Parkinson's disease: prospects for improved drug therapy.

L-Dopa has long been the mainstay of therapy for Parkinson's disease but its long-term shortcomings, principally uncoordinated, spasmodic or irregular movements (dyskinesias) and fluctuating control of motor symptoms (on/off fluctuations), are well documented. The postulated neuroprotective properties of L-deprenyl, often used as an adjunct to L-dopa, are under scrutiny and doubts have also been raised regarding its safety. Alternative therapeutic approaches are clearly needed. In this review, Jim Hagan, Derek Middlemiss, Paul Sharpe and George Poste outline some new approaches to treatment, with an emphasis on novel, selective dopamine receptor agonists. In addition, Parkinson's disease is commonly thought to be caused by the neurotoxic effects of an unidentified agent but recent data indicate a greater genetic component than previously recognized. Developments in the genetics of Parkinson's disease may provide the key to the next generation of therapeutics.

Centrally active 5-HT receptor agonists and antagonists.

Eleven subtypes of central 5-HT receptor have so far been postulated, four of which have been cloned (5-HT1A, 5-HT1C, 5-HT1D and 5-HT2) and a fifth (the 5-HT3 receptor) purified. The present review discusses the agonists and antagonists which act at these subtypes with respect to their degree of selectivity and in vivo potency. Selective agonists exist for the 5-HT1A, 5-HT1B and 5-HT3 receptors and selective antagonists for the 5-HT2 and 5-HT3 receptors.

Stereoselective blockade at the 5-HT autoreceptor and inhibition of radioligand binding to central 5-HT recognition sites by the optical isomers of methiothepin.

The enantiomers of the 5-HT autoreceptor antagonist methiothepin have been prepared and their activity as antagonists of the 5-HT autoreceptor and at the 5-HT recognition sites present in the frontal cortex of the rat have been evaluated. At the 5-HT autoreceptor, the order of potency as antagonists of 5-HT was (+)methiothepin (apparent pA2 5.95) less than (+/-)methiothepin (apparent pA2 6.62) less than or equal to (-)methiothepin (apparent pA2 6.81). At the 5-HT2 recognition site, the isomeric forms of methiothepin were potent (pIC50 approximately 8.2) and equiactive. At the subtypes of the 5-HT1 recognition sites, similar concentrations to those blocking the autoreceptor were effective and (+)methiothepin was less active than (-)methiothepin. It is concluded that the chiral association of methiothepin with the 5-HT autoreceptor provides further evidence for a pharmacological similarity between this receptor and the 5-HT1B subtype of the 5-HT1 recognition site.

Pharmacological analysis of the behavioural and thermoregulatory effects of the putative 5-HT1 receptor agonist, RU 24969, in the rat.

The roles of recognition sites for central neurotransmitters in the mediation of the behavioural effects of the putative 5-hydroxytryptamine (5-HT1) receptor agonist, RU 24969 [5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)1H indole] in the rat have been examined. The drug RU 24969 was found to have high affinity for 5-HT1A and 5-HT1B recognition sites. Hyperlocomotion, induced by RU 24969, was enhanced in animals depleted of 5-HT with 5,7-dihydroxytryptamine, suggesting an involvement of 5-HT receptors in the mediation of this behaviour. However, results of experiments with 5-HT receptor antagonists argued against the receptors being of either the 5-HT1 or 5-HT2 type. Despite the negligible affinity of RU 24969 for catecholamine receptors, hyperlocomotion induced by RU 24969 was clearly dependent on intact catecholamine systems. When hyperlocomotion was blocked by treatment with reserpine, reciprocal forepaw-treading and a flat body posture, behavioural responses which are consistent with activation of the putative 5-HT1A receptor, became evident. When animals were restrained from moving, RU 24969 dose-dependently reduced body temperature, an effect that may also be associated with activation of the 5-HT1A recognition site. Thus, although the mechanism by which RU 24969 induces hyperlocomotion is not yet established, the agonist nevertheless can induce functional responses consistent with its high affinity for the 5-HT1A recognition site.

Spironolactone causes a rapid down regulation of sigma recognition sites in guinea pig brain and liver.

The effect of dosing guinea pigs with spironolactone (100 mg/kg twice daily for 3 days) upon the sigma recognition site labelled with [3H]-DTG was investigated. Animals were dosed with 100 mg/kg spironolactone twice a day for 3 days. Spironolactone treatment caused a decrease in sigma radioligand binding in membranes prepared from liver and brain but not in adrenals or testes. Saturation analysis of [3H]-DTG radioligand binding in the brain indicated that the decrease in specifically bound [3H]-DTG binding was due to a reduction in receptor number with no change in affinity. This method for the selective depletion of brain and liver sigma recognition sites will provide a useful tool for exploring the functional significance of this site.

Effect of haloperidol and (-)-sulpiride on dopamine agonist-induced hypoactivity.

High doses of dopamine D2 receptor agonists produce hyperactivity in rodents whereas low doses suppress activity. In this study, low doses of a range of dopamine agonists were examined for their effects on locomotor activity in rats. All agonists caused a dose-related hypolocomotor effect with a rank order of potency of quinelorane > (-)-quinpirole > 7-OHDPAT > PBTO. (-)-Sulpiride (1.6-160 mumol/kg), a neuroleptic with atypical properties caused a dose-related reversal of the hypolocomotor effect produced by all four agonists whereas the typical neuroleptic haloperidol (0.005-0.16 mumol/kg) did not reverse hypolocomotion. Neither sulpiride (5-16 mumol/kg) nor haloperidol (0.005-0.16 mumol/kg) affected locomotor activity per se, although haloperidol (1.6-5 mumol/kg) did reduce locomotor activity. The different behavioural profiles shown by (-)-sulpiride and haloperidol in these tests may reflect some of the clinical characteristics of these neuroleptics. The question of whether these effects can be ascribed to differential actions at dopamine receptor subtypes will only be answered when more selective dopamine receptor antagonists are developed.

Autoradiographic localization of 5-CT-insensitive 5-HT1-like recognition sites in guinea pig and rat brain.

Quantitative autoradiographic studies, with [3H]5-HT, were used to investigate the distribution of 5-CT-insensitive 5-HT1-like (5-HT1E/1F) recognition sites in rat and guinea pig brain. For comparison and control purposes the distribution of the closely related 5-HT1D binding site, which is abundant in the guinea pig but not the rat, was also investigated, as well as total specific [3H]5-HT binding. Results from this study confirm the previously described regional distribution of the 5-HT1D binding site and also revealed a predominance of 5-CT-insensitive 5-HT1-like 5-HT1E/1F) recognition sites in the olfactory tubercle, caudate putamen, nucleus accumbens and substantia nigra of both species. Interestingly 5-CT-insensitive 5-HT1-like (5-HT1E/1F) recognition sites were particularly dense in the claustrum of the guinea pig, but not the rat.

Enhancement of 5-HT1B and 5-HT1D receptor antagonist effects on extracellular 5-HT levels in the guinea-pig brain following concurrent 5-HT1A or 5-HT re-uptake site blockade.

The effects of selective serotonin re-uptake inhibitor (SSRI), paroxetine, and 5-HT1A, 5-HT1B and 5-HT1B/1D receptor antagonists on in vivo extracellular 5-HT levels in the guinea-pig frontal cortex and dorsal hippocampus were investigated using the technique of microdialysis. The aim of the study was to further investigate the autoreceptor roles of the 5-HT1A, 5-HT1B and 5-HT1D receptors in the median vs dorsal raphe nuclei. In the frontal cortex, 5-HT1A (WAY 100635, 1 mg/kg i.p.) or 5-HT1B (SB-224289, 4 mg/kg i.p.) receptor antagonists had no effect on extracellular levels of 5-HT, whilst the mixed 5-HT1B/1D receptor antagonist (GR 127935, 0.3 mg/kg i.p) produced a significant decrease in extracellular 5-HT levels. Paroxetine (10 microM) significantly increased extracellular 5-HT levels when perfused locally into the cortex. Administration of SB-224289, followed 120 min later by WAY 100635, had no effect on extracellular 5-HT levels. In contrast, sequential administration of either WAY 100635 and GR 127935, or SB-224289 and paroxetine significantly increased extracellular 5-HT levels. In the dorsal hippocampus, whilst 5-HT1A receptor antagonism elicited by administration of WAY 100635 had no effect, both 5-HT1B and mixed 5-HT1B/1D receptor blockade significantly increased extracellular 5-HT levels. Administration of SB-224289 followed 120 min later with WAY 100635, or WAY 100635 followed 30 min later with GR 127935, potentiated the effect of the three compounds alone, significantly increasing extracellular 5-HT levels. These data demonstrate that to simultaneously increase extracellular 5-HT in both frontal cortex and dorsal hippocampus of the guinea-pig brain concurrent 5-HTA1A, 5-HT1B and 5-HT1D receptor blockade is required. Whereas in the dorsal hippocampus, 5-HT1B receptor blockade is sufficient to elicit an increase in extracellular 5-HT levels.

Importance of h5-HT1B receptor selectivity for 5-HT terminal autoreceptor activity: an in vivo microdialysis study in the freely-moving guinea-pig.

The importance of h5-HT1B receptor selectivity for 5-HT terminal autoreceptor activity was investigated with the selective h5-HT1B receptor ligands SB 219085, SB 220272, SB 224289 and SB 216641. The studies employed measurement of compound affinity and efficacy in vitro and the measurement of extracellular 5-HT in the frontal cortex of the freely-moving guinea-pig using in vivo microdialysis. All compounds had high affinity and selectivity for the h5-HT1B receptor, with SB 224289 the most selective for h5-HT1B over h5-HT1D receptors. Compounds exhibited a range of efficacies at both receptors: SB 224289 and SB 219085 were inverse agonists, SB 220272 was an antagonist and SB 216641 was a partial agonist. SB 220272, SB 216641 and SB 224289 had no effect on extracellular 5-HT following systemic administration, however, SB 219085 produced a significant increase. The SB 219085-induced increase in extracellular 5-HT was attributed to the compounds non-specific releasing properties as it was also demonstrated to increase basal release of [3H]5-HT from pre-loaded guinea-pig cortical slices. The lack of effect of the above h5-HT1B receptor selective compounds and the decrease in extracellular 5-HT elicited by the non-selective compounds GR 127935, GR125743 and methiothepin suggest that antagonism of 5-HT1D receptors may mediate this decrease in 5-HT levels. It is plausible that blockade of 5-HT1D receptors increases 5-HT levels in the raphe, this activates 5-HTtA receptors which results in an overall decrease in terminal 5-HT release. Definitive proof now awaits elucidation of the action of a selective 5-HT1D receptor antagonist.

Stereoselective actions of the isomers of metitepine at 5-HT1D receptors in the guinea pig brain.

The present studies examined the relative antagonist potencies of the optical isomers of the 5-HT receptor antagonist metitepine at the 5-HT1D binding site labelled by the novel radioligand serotonin-O-carboxymethylglycyl [125]iodotyrosinamide ([125I]GTI), and at the terminal 5-HT autoreceptor in guinea pig frontal cortex, a proposed model of 5-HT1D receptor activation. The pharmacological specificity of the [125I]GTI binding site in guinea pig frontal cortex was similar to previously published studies in the bovine cortical 5-HT1D recognition site labelled with [3H]5-HT. The (+) isomer of metitepine displaced [125I]GTI binding with a lower affinity (64 nM) than did the (-) isomer (18 nM), which was equiactive with the racemic mixture. The (-) isomer of metitepine was more effective than the (+) isomer at attenuating the inhibitory effects of 5-HT and sumatriptan at the guinea pig terminal 5-HT autoreceptor; the apparent pA2 of the (-) isomer was 8.0 (sumatriptan) and 7.7 (5-HT) while the apparent pA2 of the (+) isomer was 7.1 (sumatriptan) and 6.8 (5-HT). The (-) isomer was more effective than the (+) isomer at enhancing stimulated [3H]5-HT release. These findings support the identification of the guinea pig 5-HT terminal autoreceptor as a 5-HT1D receptor and reinforce the species homology between the 5-HT1B and 5-HT1D receptors.

Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine.

As a result of controversy in the literature regarding the classification and nomenclature of functional receptors for 5-hydroxytryptamine (5-HT), a framework for classification is proposed. The formulation of these proposals has only been made possible by the recent advent of new drug tools. It is considered that there are three main types of 5-HT receptor, two of which have been well characterised pharmacologically, using selective antagonists, and which it is proposed to name 5-HT2 and 5-HT3. These two groups broadly encompass the "D" and "M" receptors, respectively, which Gaddum identified in the guinea-pig ileum (Gaddum and Picarelli, 1957). The 5-HT2 receptor, which mediates a variety of actions of 5-HT, has been definitively shown to correlate with the 5-HT2 binding site in the brain. No binding studies in brain tissue have yet been published with radiolabelled ligands specific for 5-HT3 receptors. A number of other actions of 5-HT appear to be mediated via receptors distinct from 5-HT2 or 5-HT3 receptors. Since selective antagonists are not yet available, these receptors cannot be definitively characterised, although in many cases they do have some similarities with 5-HT1 binding sites, which are a heterogeneous entity. Criteria are proposed for tentatively classifying these receptors as "5-HT1-like" (Table 1). Definitive characterisation of these receptors will await the identification of specific antagonists. This classification of 5-HT receptors into three main groups (Table 1) is based largely, but not exclusively, on data from studies in isolated peripheral tissues where definitive classification is possible. However, it is believed that this working classification will be relevant to functional responses to 5-HT in the central nervous system.

[3H]-SB-269970 radiolabels 5-HT7 receptors in rodent, pig and primate brain tissues.

The selective 5-HT7 receptor antagonist radioligand, [3H]-SB-269970, has been reported to radiolabel the human cloned 5-HT7(a) receptor and 5-HT7 receptors in guinea pig cortex (thomas et al, 2000). Saturation analysis of [3H]-SB-269970 binding to mouse forebrain, rat cortex, pig cortex, marmoset cortex and human thalamus membranes was consistent with labelling a homogenous population of binding sites in each tissue. K(D) values for [3H]-SB-269970 binding in these tissues ranged from 0.9 to 2.3 nM, being similar to those reported for the human cloned and guinea pig cortex 5-HT7 receptors (1.3 and 1.7 nM, respectively). Bmax values for [3H]-SB-269970 binding to the mouse, rat, pig, marmoset and human brain membranes were 20, 30, 31, 14 and 68 fmoles x mg x protein(-1), respectively. For each species the profile of inhibition of [3H]-SB-269970 binding, using a number of 5-HT7 receptor agonists and antagonists, correlated well with that reported for the human cloned 5-HT7(a) receptor (correlation coefficients were 0.95, 0.94, 0.92, 0.95, 0.97 versus the mouse, rat, pig, marmoset and human tissues, respectively). In conclusion, [3H]-SB-269970 has been shown to radiolabel 5-HT7 receptors in rodent, pig and primate brain and represents a valuable tool with which to further characterise the distribution and function of 5-HT7 receptors in native tissues and elucidate their potential role in disease states.

SB 242084, a selective and brain penetrant 5-HT2C receptor antagonist.

SB 242084 has a high affinity (pKi 9.0) for the cloned human 5-HT2C receptor and 100- and 158-fold selectivity over the closely related cloned human 5-HT2B and 5-HT2A subtypes respectively. SB 242084 had over 100-fold selectivity over a range of other 5-HT, dopamine and adrenergic receptors. In studies of 5-HT-stimulated phosphatidylinositol hydrolysis using SH-SY5Y cells stably expressing the cloned human 5-HT2C receptor, SB 242084 acted as an antagonist with a pKb of 9.3, which closely resembled its corresponding receptor binding affinity. SB 242084 potently inhibited m-chlorophenylpiperazine (mCPP, 7 mgkg i.p. 20 min pre-test)-induced hypolocomotion in rats, a model of in vivo central 5-HT2C receptor function, with an ID50 of 0.11 mg/kg i.p., and 2.0 mg/kg p.o. SB 242084 (0.1-1 mg/kg i.p.) exhibited an anxiolytic-like profile in the rat social interaction test, increasing time spent in social interaction, but having no effect on locomotion. SB 242084 (0.1-1 mg/kg i.p.) also markedly increased punished responding in a rat Geller-Seifter conflict test of anxiety, but had no consistent effect on unpunished responding. A large acute dose of SB 242084 (30 mg/kg p.o.) had no effect on seizure susceptibility in the rat maximal electroshock seizure threshold test. Also, while SB 242084 (2 and 6 mg/kg p.o. 1 hr pre-test) antagonized the hypophagic response to mCPP, neither acute nor subchronic administration of the drug, for 5 days at 2 or 6 mg/kg p.o. twice daily, affected food intake or weight gain. The results suggest that SB 242084 is the first reported selective potent and brain penetrant 5-HT2C receptor antagonist and has anxiolytic-like activity, but does not possess either proconvulsant or hyperphagic properties which are characteristic of mutant mice lacking the 5-HT2C receptor.

SB-243213; a selective 5-HT2C receptor inverse agonist with improved anxiolytic profile: lack of tolerance and withdrawal anxiety.

SB-243213 (5-methyl-1-[[-2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-6-trifluoromethylindoline hydrochloride) is a new, selective 5-hydroxytryptamine (5-HT)2C receptor inverse agonist. SB-243213 has high affinity for the human 5-HT2C receptor (pK(i) 9.37) and greater than a 100-fold selectivity over a wide range of neurotransmitter receptors, enzymes and ion channels. In in vitro functional studies, SB-243213 acted as an inverse agonist at the human 5-HT2C receptor with a pK(b) of 9.8. In in vivo studies, SB-243213 was a potent inhibitor of central 5-HT2C receptor-mediated function in rats, blocking meta-chlorophenylpiperazine-induced hypolocomotion with an ID50 of 1.1 mg/kg p.o. and a long duration of action (>8 h). In rats, SB-243213 exhibited anxiolytic-like activity in both the social interaction and Geller-Seifter conflict tests. Importantly, unlike diazepam, chronic administration of SB-243213 did not result in the development of either tolerance to the anxiolytic-like effects or withdrawal anxiogenesis. Furthermore, in rodents, SB-243213 did not affect seizure threshold, did not increase body weight or induce catalepsy, but attenuated the haloperidol-induced catalepsy. SB-243213 did not affect amphetamine-, MK-801- or phencyclidine-induced hyperactivity. In conclusion, SB-243213 may possess an improved anxiolytic profile compared to benzodiazepines. SB-243213 also modulates dopaminergic transmission, lacks pro-psychotic properties and may have utility in the treatment of schizophrenia and motor disorders.

Graphics computer-aided receptor mapping as a predictive tool for drug design: development of potent, selective, and stereospecific ligands for the 5-HT1A receptor.

A conformational study of four 5-HT1A (serotonin) receptor ligands ((R-(-)-methiothepin, spiperone, (S)-(-)-propranolol, and buspirone) led to the definition of a pharmacophore and a three-dimensional map of the 5-HT1A antagonist recognition site. These models were used to design new compounds and successfully predict their potency, stereospecificity, and selectivity. For example, 8-[4-[(1,4-benzodioxan-2-ylmethyl)amino] butyl]-8-azaspiro[4.5]decane-7,9-dione (1, MDL 72832) has nanomolar affinity (pIC50 = 9.14) for the 5-HT1A binding site in rat frontal cortex. As predicted, the S-(-) enantiomer of 1 was more active than its R-(+) enantiomer (pIC50 = 9.21 and 7.66, respectively) and a naphthalene analogue of 1 displayed the expected improved selectivity.

Synthesis and 5-hydroxytryptamine antagonist activity of 2-[[2-(dimethylamino)ethyl]thio]-3-phenylquinoline and its analogues.

A series of 2-[(2-aminoethyl)thio]quinolines substituted at the 3-position with alkyl, aryl, or heteroaryl groups has been prepared in the search for novel and selective 5-HT2 antagonists. The affinity of the compounds for 5-HT1 receptor sites was measured by their ability to displace [3H]-5-HT from rat brain synaptosomes whereas the affinity for 5-HT2 receptor sites was measured by their ability to displace [3H]spiperone from synaptosomes prepared from rat brain cortex. The 5-HT2 antagonist properties of the compounds were measured in vivo by their antagonism of 5-hydroxytryptophan-induced head twitches in the mouse and by their antagonism of hyperthermia induced by fenfluramine (N-ethyl-alpha-methyl-m-(trifluoromethyl)phenethylamine hydrochloride) in the rat. The structure-activity relationships in this series are discussed and the properties of 2-[[2-(dimethylamino)ethyl]thio]-3-phenylquinoline hydrochloride (70) are highlighted.

Benz[f]isoquinoline analogues as high-affinity sigma ligands.

This paper describes the synthesis of some conformationally restricted 4-phenylpiperidine analogues and their affinities for the guinea pig cerebellum sigma recognition site ([3H]-DTG) and the rat striatum dopamine D2 receptor ([3H]-(-)-sulpiride) in order to develop potent selective sigma ligands as tools in the investigation of this site in psychosis. It was found that both hexa- and octahydrobenz[f]isoquinolines with lipophilic N-substituents had high affinities for the sigma site. Notably, trans-3-cyclohexyl-1,2,3,4,4a,5,6,10b-octahydrobenz[f]isoquinoline (26) had an affinity of 0.25 nM making it the highest affinity sigma ligand reported to date. Moreover, it is at least 10,000-fold selective over the D2 receptor and could prove to be a valuable tool in the study of sigma sites. Other analogues such as 1H-indeno[2,1-c]pyridines and 1H-benzo[3,4]cyclohepta[1,2-c]pyridines also displayed high sigma site affinity.

Spiropiperidines as high-affinity, selective sigma ligands.

A variety of achiral conformationally restricted spirocyclic piperidines have been prepared in an attempt to investigate the functional role of the central sigma recognition site. All the compounds possessed a lipophilic N-substituent incorporating either a tetralin, indan, or benzocycloheptane skeleton. Their in vitro affinity at the sigma site was assessed in radioligand displacement experiments with guinea pig cerebellum homogenates using the sigma-specific radioligand [3H]-N,N'-di-o-tolylguanidine ([3H]-DTG, [3H]-6). A study of the structure-activity relationships identified the N-butyl and N-dimethylallyl substituents as the optimum groups for high affinity and selectivity at the sigma site (e.g., 3,4-dihydro-1'-(3-methylbut-2-enyl)spiro[1H-indene-1,4'-piperidine ] (48), pIC50 = 8.9 vs [3H]-6 and greater than 10,000-fold selective over the dopamine D2 receptor). Such compounds are amongst the highest affinity sigma ligands reported to date, with excellent selectivity over the dopamine D2 receptor, and may serve as a useful tool for exploring the physiological role of the sigma site.

Structure and activity of hydrogenated derivatives of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801).

Several hydrogenated derivatives of the potent NMDA antagonist 1 have been prepared and evaluated as competitive inhibitors of [3H]-1 binding. These compounds were also tested for their ability to act as noncompetitive antagonists of NMDA in vitro. These studies indicate that two aromatic rings are not strictly required for high-affinity binding or NMDA antagonism.

Synthesis and serotonergic activity of 5-(oxadiazolyl)tryptamines: potent agonists for 5-HT1D receptors.

The synthesis and 5-HT1D receptor activity of a novel series of 5-(oxadiazolyl)tryptamines is described. Modifications of the oxadiazole 3-substituent, length of the linking chain (n), and the amine substituents are explored and reveal a large binding pocket in the 5-HT1D receptor domain. Oxadiazole substituents such as benzyl are accommodated without loss of agonist potency or efficacy. The incorporation of polar functionality on a phenyl or benzyl spacer group results in a 10-fold increase in affinity and functional potency. Optimal 5-HT1D activity is observed when the heterocycle is conjugated with the indole and the benzyl sulfonamides 20t and 20u represent some of the most potent 5-HT1D agonists known. Replacement of O for S in the heterocycle leads to a further increase in potency. Deletion of oxadiazole N-2 does not reduce activity, suggesting the requirement for only one H-bond acceptor in this location. The selectivity of these compounds for 5-HT1D receptors over other serotonergic receptors is discussed. Sulfonamide 20t shows > or = 1000-fold selectivity for 5-HT1D over 5-HT2, 5-HT1C, and 5-HT3 receptors and 10-fold selectivity with respect to 5-HT1A receptors. The functional activity of this series of compounds is studied and demonstrates high 5-HT1D receptor potency and efficacy comparable to that of 5-HT.