Margaret M. Schweri

[3H]Threo‐(±)‐Methylphenidate Binding to 3,4‐Dihydroxyphenylethylamine Uptake Sites in Corpus Striatum: Correlation with the Stimulant Properties of Ritalinic Acid Esters

Abstract: Saturable and stereoselective binding sites for [3H]threo‐(±)‐methylphenidate were characterized in rat brain membranes. The highest density of [3H]threo‐(±)‐ methylphenidate binding sites was found in the synapto somal fraction of corpus striatum. Scatchard analysis revealed a single class of noninteracting binding sites with an apparent dissociation constant (KD) of 235 nM and a maximum number of binding sites (Bmax) of 13.4 pmol/mg protein. Saturable, high‐affinity binding of [3H]threo‐(±)‐methylphenidate to striatal synaptosomal membranes was dependent on the presence of sodium ions. A good correlation (r = 0.88; p < 0.001) was observed between the potencies of various psychotropic drugs in displacing [3H]threo‐(±)‐methylphenidate from these sites and their potencies as inhibitors of [3H]3,4‐dihydroxyphenylethyl‐ amine ([3H]dopamine) uptake into striatal synaptosomes. A good correlation (r = 0.85; p < 0.001) was also observed between the potencies of a series of ritalinic acid esters in inhibiting [3H]threo‐(±)‐methylphenidate binding to striatal synaptosomal membranes and their potencies as motor stimulants in mice. These observations suggest that the binding sites for [3H]threo‐(±)‐methyl‐phenidate described here are associated with a dopamine uptake or transport complex, and that these sites may mediate the motor stimulant properties of ritalinic acid esters such as methylphenidate.

Blockade of 3-carbomethoxy-β-carboline induced seizures by diazepam and the benzodiazepine antagonists, Ro 15-1788 and CGS 8216

The benzodiazepine antagonists Ro 15-1788 and CGS 8216 blocked the clonic and tonic convulsions elicited by 3-carbomethoxy-beta-carboline (beta-CCM). The PD50 values for Ro 15-1788, CGS 8216, and diazepam were: 2.0, 0.6, and 2.0 mg/kg, respectively. Neither Ro 15-1788 nor CGS 8216 potentiated the effect of a threshold convulsant dose of beta-CCM. Moreover, these benzodiazepine antagonists neither attenuated nor potentiated the tremorigenic actions of another beta-carboline, harmaline. Diazepam, however, considerably reduced the tremorigenic actions of this drug.

Effects of methylphenidate analogues on phenethylamine substrates for the striatal dopamine transporter: potential as amphetamine antagonists?

Abstract : Methylphenidate (MPD) was found to inhibit competitively the striatal dopamine transporter (DAT) and bind at sites on the DAT in common with both cocaine (a non‐substrate site ligand) and amphetamine (a substrate site ligand). Some methylphenidate analogues modified on the aromatic ring and/or at the nitrogen were tested to determine whether the profile of inhibition could be altered. None was found to stimulate the release of dopamine in the time frame (≤60 s) of the experiments conducted, and each of the analogues tested was found to noncompetitively inhibit the transport of dopamine. It was found that halogenating the aromatic ring with chlorine (threo‐3,4‐dichloromethylphenidate hydrochloride ; compound 1) increased the affinity of MPD to inhibit the transport of dopamine. A derivative of MPD with simultaneous, single methyl group substitutions on the phenyl ring and at the nitrogen (threo‐N‐methyl‐4‐methylphenidate hydrochloride ; compound 2) bound at a site in common with MPD. A benzyl group positioned at the nitrogen (threo‐N‐benzylmethylphenidate hydrochloride ; compound 3) imparted properties to the inhibitor in which binding at substrate and non‐substrate sites could be distinguished. This analogue bound at a mutually interacting site with that of methylphenidate and had a Kint value of 4.29 μM. Furthermore, the N‐substituted analogues (compounds 2 and 3), although clearly inhibitors of dopamine transport, were found to attenuate dramatically the inhibition of dopamine transport by amphetamine, suggesting that the development of an antagonist for substrate analogue drugs of abuse may be possible.

Pharmacokinetic and pharmacodynamic factors contributing to the convulsant action of β-carboline-3-carboxylic acid esters

Abstract Both the methyl ester of β-carboline-3-carboxylic acid and the 6, 7-dimethoxy-4-ethyl derivative of this compound are potent convulsants in rodents, while the ethyl ester of β-carboline-3-carboxylic acid does not cause convulsions, even when administered at very high doses. The rate of degradation of these compounds by rat plasma ( in vitro ) parallels their potencies as convulsants. In contrast, 3-carboethoxy-β-carboline was found to potently elicit tonic and clonic convulsions in the squirrel monkey ( Saimiri sciureus ). Furthermore, the rate of degradation of 3-carboethoxy-β-carboline in monkey plasma ( in vitro ) is negligible compared with rats. No significant differences were observed in either the potency or efficacy of GABA to inhibit [ 3 H] β-carboethoxy-β-carboline binding in rat and monkey brain. These data strongly suggest that pharmacokinetic, as well as pharmacodynamic, factors may determine the pharmacologic profile of these β-carboline-3-carboxylic acid esters.

3-carboethoxy-β-carboline (β-CCE) elicits electroencephalographic seizures in rats: Reversal by the benzodiazepine antagonist CGS 8216

Abstract Intravenous administration of 3-carboethoxy-β-carboline (β-CCE, 10 mg/kg) to rats resulted in multiple bursts of rhythmic waves (2–4 second duration, 5–7 Hz) with amplitudes of 100–250 μV. Pretreatment of animals with the benzodiazepine receptor antagonist CGS 8216 prevented the electroencephalographic seizures elicited by β-CCE. This dose of CGS 8216 did not produce any electroencephalographic abnormalities when administered alone. These observations suggest that the electroencephalographic seizures elicited by β-CCE are mediated via an interaction with benzodiazepine receptors. An in vitro study of the rate of degradation of β-CCE and 3-carbomethoxy-β-carboline (β-CCM) in rat plasma demonstrated that the rate of degradation of the former compound was three times more rapid than the latter. These observations, taken together with previous studies demonstrating that parenteral administration of β-CCM elicits tonic and clonic seizures, suggests that pharmacokinetic factors may be involved in defining the pharmacologic profile of β-carboline-3-carboxylic acid esters.

Can stimulant binding and dopamine transport be differentiated? Studies with GBR 12783 derivatives.

Both 3- and 4-substituted GBR 12783 derivatives were synthesized in an effort to create site-directed cocaine antagonists. The potencies of these compounds to inhibit stimulant ([3H]WIN 35,428) binding and synaptosomal [3H]dopamine uptake were determined and compared with a large number of compounds assayed under identical experimental conditions. Three groups of compounds were identified which affected stimulant binding and dopamine transport to varying degrees. The 3-substituted GBR 12783 derivatives resembled the known non-amphetamine stimulant agents, in that they were approximately equipotent in inhibiting [3H]WIN 35,428 binding and [3H]dopamine transport. The 4-substituted GBR 12783 derivatives formed a second group that was approximately seven times more potent in the inhibition of [3H]dopamine uptake compared to [3H]WIN 35,428 binding. The third group, approximately one hundred-fold more potent at inhibiting [3H]dopamine uptake than stimulant binding, consisted mainly of substrates for the dopamine transporter. Although these GBR derivatives did not meet the criteria for potential cocaine antagonists, the results demonstrate that slight modifications of the molecular structure of a stimulant drug can differentially affect binding and transport. This finding holds great promise for the eventual development of a true pharmacological antagonist of cocaine.

Synthesis and pharmacology of irreversible affinity labels as potential cocaine antagonists: aryl 1,4-dialkylpiperazines related to GBR-12783

As part of a program aimed at designing irreversible antagonists of the stimulant and reinforcing properties of cocaine, derivatives of GBR-12783 containing electrophilic substituents were synthesized. GBR-12783, a potent and selective inhibitor of both stimulant binding and dopamine transport, was modified to incorporate either isothiocyanate or maleimido groups at the meta- or para-positions in one phenyl ring of the geminal diphenyl portion of the molecule. The effect of these compounds, as well as their respective amino- or nitro-substituted precursors, on stimulant binding to rat striatal tissue was studied using the [3H]methylphenidate radioreceptor assay. Under the assay conditions used, the compounds were found to have IC50s (nM) ranging from 11.9 (m-nitro) to 1677 (p-maleimido); the parent compound, GBR-12783, had an IC50 of 12.0. Using a washout technique (repeated washing with 100 mM KCl) which completely removed the tightly bound, but reversible GBR-12783, both the m- and p-isothiocyanate compounds were found to irreversibly inhibit binding of [3H]methylphenidate to the stimulant recognition site. The m-maleimido derivative also irreversibly inhibited binding, albeit with lower efficacy than was observed with the isothiocyanate compounds. Neither the p-maleimido, nor the amino or nitro intermediates, were capable of irreversible inhibition.