subjected to a procedure by which Silastic tubes containing testosterone (T) or estradiol-17 beta (E) were subcutaneously implanted. All 88-week old control animals had bilateral IC tumors, whereas 100% of the T-, E- and LH-RHa-treated rats at week 88 were tumor free. These treated rats showed significantly reduced peripheral LH levels compared to the control animals. No hyperplasia or tumors of the IC were noted in 70-week old animals receiving T for 28 weeks, while all age-matched untreated animals showed IC tumors or hyperplasia. The IC tumors in rats 69 weeks of age decreased in size in response to 4-week treatment involving the subcutaneous implantation of tubes containing T. After removal of the tubes, however, the tumors once again increased in size. Reduced and subsequently elevated serum LH levels were also observed with this treatment. These changes in tumor mass volume were mainly due to a change in the tumor cell volume which was possibly related to LH levels, i.e., large cells were predominant under relatively high peripheral LH levels and intermediate cells were predominant under low LH levels. Quantitative analyses of the in vitro products formed from [3H]progesterone and [14C]4-androstenedione in tissue-homogenates revealed a low 17 alpha-hydroxylase activity and a high 5 alpha-reductase activity in the IC tumors compared to those activities in the testicular tissues; in the tumors, low production of T and high production of 5 alpha-metabolites of progesterone were observed. From these data, it seems reasonable to conclude that sufficient levels of LH are essential for the induction of IC tumors or hyperplasia and the maintenance of tumor mass, and that the low production of T in the tumors is due, at least in part, to decreased 17 alpha-hydroxylase and increased 5 alpha-reductase activities under active steroidogenesis.

Circadian rhythm in hypotensive effect of sodium nitroprusside in rats and its relevance to sympathetic nervous activity.

Circadian rhythmicity in the hypotensive effects of sodium nitroprusside (SNP) was determined to characterize the rhythmicity in hypotension mediated by nitric oxide (NO) donor in rats. When SNP was infused for 90 seconds every hour for 48 hours and the mean blood pressure was determined automatically by telemetry under light-dark conditions (LD), the degree of SNP-induced hypotension was shown to be minimal at the onset of the dark phase and to have marked circadian rhythmicity. The possible relationship between the circadian rhythm of the sympathetic nervous system (SNS) activity and SNP-induced hypotension was examined under LD conditions. The SNS activity assessed by blood pressure beat-to-beat variability analysis using the maximum entropy method (MEM) was higher at the preinfusion time at the onset of the dark phase than during the middle of the light phase. In addition, pretreatment with an alpha-blocker, phentolamine, followed by SNP infusion at the onset of the dark phase restored the SNP-induced hypotension and consequently dampened the daily variation in the degree of SNP-induced hypotension. The circadian rhythmicity determined by MEM was weakened, but persisted, in constant dark conditions (DD), suggesting partial involvement of endogenously driven circadian rhythms. In conclusion, the hypotensive effect of hourly infused SNP in rats was decreased in the dark phase in LD, especially at the onset of the dark phase, and clearly showed circadian rhythmicity in both LD and DD. The SNP-induced hypotension may be affected by rapid activation of the SNS at the onset of the dark phase in LD, and regulation of the circadian rhythm in SNP-induced hypotension in rats may be affected by both exogenous light stimuli and the endogenous biological clock.

Collaborative project to establish the optimal period and parameters for detection of reproductive toxicity in male rats--effects of compound T on male fertility.

To assess the optimal dosing period and parameters for measurement of effects on male fertility, Compound T was administered to male Jcl:Wistar rats at dosage levels of 0, 2, 10 and/or 50 mg/kg/day for 4 weeks (Experiment 1) or for 9 weeks (Experiment 2). Experiment 1: In the 50 mg/kg group, the ventral prostate weight was low when compared to the control value, and desquamation of round spermatids was observed in the testes. Experiment 2: When treated males were mated with untreated females after dosing for 9 weeks (the first mating), the fertility index was slightly lowered and preimplantation loss was significantly elevated in the 50 mg/kg group as compared to the control values. In this treatment group, serum testosterone level at 2.5 hours after dosing was significantly decreased after dosing for 12 weeks, and degeneration of spermatids/spermatocytes in the testis and epididymis was observed after dosing for 13 weeks. After a recovery period of for 6 weeks, remating resulted in copulatory and fertility indices and cesarean section data which were comparable in all groups. In conclusion, there were no differences in toxicity relevant to male fertility between 4 and 9 weeks of dosing, and it is considered that the observed changes resulted from decreased function of Sertoli cells due to depressed production/secretion of testosterone.

Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats 24). Testicular toxicity of boric acid after 2- and 4-week administration periods.

To assess whether or not male reproductive toxicity can be evaluated in a 2-week administration study, boric acid was administered daily by oral gavage to male Jcl:Wistar rats at dosage levels of 0, 300 and 500 mg/kg for 2 and 4 weeks, and the results obtained with the two different treatment schedules were compared. After a 2-week administration, decreased testis weights were observed in the 500 mg/kg group. Histopathologically, exfoliation of round spermatids, retention of step 19 spermatids and increased numbers of residual body-like structures in the seminiferous tubules and cell debris in the cranial epididymal ducts were observed in the 300 and 500 mg/kg groups. Distorted cytoplasmic lobes of step 19 spermatids, debris in the seminiferous tubules and focal atrophy of the seminiferous tubules with multinucleated giant cells formation and necrosis of spermatocytes were also observed in the 500 mg/kg group. After a 4-week administration, testis and epididymis weights were decreased in the 300 and 500 mg/kg groups. Histopathological changes in the 300 mg/kg group were similar to those found in the 300 and 500 mg/kg groups after a 2-week administration. Diffuse atrophy of the seminiferous tubules was additionally observed in the 500 mg/kg group. These results suggest that 2 weeks is a sufficient treatment period for the detection of the testicular toxicity caused by boric acid.

Role of angiotensin II in reflex tachycardia during hypotension caused by a calcium channel blocker.

Blood pressure and heart rate were measured by telemetry in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) to investigate the contribution of angiotensin II to the reflex tachycardia resulting from exaggerated hypotension caused by a high dose of a calcium channel blocker. Pre-treatment with TCV-116, an angiotensin II AT1 receptor antagonist, or enalapril partially attenuated the reflex tachycardia induced by manidipine, but TCV-116 had almost no effect on the sinus tachycardia induced by isoproterenol. The suppressive effects of TCV-116 against the reflex tachycardia tended to be more obvious in WKY than in SHR, though the difference was not statistically significant. Concurrent administration of propranolol almost completely inhibited both the reflex tachycardia and the sinus tachycardia in SHR and WKY, indicating that the sympathetic nervous system contributes to both types of tachycardia. We demonstrated that angiotensin II may be involved in the reflex tachycardia induced by calcium channel blockers probably via activation of some component of the sympathetic nervous system other than postsynaptic factors at the sinus node.

Circadian and short-term variabilities in blood pressure and heart rate measured by telemetry in rabbits and rats.

Blood pressure (BP) and heart rate (HR) were measured by telemetry in conscious unrestrained rabbits to clarify the profile of their variabilities. The variabilities were assessed for two periods, 24 h (circadian rhythm) and 1 h (short-term variability), and compared with those in rats. BP and HR in rabbits were lower than those in rats but the circadian rhythms in rabbits showed nocturnal patterns as is the case in rats. In contrast, short-term variabilities in BP in rabbits were considerably larger than those in rats. The short-term variability in BP in rabbits was suppressed by alpha-adrenergic blocking without changes in basal values but not by beta-adrenergic blocking or angiotensin converting enzyme inhibition. These results demonstrate the need to take the unique characteristics of short-term variability in BP in rabbit into consideration when the circadian rhythm is focused on and that the short-term variability in BP in rabbits is caused mainly by activation of alpha-adrenergic receptors.

Characterization of the high sensitivity of rabbits to the effects of TCV-116, an angiotensin II receptor antagonist.

A high incidence of maternal toxicity in rabbits characterized by uremia and death was observed when TCV-116, a novel angiotensin II subtype-1 (AT1) receptor antagonist, was orally administered to pregnant rabbits at dosage levels of 3 mg/kg/day or more. The effects of TCV-116 on blood pressure in nonpregnant or male rabbits and rats and on blood chemistry, renal circulation, and plasma renin activity in nonpregnant or male rabbits were examined to characterize the toxicity in rabbits. In a 2-week repeated dose study, most nonpregnant female rabbits receiving 3 or 100 mg/kg/day died or were sacrificed in a moribund state, indicating that toxicity could be caused independently of pregnancy. When these rabbits became moribund, marked hypotension, accompanied by increases in plasma concentrations of urea nitrogen, creatinine, and potassium, was observed, suggesting uremia. In a single-dose study, blood pressure in rabbits was decreased after administration of 10 or 100 mg/kg of TCV-116, and the hypotension was more marked and sustained than that in rats, as was the case with 30 mg/kg of enalapril. The sustained pharmacological effect in rabbits was also confirmed with regard to decreases in effective renal plasma flow and the glomerular filtration rate and increased plasma renin activity. Species differences in the hypotensive effect and mortality could not be explained by toxicokinetic data for the active metabolite of TCV-116 in various species, which supported a possibility that the differences in toxicity may be related to the species difference in sensitivity to the pharmacological effect of TCV-116. We conclude that the specific maternal toxicity of TCV-116 in rabbits may be mainly due to the higher sensitivity of rabbits to the pharmacological effects and is caused by marked and sustained hypotension resulting in the decrease in glomerular filtration rate, uremia, and death.

Spontaneous lesions in cynomolgus monkeys used in toxicity studies.

Spontaneous lesions in wild-caught, laboratory-maintained cynomolgus monkeys used in drug-toxicity studies were examined histopathologically in an effort to better distinguish toxic changes from spontaneous lesions and assess the toxicity of drugs more exactly. In the liver and kidney, where toxic changes are observed frequently, many spontaneous lesions were observed. Infiltration of mononuclear cells, vacuolization of the hepatocytes, dilatation of the renal tubules, and vacuolization of the renal epithelia were observed at a relatively high frequency. It is considered important to examine these changes carefully, because they closely resemble the changes recognized as toxic. Deposition of brownish pigment was observed in various organs such as the liver, kidney, spleen, intestinal tract, lung and brain, however the type of pigment differed among the organs, and histochemical examination revealed anthracosis or accumulation of hemosiderin, or melanin. Since the monkeys were caught in the wild, many parasitic lesions were observed especially in the large intestine and liver. Helminthous worms were frequently observed in the granulomas in the large intestine, however, no parasites were observed in the granulomas in the liver. Such lesions in the liver may be misinterpreted as toxic changes, when only scars or inflammatory lesions are observed.

Effect of sulpiride-induced hyperprolactinaemia on catecholamine turnover and LRH concentration in the medial basal hypothalamus of rats.

In order to clarify the mechanism of hyperprolactinaemic anovulation, the medial basal hypothalamic (MBH) catecholamine (CA) turnover and LRH concentration, and the serum levels and pituitary contents of gonadotrophins and prolactin (Prl) in hyperprolactinaemic female rats were examined. Hyperprolactinaemia (HPrl) was produced by oral administration of sulpiride for 10 consecutive days; each measurement made on the sulpiride-treated rats was compared with that of control dioestrus rats. Prl, LH, FSH and LRH were determined by radioimmunoassay; CA turnover, as assessed by the accumulation of CA following monoamine oxidase inhibition, was assayed by high performance liquid chromatography with electrochemical detection. Sulpiride treatment induced (1) an increase in the serum Prl and a decrease in the serum LH, (2) an increase in the pituitary FSH and LH contents, (3) an increase in the MBH LRH concentration, and (4) an increase in the MBH dopamine (DA) turnover. These results suggest that HPrl may induce anovulation by impaired LH secretion which was caused by the suppression of LRH release due to an increase in DA turnover in the MBH.

Immunohistological study on malignant fibrous histiocytoma.

Malignant fibrous histiocytoma (MFH) shows a mixed proliferation of both fibroblastic and histiocytic cells. Because of their complex morphologic appearances, the nature of truly neoplastic cells in MFH has been controversial. In the present study, immunoperoxidase method (PAP method) was used to examine the intracytoplasmic lysozyme (LY), alpha-1-antitrypsin (A1AT), fibronectin (FN), and polyvalent immunoglobulin (PI) in the fibroblastic and histiocytic cells. Twenty-three cases with MFH were histologically divided into three groups; predominantly fibroblastic type (Group I; 5 cases), mixed fibroblastic and histiocytic type (Group II; 15 cases), and almost pure histiocytic type (Group III; 3 cases). Fibroblastic cells showed a strong positive reaction for LY and A1AT, suggesting the histiocytic nature, while the proliferating cells in Group II were more intensely stained by each of the antibodies than in Groups I and III. Enzyme histochemical examinations on fresh materials were available in 3 cases. These findings suggest that proliferating cells in MFH possess a histiocyte nature.

[Beneficial effects of the combination of idebenone and manidipine 2HCl on neurological deficits and histological changes following cerebrovascular lesions in stroke-prone spontaneously hypertensive rats].

We investigated the effects of the combination of idebenone, an energy metabolism enhancer, and manidipine 2HCl, a dihydropyridine-derivative calcium antagonist, on neurological deficits and histological changes in the brain and kidneys of stroke-prone spontaneously hypertensive rats (SHRSP) with cerebrovascular lesions (stroke). The SHRSP were kept on a 1% NaCl solution as their drinking water to synchronize the onset of stroke. After the onset of stroke symptoms, the salt solution was replaced with tap water. On the day following the onset of stroke, idebenone (50 mg/kg), manidipine 2HCl (2 mg/kg) or a combination of idebenone (50 mg/kg) and manidipine 2HCl (2 mg/kg) was administered orally once a day for 3 weeks. In the combination group and manidipine 2HCl-treated group, the neurological deficits after the onset of stroke were ameliorated during the entire experimentalperiod. Especially, the combination significantly decreased the number of days with severe neurological deficits as compared to the control group. The combination and manidipine 2HCl significantly recovered the decrease in body weight and ameliorated the increase of brain weight, which was mainly caused by edema, significantly as compared to the control group. Manidipine 2HCl ameliorated the histological changes in the brain. In the combination group, the histological changes in both the brain and the kidneys were ameliorated. In conclusion, the combination of idebenone and manidipine 2HCl significantly ameliorated the neurological deficits and the histological changes in the brain and the kidney of SHRSP with stroke as compared to each individual treatment. We concluded that manidipine 2HCl enhances the therapeutic effect of idebenone in the treatment of cerebrovascular diseases.