Randomised clinical trial: exploratory phase 2 study of ONO-2952 in diarrhoea-predominant irritable bowel syndrome.

BACKGROUND: ONO-2952 is a novel and selective inhibitor of translocator protein 18 kDa that reduces stress-induced defecation and visceral hyperalgesia in rat models. AIM: To evaluate the efficacy and safety of ONO-2952 in females with irritable bowel syndrome with diarrhoea in an exploratory proof-of-concept study. METHODS: A randomised, double-blind, placebo-controlled study was conducted at 49 US centres. Two hundred subjects with irritable bowel syndrome with diarrhoea (Rome III criteria) were randomised to ONO-2952 20 mg, or 60 mg, or placebo. Subjects recorded irritable bowel syndrome symptoms daily during a 2-week baseline period, the 4-week treatment period and for 4 weeks post-treatment. The co-primary endpoints were change from baseline to week 4 in abdominal pain, stool consistency and stool frequency. RESULTS: Improvements in irritable bowel syndrome symptoms were seen with ONO-2952 over placebo in per-protocol analyses for all three co-primary endpoints, but these did not reach statistical significance at the 5% level. The largest improvement was seen with ONO-2952 60 mg. ONO-2952 was well tolerated with a safety profile similar to that of placebo. Most adverse events were mild or moderate in severity and not treatment related. CONCLUSION: ONO-2952 showed evidence of clinical efficacy in reducing irritable bowel syndrome-related symptoms in female subjects with irritable bowel syndrome with diarrhoea, and further evaluation is, therefore, warranted to assess its potential as a treatment for irritable bowel syndrome with diarrhoea (NCT01844180).

A dual CysLT1/2 antagonist attenuates allergen-induced airway responses in subjects with mild allergic asthma.

BACKGROUND: The cysteinyl leukotrienes (cysLTs) play a key role in the pathophysiology of asthma. In addition to functioning as potent bronchoconstrictors, cysLTs contribute to airway inflammation through eosinophil and neutrophil chemotaxis, plasma exudation, and mucus secretion. We tested the activity of the dual cysLT1/2 antagonist, ONO-6950, against allergen-induced airway responses. METHODS: Subjects with documented allergen-induced early (EAR) and late asthmatic response (LAR) were randomized in a three-way crossover study to receive ONO-6950 (200 mg) or montelukast (10 mg) or placebo q.d. on days 1-8 of the three treatment periods. Allergen was inhaled on day 7 two hours postdose, and forced expiratory volume in 1 s (FEV1 ) was measured for 7 h following challenge. Sputum eosinophils and airway hyperresponsiveness were measured before and after allergen challenge. The primary outcome was the effect of ONO-6950 vs placebo on the EAR and LAR. RESULTS: Twenty-five nonsmoking subjects with mild allergic asthma were enrolled and 20 subjects completed all three treatment periods per protocol. ONO-6950 was well tolerated. Compared to placebo, ONO-6950 significantly attenuated the maximum % fall in FEV1 and area under the %FEV1 /time curve during the EAR and LAR asthmatic responses (P < 0.05) and allergen-induced sputum eosinophils. There were no significant differences between ONO-6950 and montelukast. CONCLUSIONS: Attenuation of EAR, LAR, and airway inflammation is consistent with cysLT1 blockade. Whether dual cysLT1/2 antagonism offers additional benefit for treatment of asthma requires further study.

Interleukin-2 modulates the responsiveness to angiotensin II in cultured vascular smooth muscle cells.

Preincubation with interleukin-2 (IL-2), a T cell-derived cytokine, enhanced the increase in intracellular Ca2+ ([Ca2+]i) induced by angiotensin II (AII) in vascular smooth muscle cells (VSMC). IL-2 itself did not affect the basal [Ca2+]i level or the maximal response of [Ca2+]i increase induced by AII. Furthermore, IL-2-induced enhancement was not observed in the absence of extracellular Ca2+, suggesting that IL-2 enhances Ca2+ influx induced by AII. IL-2 also enhanced the stimulation of DNA synthesis induced by AII, although IL-2 alone did not stimulate DNA synthesis. Genistein, an inhibitor of protein tyrosine kinases, significantly inhibited IL-2-induced enhancement of both Ca2+ influx and DNA synthesis induced by AII. A neutralizing antibody against heparin-binding epidermal growth factor-like growth factor (HB-EGF) partially inhibited IL-2-induced enhancement of DNA synthesis induced by AII. These findings suggest that autocrine HB-EGF is partially involved in the mechanism of IL-2-induced enhancement of DNA synthesis. On the other hand IL-2 stimulated both glycosaminoglycan (GAG) and prostacyclin syntheses and enhanced the stimulation of both GAG and prostacyclin syntheses induced by AII. Therefore, IL-2 may play important roles in the pathogenesis of atherosclerosis and vascular disease by modulating the responsiveness to AII in VSMC.

Elastase enhances cAMP accumulation and the inhibition of DNA synthesis induced by OP-41483, a stable prostacyclin analogue, in vascular smooth muscle cells.

To define the physiological roles of elastase in the vascular wall, we examined whether elastase at low concentrations can modulate the proliferation of vascular smooth muscle cells (VSMC). Elastase itself at low concentrations from 1 to 50 ng/ml inhibited DNA synthesis dose-dependently in VSMC. However, phenylmethylsulfonyl fluoride-inactivated elastase failed to induce this inhibition. OP-41483, a stable analogue of prostacyclin, inhibited DNA synthesis and stimulated accumulation of cAMP in VSMC. Preincubation of VSMC for 24 h with 50 ng/ml elastase enhanced both inhibition of DNA synthesis and the accumulation of cAMP induced by OP-41483. Preincubation of VSMC with 12-O-tetradecanoylphorbol 13-acetate, an activator of protein kinase C (PKC), also enhanced cAMP accumulation induced by OP-41483. On the other hand, elastase failed to enhance OP-41483-induced cAMP accumulation in PKC down-regulated cells. Furthermore, coincubation with chelerythrine, an inhibitor of PKC, inhibited the enhancement of cAMP accumulation induced by preincubation with elastase. These results suggest that elastase at low concentrations can enhance the inhibition of VSMC proliferation induced by prostacyclin through the activation of protein kinase C.

Effects of an angiotensin II receptor antagonist, CV-11974, on angiotensin II-induced increases in cytosolic free calcium concentration, hyperplasia, and hypertrophy of cultured vascular smooth muscle cells.

The effects of CV-11974, a potent nonpeptide antagonist of the angiotensin II (AII) type-1 receptor (AT1), on cytosolic free calcium concentration ([Ca2+]i), hyperplasia, and hypertrophy of cultured vascular smooth muscle cells (VSMC) from rat aorta were studied. [Ca2+]i was measured by fura 2, and hyperplasia and hypertrophy were determined by incorporation of [3H]thymidine and [3H]leucine, respectively. CV-11974 had no effect on [Ca2+]i itself, but suppressed 10(-7) M AII-induced increase in [Ca2+]i dose dependently at concentrations from 10(-10) M and completely at 10(-7) M. CV-11974 suppressed both Ca2+ release from intracellular Ca2+ stores and Ca2+ influx from the extracellular space. However, CV-11974 had no effect on the increases in [Ca2+]i induced by prostaglandin F2 alpha (PGF2 alpha), a potent vasoconstrictor, or ionomycin, a Ca2+ ionophore. These results indicate that the suppressive effects of CV-11974 act on the binding of AII and its specific receptors. AII 10(-7) M increased the synthesis of DNA and protein to 1.5 and 1.7 times the control values, respectively. CV-11974 had no effect on synthesis of DNA or protein, but suppressed the AII-stimulated synthesis of DNA and protein dose dependently at concentrations > or = 10(-8) and 10(-10) M, respectively and completely at 10(-6) M. These results indicate that AII increases [Ca2+]i and synthesis of DNA and protein in VSMC through activation of AT1. CV-11974 showed no partial agonistic effects on AII. Thus, CV-11974 may act not only as an antihypertensive agent, but also as an inhibitor of vascular injury stimulated by AII.

Effects of beraprost sodium, a stable analogue of prostacyclin, on hyperplasia, hypertrophy and glycosaminoglycan synthesis of rat aortic smooth muscle cells.

The effects of beraprost sodium, a stable analogue of prostacyclin, on the syntheses of DNA, protein and glycosaminoglycans (GAG) of cultured vascular smooth muscle cells (SMC) were studied. SMC were isolated from the thoracic aorta of male Wistar rats. The syntheses of DNA, protein and GAG of SMC were determined by incorporations of [3H]thymidine, [3H]leucine and [35S]sulfuric acid, respectively. Insulin at a concentration of 10(-6) M stimulated DNA synthesis 4 fold compared to control. Beraprost sodium suppressed the insulin-stimulated DNA synthesis dose-dependently at concentrations greater than 10(-7) M and suppressed it by 68% at 10(-5) M. Platelet derived growth factor (PDGF) at a concentration of 20 ng/ml stimulated DNA synthesis 6 fold compared to control. Beraprost sodium suppressed the PDGF-stimulated DNA synthesis dose-dependently at concentrations greater than 10(-7) M and suppressed it by 51% at 10(-5) M. Beraprost sodium suppressed GAG synthesis dose-dependently at concentrations greater than 10(-7) M and suppressed it by 49% at 10(-5) M. However, beraprost sodium at concentrations up to 10(-5) M did not affect protein synthesis. These results indicate that beraprost sodium suppressed the proliferation and GAG synthesis of SMC but did not affect hypertrophy. Beraprost sodium may be a potent antiarteriosclerotic agent through suppression of hyperplasia of SMC and modification of matrix protein.

[A case of Hashimoto's thyroiditis associated with renal tubular acidosis, Sjögren syndrome and empty sella syndrome].

This report describes a 48-year old female patient with Hashimoto's thyroiditis, distal-type renal tubular acidosis (d-RTA), Sjögren syndrome (SjS), and empty sella syndrome (ESS). She has been receiving replacement of thyroxine for Hashimoto's thyroiditis since 1967. She felt muscle weakness and numbness in the extremities and was found to have low serum potassium (2.9 mEq/l) in 1987. Since then she has been administrated potassium chloride orally. She was admitted to our hospital because of recurrence of muscle weakness and numbness of the extremities in November 1990. Laboratory examination revealed that her serum levels of antimicrosomal antibody and anti-thyroglobulin antibody were highly positive (MCHA: x 2(10) x 100, and TGHA: x 100). Furthermore, she was revealed to have 1) d-RTA by oral tolerance tests with the administration of NH4Cl and NaHCO3, 2) SjS by Schirmer test and sialography, and 3) ESS by computed tomography and magnetic resonance imaging examinations of the pituitary. Association of Hashimoto's thyroiditis, d-RTA, SjS and ESS in this case may possibly be caused by common autoimmune mechanism.

[Abnormalities in acid-base balance in the elderly].

Physiological decline in the ability to adjust acid-base balance and increase the incidence of diseases with aging, modifies pathophysiological and clinical features of acid-base disturbance in the elderly. Regulation of pH ultimately depends on the kidney and lung, however, the ability of the two organs is decreased with physiological aging. Moreover, the elderly are more prone to suffer from renal insufficiency and/or chronic obstructive pulmonary disease. Furthermore, medication with various drugs, such as diuretics, often affect the acid-base balance in the elderly. This paper describes the characteristics of the abnormalities in acid-base balance in the elderly, including metabolic acidosis and alkalosis, and respiratory acidosis and alkalosis.

Effects of manidipine hydrochloride on proliferation and glycosaminoglycan synthesis of cultured vascular smooth muscle cells.

The effects of manidipine hydrochloride, a calcium channel blocker, on the proliferation and the synthesis of glycosaminoglycans (GAG) in cultured rat aortic vascular smooth muscle cells (VSMC) were studied. Mandipine hydrochloride suppressed the DNA synthesis of VSMC dose dependently at concentrations of more than 10(-8) M. Mandipine hydrochloride 10(-6) M suppressed proliferation of VSMC to 50% of the control value. Manidipine hydrochloride stimulated the synthesis of GAG at concentrations above 10(-11) M. Manidipine hydrochloride 10(-8) M stimulated synthesis of GAG to 450% of control. Our findings suggest that because mandipine hydrochloride suppresses proliferation and stimulates GAG synthesis of VSMC, it may be an anti-arteriosclerotic agent.

[The influence of aging on parathyroid hormone-induced enhancement in (Ca2+ + Mg2+)-ATPase activity of rabbit renal basolateral membrane].

We studied the influence of aging on the stimulatory effect of parathyroid hormone (PTH) to (Ca2+ + Mg2+)-ATPase activity of rabbit renal basolateral membrane (BLM). Stimulated by PTH, (Ca2+ + Mg2+)-ATPase activity of BLM from aged rabbits was lower than those from young rabbits. However, the stimulatory effect of cyclic AMP (cAMP) showed no differences in terms of (Ca2+ + Mg2+)-ATPase activity in BLM from aged and young rabbits. As cAMP production in BLM caused by PTH stimulation was not measured, it is unclear whether cAMP production is different in BLM from young and aged rabbits. From these results, we concluded that in BLM from aged rabbit, the lower effect of PTH on (Ca2+ + Mg2+)-ATPase is due to lower production of cAMP or some other disorder of the PTH signal transduction mechanism. As (Ca2+ + Mg2+)-pump plays an important role in Ca2+ reabsorption in kidney, its lower activity in aged rabbit may be one reason for aberration of renal calcium handling.

Interleukin-6 stimulates proliferation of cultured vascular smooth muscle cells independently of interleukin-1 beta.

The effects of interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) on proliferation of cultured vascular smooth muscle cells (VSMCs) were investigated. Treatment with IL-6 caused a rapid increase in the c-myc mRNA level, and resulted in increases in DNA synthesis and cell number. IL-1 beta stimulated the DNA synthesis of the cells. EGF showed synergistic and PDGF or IL-1 beta showed additive effects with IL-6 on the DNA synthesis. These results suggest that IL-6, independently of IL-1 beta, may be important in the proliferation of VSMCs.

[Case report of a patient with pseudohypoparathyroidism associated with slight increase in serum level of unconjugated bilirubin].

We treated a 29-year-old male patient with pseudohypoparathyroidism type I, who showed a slight increase in serum indirect bilirubin without any signs of liver dysfunction. Serum levels of total, direct and indirect bilirubins were 2.4, 0.7 and 1.7mg/dl, respectively (normal ranges: 0.2-0.8, 0-0.2 and 0.2-0.6mg/dl, respectively). The cause of the increases in serum bilirubin levels was not clear; however, hemolytic anemia, hereditary unconjugated hyperbilirubinemia or ineffective erythropoiesis were ruled out as causes for the increase, since 1) his serum level of haptoglobin was normal, 2) increase in serum level of indirect bilirubin 120 minutes after the infusion of 50mg nicotinic acid was within the normal range, and 3) severe anemia was not observed. Osmotic fragility of his circulating red blood cells was also within normal range. Three other patients with pseudohypoparathyroidism visiting our clinic also showed slightly high levels of serum indirect bilirubin, although four outpatients with idiopathic hypoparathyroidism showed no such abnormality. Abnormality in the responsiveness to parathyroid hormone and/or to that in the cyclic AMP productivity in this disease may cause the increase in the circulating unconjugated bilirubin.

Effects of 1,25-dihydroxyvitamin D3 on the syntheses of DNA and glycosaminoglycans by rat aortic smooth muscle cells in vitro.

Studies were made on the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the syntheses of DNA and glycosaminoglycans (GAG) by rat aortic smooth muscle cells (SMC) in vitro. DNA synthesis in cell cultures without fetal calf serum (FCS) was stimulated by incubation for 24 hr with 1,25-(OH)2D3 at concentrations of more than 10(-12) M, stimulation being maximal at a concentration of 10(-8) M. On the other hand, GAG synthesis was inhibited dose-dependently by 1,25-(OH)2D3 at concentrations of more than 10(-11) M. Other vitamin D3 metabolites had similar, but weaker effects on the syntheses of DNA and GAG by SMC, which were proportional to their affinities for the 1,25-(OH)2D3 receptor. These effects of 1,25-(OH)2D3 were not seen after short-term incubation (1 hr). These findings suggested that 1,25-(OH)2D3 stimulated the proliferation of SMC independent of growth factors in FCS, and that its effects were dependent on its specific receptor. Excess 1,25-(OH)2D3 might cause arteriosclerosis not only by stimulating proliferation but also by suppressing GAG synthesis.

The action of low density lipoprotein on vascular smooth muscle cells involves increase in intracellular pH.

The effect of low density lipoprotein (LDL) on the intracellular pH (pHi) of vascular smooth muscle cells (VSMC) was investigated using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF). LDL and apoprotein B (apo-B), a binding protein for the LDL receptor, caused transient acidification followed by Na(+)-dependent and amiloride-sensitive alkalization of the cells due to stimulation of Na+/H+ exchanger. NH4Cl also caused intracellular alkalization, but independently of extracellular Na+. LDL, apo-B and NH4Cl all stimulated thymidine incorporation. These results indicate that the binding of LDL to its receptor stimulates Na+/H+ exchanger, resulting in alkalization of VSMC and suggest that this may function as a massage in stimulation of DNA synthesis evoked by LDL.

Endothelin stimulates Na+/H+ exchange in vascular smooth muscle cells.

The effect of endothelin (ET) on the intracellular pH (pHi) of vascular smooth muscle cells (VSMC), was investigated using a fluorescent pH indicator 2',7'-bis(carboxyethyl)carboxyfluorescein (BCECF). ET at concentrations of over 10(-9) M caused dose-dependent transient acidification followed by Na(+)-dependent and amiloride-sensitive alkalization of the cells due to stimulation of Na+/H+ exchange. The alkalization induced by ET was Ca2(+)-dependent and was inhibited by a calcium channel blocker, nicardipine. Pretreatment with H-7, an inhibitor of protein kinase C, also inhibited the ET-induced cell alkalization. These results indicate that ET stimulates Na+/H+ exchange, resulting in alkalization of VSMC and that this ET-induced cell-alkalization is probably linked to Ca2+ influx and activation of protein kinase C.

Interleukin-6 stimulates c-myc expression and proliferation of cultured vascular smooth muscle cells.

The effects of interleukin-6 (IL-6), a cytokine recently found to be secreted by monocytes and macrophages, on c-myc expression and proliferation of cultured vascular smooth muscle cells (VSMC) were investigated. Treatment with IL-6 caused rapid increase in the c-myc mRNA level of VSMC. It also stimulated DNA synthesis and proliferation of the cells significantly and dose-dependently at concentrations of more than 10 U/ml. These results suggest that IL-6 may be important in the proliferation of VSMC, which is a key event in the development of arteriosclerosis, as a factor mediating immune cell-VSMC interaction.

Comparison of the inhibitions of proliferation of normal and psoriatic fibroblasts by 1 alpha,25-dihydroxyvitamin D3 and synthetic analogues of vitamin D3 with an oxygen atom in their side chain.

The inhibitory effects of 1 alpha,25-(OH)2D3 and synthetic oxa-derivatives of vitamin D3 on growth of normal and psoriatic fibroblasts in culture were compared. Proliferation of normal fibroblasts was strongly inhibited by these new compounds in the following order: 22-oxa-1 alpha,25-(OH)2D3 greater than 22-oxa-1 alpha-(OH)D3 greater than 1 alpha,25-(OH)2D3 greater than 20-oxa-1 alpha,25-(OH)2D3. 22-Oxa-1 alpha,25-(OH)2D3 was about 10-times more inhibitory than 1 alpha,25-(OH)2D3. Proliferation of psoriatic fibroblasts was not inhibited by 1 alpha,25-(OH)2D3 at concentrations of up to 10(-6) M, but was suppressed by 10(-8)-10(-6) M 22-oxa-1 alpha,25-(OH)2D3 and 10(-6) M 22-oxa-1 alpha-(OH)D3. These results suggest that oxa-derivatives of vitamin D3, especially 22-oxa-1 alpha,25-(OH)2D3, should be useful in further studies on the cause and treatment of psoriasis.

Effects of valinomycin on calcium mobilization in vascular smooth muscle cells induced by angiotensin II.

The effect of the specific potassium (K+) ionophore valinomycin on increase in intracellular calcium concentration [( Ca2+]i) was studied in vascular smooth muscle cells (VSMC). Valinomycin at more than 10(-9) M dose-dependently suppressed phasic increase in [Ca2+]i in VSMC induced by angiotensin II (AII) in both control and Ca2+-free solution, indicating that it suppressed the release of Ca2+ from intracellular Ca2+ stores. Nicorandil and cromakalim, which are both K+ channel openers, also suppressed the increases in [Ca2+]i induced by AII in the Ca2+ free solution. However, valinomycin did not suppress AII-induced production of inositol 1,4,5-trisphosphate (IP3), which is known to mediate the release of Ca2+. These results indicate that decrease of intracellular K+ induced by valinomycin suppressed the release of Ca2+ from intracellular Ca2+ stores induced by IP3.