Peter C. Waldmeier

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE, THE PUTATIVE TARGET OF THE ANTIAPOPTOTIC COMPOUNDS CGP 3466 AND R-(-)-DEPRENYL

R-(−)-Deprenyl (Selegiline) represents one of the drugs currently used for the treatment of Parkinson’s disease. This compound was shown to protect neurons or glias from programmed cell death in a variety of models. The mechanism of action of neuroprotection as well as inhibition of apoptosis remains elusive. CGP 3466 is a structurally related analog ofR-(−)-deprenyl that exhibits virtually no monoamine oxidase type B inhibiting activity but is neuroprotective in the picomolar concentration range. We showed specific binding of CGP 3466 to glyceraldehyde-3-phosphate dehydrogenase by affinity binding, by affinity labeling, and by means of BIAcore® technology. Apoptosis assays based on the human neuroblastoma cell line PAJU established the importance of this interaction for mediating drug-induced inhibition of programmed cell death.

Cyclophilin D as a Drug Target

The mitochondrial permeability transition (MPT) plays an important role in damage-induced cell death, and agents inhibiting the MPT may have a therapeutic potential for treating human conditions such as ischemia/reperfusion injury, trauma, and neurodegenerative diseases. The mitochondrial matrix protein, cyclophilin D (CYP D), a member of a family of highly homologous peptidylprolyl cis-trans isomerases (PPIases), plays a decisive role in MPT, being an integral constituent of the MPT pore. Other putative MPT pore proteins include the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC). In an alternative model, the MPT pore is formed by clusters of misfolded membrane proteins outlining aqueous channels that are regulated by CYP D and other chaperone-like proteins. Like cyclophilin A (CYP A) and other cyclophilin family members, CYP D is targeted by the immunosuppressant cyclosporin A (CsA). CsA is cytoprotective in many cellular and animal models, but protection may result from either inhibition of the MPT through an interaction with CYP D or inhibition of calcineurin-mediated dephosphorylation of BAD through an interaction with CYP A. The relevance of MPT inhibition by CsA for its cytoprotective effects is well documented in many cellular models. Mechanisms of action in vivo are more difficult to define, and accordingly the evidence is as yet less compelling in in vivo animal models of ischemia/reperfusion injury, trauma and neurodegenerative diseases. Notwithstanding, CYP D is a drug target of high interest. Structural considerations suggest feasibility of designing CYP D ligands without immunosuppressant properties. This is highly desirable, since they have the potential of being useful therapeutic agents in a variety of disease states. It might be a tougher challenge to obtain compounds specific for CYP D vs. other cyclophilins, and/or of small molecular weight, allowing brain penetration to make them suitable for treating neurodegenerative diseases.

Serotonin-dopamine interactions in the nigrostriatal system

A study was made of the effects of serotonin uptake inhibition and receptor blockade on the increase in rat striatal homovanillic acid and 3,4-dihydroxyphenylacetic acid and on some behavioural responses induced by haloperidol. The serotonin uptake inhibitors CGP 6085 A (4-(5,6-dimethyl-2-benzofuranyl)-piperidine-HCl), citalopram (Lu 10-171), fluoxetine (Lilly 110140), and clomipramine potentiated the increase in striatal deaminated dopamine metabolites after the neuroleptic. In contrast, the serotonin antagonists methysergide, mianserin and cinanserin antagonized the acceleration of dopamine turnover induced by haloperidol. The catalepsy induced by this neuroleptic was potentiated by CGP 6085 A and citalopram. These 5-HT uptake inhibitors also potentiated the antagonism by haloperidol of apomorphine-induced stereotypies. These results seem to make it worthwhile to test a combination of haloperidol and a serotonin antagonist in schizophrenic patients to see whether the ratio of the therapeutic effect to the extrapyramidal side effects can be improved.

Specific γ‐hydroxybutyrate‐binding sites but loss of pharmacological effects of γ‐hydroxybutyrate in GABAB(1)‐deficient mice

γ‐Hydroxybutyrate (GHB), a metabolite of γ‐aminobutyric acid (GABA), is proposed to function as a neurotransmitter or neuromodulator. γ‐Hydroxybutyrate and its prodrug, γ‐butyrolactone (GBL), recently received increased public attention as they emerged as popular drugs of abuse. The actions of GHB/GBL are believed to be mediated by GABAB and/or specific GHB receptors, the latter corresponding to high‐affinity [3H]GHB‐binding sites coupled to G‐proteins. To investigate the contribution of GABAB receptors to GHB actions we studied the effects of GHB in GABAB(1)−/− mice, which lack functional GABAB receptors. Autoradiography reveals a similar spatial distribution of [3H]GHB‐binding sites in brains of GABAB(1)−/− and wild‐type mice. The maximal number of binding sites and the KD values for the putative GHB antagonist [3H]6,7,8,9‐tetrahydro‐5‐hydroxy‐5H‐benzocyclohept‐6‐ylidene acetic acid (NCS‐382) appear unchanged in GABAB(1)−/− compared with wild‐type mice, demonstrating that GHB‐ are distinct from GABAB‐binding sites. In the presence of the GABAB receptor positive modulator 2,6‐di‐tert‐butyl‐4‐(3‐hydroxy‐2,2‐dimethyl‐propyl)‐phenol GHB induced functional GTPγ[35S] responses in brain membrane preparations from wild‐type but not GABAB(1)−/− mice. The GTPγ[35S] responses in wild‐type mice were blocked by the GABAB antagonist [3‐[[1‐(S)‐(3,4dichlorophenyl)ethyl]amino]‐2‐(S)‐hydroxy‐propyl]‐cyclohexylmethyl phosphinic acid hydrochloride (CGP54626) but not by NCS‐382. Altogether, these findings suggest that the GHB‐induced GTPγ[35S] responses are mediated by GABAB receptors. Following GHB or GBL application, GABAB(1)−/− mice showed neither the hypolocomotion, hypothermia, increase in striatal dopamine synthesis nor electroencephalogram delta‐wave induction seen in wild‐type mice. It, therefore, appears that all studied GHB effects are GABAB receptor dependent. The molecular nature and the signalling properties of the specific [3H]GHB‐binding sites remain elusive.

Alteration of central alpha2- and beta-adrenergic receptors in the rat after DSP-4, a selective noradrenergic neurotoxin

A peripheral injection of DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine] produced a marked, selective, and lasting depletion of norepinephrine in certain regions of the rat central nervous system. This depletion at 10 days after injection was associated with regional alterations in some, but not all, adrenergic binding sites (receptors) as determined by in vitro [3H]prazosin (alpha 1), [3H]p-aminoclonidine (alpha 2), and [3H]dihydroalprenolol (beta) binding. The neocortical alpha 1-receptor was not changed. The alpha 2-receptor in several regions was altered as indicated by an increase in ligand affinity; additionally, the density of this receptor was slightly decreased in some regions. Depending on the region, the beta-receptor either increased in density or was unchanged. The increased density of this receptor in neocortex corresponded to an increased activity of isoproterenol-sensitive adenylate cyclase. These two changes were not affected by subchronic treatment with desipramine, a norepinephrine uptake inhibitor. The changes were, however, partially or completely reversed by subchronic administration of clenbuterol, a centrally-acting beta-receptor agonist. The dopaminergic receptor in various regions was unaltered as assessed by in vivo and/or in vitro binding of [3H]spiperone. The in vivo binding of this ligand also indicated that the serotoninergic receptor in frontal neocortex was unchanged. Assessment of adrenergic receptors in neocortex at 50 days after injection indicated only the above affinity change of the (presumably postsynaptic) alpha 2-receptor. The alpha 1-receptor remained unaltered. The density of the beta-receptor had normalized, as had the activity of isoproterenol-sensitive adenylate cyclase. Implicit in these findings is the following rank order of receptor sensitivity to chronic norepinephrine depletion: alpha 2 greater than beta greater than alpha 1. The use of DSP-4 has clear advantages over other methods of depleting central norepinephrine. This neurotoxin can be administered by intraperitoneal injection, the depletion of norepinephrine can be readily checked by absence of the post-decapitation reflex, and the changes in other neurotransmitter concentrations are relatively minor or nonexistent. The alteration of alpha 2- and beta-receptors, as a consequence of DSP-4 treatment, may form the basis of a new animal model of adrenergic receptor supersensitivity. Such a model may clarify the importance of these central receptors to physiological and behavioral processes.

Preferential deamination of dopamine by an A type monoamine oxidase in rat brain.

SummaryThe effect of graded closes of clorgyline, a preferential inhibitor of MAO A, and of deprenil, a preferential inhibitor of MAO B, on the activities of serotonin-deaminating MAO (MAO A) of dopamine-deaminating MAO, and of phenethylamine-deaminating MAO, (MAO B), in rat corpus striatum were compared with the effects of the drugs on striatal levels of homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC).The dose-response curves for the two last-mentioned dopamine metabolites closely follow those for MAO A and dopamine-deaminating activity, whether clorgyline or deprenil was used as MAO inhibitor.In addition, the effect of these drugs on dopamine levels and on the accumulation of 3H-dopamine + 3H-methoxytyramine formed from 3H-DOPA in rat whole brain was analysed. In contrast to the marked increases caused by clorgyline, the effects of deprenil were negligible. In reserpinized rats, clorgyline potentiated the effect of l-DOPA on motor activity; deprenil did not.These results suggest that the deamination of dopamine in vivo is almost entirely effected by MAO A.

The Nonimmunosuppressive Cyclosporin Analogs NIM811 and UNIL025 Display Nanomolar Potencies on Permeability Transition in Brain-Derived Mitochondria.

Cyclosporin A (CsA) is highly neuroprotective in several animal models of acute neurological damage and neurodegenerative disease with inhibition of the mitochondrial permeability transition (mPT) having emerged as a possible mechanism for the observed neuroprotection. In the present study, we have evaluated two new nonimmunosuppressive cyclosporin analogs NIM811 (Novartis) and UNIL025 (Debiopharm) for their ability to inhibit mPT in rat brain-derived mitochondria. Both NIM811 and UNIL025 were found to be powerful inhibitors of calcium-induced mitochondrial swelling under energized and deenergized conditions, and the maximal effects were identical to those of native CsA. The potencies of mPT inhibition by NIM811 and UNIL025 were stronger, with almost one order of magnitude higher potency for UNIL025 compared to CsA, correlating to their respective inhibitory action of cyclophilin activity. These compounds will be instrumental in the evaluation of mPT as a central target for neuroprotection in vivo.

Oxaprotiline, a noradrenaline uptake inhibitor with an active and an inactive enantiomer

Abstract The new antidepressant drug, oxaprotiline, and its two enantiomers were investigated with respect to their effects on noradrenaline (NA) and serotonin (5-HT) uptake in vitro and in vivo after acute and repeated treatment. Moreover, the alpha-adrenolytic effects in vitro were also studied. Oxaprotiline proved to be a highly potent and selective inhibitor of NA uptake in rat synaptosomal preparations in vitro and in the rat heart and brain in vivo The NA uptake-inhibiting properties were found to be confined entirely to the (+)- or S-enantiomer: (−)- or R-oxaprotiline, the absolute configuration of which corresponds to that of the naturally occurring (−)-NA, was about 1000 times less potent than the (+)-form in vitro , and was inactive in vivo at doses exceeding the ED 50 of the latter 100-fold. The selectivity of oxaprotiline with respect to NA uptake inhibition was retained after 10 daily administrations. No sign of cumulation or attenuation of the effect was evident. No uptake-inhibiting effect of (−)-oxaprotiline appeared after 10 daily administrations of high doses, indicating that no racemization occurred in the organism. The α 1 -adrenoceptor antagonistic effect of oxaprotiline, as determined by the ability to displace [ 3 H]prazosin, was in the range of that of imipramine, the (−)-enantiomer being somewhat more potent than the (+)-form. In contrast to imipramine, oxaprotiline was devoid of α 2 -adrenoceptor antagonistic effects, as judged by the ability to affect the impulse-related release of [ 3 H]NA from rat cortical slices. Since oxaprotiline proved to be an effective antidepressant, clinical testing of its two enantiomers might be helpful with respect to the validation of the catecholamine hypothesis of depression. Moreover, in animal studies, they might help to determine which effects of antidepressants are related to NA uptake inhibition and which are not.

GABA and glutamate release affected by GABAB receptor antagonists with similar potency : no evidence for pharmacologically different presynaptic receptors

1 The effects of a series of nine GABAB receptor antagonists of widely varying potencies on electrically stimulated release from cortical slices of [3H]‐GABA in the absence or presence of 10 μm of the GABABagonist, (−)−baclofen and of endogenous glutamate in the presence of (−)−baclofen were compared. 2 The concentrations of the compounds half maximally increasing [3H]‐GABA release (EC50s) at a stimulation frequency of 2 Hz correlated well with the IC50 values obtained from the inhibition of the binding of the agonist, [3H]‐CGP 27492, to GABAB receptors in rat brain membranes (rank order of potency: CGP 56999 A ≥ CGP 55845 A > CGP 52432 ≥ CGP 56433 A > CGP 57034 A > CGP 57070 A ≥ CGP 57976 > CGP 51176 > CGP 35348). 3 Likewise, the concentrations causing half‐maximal increases of [3H]‐GABA in the absence or presence of (−)−baclofen, and of endogenous glutamate in the presence of (−)−baclofen, correlated well with each other. Reports in the literature suggesting the CGP 35348 exhibits a 70 fold preference for inhibition of (−)−baclofens effects on glutamate over [3H]‐GABA release, and that CGP 52432 shows a 100 fold preference in the opposite sense, could not be confirmed in our model. 4 Therefore, our results suggest that, if there are pharmacological differences between GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings in the rat cortex, they are not revealed by this series of compounds of widely different potencies. 5 In particular, our results with CGP 35348 and CGP 52432 do not support the hypothesis that GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings represent subtypes with different pharmacology.

Prospects for antiapoptotic drug therapy of neurodegenerative diseases.

The evidence for a role of apoptosis in the neurodegenerative diseases, Alzheimers disease (AD), Parkinsons disease (PD) and amyotrophic lateral sclerosis (ALS), and in the more acute conditions of cerebral ischemia, traumatic brain injury (TBI), and spinal cord injury (SCI) is reviewed with regard to potential intervention by means of small antiapoptotic molecules. In addition, the available animal models for these diseases are discussed with respect to their relevance for testing small antiapoptotic molecules in the context of what is known about the apoptotic pathways involved in the diseases and the models. The principal issues related to pharmacotherapy by apoptosis inhibition, i.e., functionality of rescued neurons and potential interference with physiologically occurring apoptosis, are pointed out. Finally, the properties of a number of small antiapoptotic molecules currently under clinical investigation are summarized. It is concluded that the evidence for a role of apoptosis at present is more convincing for PD and ALS than for AD. In PD, damage to dopaminergic neurons may occur through oxidative stress and/or mitochondrial impairment and culminate in activation of an apoptotic, presumably p53-dependent cascade; some neurons experiencing energy failure may not be able to complete apoptosis, end up in necrosis and give rise to inflammatory processes. These events are reasonably well reflected in some of the PD animal models, notably those involving 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone. In sporadic ALS, an involvement of pathways involving p53 and Bcl-2 family members appears possible if not likely, but is not established. The issue is important for the development of antiapoptotic compounds for the treatment of this disease because of differential involvement of p53 in different mutant superoxide dismutase (SOD) mice. Most debated is the role of apoptosis in AD; this implies that little is known about potentially involved pathways. Moreover, there is a lack of suitable animal models for compound evaluation. Apoptosis or related phenomena are likely involved in secondary cell death in cerebral ischemia, TBI, and SCI. Most of the pertinent information comes from animal experiments, which have provided some evidence for prevention of cell death by antiapoptotic treatments, but little for functional benefit. Much remains to be done in this area to explore the potential of antiapoptotic drugs. There is a small number of antiapoptotic compounds in clinical development. With some of them, evidence for maintenance of functionality of the rescued neurons has been obtained in some animal models, and the fact that they made it to phase II studies in patients suggests that interference with physiological apoptosis is not an obligatory problem. The prospect that small antiapoptotic molecules will have an impact on the therapy of neurodegenerative diseases, and perhaps also of ischemia and trauma, is therefore judged cautiously positively.