Greg C. Rigdon

Differential effects of apomorphine on prepulse inhibition of acoustic startle reflex in two rat strains

Apomorphine disruption of prepulse inhibition (PPI) has been proposed as an animal model of sensorimotor gating deficits exhibited by schizophrenics. The effects of apomorphine on PPI of the acoustic startle reflex in male rats of Wistar and CD (Sprague-Dawley derived) strains were compared under identical test conditions. In Wistar rats, subcutaneous administration of 0.25–1.0 mg/kg apomorphine blocked PPI without affecting startle amplitude. In CD rats, apomorphine (0.3–3.0 mg/kg, SC) had no effect on PPI, but increased startle amplitude. Therefore, choice of rat strain is an important factor in the design of experiments studying apomorphine effects on PPI.

KCNQ potassium channels: drug targets for the treatment of epilepsy and pain

Epilepsy and neuropathic pain are disorders characterised by excessive neuronal activity. These disorders are currently managed by drugs that are capable of dampening neuronal excitability, including voltage-gated sodium channel blockers, voltage-operated calcium channel modulators and modulators of inhibitory GABAergic neurotransmission. However, these drugs are rarely 100% efficacious and their use is often associated with limiting side effects. Thus, there is a clear medical need for novel agents to treat these diseases. One potential mechanism that has not yet been exploited is potassium (K+) channel opening. A significant (and growing) body of genetic, molecular, physiological and pharmacological evidence now exists to indicate that KCNQ-based currents represent particularly interesting targets for the treatment of diseases such as epilepsy and neuropathic pain. Evidence supporting these K+ channels as novel drug targets will be reviewed in the following article. Worldwide patent activity relating to KCNQ channels and KCNQ-modulating drugs and their uses will also be summarised.

N-Pyridyl and Pyrimidine Benzamides as KCNQ2/Q3 Potassium Channel Openers for the Treatment of Epilepsy

A series of N-pyridyl benzamide KCNQ2/Q3 potassium channel openers were identified and found to be active in animal models of epilepsy and pain. The best compound 12 [ICA-027243, N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide] has an EC50 of 0.38 μM and is selective for KCNQ2/Q3 channels. This compound was active in several rodent models of epilepsy and pain but upon repeated dosing had a number of unacceptable toxicities that prevented further development. On the basis of the structure-activity relationships developed around 12, a second compound, 51, [N-(2-chloro-pyrimidin-5-yl)-3,4-difluoro-benzamide, ICA-069673], was prepared and advanced into a phase 1 clinical study. Herein, we describe the structure-activity relationships that led to the identification of compound 12 and to the corresponding pyrimidine 51.

Is clozapine selective for the dopamine D4 receptor

Binding of 3H-spiperone and 3H-raclopride to membranes of cells stably-transfected with a human dopamine D2 receptor clone was investigated, as was that of 3H-spiperone to those stably-transfected with a human D4 receptor clone. 3H-spiperone and 3H-raclopride labeled the same number of sites in the D2 receptor preparation. The inhibition of binding by clozapine, spiperone, (-) eticlopride, haloperidol and the novel substituted benzamide 1192U90 was also investigated. Clozapine and 1192U90 showed greater inhibition of 3H-raclopride binding than 3H-spiperone binding to the D2 receptor. Comparison with inhibition of 3H-spiperone binding to the D4 receptor revealed that clozapine and 1192U90 displayed apparent selectivity (as assessed by Ki ratios) for the D4 receptor when compared with binding of 3H-spiperone, but not 3H-raclopride, to the D2 receptor.

1192U90 in animal tests that predict antipsychotic efficacy, anxiolysis, and extrapyramidal side effects

1192U90 was developed on the assumption that antagonism of 5-HT2 receptors efficacy yields more potently than D2 receptors against positive and negative symptoms of schizophrenia with minimal liability for extrapyramidal side effects (EPSs), and that 5-HT1A agonism further reduces EPSs and provides anxiolytic and antidepressant activity. 1192U90 was submitted to four tests that predict antipsychotic efficacy (antagonism of apomorphine-induced climbing in mouse, antagonism of apomorphine-induced circling in rats with unilateral 6-OHDA lesions, antagonism of amphetamine-induced hyperlocomotion in rat, and inhibition of conditioned avoidance in rat), two tests of 5-HT2 function (antagonism of 5-MeODMT-induced head twitches in mouse and antagonism of 5-HTP-induced wet dog shakes in rat), and three tests that predict EPS liability (antagonism of apomorphine-induced stereotypy in mouse and rat and induction of catalepsy in mouse). ED50s (mg/kg PO) were as follows: climbing 10.1, circling 7.9, hyperlocomotion 6.6, and avoidance 5.7; head twitches 5 and wet dog shakes 4.6; stereotypy in mouse 91.1, stereotypy in rat 133.4, and catalepsy 192.4. The ratio of ED50 for stereotypy antagonism to ED50 for climbing antagonism was 9 (compared to 4, 3, and 4 for clozapine, risperidone, and haloperidol). The ratio of ED50 for catalepsy induction to ED50 for climbing antagonism was 19 (compared to 7, 2, and 17 for clozapine, risperidone, and haloperidol). 1192U90 was also submitted to three tests that predict anxiolysis: It produced only a small increase in punished lever pressing for food in rat (Geller-Seifter conflict test), which is specific for rapid-onset efficacy, but produced large increases in punished key pecking for food in pigeon and cork gnawing in rat, which identify the delayed onset 5-HT1A agonists such as buspirone. The results suggest that 1192U90 would be effective for positive and negative symptoms of schizophrenia, with minimal liability for EPSs, and may also have anxiolytic properties.

Anti-haemolytic effect of senicapoc and decrease in NT-proBNP in adults with sickle cell anaemia

Dear Editor: The Gardos channel inhibitor senicapoc is proven to diminish haemolytic anaemia in a Phase II study conducted in adults with sickle cell anaemia (Ataga, et al 2008), and later confirmed in a Phase III study in sickle cell disease subjects as reported in a recent article in the British Journal of Haematology (Ataga, et al 2011). Brain natriuretic peptide is a hormone secreted by ventricular cardiomyocytes in response to stretch (Levin, et al 1998). The plasma level of its propeptide (NT-proBNP) provides a convenient biomarker of cardiac stress, correlating in sickle cell subjects with pulmonary hypertension proven by pulmonary artery catheterization, or estimated by noninvasive Doppler echocardiography (Machado, et al 2006). Increased NT-proBNP in sickle cell adults is associated with lower haemoglobin, high serum lactate dehydrogenase (LDH) and other laboratory variables (Machado, et al 2006, Mekontso Dessap, et al 2008, Mokhtar, et al 2010, van Beers, et al 2008, Voskaridou, et al 2007). Because senicapoc is proven to increase haemoglobin and decrease LDH in sickle cell anaemia (Ataga, et al 2008), we hypothesized that NT-proBNP levels would fall in sickle cell anaemia subjects who respond to senicapoc treatment with increased haemoglobin level. We obtained local regulatory approval to study coded plasma samples from the 2008 Phase II senicapoc trial (Ataga, et al 2008). We procured archived plasma samples from that trial, and measured NT-proBNP by the same standard clinical laboratory assay we have previously reported in sickle cell anaemia (Machado, et al 2006). In the 53 treated subjects, NT-proBNP levels declined by a nonsignificant degree. However, when the analysis was restricted to the 35 subjects (66%) who responded to senicapoc with a rise in total Hb of at least 5 gm/L, significant changes were observed in NT-proBNP and other markers. Subjects who responded to 6mg daily or 10mg daily were analyzed together as a single group. Among these 35 senicapoc responders, baseline NT-proBNP fell by 26% (geometric mean 97 vs. 72 ng/l, p<0.01, paired t-test, log transformed values; Fig. 1). Haemoglobin levels increased by 13% (mean 79 ± 2 vs. 89 ± 2 gm/L, mean ± SEM, p<0.001, paired t-test; Fig. 2A), as expected in this group defined by senicapoc-induced rise in haemoglobin. Serum LDH levels fell 22% (geometric mean 466 vs. 365 iu/l, p<0.001, paired t-test, log transformed values; Fig. 2B), serum bilirubin levels dropped 35% (67.2 vs. 44.5 µmol/l, p<0.001; Fig. 2C), and percentage reticulocytes declined (12.5 vs. 8.6%, p<0.001; Fig. 2D). These results suggest that senicapoc simultaneously diminished both haemolysis and NT-proBNP in a subgroup of subjects. Figure 1 NT-proBNP levels before and after senicapoc therapy. White bar indicates baseline plasma NT-proBNP level prior to initiating oral senicapoc therapy in 35 adults with sickle cell anaemia. The black bar indicates plasma NT-proBNP level on day 85 of senicapoc ... Figure 2 Indicators of haemolytic severity before and after senicapoc therapy. White bars indicate baseline values prior to initiating oral senicapoc therapy in 35 adults with sickle cell anaemia. The black bars indicate values on day 85 of senicapoc therapy in ... A limitation in our study is that only two-thirds of these senicapoc subjects had a sufficient increase in haemoglobin to observe the linked decrease in NT-proBNP, and inclusion of the senicapoc nonresponders obscures this NT-proBNP change. This study was also not prospectively designed to determine the efficacy of senicapoc in reducing NT-proBNP, and the results should be interpreted with caution. However, the findings support important concepts linking haemolytic anaemia to elevated pulmonary arterial pressure. A similar decline in NT-proBNP also has been reported in patients with another chronic haemolytic anaemia, paroxysmal nocturnal hemoglobinuria, in whom hemolysis was reduced by use of the drug eculizumab (Hill, et al 2010). We also have reported downward trends in NT-proBNP and significant decline in pulmonary artery systolic pressure estimated by Doppler echocardiography in several sickle cell anaemia patients in whom hydroxycarbamide produced robust increase in foetal haemoglobin and significant reduction in serum LDH (Olnes, et al 2009). Severity of chronic haemolytic anaemia is linked to high levels of the functional cardiac marker NT-proBNP, and three mechanistically different pharmacological interventions that attenuate intravascular hemolysis also reduced NT-proBNP in these two pathologically distinct haemolytic anaemias. Our result suggests that reducing hemolysis in sickle cell anaemia results in lower NT-proBNP, previously shown to serve in this population more as a biomarker of mean pulmonary artery pressure than of left ventricular dysfunction (Machado, et al 2006). Additional research directed at reducing hemolysis is warranted as a potential means to reduce NT-proBNP and improve pulmonary hypertension in sickle cell disease.

Biochemical and pharmacologic properties of 2614w94, a reversible, competitive inhibitor of monoamine oxidase-A

2614W94 [3‐(1‐trifluoromethyl)ethoxyphenoxathiin 10,10‐dioxide] is a selective, reversible inhibitor of monoamine oxidase‐A with a competitive mechanism of inhibition and a Ki value of 1.6 nM with serotonin as substrate. In pretreated rats, the ED50 value after single oral dosing was 1.7 mg/kg, similar to an ED50 value of 1.1 mg/kg estimated in the 5‐hydroxytryptophan potentiation test. Maximal inhibition of monoamine oxidase‐A (MAO‐A) was observed by 0.5 h after dosing, suggesting rapid transport to brain. Inhibition in brain was maintained for several hours, followed by a gradual reversal with a half‐time of 7.2 h. Brain levels of parent compound were higher than plasma levels at all times after dosing. No significant inhibition of MAO‐B was observed. After preincubation of MAO with 2614W94 at 37°C, the inhibition was reversed by dialysis. Concentrations of serotonin, norepinephrine, and dopamine were clearly elevated in brains of rats after single oral doses, whereas levels of MAO metabolites were decreased. In a rat model designed to show blood pressure elevations in response to a threshold dose of orally administered tyramine, 2614W94 compared well with moclobemide, an MAO‐A selective inhibitor that has not been associated with problems relating to dietary tyramine. The two stereoisomers of 2614W94 were both potent MAO‐A inhibitors. In vitro and in vivo properties of 2614W94 suggest that this compound and its close analogs are among the most potent MAO‐A inhibitors known and that they may have therapeutic potential as safe new antidepressant/anxiolytic agents. Drug Dev. Res. 45:1–9, 1998.