CGP 35348: a centrally active blocker of GABAB receptors

The biochemical, electrophysiological and pharmacological properties of the new GABAB receptor blocker CGP 35348 are described. In a variety of receptor binding assays CGP 35348 showed affinity for the GABAB receptor only. CGP 35348 had an IC50 of 34 microM at the GABAB receptor. The compound antagonized (100, 300, 1000 microM) the potentiating effect of L-baclofen on noradrenaline-induced stimulation of adenylate cyclase in rat cortex slices. In electrophysiological studies CGP 35348 (10, 100 microM) antagonized the effect of L-baclofen in the isolated rat spinal cord. In the hippocampal slice preparation CGP 35348 (10, 30, 100 microM) blocked the membrane hyperpolarization induced by D/L-baclofen (10 microM) and the late inhibitory postsynaptic potential. CGP 35348 appeared to be 10-30 times more potent than the GABAB receptor blocker phaclofen. Ionophoretic and behavioural experiments showed that GABAB receptors in the brain were blocked after i.p. administration of CGP 35348. This compound may be of considerable value in elucidating the roles of brain GABAB receptors.     

Pharmacologically distinct GABAB receptors that mediate inhibition of GABA and glutamate release in human neocortex

1. The release of endogenous γ-aminobutyric acid (GABA) and glutamic acid in the human brain has been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery to reach deeply located tumours.
2. The basal outflows of GABA and glutamate from superfused synaptosomes were largely increased during depolarization with 15 mM KCl. The K⁺-evoked overflows of both amino acids were almost totally dependent on the presence of Ca²⁺ in the superfusion medium.
3. The GABA_B receptor agonist (−)-baclofen (1, 3 or 10 μM) inhibited the overflows of GABA and glutamate in a concentration-dependent manner. The inhibition caused by 10 μM of the agonist ranged from 45–50%.
4. The effect of three selective GABA_B receptor antagonists on the inhibition of the K⁺-evoked GABA and glutamate overflows elicited by 10 μM (−)-baclofen was investigated. Phaclofen antagonized (by about 50% at 100 μM; almost totally at 300 μM) the effect of (−)-baclofen on GABA overflow but did not modify the inhibition of glutamate release. The effect of (−)-baclofen on the K⁺-evoked GABA overflow was unaffected by 3-amino-propyl (diethoxymethyl)phosphinic acid (CGP 35348; 10 or 100 μM); however, CGP 35348 (10 or 100 μM) antagonized (−)-baclofen (complete blockade at 100 μM) at the heteroreceptors on glutamatergic terminals. Finally, [3-[[(3,4-dichlorophenyl) methyl]amino]propyl] (diethoxymethyl) phosphinic aid (CGP 52432), 1 μM, blocked the GABA_B autoreceptor, but was ineffective at the heteroreceptors. The selectivity of CGP 52432 was lost at 30 μM, as the compound, at this concentration, inhibited completely the (−)-baclofen effect both on GABA and glutamate release.
5. It is concluded that GABA and glutamate release evoked by depolarization of human neocortex nerve terminals can be affected differentially through pharmacologically distinct GABA_B receptors.

     

Potentiation of metabotropic GABAB receptors by L-amino acids and dipeptides in rat neocortex

Selected neutral l-α-amino acids, and their dipeptides, were reversible, stereospecific, potentiators of GABA_B receptor-mediated hyperpolarizing responses to baclofen (3-100 μM) in rat neocortical slices. These responses were sensitive to the GABA_B receptor antagonist (+)-(S)-5,5-dimethylmorpholinyl-2-acetic acid (Sch50911) (30 μM). Most potent were l-Leu, l-Ile and l-Phe, as were the dipeptides l-Phe-Phe and l-Phe-Leu, and less potent were l-Met, l-Val, l-Cys, l-Cystine, l-Tyr, l-Thr, l-Arg and l-Ser. Inactive were l-Trp, l-His, l-Lys and l-Pro. These potentiators gave leftward shifts of the baclofen concentration-response curves with a Hill slope of 2, and a marked increase in the maximal hyperpolarizing responses. Selected l-amino acids and dipeptides are a class of naturally occurring GABA_B potentiators, which may be allosteric modulators.     

Stimulation of GABAB receptors increases the expression of the proenkephalin gene in slice cultures of rat neocortex

In rat neocortex the proenkephalin gene is expressed in GABAergic interneurons. Immunocytochemistry and in situ hybridisation show only a small number of cells in layers II to VI which express the gene. In organotypic slices of rat neocortex, the GABAA receptor inhibitor bicuculline methiodide enhances the expression of the gene in numerous cells. In the present study, we have investigated how GABA regulates the expression of the proenkephalin gene. The GABAA receptor antagonist bicuculline methiodide and the inhibitor of ligand-gated Cl- channels picrotoxin strongly enhanced the expression of the gene in numerous cells which were arranged in neocortical layers II/III and V/VI. Since bicuculline methiodide can also block Ca(++)-activated K+ channels, the possible involvement of such channels was tested. However, apamin which blocks only Ca(++)-activated K+ channels had no effect on the expression of the proenkephalin gene indicating that the effect of bicuculline methiodide was due to inhibition of GABAA receptors. In addition, the GABAB receptor agonist baclofen increased the neocortical expression of the proenkephalin gene mainly in cells located in layers V/VI of the neocortex. The effect of baclofen was inhibited by the GABAB receptor antagonists CGP35348 and CGP52432. Also muscimol, an agonist at GABAA receptors, enhanced the expression of the proenkephalin gene. This effect was blocked by CGP52432 confirming previous observations that muscimol can also stimulate GABAB receptors. Our results indicate that GABA can regulate the expression of the opioid peptide in neocortical neurons in a bidirectional manner. The expression is suppressed via GABAA and enhanced via GABAB receptors.     

GABAB receptor-mediated inhibition of the neurogenic vasopressor response in the pithed rat.

1. The effects of gamma-aminobutyric acid (GABA) and related drugs on the vasopressor response induced by electrical stimulation (single pulse of 30 V and 1 ms) of the preganglionic sympathetic nerve fibres or by injection of noradrenaline 0.3 nmol kg-1 were studied in the pithed rat. 2. The electrically-induced increase in diastolic blood pressure was inhibited by GABA and the GABAB-receptor agonist R-(--)-baclofen but was not affected by its S-(+)-enantiomer and by the GABAA-receptor agonists muscimol and 3-aminopropane sulphonic acid. 3. The dose-response curve of R-(--)-baclofen for its inhibitory effect on the electrically-induced vasopressor response was shifted to the right by the GABAB-receptor antagonist 2-hydroxysaclofen, but was not affected by the GABAA-receptor antagonist bicuculline. 2-Hydroxysaclofen and bicuculline by themselves did not affect the electrically-induced vasopressor response. 4. The increase in diastolic blood pressure induced by exogenous noradrenaline was not affected by the GABA-related drugs, which also had no (or very slight) effects on the basal diastolic blood pressure. 5. It is concluded that GABA inhibits catecholamine release in the resistance vessels of the rat via GABAB-receptors, probably located presynaptically on the postganglionic sympathetic nerve fibres.     

The H current blocker ZD7288 decreases epileptiform hyperexcitability in the rat neocortex by depressing synaptic transmission

Neurons respond to intracellular injection of hyperpolarizing current pulses by generating depolarizing sags contributed by a cation current termed Ih. Ih modulates neuron excitability and rhythmicity. It is, however, unclear whether the net effect of changing Ih leads to facilitation or depression of cortical epileptiform activity. Here, we addressed this issue by using field and intracellular recordings to study the effects of ZD7288 (10-100 microM), a bradycardic agent known to abolish Ih, on the epileptiform discharges (duration = 2.5 +/- 0.3 s, mean +/- SEM; interval of occurrence = 34.2 +/- 3.3 s, n = 30 slices) induced in rat neocortical slices by 4-aminopyridine and GABA receptor antagonists. ZD7288 abolished the depolarizing sags seen during injection of intracellular hyperpolarizing current pulses while increasing resting membrane potential and apparent input resistance. These effects, which were fully established with 10 microM ZD7288, were associated with a dose-dependent decrease in the occurrence of spontaneous epileptiform events and a reduction in their duration (the latter change occurring at doses > 20 microM). ZD7288 also caused a dose-dependent decrease of background postsynaptic potentials. Finally, ZD7288 could depress epileptiform activity during Cs+ pre-treatment, a procedure known to block Ih. These data indicate that ZD7288 hampers neocortical epileptiform synchronization, but also suggest that most of this action reflects the ability of ZD7288 to decrease synaptic transmission.     

Nilvadipine, a new calcium channel blocker, reduces ischemic brain injury in rats.

The effects of nilvadipine, a dihydropyridine type calcium channel blocker, on cerebral infarction induced by focal brain ischemia was studied in rats. The area of infarction was measured 24 hr after occlusion of the middle cerebral artery (MCA) in spontaneously hypertensive rats using triphenyltetrazolium chloride. Nilvadipine, given immediately after MCA occlusion, reduced the area of infarction significantly at doses of 0.32 mg/kg (i.p.) and 3.2, 10 and 32 mg/kg (p.o.). Nicardipine suppressed the area of infarction at a dose of 32 mg/kg (p.o.). The results suggest that nilvadipine is effective against ischemic brain injury.     

Homotaurine: a GABAB antagonist in guinea-pig ileum.

Homotaurine (3-aminopropane sulphonic acid) did not inhibit the twitch response in guinea-pig ileum longitudinal muscle whilst gamma-aminobutyric acid (GABA) and (-)-baclofen evoked dose-dependent inhibitions. The inhibitory effects of GABA and (-)-baclofen were prevented in the presence of homotaurine 2 X 10(-4) and 10(-3) M. The log dose-effect curves of GABA and (-)-baclofen were shifted in a parallel manner compatible with competitive antagonism. The pA2 of homotaurine with GABA (4.22 +/- 0.05) and (-)-baclofen (4.26 +/- 0.1) were the same. Homotaurine did not antagonize the inhibitory effects of morphine (ED50 4 X 10(-7) M), noradrenaline (ED50 10(-6) M) or ATP (ED50 1.5 X 10(-5) M). The inferior homologue of homotaurine, taurine 10(-3) M, did not modify the inhibitory effects of GABA and (-)-baclofen. Picrotoxin 5 X 10(-5) M antagonized GABAA receptor-mediated contraction but did not affect GABAB receptor-mediated inhibition. At the same concentration the drug did not influence the antagonistic action of homotaurine, thus showing no GABAA receptor-mediated interference. It may be concluded that homotaurine is a competitive antagonist of GABAB mediated effects in the guinea-pig ileum.     

GABAB receptor: a site of therapeutic benefit

Although the presence of functional GABAB receptors in mammalian brain has been known for more than 20 years, there is still only one therapeutic agent in use, baclofen, which mediates its effects directly via this receptor. However, activation of this receptor can produce numerous effects that might be amenable to drug development. Evidence from preclinical studies also suggests that antagonism of the GABAB receptor produces beneficial clinical effects.     

CGP 44532, a GABAB receptor agonist, is hedonically neutral and reduces cocaine-induced enhancement of reward

Drugs that alter the function of gamma-aminobutyric acid (GABA) neurotransmission seem to reduce cocaine reinforcement, and as such may be useful in pharmacologically treating cocaine addiction. In the present experiment, the anti-cocaine effects of CGP 44532, a phosphinic acid analogue of GABA, and a highly selective GABA(B) receptor agonist were examined in male Sprague-Dawley rats using brain stimulation reward (BSR) paradigm. In this method, the relationship between the rate of bar pressing and the frequency of stimulation pulses was analyzed in two measures: the maximum rate of responding (MAX) and the frequency necessary to sustain half maximal rate of responding known as the locus of rise (LOR). CGP 44532 was found to be hedonically neutral without producing any measurable effects on performance (MAX). It also dose-dependently reduced cocaine-induced BSR enhancement, in the order of 15-31%, as shown by progressive shifts in LOR towards baseline. Thus, in theory, administration of CGP 44532 might reduce cocaine's hedonic effects, while also maintaining patient compliance. Whether this agent would also be effective at curbing craving, a long-term consequence of drug abuse, remains to be determined.     

CGP7930: a positive allosteric modulator of the GABAB receptor

CGP7930 (3-(3',5'-Di-tert-butyl-4'-hydroxy)phenyl-2,2-dimethylpropanol) is a positive allosteric modulator of the metabotropic GABAB receptor. CGP7930 has been found to modulate the GABAB receptor in the open, or high affinity, state increasing agonist affinity for the receptor and signal transduction efficacy following agonist stimulation. The GABAB heteromeric subunit B2, involved in signal transduction but not ligand binding, seems to be the site of action of CGP7930 and similar allosteric modulators. When administered alone in na?ve animals, CGP7930 acts as an anxiolytic in rodents without other overt behavioral effects and has also been demonstrated to reduce self-administration of nicotine, cocaine, or alcohol in rodents, suggesting that "fine tuning" of the GABAB receptor by positive allosteric modulators may be able to regulate abuse of these drugs. Baclofen, the GABAB agonist, is currently finding use in treating addiction and various other disorders, but this can result in off-target effects and tolerance. CGP7930 when co-administered with baclofen enhances its potency, which could in theory minimize deleterious effects. Further study of CGP7930 is required, but this compound, and others like it, holds potential in a clinical setting.     

Eplerenone: a selective aldosterone blocker

Recent studies suggest that aldosterone may play a larger role than once appreciated in normal physiologic function and cardiovascular disease. Some of the adverse cardiovascular effects that have been described include cardiac and vascular fibrosis, vascular necrosis and inflammation, impaired endothelial function, reduced fibrinolysis, hypertension, left ventricular hypertrophy (LVH), congestive heart failure, and cardiac arrhythmias. In light of these findings, the ability to block the actions of aldosterone has gained increased therapeutic importance. Eplerenone is a selective aldosterone receptor blocker that displays little interaction with androgen and progesterone receptors. Eplerenone has already been approved for the treatment of systemic hypertension and has been evaluated in numerous hypertension subgroups, including patients with low plasma renin activity; diabetes; LVH; uncontrolled blood pressure while receiving monotherapy with angiotensin-converting enzyme inhibitors, angiotensin-receptor blockers, calcium channel blockers, or beta-blockers; and in black patients. Results of these trials indicate that eplerenone lowers blood pressure and reduces end-organ damage. Further proof of the therapeutic importance of mineralocorticoid receptor blockade comes from the eplerenone post acute myocardial infarction survival and efficacy study (EPHESUS). In this large-scale clinical outcome trial, eplerenone was shown to reduce total mortality by 15% as well as the combined endpoint of cardiovascular mortality/cardiovascular hospitalization by 13% when administered at a mean of 7.3 days post myocardial infarction to patients with evidence of systolic left ventricular dysfunction and symptoms of heart failure. Eplerenone is well tolerated, with an adverse effect profile comparable to placebo. The advent of selective aldosterone blockers, such as eplerenone, should prove to be of great therapeutic value in hypertension control and prevention of cardiovascular disease and associated end-organ damage.     

Novel GABAB receptor positive modulators: a patent survey

Importance of the field: Positive allosteric modulators of GABA_B receptors may have a similar potential as positive modulators of GABA_A receptors, the benzodiazepines discovered in 1957. The discovery of positive allosteric modulators of GABA_B receptors at Novartis in Basel in 2000 opened the way to search for compounds, which activate GABA_B receptors without the drawbacks of full agonists, such as desensitization, tolerance, muscle-relaxant effects, hypothermia, and central and gastrointestinal side effects.

Areas covered in this review: Numerous animal experiments point out that several indications can be addressed with positive modulators of GABA_B receptors, such as depression, anxiety, schizophrenia, neuropathic and chronic pain and treatment of craving for drugs of abuse, such as alcohol, cocaine and nicotine. Peripherally acting compounds may be valuable drugs for the treatment of gastroesophageal reflux disease and irritable bowel syndrome.

What the reader will gain: An overview on 19 patents in this field, of the different scaffolds for positive modulators of GABA_B receptors and of the major players in the field.

Take home message: The search for subtype selective benzodiazepine receptor ligands has proved to be extremely difficult. Positive modulators of GABA_B receptors may provide novel anxiolytic drugs faster.     

PARP inhibition reduces acute colonic inflammation in rats

Poly(ADP-ribose) polymerases (PARP) comprise a family of enzymes which catalyse poly(ADP-ribosyl)ation of DNA-binding proteins. Multiple researches indicate the importance of PARP in promoting cell recruitment and thereby inducing organ injury in various forms of inflammation, such as colitis. We have evaluated the effects of two PARP inhibitors, nicotinamide and 1,5-dihydroxyisoquinoline, in acute colitis induced by trinitrobenzensulfonic acid (TNBS) in rats. Nicotinamide (20-40 mg/kg) and 1,5-dihydroxyisoquinoline (4-8 mg/kg) were administered 48, 24 and 1 h prior to the induction of colitis as well as 24 h later. 48 h after colitis induction the lesions were blindly scored and quantified as ulcer index. Histological study and colonic inflammation were assessed by gross appearance and myeloperoxidase (MPO) activity. Prostaglandin E2 (PGE2) synthesis and, cyclooxygenase-1 and cyclooxygenase-2 expressions by Western blotting and immunohistochemistry were also performed. Inflammation following TNBS induction was characterized by increased colonic wall thickness, oedema, diffuse inflammatory cells infiltration in the mucosa and necrosis. Furthermore, increased MPO activity, cyclooxygenase-2 expression and PGE2 synthesis were significantly augmented after TNBS instillation. On the contrary, treatment with 1,5-dihydroxyisoquinoline significantly reduced the degree of colon injury and also caused a substantial reduction in the rise in MPO activity, in the increase of staining for cyclooxygenase-2, as well as in the up-regulation of PGE2 caused by TNBS in the colon. Although nicotinamide significantly did not reduce macroscopic damage, it decreased both MPO activity and PGE2 colonic levels. In conclusion, we demonstrated that PARP inhibition can exert beneficial effects in experimental colitis and may, therefore, be useful in the treatment of ulcerative colitis.     

Autoreceptor-mediated regulation of GABA release: role of uptake inhibition and effects of novel GABAB antagonists

Summary While the role of GABA_B autoreceptors in the regulation of GABA release in synaptosomes and brain slices is well established, little is known about their role in vivo. Doubts have arisen because there is an apparent discrepancy between the frequencies at which GABA neurons fire and the frequency range within which autoreceptor regulation is observed in vitro. To see whether this apparent mismatch could be due to the use of a GABA uptake inhibitor in the release experiments in slices, we have compared the frequency dependencies of GABA release in the presence and absence of uptake inhibition. Beforehand, the previously incomplete frequency curve in the presence of uptake inhibition was extended at the lower end. To achieve this, stimulation was performed by means of groups of 4 pseudo-one-pulses (POP's) at inter-POP intervals corresponding to frequencies of 0.015625-0.5 Hz. It could be shown that activation of the GABA_B autoreceptor by endogenously released GABA begins at a stimulation frequency as low as 0.0625 Hz. Experiments with the antagonist, CGP 35348, at inter-POP intervals of 1 min, at which the preceding POP has no longer an effect on GABA release during the next one, showed that basal release alone already substantially activated the autoreceptor. The frequency dependence in the absence as compared to the presence of uptake inhibition was shifted towards higher frequencies by a factor of 4. We do not consider this enough to remove our doubts about the in vivo operativity of GABA_B autoreceptors.Further experiments in the presence of uptake inhibition were made to see whether the expectation was met that autoreceptor-mediated regulation of GABA release could be blocked out by sufficiently high concentrations of potent antagonists. As judged from the frequency dependence in the presence of 1 mol/l of the potent compound CGP 55845 as well as from the similarity of the maximal increases of GABA release caused at 0.125 and 2 Hz by various other potent, novel GABA_B antagonists, this was not the case. Possible explanations are the occurrence of an agonist and an antagonist state of the autoreceptor prevailing at low GABA concentrations or, less likely, the occurrence of two autoreceptor subtypes with different relative sensitivities towards GABA and baclofen on the one hand and towards aminophosphonous acid antagonists on the other.Finally, it was shown that also in the absence of uptake inhibition, regulation of GABA release was mediated entirely by GABA_B autoreceptors. Both muscimol and bicuculline at 1 and 10 mol/1 were without effect. Correspondence to P. Waldmeier at the above address     

Myxothiazol: a reversible blocker of the cell cycle

Myxothiazol, a potent inhibitor of the cytochrome bc1 oxidoreductase, was shown by the use of flow cytometry to block reversibly the late G1/S phase of the cell cycle of human lymphoblastic T-cell line Jurkat (clone 886) at concentrations of 0.5 microgram/ml. These observations are compared to those of other drugs, such as antimycin, which effect the respiratory chain, and with O2-deficiency.     

The angiotensin receptor blocker losartan reduces coronary arteriole remodeling in type 2 diabetic mice

Cardiovascular complications are a leading cause of morbidity and mortality in type 2 diabetes mellitus (T2DM) and are associated with alterations of blood vessel structure and function. Although endothelial dysfunction and aortic stiffness have been documented, little is known about the effects of T2DM on coronary microvascular structural remodeling. The renin–angiotensin–aldosterone system plays an important role in large artery stiffness and mesenteric vessel remodeling in hypertension and T2DM. The goal of this study was to determine whether the blockade of AT₁R signaling dictates vascular smooth muscle growth that partially underlies coronary arteriole remodeling in T2DM. Control and db/db mice were given AT₁R blocker losartan via drinking water for 4 weeks. Using pressure myography, we found that coronary arterioles from 16-week db/db mice undergo inward hypertrophic remodeling due to increased wall thickness and wall-to-lumen ratio with a decreased lumen diameter. This remodeling was accompanied by decreased elastic modulus (decreased stiffness). Losartan treatment decreased wall thickness, wall-to-lumen ratio, and coronary arteriole cell number in db/db mice. Losartan treatment did not affect incremental elastic modulus. However, losartan improved coronary flow reserve. Our data suggest that Ang II–AT₁R signaling mediates, at least in part, coronary arteriole inward hypertrophic remodeling in T2DM without affecting vascular mechanics, further suggesting that targeting the coronary microvasculature in T2DM may help reduce cardiac ischemic events.     

The broad-spectrum cation channel blocker pinokalant (LOE 908 MS) reduces brain infarct volume in rats: a temperature-controlled histological study

Activation of cation channels conducting Ca2+, Na+ and K+ is involved in the pathogenesis of infarction in experimental focal cerebral ischaemia. Pinokalant (LOE 908 MS) is a novel broad-spectrum inhibitor of several subtypes of such channels and has previously been shown to improve the metabolic and electrophysiologic status of the ischemic penumbra and to reduce lesion size on magnetic resonance images in the acute phase following middle cerebral artery occlusion in rats. The purpose of the present study was to investigate whether these beneficial effects of pinokalant are translated into permanent neuroprotection in terms of a reduction in infarct size one week after middle cerebral artery occlusion in rats. Halothane-anaesthetized male Wistar rats subjected to permanent distal middle cerebral artery occlusion were randomly assigned to one of two treatment groups: 1) Control (vehicle intravenous loading dose followed by infusion); 2) Pinokalant (0.5 mg/kg intravenous loading dose followed by infusion of 1.25 mg/kg/hr). Infusions started 30 min. after middle cerebral artery occlusion and were continued for 24 hr. Body temperature and mean arterial blood pressure were monitored by telemetry during this period and the spontaneous temperature after course in control rats established in other experiments was imitated. Seven days later histological brain sections were prepared and the infarct volumes measured. Body temperature did not differ between the groups. Mean arterial blood pressure was slightly higher in the pinokalant group. Pinokalant treatment significantly reduced cortical infarct volume from 33.8+/-15.8 mm3 to 24.5+/-13.1 mm3 (control group versus pinokalant group, P=0.017, t-test). Taking the effective drug plasma concentration established in other experiments into account revealed that in rats with plasma concentrations within the therapeutic interval, infarct volumes were further reduced to 17.9+/-7.5 mm3 (P<0.005).     

N-o-Methoxyphenylpiperazine: a simple blocker of dopaminergic receptors in the brain

N-o-Methoxyphenylpiperazine (MPP) is a moderately effective in vivo blocker of dopaminergic receptors. Its ability to increase the concentration of rat brain homovanillic acid (HVA) and the resulting time course for HVA were similar to the actions of clozapine. The increased concentration of HVA did not result from decreased outflow from brain because HVA also rapidly decreased after a subsequent injection of pargyline. MPP blocked the circling behaviour caused by apomorphine in mice with a unilateral striatal lesion, and MPP and apomorphine reciprocally blocked the occurrence of stereotypy and increased HVA in rats. Diazepam partially prevented the MPP-induced elevation of HVA. Thus, both biochemical and pharmacological evidence indicate the dopaminergic blocking action of MPP.