Potent non-hydroxamate inhibitors of histone deacetylase-8: Role and scope of an isoindolin-2-yl linker with an α-amino amide as the zinc-binding unit.

A series of potent inhibitors of histone deacetylase-8 (HDAC8) is described that contains an α-amino amide zinc-binding unit and a substituted isoindolinyl capping group. The presence of a 2,4-dichlorophenyl unit located in the acetate-release cavity was shown to confer a gain of approx. 4.3 kJ mol-1 in binding energy compared to a phenyl group, and the isoindoline linker has approx. 5.8 kJ mol-1 greater binding energy than the corresponding tetrahydroisoquinoline ring system. In a series of 5-substituted isoindolin-2-yl inhibitors, a 5-acetylamino derivative was found to be more potent than the 5-unsubstituted lead HDAC8 inhibitor (increase in binding energy of 2.0 kJ mol-1, ascribed to additional binding interactions within the Nε-acetyl-l-lysine binding tunnel in HDAC8, including hydrogen bonding to Asp101. Tolerance of a 5-substituent (capping group) on the isoindoline ring has been demonstrated, and which in some cases confers improved enzyme inhibition, the HDAC8 substrate-binding region providing a platform for additional interactions.

Antihistamine Effect of a Pure Bioactive Compound Isolated from Slug (Diplosolenodes occidentalis) Material.

OBJECTIVE: Folklore claims of the therapeutic effect of garden slug (Diplosolenodes occidentalis) extract used to relieve bronchoconstriction in asthmatic individuals were never validated scientifically. The aim of this study was to isolate the pure bioactive compound from slug extract causing this effect. METHODS: The crude ground material was prepared in ethanol and after filtration, separation by flash column chromatography method was done. The structure was elucidated by data from hydrogen and carbon nuclear magnetic resonance (NMR) profiles. The bioactive compound was assessed for dose-dependent response effects on guinea pig tracheal smooth muscle pre-contracted with histamine. Receptor specificity studies were done by using HTMT dimaleate (H1 agonist). The type of antagonism was also identified. RESULTS: The pure component isolated from garden slug material was identified by spectral studies as glyceryl trilinolenate (GT). It caused dose-dependent relaxation in guinea pig tracheal smooth muscle strips pre-contracted with histamine, it acted via H1 type receptors and showed non-competitive antagonism. CONCLUSION: Glyceryl trilinolenate produced dose-dependent relaxation in tracheal smooth muscle strips in the presence of the agonist histamine. Glyceryl trilinolenate displayed non-competitive antagonism at H1 receptors in the trachea. This agent was able to alleviate bronchoconstriction in individuals presenting with atopic asthma in rural agricultural areas in Jamaica (verbal communications). It is possible that GT can be useful therapeutically to produce tracheal smooth muscle relaxation in individuals presenting with atopic asthma.

A distal regulatory region of a class I human histone deacetylase.

Histone deacetylases (HDACs) are key enzymes in epigenetics and important drug targets in cancer biology. Whilst it has been established that HDACs regulate many cellular processes, far less is known about the regulation of these enzymes themselves. Here, we show that HDAC8 is allosterically regulated by shifts in populations between exchanging states. An inactive state is identified, which is stabilised by a range of mutations and resembles a sparsely-populated state in equilibrium with active HDAC8. Computational models show that the inactive and active states differ by small changes in a regulatory region that extends up to 28 Å from the active site. The regulatory allosteric region identified here in HDAC8 corresponds to regions in other class I HDACs known to bind regulators, thus suggesting a general mechanism. The presented results pave the way for the development of allosteric HDAC inhibitors and regulators to improve the therapy for several disease states.

Six-shogaol inhibits production of tumour necrosis factor alpha, interleukin-1 beta and nitric oxide from lipopolysaccharide-stimulated RAW 264.7 macrophages.

OBJECTIVE: We previously reported that 6-shogaol, a phenolic compound from ginger has antiinflammatory properties in a Complete Freund's Adjuvant (CFA) model of mono-arthritic rats. In the present study, we investigated the effects of 6-shogaol on the production of inflammatory mediators from lipopolysaccharide (LPS) activated RAW 264.7 macrophages. These mediators (TNF-alpha, IL-1-beta and NO) and their output from macrophages are involved in various pathophysiological events of chronic inflammation and arthritis. METHODS: Effects of 6-shogaol were investigated on the production of the mediators TNF-alpha, IL-1-beta and NO (measured as nitrate)from macrophages. Lipopolysaccharide activated RAW 264.7 macrophages were cultured in the presence and absence of 6-shogaol (2 microM, 10 microM and 20 microM) and ELISA was used to quantify the output of the mediators. RESULTS: 6-shogoal (2 microM, 10 microM and 20 microM) significantly inhibited the production of nitric oxide (NO), IL-1beta and TNF-alpha from the LPS activated RAW264.7 macrophages. CONCLUSION: The results suggest that macrophages are targets for the anti-inflammatory effects of 6-shogaol. Also, the inhibitory effects against TNF-alpha, IL-1beta and NO production from LPS activated macrophages are cellular mechanisms by which 6-shogaol produced its anti-inflammatory effects. These mechanisms provide an explanation of the protection by 6-shogaol against development of joint inflammation and cartilage degradation in CFA induced mono-arthritis that we previously demonstrated (1). Based on these results with 6-shogaol, there is evidence that it exhibits exploitable anti-inflammatory properties.

The effect of phytic acid on the levels of blood glucose and some enzymes of carbohydrate and lipid metabolism.

In this study, six groups of rats were fed as follows: Groups 1 and 2 were fed formulated diets supplemented with zinc or without zinc respectively. Groups 3 and 4 were fed formulated diets supplemented with zinc plus phytic acid extracted from sweet potato (Ipomea batatas) or commercial phytic acid respectively. Groups 5 and 6 were fed formulated diets supplemented with phytic acid extract from sweet potato or commercial phytic acid respectively. The animals were fed for three weeks and then sacrificed The activities of key enzymes of carbohydrate and lipid metabolism as well as transaminases in the liver were determined. Blood glucose level was also assessed. Phytic acid extract consumption from sweet potato and commercial phytic acid plus zinc supplement lowered blood glucose levels. There was no significant change in the activity of 6-phosphogluconate dehydrogenase among the groups. Similarly, phytic acid supplementation showed no significant decrease in the activity of pyruvate kinase compared to the group fed formulated diets. There was a significant increase in the activity of glucose-6-phosphate dehydrogenase in the groups fed phytic extract from sweet potato compared to the other groups. The activities of malic enzyme and ATP-citrate lyase in this study were not significantly altered among the groups. There is a lowering of blood glucose levels which is desirable for diabetics who consume sweet potato diets. The changes in some of the hepatic metabolic enzymes are geared towards compensating for the decreased glycolytic responses.

Spinal superfusion of dopamine excites renal sympathetic nerve activity.

Chloralose-anesthetized rats, spinalized at C1, were used to investigate the effects of spinal infusion of dopamine on renal sympathetic nerve activity (RSNA). A subarachnoid spinal superfusion technique was used to localize dopamine in the spinal cord while renal sympathetic nerve activity was recorded from the left renal nerve. Dopamine (25-200 pmol) produced dose-dependent increases in renal sympathetic nerve activity (35 +/- 5% - 77 +/- 6%) and mean arterial blood pressure (25 +/- 5 mmHg - 38 +/- 2 mmHg). This increase in renal sympathetic nerve activity was potentiated by pretreatment with disulfiram (0.67 mmol/kg, interscapularly). Superfusion of equivalent doses of norepinephrine (NE) (25-50 pmol) had no effect or (100 pmol NE) inconsistent effect on renal sympathetic nerve activity. Intravenous injection of dopamine (25-200 pmol) produced no changes in renal sympathetic nerve activity and small increases of (5-7 mmHg) in mean blood pressure. Spinally superfused haloperidol (3 nmol) inhibited the dopamine-induced excitation of renal sympathetic nerve activity, but superfused phentolamine (3 nmol) potentiated the response. The magnitude of renal nerve excitation (RNE), elicited by electrical stimulation of points between lamina four and seven and the adjacent white matter of the cervical cord, was reduced to 60% of control by alpha-methyl-p-tyrosine (0.25 mumol, spinally) and was restored to 85% of control by dopamine (25 pmol) but not by NE (25 pmol). However, the magnitude of renal nerve excitation, elicited by stimulation of the same cervical area, was unaffected by pretreatment with disulfiram interscapularly. Therefore, it is concluded that dopamine itself may be an important neurotransmitter of a spinal system modulating renal sympathetic nerve activity.

Interaction between the baroreflex and anterior hypothalamic stimulation. Demonstration of a noradrenergic involvement.

Cats, anaesthetized with alpha-chloralose, were used to investigate an interaction between the baroreceptor reflex and stimulation of the anterior hypothalamus (AH). In these cats, stimulation of the anterior hypothalamus gave autonomic responses that characterize the defense reaction. Stimulation of the anterior hypothalamus also interacted with the baroreceptor reflex without altering the effectiveness of the vasodepressor component of the reflex (induced by stimulation of the aortic nerves). However, in spinal cats the bradycardia component (induced by angiotensin at 0.097 nmol/kg pe min, i.v.) was inhibited by stimulation of the anterior hypothalamus and the inhibition was augmented by pretreatment with phentolamine (17.7 nmol) in the nucleus tractus solitarius (NTS). Pretreatment with phentolamine (17.7 nmol) in the nucleus tractus solitarius also reduced the sensitivity of the bradycardia component of the reflex and attenuated (3 and 6 nmol) the induced inhibition of the bradycardia induced by stimulation of the anterior hypothalamus. This attenuation induced by NE was abolished by pretreatment with phentolamine (17.7 nmol), but not by pretreatment with sotalol (16 nmol) or haloperidol (12 nmol) in the nucleus tractus solitarius. Additionally, NE was released from the nucleus tractus solitarius during activation of the baroreceptor and during stimulation of the anterior hypothalamus in cats with intact baroreceptor nerves, but not in baroreceptor-denervated cats. Moreover, the baroreceptor reflex attenuated the pressor response evoked by stimulation of the anterior hypothalamus, and also caused the release of NE from the anterior hypothalamus. At this site, injection of NE (3 and 6 nmol) attenuated the pressor response evoked by stimulation of the anterior hypothalamus and this attenuation was abolished by pretreatment with phentolamine (17.7 nmol), but not by pretreatment with sotalol (16 nmol) or haloperidol (12 nmol) in the anterior hypothalamus. It is concluded that NE participates at the nucleus tractus solitarius and the anterior hypothalamus as an attenuator in the interaction between the baroreflex and stimulation of nerve fibers in the anterior hypothalamus that mediate the defense reaction.

The spinal course and medullary termination of myelinated renal afferents in the rat.

Myelinated afferent fibers, recorded in the left renal nerve of rats, were antidromically activated by discrete electrical stimulation of the cervical spinal cord and the caudal medulla. The lowest thresholds for activation of these fibers were found in the most medial portion of the ipsilateral fasciculus gracilis. This region of minimum threshold continued rostrad through the nucleus commissuralis. Based on threshold vs depth contours, fibers appeared to terminate in the ipsilateral nucleus gracilis and nucleus solitarius. Myelinated fibers could be activated by punctate pressure on the renal hilus. Action potentials generated by hilar pressure collided with antidromically-conducted action potentials elicited by electrical stimulation at cervical levels. We conclude that myelinated renal afferents carry information from intrarenal receptors, via the dorsal column system, to both visceral afferent and dorsal column nuclei.

Characteristics of impulsive suicide attempts and attempters.

Suicide attempts often are impulsive, yet little is known about the characteristics of impulsive suicide. We examined impulsive suicide attempts within a population-based, case-control study of nearly lethal suicide attempts among people 13-34 years of age. Attempts were considered impulsive if the respondent reported spending less than 5 minutes between the decision to attempt suicide and the actual attempt. Among the 153 case-subjects, 24% attempted impulsively. Impulsive attempts were more likely among those who had been in a physical fight and less likely among those who were depressed. Relative to control subjects, male sex, fighting, and hopelessness distinguished impulsive cases but depression did not. Our findings suggest that inadequate control of aggressive impulses might be a greater indicator of risk for impulsive suicide attempts than depression.

Unstable diabetic state produced by a small dose of streptozotocin in rats.

A diabetic state was induced with a single intraperitoneal dose (45 mg/kg) of streptozotocin in rats. Their fasting blood glucose concentrations oscillated between 12.7 +/- 1.9 mmol/l and 4.6 +/- 0.6 mmol/l during 35 days of monitoring. Their body weights were also reduced, while controls gained weight, although food consumption was not significantly different. Also, within the first 1/2-hour of the oral glucose tolerance test, blood glucose concentration increased in the diabetic and the control rats, but only in the control rats was there a simultaneous increase in serum IRI concentration (7.2 +/- 8 x 10(2) pmol/l to 27.0 +/- 5.2 x 10(2) pmol/l) which, like the blood glucose concentration, subsequently fell to fasting level in the control rats. In the diabetic rats, however, it was not until the following hour of the tolerance test that serum IRI concentration increased (3.4 +/- 0.3 x 10(2) pmol/l to 65.0 +/- 12.5 x 10(2) pmol/l) and blood glucose concentration began to fall. By the end of the test in the diabetic rats, blood glucose concentration fell but remained significantly higher than the control value. Additionally, no pancreatic tumours were identified in these diabetic rats. The results therefore suggest that an unstable diabetic state was produced by streptozotocin because the threshold for insulin secretion by glucose was increased, while the production of insulin by the pancreas was not significantly affected.

Demonstration of bioequivalence between ventasol syrup and ventolin syrup in humans and in dogs.

Ventasol syrup, a new locally produced salbutamol formulation, was compared with the standard salbutamol formulation, ventolin syrup, for determination of oral bioequivalence in stable asthmatic human subjects and in dogs. On separate occasions, each subject received a single 10 ml oral dose of each formulation containing 4 mg salbutamol. In the human subjects, statistically similar peak plasma concentrations of salbutamol were obtained (196 +/- 7 ng/ml for ventasol syrup and 185 +/- 6 ng/ml for ventolin syrup) 3 hours after oral administration of the formulations. From the ratio of the AUCo-infinity for the formulations (1.04), a relative oral bioavailability of 104%, indicating equivalent total salbutamol output, was also obtained in the human subjects. Similarly, in the dogs, the formulations produced statistically equivalent peak plasma concentrations of salbutamol (259 +/- 24ng/ml for ventasol syrup and 285 +/- 29ng/ml for ventolin syrup) 3 hours after oral administration. Also, from the ratio of the AUCo-infinity for the formulations (1.02), a relative oral bioavailability of 102%, indicating similar total salbutamol output, was obtained in the dogs. From these results, it is concluded that oral bioequivalence between ventasol syrup and ventolin syrup was demonstrated in human subjects and in dogs.

Streptozotocin alters pancreatic beta-cell responsiveness to glucose within six hours of injection into rats.

A 24-hour glycaemic profile following streptozotocin (80 mg/kg. i.p.) injection was investigated in fasted rats. The most prominent changes in blood glucose were hyperglycaemia associated with low levels of plasma insulin after two hours followed by hypoglycaemia associated with high levels of plasma insulin after six hours; subsequently hyperglycaemia progressively developed and this was associated with decreasing levels of plasma insulin. Further probing revealed that at two hours after streptozotocin injection, the pancreatic beta-cells could not respond to an oral glucose load while, at six hours after, there was an apparent return of beta-cell responsiveness, but subsequently beta-cell responsiveness was progressively lost and histological examination revealed cellular damage. From these results, it is concluded that within six hours of injection, streptozotocin initiates pancreatic beta-cell damage which leads to the development of diabetes mellitus.

Evidence that guanylate cyclase is involved in modulating the resting tension and neurally-induced contraction of the guinea pig tracheal muscle.

Electrical field stimulation of guinea-pig tracheal muscle strips produced a frequency-dependent biphasic response consisting of an initial cholinergic contraction followed by relaxation. Both phases of the response were of neural origin. In the presence of methylene blue, a guanylate cyclase inhibitor, the resting tension and the contraction were increased, but the accompanying relaxation was inhibited. However, in the presence of sodium nitroprusside, a guanylate cyclase activator, the resting tension was reduced and the contraction was inhibited, but the relaxation was prolonged and increased. Similarly, in the presence of either 3-isobutyl-1-methylxanthine, which promotes cyclic guanosine monophosphate (cGMP) accumulation, or 8-bromo-cGMP, an analogue of cGMP, the resting tension was reduced and the contraction was inhibited but the relaxation was prolonged and increased. From these results, it is concluded that guanylate cyclase is involved in modulating the resting tension and the neurally-induced contraction of guinea-pig tracheal muscle.

Activation of guanylate cyclase in the guinea-pig trachea reduces contractile responses of the smooth muscle.

Guinea-pig tracheal strips were used to investigate whether activation of guanylate cyclase in the trachea can reduce the contractile responses of the smooth muscle. Guanylate cyclase was activated by glyceryl trinitrate and a combination of sodium nitrite and ascorbic acid. These activators inhibited tracheal smooth muscle contractions produced by acetylcholine, histamine and electrical field stimulation. However, in the presence of methylene blue, a guanylate cyclase inhibitor, tracheal smooth muscle contractions were not inhibited by the activators. But, in the presence of propranolol, which blocked inhibition mediated by beta-adrenoceptor, both glyceryl trinitrate and the sodium nitrite/ascorbic acid combination were still capable of inhibiting tracheal smooth muscle contractions. Additionally, methylene blue inhibited tracheal smooth muscle relaxation that was electrically induced. These results suggest that the inhibitory action mediated by activated guanylate cyclase may be a mechanism for regulating tracheal smooth muscle contractile responses.

Isolation of a muscarinic alkaloid with ocular hypotensive action from Trophis racemosa.

A muscarinic alkaloid with a quaternary nitrogen was isolated from Trophis racemosa. Aqueous solutions (0.5%-2%) of the chloride salt of the alkaloid produced dose-dependent reductions of intra-ocular pressure ranging from 6.6 +/- 0.7 mmHg to 15.7 +/- 0.3 mmHg, (p < 0. 001, n = 5) in dogs. Atropine (0.1 mL of a 1% solution) and pirenzepine at a non selective antagonist dose (0.1 mL of 0.5% solution) for M(1) and M(3) receptors blocked the reduction of intra-ocular pressure, but alpha-adrenoceptor blockade with phenoxybenzamine (0.1 mL of a 1% solution) did not block the reduction of intra-ocular pressure. On the isolated guinea-pig ileum and trachea, the alkaloid produced contractions which were inhibited by atropine (6 x 10(-7) M or 0.4 microg/mL) and by pirenzepine at a non-selective antagonist dose (3.1 x 10(-6) M or 1.3 microg/mL) for M(1) and M(3) receptors. But neither selective blockade of M(2) receptors with gallamine (1.7 x 10(-6) M or 1.5 microg/mL) nor selective blockade of M(1) receptors with pirenzepine (7 x 10(-9) M or 3 ng/mL) inhibited the alkaloid-induced contractions. There was also no inhibition of the alkaloid-induced contractions in the presence of ganglionic nicotinic receptor blockade with pentolinium (5.6 x 10(-7) M or 0.3 microg/mL) and hexamethonium (1.7 x 10(-6) M or 0.6 microg/mL), but nicotine-induced contractions were inhibited by these ganglionic blockers. These results suggest that a muscarinic alkaloid from Trophis racemosa produced ocular hypotension via M(3) receptor stimulation in dogs.