DBH Inhibitor Research Articles

Chronic loss of noradrenergic tone produces β-arrestin2-mediated cocaine hypersensitivity and alters cellular D2 responses in the nucleus accumbens.

Cocaine blocks plasma membrane monoamine transporters and increases extracellular levels of dopamine (DA), norepinephrine (NE) and serotonin (5-HT). The addictive properties of cocaine are mediated primarily by DA, while NE and 5-HT play modulatory roles. Chronic inhibition of dopamine β-hydroxylase (DBH), which converts DA to NE, increases the aversive effects of cocaine and reduces cocaine use in humans, and produces behavioral hypersensitivity to cocaine and D2 agonism in rodents, but the underlying mechanism is unknown. We found a decrease in β-arrestin2 (βArr2) in the nucleus accumbens (NAc) following chronic genetic or pharmacological DBH inhibition, and overexpression of βArr2 in the NAc normalized cocaine-induced locomotion in DBH knockout (Dbh -/-) mice. The D2/3 agonist quinpirole decreased excitability in NAc medium spiny neurons (MSNs) from control, but not Dbh -/- animals, where instead there was a trend for an excitatory effect. The Gαi inhibitor NF023 abolished the quinpirole-induced decrease in excitability in control MSNs, but had no effect in Dbh -/- MSNs, whereas the Gαs inhibitor NF449 restored the ability of quinpirole to decrease excitability in Dbh -/- MSNs, but had no effect in control MSNs. These results suggest that chronic loss of noradrenergic tone alters behavioral responses to cocaine via decreases in βArr2 and cellular responses to D2/D3 activation, potentially via changes in D2-like receptor G-protein coupling in NAc MSNs.

Etamicastat, a new dopamine-ß-hydroxylase inhibitor, pharmacodynamics and metabolism in rat

Despite the importance of sympathetic nervous system in pathophysiological mechanisms of cardiac heart failure and essential hypertension, therapy specifically targeting the sympathetic nervous system is currently underutilized. Etamicastat is a novel dopamine-ß-hydroxylase (DBH) inhibitor that is oxidized into BIA 5-965 and deaminated followed by oxidation to BIA 5-998, which represents 13% of total etamicastat and quantified metabolites. However, the primary metabolic pathway of etamicastat in rats was found to be the N-acetylation (BIA 5-961), which represents 44% of total etamicastat and quantified metabolites. Trace amounts of BIA 5-961 de-sulfated and S-glucuronide were also detected. All the main metabolites of etamicastat inhibited DBH with IC50 values of 306 (228, 409), 629 (534, 741), 427 (350, 522)nM for BIA 5-965, BIA 5-998 and BIA 5-961, respectively. However, only etamicastat (IC50 of 107 (94; 121)nM) was able to reduce catecholamine levels in sympathetic nervous system innervated peripheral tissues, without effect upon brain catecholamines. Quantitative whole body autoradiography revealed a limited transfer of etamicastat related radioactivity to brain tissues and the mean recovery of radioactivity was ~90% of the administered radioactive dose, eliminated primarily via renal excretion over 5 days. The absolute oral bioavailability of etamicastat was 64% of the administered dose. In conclusion, etamicastat is a peripheral selective DBH inhibitor mainly N-acetylated in the aminoethyl moiety and excreted in urine. Etamicastat main metabolites inhibit DBH, but only etamicastat demonstrated unequivocal pharmacological effects as a DBH inhibitor with impact upon the activity of the sympathetic nervous system under in vivo conditions.

Effects of pharmacologic dopamine β-hydroxylase inhibition on cocaine-induced reinstatement and dopamine neurochemistry in squirrel monkeys.

Disulfiram has shown promise as a pharmacotherapy for cocaine dependence in clinical settings, although it has many targets, and the behavioral and molecular mechanisms underlying its efficacy are unclear. One of many biochemical actions of disulfiram is inhibition of dopamine β-hydroxylase (DBH), the enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic neurons. Thus, disulfiram simultaneously reduces NE and elevates DA tissue levels in the brain. In rats, both disulfiram and the selective DBH inhibitor nepicastat block cocaine-primed reinstatement, a paradigm which is thought to model some aspects of drug relapse. This is consistent with some clinical results and supports the use of DBH inhibitors for the treatment of cocaine dependence. The present study was conducted to confirm and extend these results in nonhuman primates. Squirrel monkeys trained to self-administer cocaine were pretreated with disulfiram or nepicastat prior to cocaine-induced reinstatement sessions. Neither DBH inhibitor altered cocaine-induced reinstatement. Unexpectedly, nepicastat administered alone induced a modest reinstatement effect in squirrel monkeys, but not in rats. To investigate the neurochemical mechanisms underlying the behavioral results, the effects of DBH inhibition on extracellular DA were analyzed in the nucleus accumbens (NAc) using in vivo microdialysis in squirrel monkeys. Both DBH inhibitors attenuated cocaine-induced DA overflow in the NAc. Hence, the attenuation of cocaine-induced changes in accumbal DA neurochemistry was not associated with altered cocaine-seeking behavior. Overall, the reported behavioral effects of DBH inhibition in rodent models of relapse did not extend to nonhuman primates under the conditions used in the current studies.

Dopamine β-Hydroxylase Inhibitors Enhance the Discriminative Stimulus Effects of Cocaine in Rats

Inhibitors of dopamine β-hydroxylase (DBH), the enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic cells, have shown promise for the treatment of cocaine abuse disorders. However, the mechanisms underlying the beneficial effects of these compounds have not been fully elucidated. We used the drug discrimination paradigm to determine the impact of DBH inhibitors on the interoceptive stimulus properties of cocaine. Sprague-Dawley rats were trained to discriminate cocaine (5.6 mg/kg) from saline using a multicomponent, food-reinforced discrimination procedure. On test days, subjects were pretreated with the nonselective DBH inhibitor disulfiram (0-100.0 mg/kg i.p.) or the selective DBH inhibitor nepicastat (0-56.0 mg/kg i.p.) 2 hours prior to a test session either alone or in combination with cumulatively administered cocaine (0-5.6 mg/kg i.p.). Neither disulfiram nor nepicastat substituted for the cocaine stimulus when tested up to doses that nonspecifically reduced responding. However, in combination studies, pretreatment with either disulfiram or nepicastat produced leftward shifts in the cocaine dose-response function and also conferred cocaine-like stimulus effects to the selective NE transporter inhibitor, reboxetine (0.3-5.6 mg/kg i.p.). These results indicate that pharmacological inhibition of DBH does not produce cocaine-like interoceptive stimulus effects alone, but functionally enhances the interoceptive stimulus effects of cocaine, possibly due to facilitated increases in DA released from noradrenergic terminals. These findings suggest that DBH inhibitors have low abuse liability and provide support to clinical reports that some subjective effects produced by cocaine, particularly aversive effects, are enhanced after DBH inhibition.

Abstract 454: Hypertension Control by Decrease of Sympathetic Hyperactivity in SHR Rats: Comparison Between Renal Denervation and Dopamine ß-hydroxylase Inhibition

Activation of the sympathetic nervous system has been described in the development and progression of systemic hypertension. Recently, catheter-based renal nerve ablation for treatment of drug-resistant hypertension has been developed. An alternative strategy for modulation of sympathetic nerve function is to reduce the biosynthesis of norepinephrine (NE) via inhibition of dopamine β-hydroxylase (DβH), the enzyme that catalysis the conversion of dopamine (DA) to NE in sympathetic nerves. Renal denervation was assessed in the rat by transient (90 sec) ischemia of renal tissues. Bilateral renal artery occlusion (RAO) was assessed in spontaneously hypertensive rats (SHR) to evaluate the effect of renal denervation on NE levels and blood pressure throughout a 28-day period. Oral treatment with the selective peripheral DβH inhibitor etamicastat (30 mg/Kg body weight) was performed in a second cohort of SHR and NE levels and blood pressure was assessed. Renal denervation resulted in marked depletion of NE levels, selective for the renal sympathetic innervations, 5 days after RAO in renal cortex (16.5±7.7% of control values, P<0.0001). Decreased NE levels persisted up to 28 days after RAO (40.8±19.4% of control values, P<0.0001). No differences in left ventricle NE levels were observed. Renal denervation significantly decreased systolic blood pressure (SBP) 5 days (165±6 vs. 187±2 mmHg, P<0.05) and 14 days post-surgery (194±3 vs. 204±2 mmHg, P<0.05), but had no effect on heart rate. Systolic blood pressure recovered to basal values 28 days post-surgery. Likewise, etamicastat treatment resulted in a depletion of NE levels in both renal cortex (70.9±9.1% of control values, P<0.05) and left ventricle (55.8±3.9% of control values, P<0.01). Etamicastat treatment decreased SBP (228±7 vs. 174±9 mmHg, P<0.001) without effects on heart rate. In conclusion, these results suggest that renal denervation and DβH inhibition similarly decreases sympathetic hyperactivity and high blood pressure in the SHR. Downregulation of sympathetic drive with DβH inhibitors may be useful in the treatment of hypertension.

Abstract 425: Beneficial Effects of BIA 5-1058 in a Genetic Model of Salt-sensitive Hypertension and Heart Failure

Hyperactivity of Sympathetic Nervous System (SNS) is implicated in numerous cardiovascular diseases. One strategy to slow down the SNS drive is to reduce the biosynthesis of norepinephrine (NE) via inhibition of dopamine-β-hydroxylase (DβH), the enzyme that catalyses the conversion of dopamine (DA) to NE in sympathetic nerves. BIA 5-1058 is a new DβH inhibitor that decreases NE and increases DA levels in peripheral sympathetically innervated tissues. It was previously shown that BIA 5-1058 dose-dependently reduces blood pressure (BP) in spontaneously hypertensive rats. The Dahl salt-sensitive (Dahl/SS) rat is known to develop systemic hypertension, over 200 mmHg, due to combined pressure and volume overload, which results in compensated left ventricle (LV) hypertrophy after five to six weeks and cardiac failure with LV dilation and contractile dysfunction after 10 to 12 weeks of high-salt (HS) diet. The aim of the present study was to evaluate the effect of BIA 5-1058 in salt-induced hypertension and heart failure in the Dahl/SS rat. To evaluate the effect of acute BIA 5-1058 treatment on hypertension, six Dahl/SS rats were implanted with telemetry device TA11PA-C40 (DSI) and fed a HS (8% NaCl) diet. Salt-induced hypertensive animals were used to evaluate two different doses (10 and 100 mg/Kg) of BIA 5-1058 on BP. Afterwards, a second cohort of rats were used to evaluate the effect of prolonged BIA 5-1058 treatment (30 mg/Kg/day) on the survival rate of Dahl/SS rats fed a HS diet. The reduction in the sympathetic tone attained by DβH inhibition with BIA 5-1058 showed a dose- and time-dependent effect on systolic (SBP) and diastolic (DBP) blood pressure. The maximum reduction on SBP was -16.1 and -22.9 mmHg for 10 and 100 mg/Kg, respectively. Likewise, the maximum reduction on DBP was -18.8 and -23.3 mmHg for 10 and 100 mg/Kg, respectively. Survival rate of Dahl/SS rats fed a HS diet demonstrated that BIA 5-1058, beyond BP reduction, shows beneficial effects to the heart of Dahl/SS rats fed a HS diet with a significant 19-day increase in median survival. In conclusion, the use of DβH inhibitors, like BIA 5-1058, is a promising approach to treat hypertension, heart failure and others cardiovascular diseases where a reduction in the sympathetic tone has beneficial effects.

Abstract 532: BIA 5-1058, Beyond Blood Pressure, Improves Cardiometabolism and Decrease End-organ Damage

The sympathetic nervous system (SNS) can alter blood pressure (BP) by modulation of cardiac output, peripheral vascular resistance and renal function. One strategy for controlling sympathetic nerve function is to reduce the biosynthesis of norepinephrine (NE) via inhibition of dopamine β-hydroxylase (DβH). BIA 5-1058 is a new peripheral DβH inhibitor that decreases NE levels in sympathetically innervated tissues and slows down the drive of SNS. In order to evaluate the cardiometabolic effects of BIA 5-1058 in aged spontaneously hypertensive rats (SHR), 12 male SHR 50-week-old were randomized into two groups and one group was treated with BIA 5-1058 (30 mg/Kg/day) mixed in the diet for 9 weeks. During week 8 of treatment, blood pressure (BP) and heart rate (HR) were measured by tail cuff. At the end of the study, 24-hour urine and plasma was collected and organs weight was recorded. BIA 5-1058 treatment reduced systolic BP (224±5 vs 183±8 mmHg, p<0.05) with no significant effect on HR. The heart/body weight ratio was decreased in animals treated with BIA 5-1058 (3.66±0.08 vs 3.45±0.04 mg/g, p<0.05), while the ratio kidney/body weight was unchanged. BIA 5-1058 significantly decreased plasma levels of the inflammatory markers CRP (447.3±7.1 vs 401.2±7.1 μg/ml, p<0.05), MCP-1 (90.3±10.5 vs 58.2±9.0 pg/ml, p<0.05) and ASAT (91.7±8.0 vs 67.3±4.0 U/l, p<0.05) but no significant effect on ALAT was observed. Concerning lipid metabolism, there was a decrease in plasma triglycerides (0.94±0.05 vs. 0.70±0.05 mmol/l, p<0.05) and free fatty acids levels (0.23±0.03 vs 0.11±0.01 mmol/l, p<0.05) induced by BIA 5-1058, while total cholesterol plasma levels was similar in both groups. BIA 5-1058 significantly reduced the 24-hour urine excretion (13.5±1.6 vs 8.9±1.2 ml, p<0.05), but had no significant effect in the amount of protein excreted in urine nor in the creatinine clearance rate. In conclusion, the new DBH inhibitor, BIA 5-1058, presents cardiometabolic benefits in aged SHR.

Xanthoangelol and 4-Hydroxyderricin Are the Major Active Principles of the Inhibitory Activities against Monoamine Oxidases on Angelica keiskei K

Monoamine oxidase inhibitors (MAOI) have been widely used as antidepressants. Recently, there has been renewed interest in MAO inhibitors. The activity-guided fractionation of extracts from Angelica keiskei Koidzumi (A. keiskei K.) led to the isolation of two prenylated chalcones, xanthoangelol and 4-hydroxyderricin and a flavonoid, cynaroside. These three isolated compounds are the major active ingredients of A. keiskei K. to inhibit the MAOs and DBH activities. Xanthoangelol is a nonselective MAO inhibitor, and a potent dopamine β-hydroxylase (DBH) inhibitor. IC50 values of xanthoangelol to MAO-A and MAO-B were calculated to be 43.4 μM, and 43.9 μM. These values were very similar to iproniazid, which is a nonselective MAO inhibitor used as a drug against depression. The IC50 values of iproniazid were 37 μM, and 42.5 μM in our parallel examination. Moreover, IC50 value of xanthoangelol to DBH was calculated 0.52 μM. 4-Hydroxyderricin is a potent selective MAO-B inhibitor and also mildly inhibits DBH activity. The IC50 value of 4-hydroxyderricin to MAO-B was calculated to be 3.43 μM and this value was higher than that of deprenyl (0.046 μM) used as a positive control for selective MAO-B inhibitor in our test. Cynaroside is a most potent DBH inhibitor. The IC50 value of cynaroside to DBH was calculated at 0.0410 μM. Results of this study suggest that the two prenylated chalcones, xanthoangelol and 4-hydroxyderricin isolated from A. keiskei K., are expected for potent candidates for development of combined antidepressant drug. A. keiskei K. will be an excellent new bio-functional food material that has the combined antidepressant effect.

The dopamine β-hydroxylase inhibitor, nepicastat, suppresses chocolate self-administration and reinstatement of chocolate seeking in rats

Craving for chocolate is a common phenomenon, which may evolve to an addictive-like behaviour and contribute to obesity. Nepicastat is a selective dopamine β-hydroxylase (DBH) inhibitor that suppresses cocaine-primed reinstatement of cocaine seeking in rats. We verified whether nepicastat was able to modify the reinforcing and motivational properties of a chocolate solution and to prevent the reinstatement of chocolate seeking in rats. Nepicastat (25, 50 and 100 mg/kg, intraperitoneal) produced a dose-related inhibition of operant self-administration of the chocolate solution in rats under fixed-ratio 10 (FR10) and progressive-ratio schedules of reinforcement, measures of the reinforcing and motivational properties of the chocolate solution, respectively. The effect of nepicastat on the reinstatement of chocolate seeking was studied in rats in which lever-responding had been extinguished by removing the chocolate solution for approximately 8 d. Nepicastat dose-dependently suppressed the reinstatement of lever-responding triggered by a 'priming' of the chocolate solution together with cues previously associated with the availability of the reward. In a separate group of food-restricted rats trained to lever-respond for regular food pellets, nepicastat reduced FR10 lever-responding with the same potency as for the chocolate solution. Spontaneous locomotor activity was not modified by nepicastat doses that reduced self-administration of the chocolate solution and regular food pellets and suppressed the reinstatement of chocolate seeking. The results indicate that nepicastat reduces motivation to food consumption sustained by appetite or palatability. Moreover, the results suggest that DBH inhibitors may be a new class of pharmacological agents potentially useful in the prevention of relapse to food seeking in human dieters.

Modulation of the discriminative‐stimulus effects of cocaine by dopamine β‐hydroxlyase inhibitors in rats

Inhibitors of dopamine β‐hydroxylase (DBH), the enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic cells, have shown promise for the treatment of cocaine dependence, yet their effects on the interoceptive‐stimulus effects of cocaine are not well characterized. The current experiments examined the impact of DBH inhibitors on the discriminative‐stimulus effects of cocaine. Sprague‐Dawley rats (n=8) were trained to discriminate 5.6 mg/kg cocaine from saline using a multi‐component, food‐reinforced discrimination procedure. On test days, subjects were pretreated with the nonselective DBH inhibitor disulfiram (0 – 100.0 mg/kg i.p.) or the selective DBH inhibitor nepicastat (0 – 56.0 mg/kg i.p.) 2 hr prior to a session in which cocaine (0 – 5.6 mg/kg) was cumulatively administered across components. Both disulfiram and nepicastat failed to generalize to cocaine when tested up to doses that significantly reduced responding. However, in combination studies, both disulfiram and nepicastat produced dose‐dependent leftward shifts in the cocaine dose‐response function. These results suggest that pharmacological inhibition of DBH does not produce cocaine‐like interoceptive‐stimulus effects alone, but functionally enhances the interoceptive‐stimulus effects of cocaine, possibly due to facilitated increases in DA originating from NE terminals. Support: DA015040 and DA027535.

814 ANTIHYPERTENSIVE EFFECT OF BIA 5–1058 A NEW SELECTIVE PERIPHERAL DOPAMINE β-HYDROXYLASE INHIBITOR

Background: Sympathetic nervous system tone alters blood pressure by modulation of cardiac output, peripheral vascular resistance and renal function. One strategy for controlling sympathetic nerve function is reduction of biosynthesis of norepinephrine (NE) via inhibition of dopamine β-hydroxylase (DβH; EC 1.14.17.1), the enzyme that catalyses the conversion of dopamine (DA) to NE in sympathetic nerves. BIA 5-1058 is a reversible DβH inhibitor that decreases NE levels in peripheral sympathetically innervated tissues, without effect in brain tissues. Methods: In telemetry transmitter implanted SHR single administration of BIA 5-1058 demonstrates dose (3, 30 and 100 mg/Kg) and time dependent blood pressure effects with no significant effect on heart rate (HR) and total activity monitored over 96-hours. Results: The maximum reductions of systolic blood pressure (SBP) were -10.8, -21.1 and -35.2 mmHg for 3, 30 and 100 mg/Kg, respectively and diastolic blood pressure (DBP) were -9.9, -18.4 and -24.8 mmHg, respectively. Antihypertensive effects of BIA 5-1058 (30 mg/Kg) was further evaluated in combination with efficacious doses of well-known antihypertensive drugs, like the ACE inhibitor captopril, the AT1 receptor antagonist losartan, the diuretic hydrochlorothiazide, beta-blocker metoprolol, the alpha-1 receptor antagonist prazosin, and the calcium channel blocker diltiazem. All drugs were administered orally (single dose) in a cross-over design and the effect was monitored for 72 hours. Combination of BIA 5-1058 the tested antihypertensive drugs caused a stronger and prolonged blood pressure decrease than any single compound. Conclusions: In conclusion, peripheral DβH inhibitors can be used, alone or in combination with others antihypertensive drugs, to reduce blood pressure.

Abstract 291: Antihypertensive Effect Of Bia 5-1058 a New Selective Peripheral Dopamine β-hydroxylase Inhibitor

The sympathetic nervous system can alter blood pressure by modulation of cardiac output, peripheral vascular resistance and renal function. One strategy for controlling sympathetic nerve function is to reduce the biosynthesis of norepinephrine (NE) via inhibition of dopamine β-hydroxylase (DβH; EC 1.14.17.1 ), the enzyme that catalyses the conversion of dopamine (DA) to NE in sympathetic nerves. BIA 5-1058 is a reversible DβH inhibitor that decreases NE levels in peripheral sympathetically innervated tissues slowing down sympathetic nervous system drive, without effect in brain tissues. In freely moving SHR implanted with radio-telemetry transmitters single administration of BIA 5-1058 showed a dose (3, 30 and 100 mg/Kg) and time dependent effect on blood pressure with no significant effect on heart rate (HR) and total activity monitored over a 96-hour period. The maximum reduction on systolic blood pressure (SBP) was -10.8, -21.1 and -35.2 mmHg for 3, 30 and 100 mg/Kg, respectively and the maximum reduction on diastolic blood pressure (DBP) was -9.9, -18.4 and -24.8 mmHg for 3, 30 and 100 mg/Kg, respectively. The antihypertensive effect of BIA 5-1058 (30 mg/Kg) was further evaluated in combination with efficacious doses of well-known antihypertensive drugs, like the ACE inhibitor captopril, the AT1 receptor antagonist losartan, the diuretic hydrochlorothiazide, beta-blocker metoprolol, the alpha-1 receptor antagonist prazosin, and the calcium channel blocker diltiazem. All drugs were administered orally (single dose) in a cross-over design and the effect was monitored for 72 hours. The combination of BIA 5-1058 with any of the tested antihypertensive drugs caused a stronger and prolonged blood pressure decrease than any of the compounds alone.In conclusion, peripheral DβH inhibitors can be used, alone or in combination with others antihypertensive drugs, to reduce blood pressure.

Monoamine Oxidase and Dopamine β-Hydroxylase Inhibitors from the Fruits of Gardenia jasminoides

This research was designed to determine what components of Gardenia jasminoides play a major role in inhibiting the enzymes related antidepressant activity of this plant. In our previous research, the ethyl acetate fraction of G. jasminosides fruits inhibited the activities of both monoamine oxidase-A (MAO-A) and monoamine oxidase-B (MAO-B), and oral administration of the ethanolic extract slightly increased serotonin concentrations in the brain tissues of rats and decreased MAO-B activity. In addition, we found through in vitro screening test that the ethyl acetate fraction showed modest inhibitory activity on dopamine-β hydroxylase (DBH). The bioassay-guided fractionation led to the isolation of five bio-active compounds, protocatechuic acid (1), geniposide (2), 6'-O-trans-p-coumaroylgeniposide (3), 3,5-d-ihydroxy-1,7-bis (4-hydroxyphenyl) heptanes (4), and ursolic acid (5), from the ethyl acetate fraction of G. jasminoides fruits. The isolated compounds showed different inhibitory potentials against MAO-A, -B, and DBH. Protocatechuic acid showed potent inhibition against MAO-B (IC50 300 μmol/L) and DBH (334 μmol/L), exhibiting weak MAO-A inhibition (2.41 mmol/L). Two iridoid glycosides, geniposide (223 μmol/L) and 6'-O-trans-p-coumaroylgeniposide (127μmol/L), were selective MAO-B inhibitor. Especially, 6'-O-trans-p-coumaroylgeniposide exhibited more selective MAO-B inhibition than deprenyl, well-known MAO-B inhibitor for the treatment of early-stage Parkinson’s disease. The inhibitory activity of 3,5-di-hydroxy-1,7-bis (4-hydroxyphenyl) heptane was strong for MAO-B (196 μmol/L), modest for MAO-A (400 μmol/L), and weak for DBH (941 μmol/L). Ursolic acid exhibited significant inhibition of DBH (214 μmol/L), weak inhibition of MAO-B (780 μmol/L), and no inhibition against MAO-A. Consequently, G. jasminoides fruits are considerable for development of biofunctional food materials for the combination treatment of depression and neurodegenerative disorders.

Safety, Tolerability, and Pharmacokinetics of Etamicastat, a Novel Dopamine-β-Hydroxylase Inhibitor, in a Rising Multiple-Dose Study in Young Healthy Subjects

Background: Activation of the sympathetic nervous system is an important feature in hypertension and congestive heart failure. A strategy for directly modulating sympathetic nerve function is to reduce the biosynthesis of norepinephrine (noradrenaline) via inhibition of dopamine-β-hydroxylase (DβH). Objective: To assess the safety, tolerability, and pharmacokinetics of etamicastat (BIA 5–453), a new DβH inhibitor, following repeated dosing. Methods: A double-blind, randomized, placebo-controlled study was conducted in healthy young male volunteers. Participants received once-daily doses of placebo or etamicastat 25, 50, 100, 200, 400, or 600 mg, for 10 days. Results: Etamicastat underwent N-acetylation to its metabolite BIA 5–961. Etamicastat and BIA 5–961 maximum concentrations were achieved at 1–3 and 2–4 hours, respectively, after dosing. Elimination half-lives ranged from 18.1 to 25.7 hours for etamicastat and 6.7 to 22.5 hours for BIA 5–961. Both etamicastat and BIA 5-961 followed linear pharmacokinetics. The extent of systemic exposure to etamicastat and BIA 5–961 increased in an approximately dose-proportional manner, and steady-state plasma concentrations were attained up to 9 days of dosing. Etamicastat accumulated in plasma following repeated administration. The mean observed accumulation ratio was 1.3–1.9 for etamicastat and 1.3–1.6 for BIA 5–961. Approximately 40%of the etamicastat dose was recovered in urine in the form of parent compound and BIA 5–961. There was a high variability in pharmacokinetic parameters, attributable to different N-acetyltransferase-2 (NAT2) phenotype. Urinary excretion of norepinephrine decreased following repeated administration of etamicastat. Etamicastat was generally well tolerated. There was no serious adverse event or clinically significant abnormality in clinical laboratory tests, vital signs, or ECG parameters. Conclusion: Etamicastat was well tolerated. Etamicastat undergoes N-acetylation, which is markedly influenced by NAT2 phenotype. NAT2 genotyping could be a step toward personalized medicine for etamicastat. Trial Registration: EudraCT No. 2007-004142-33

Safety, Tolerability, and Pharmacokinetics of Etamicastat, a Novel Dopamine-β-Hydroxylase Inhibitor, in a Rising Multiple-Dose Study in Young Healthy Subjects

Background: Activation of the sympathetic nervous system is an important feature in hypertension and congestive heart failure. A strategy for directly modulating sympathetic nerve function is to reduce the biosynthesis of norepinephrine (noradrenaline) via inhibition of dopamine-β-hydroxylase (DβH).Objective: To assess the safety, tolerability, and pharmacokinetics of etamicastat (BIA 5–453), a new DβH inhibitor, following repeated dosing.Methods: A double-blind, randomized, placebo-controlled study was conducted in healthy young male volunteers. Participants received once-daily doses of placebo or etamicastat 25, 50, 100, 200, 400, or 600 mg, for 10 days.Results: Etamicastat underwent N-acetylation to its metabolite BIA 5–961. Etamicastat and BIA 5–961 maximum concentrations were achieved at 1–3 and 2–4 hours, respectively, after dosing. Elimination half-lives ranged from 18.1 to 25.7 hours for etamicastat and 6.7 to 22.5 hours for BIA 5–961. Both etamicastat and BIA 5-961 followed linear pharmacokinetics. The extent of systemic exposure to etamicastat and BIA 5–961 increased in an approximately dose-proportional manner, and steady-state plasma concentrations were attained up to 9 days of dosing. Etamicastat accumulated in plasma following repeated administration. The mean observed accumulation ratio was 1.3–1.9 for etamicastat and 1.3–1.6 for BIA 5–961. Approximately 40%of the etamicastat dose was recovered in urine in the form of parent compound and BIA 5–961. There was a high variability in pharmacokinetic parameters, attributable to different N-acetyltransferase-2 (NAT2) phenotype. Urinary excretion of norepinephrine decreased following repeated administration of etamicastat. Etamicastat was generally well tolerated. There was no serious adverse event or clinically significant abnormality in clinical laboratory tests, vital signs, or ECG parameters.Conclusion: Etamicastat was well tolerated. Etamicastat undergoes N-acetylation, which is markedly influenced by NAT2 phenotype. NAT2 genotyping could be a step toward personalized medicine for etamicastat.Trial Registration: EudraCT No. 2007-004142-33

Disulfiram Attenuates Drug-Primed Reinstatement of Cocaine Seeking via Inhibition of Dopamine β-Hydroxylase

The antialcoholism medication disulfiram (Antabuse) inhibits aldehyde dehydrogenase (ALDH), which results in the accumulation of acetaldehyde upon ethanol ingestion and produces the aversive 'Antabuse reaction' that deters alcohol consumption. Disulfiram has also been shown to deter cocaine use, even in the absence of an interaction with alcohol, indicating the existence of an ALDH-independent therapeutic mechanism. We hypothesized that disulfiram's inhibition of dopamine β-hydroxylase (DBH), the catecholamine biosynthetic enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic neurons, underlies the drug's ability to treat cocaine dependence. We tested the effects of disulfiram on cocaine and food self-administration behavior and drug-primed reinstatement of cocaine seeking in rats. We then compared the effects of disulfiram with those of the selective DBH inhibitor, nepicastat. Disulfiram, at a dose (100 mg/kg, i.p.) that reduced brain NE by ∼40%, did not alter the response for food or cocaine on a fixed ratio 1 schedule, whereas it completely blocked cocaine-primed (10 mg/kg, i.p.) reinstatement of drug seeking following extinction. A lower dose of disulfiram (10 mg/kg) that did not reduce NE had no effect on cocaine-primed reinstatement. Nepicastat recapitulated the behavioral effects of disulfiram (100 mg/kg) at a dose (50 mg/kg, i.p.) that produced a similar reduction in brain NE. Food-primed reinstatement of food seeking was not impaired by DBH inhibition. Our results suggest that disulfiram's efficacy in the treatment of cocaine addiction is associated with the inhibition of DBH and interference with the ability of environmental stimuli to trigger relapse.

Effects of disulfiram and dopamine beta-hydroxylase knockout on cocaine-induced seizures

The antialcoholism drug disulfiram has shown recent promise as a pharmacotherapy for treating cocaine dependence, probably via inhibition of dopamine β-hydroxylase (DBH), the enzyme that catalyzes the conversion of dopamine (DA) to norepinephrine (NE). We previously showed that DBH knockout ( Dbh −/−) mice, which lack NE, are susceptible to seizures and are hypersensitive to the psychomotor, rewarding, and aversive effects of cocaine, suggesting that disulfiram might exacerbate cocaine-induced seizures (CIS) by inhibiting DBH. To test this, we examined CIS in wild-type and Dbh −/− mice following administration of disulfiram or the selective DBH inhibitor nepicastat. We found that Dbh genotype had no effect on CIS probability or frequency, whereas disulfiram, but not nepicastat, increased the probability of having CIS in both wild-type and Dbh −/− mice. Both disulfiram and nepicastat increased CIS frequency in wild-type but not Dbh −/− mice. There were no genotype or treatment effects on serum cocaine levels, except for an increase in disulfiram-treated Dbh −/− mice at the highest dose of cocaine. These results suggest that disulfiram enhances CIS via two distinct mechanisms: it both increases CIS frequency by inhibiting DBH and increases CIS frequency in a DBH-independent manner.

Genetic or pharmacological blockade of noradrenaline synthesis enhances the neurochemical, behavioral, and neurotoxic effects of methamphetamine

N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) lesions of the locus coeruleus, the major brain noradrenergic nucleus, exacerbate the damage to nigrostriatal dopamine (DA) terminals caused by the psychostimulant methamphetamine (METH). However, because noradrenergic terminals contain other neuromodulators and the noradrenaline (NA) transporter, which may act as a neuroprotective buffer, it was unclear whether this enhancement of METH neurotoxicity was caused by the loss of noradrenergic innervation or the loss of NA itself. We addressed the specific role of NA by comparing the effects of METH in mice with noradrenergic lesions (DSP-4) and those with intact noradrenergic terminals but specifically lacking NA (genetic or acute pharmacological blockade of the NA biosynthetic enzyme dopamine beta-hydroxylase; DBH). We found that genetic deletion of DBH (DBH-/- mice) and acute treatment of wild-type mice with a DBH inhibitor (fusaric acid) recapitulated the effects of DSP-4 lesions on METH responses. All three methods of NA depletion enhanced striatal DA release, extracellular oxidative stress (as measured by in vivo microdialysis of DA and 2,3-dihydroxybenzoic acid), and behavioral stereotypies following repeated METH administration. These effects accompanied a worsening of the striatal DA neuron terminal damage and ultrastructural changes to medium spiny neurons. We conclude that NA itself is neuroprotective and plays a fundamental role in the sensitivity of striatal DA terminals to the neurochemical, behavioral, and neurotoxic effects of METH.

Effects of dopamine beta-hydroxylase inhibition with nepicastat on the progression of left ventricular dysfunction and remodeling in dogs with chronic heart failure.

Inhibition of dopamine beta-hydroxylase (DBH) results in a decrease in norepinephrine synthesis. The present study was a randomized, blinded, placebo-controlled investigation of the long-term effects of therapy with the DBH inhibitor nepicastat (NCT) on the progression of left ventricular (LV) dysfunction and remodeling in dogs with chronic heart failure (HF). Moderate HF (LV ejection fraction [LVEF] 30% to 40%) was produced in 30 dogs by intracoronary microembolization. Dogs were randomized to low-dose NCT (0.5 mg/kg twice daily, n=7) (L-NCT), high-dose NCT (2 mg/kg twice daily, n=7) (H-NCT), L-NCT plus enalapril (10 mg twice daily, n=8) (L-NCT+ENA), or placebo (PL, n=8). Transmyocardial (coronary sinus-arterial) plasma norepinephrine (tNEPI), LVEF, end-systolic volume, and end-diastolic volume were measured before and 3 months after initiating therapy. tNEPI levels were higher in PL compared with NL (86+/-20 versus 13+/-14 pg/mL, P:<0.01). L-NCT alone and L-NCT+ENA reduced tNEPI toward normal (28+/-4 and 39+/-17 pg/mL respectively), whereas HD-NCT reduced tNEPI to below normal levels (3+/-10 pg/mL). In PL dogs, LVEF decreased but was unchanged with L-NCT and increased with L-NCT+ENA. L-NCT and L-NCT+ENA prevented progressive LV remodeling, as evidenced by lack of ongoing increase in end-diastolic volume and end-systolic volume, whereas H-NCT did not In dogs with HF, therapy with L-NCT prevented progressive LV dysfunction and remodeling. The addition of ENA to L-NCT afforded a greater increase in LV systolic function. NCT at doses that normalize tNEPI may be useful in the treatment of chronic HF.

Dopamine- -Hydroxylase Inhibition: A Novel Sympatho-Modulatory Approach for the Treatment of Congestive Heart Failure

Abstract: Pre-clinical and clinical studies suggest that chronic sympathetic activation in congestive heart failure (CHF) is a maladaptive response which accelerates the progressive worsening of the disease. Consequently, therapeutic interventions which inhibit sympathetic nerve function are likely to favorably alter the natural course of the disease. Indeed, recent clinical studies have shown that treatment with carvedilol, a P­ blocker, reduces mortality and the risk of death and hospitalization. The therapeutic value of P-blockers, however, may be limited by their propensity to cause acute hemodynamic deterioration which results from abrupt withdrawal of sympathetic support. Thus, although the intrnuuction or P- blockers represents an important advance in the treatment of CHF, a better tolerated means of modulating the sympathetic nervous system would be highly desirable. An alternative strategy for directly modulating sympathetic nerve function is to inhibit the biosynthesis of norepinephrine (NE) via inhibition of dopamine-P-hydroxylase (DBH), the enzyme which catalyzes the conversion of dopamine (DA) to NE in sympathetic nerves. This approach may have the following three merits over P-blockade. First, this class of drugs would be expected to produce gradual modulation, as opposed to abrupt blockade, of sympathetic nerve function and, consequently, would not be associated with acute hemodynamic worsening thereby obviating the need for dose-titration. Second, from a theoretical standpoint, DBH inhibitors, at low doses, would preferentially inhibit NE release in the heart £ince the storage pool of NE in this organ is selectively depleted in CHF. Lastly, inhibition of DBH would augment the levels of DA which, via agonism of dopamine receptors, could have beneficial effects on renal function. Nepicastat is a novel, selective and potent (IC50 = 9 nM) inhibitor of DBH. Preclinical studies have shown that nepicastat produces gradual modulation of catecholamine levels (reduction in NE and elevation of DA and• DA/NE ratio) in cardiovascular tissues and plasma, attenuates sympathetically-mediated cardiovascular responses and also has salutary effects on renal function. In a canine heart failure model, normalization of transmyocardial norepinephrine balance with nepicastat retards the process of ventricular dilation and prevents progressive worsening of cardiac functiun. Early short-term clinical studies in CHF patients have shown that nepicastat is well tolerated and produces significant and dose-dependent increases in plasma DA/NE concentrations. In conclusion, DBH inhibition, with drugs such as nepicastat, may be a better-tolerated mechanism for producing sympatho-inhibition and thus represents a promising therapy for CHF.