Multiple Binding Sites for [\u3csup\u3e125\u3c/sup\u3eI]RTI-121 and Other Cocaine Analogs in Rat Frontal Cerebral Cortex

In an effort to identify novel binding sites for cocaine and its analogs, we carried out binding studies with the high-affinity and selective ligand [125I]RTI-121 in rat frontal cortical tissue. Very low densities of binding sites were found. Saturation analysis revealed that the binding was to both high- and low-affinity sites. Pharmacological competition studies were carried out with inhibitors of the dopamine, norepinephrine, and serotonin transporters. The various transporter inhibitors inhibited the. binding of 15 pM [125I]RTI-121 in a biphasic fashion following a two-site binding model. The resultant data were complex and did not suggest a simple association with any single transporter. Correlational analysis supported the following hypothesis: [125I] RTI-121 binds to known transporters and not to novel sites; these include dopamine, norepinephrine, and serotonin transporters. Immunoprecipitation of transporters photoaffinity labeled with [125]RTI-82 and subsequent analysis of SDS-page gels revealed the presence of authentic dopamine transporters in these samples; displacement of the photoaffinity label occurred with a typical dopamine transporter pharmacology. These data are compatible with the binding properties of RTI- 121 and the presence of several known transporters in the tissue studied

Highly selective chiral N-substituted 3alpha-[bis(4'-fluorophenyl)methoxy]tropane analogues for the dopamine transporter: synthesis and comparative molecular field analysis

In a continuing effort to further characterize the role of the dopamine transporter in the pharmacological effects of cocaine, a series of chiral and achiral N-substituted analogues of 3alpha-[bis(4'-fluorophenyl)methoxy]tropane (5) has been prepared as potential selective dopamine transporter ligands. These novel compounds displaced [(3)H]WIN 35,428 binding from the dopamine transporter in rat caudate putamen with K(i) values ranging from 13. 9 to 477 nM. Previously, it was reported that 5 demonstrated a significantly higher affinity for the dopamine transporter than the parent drug, 3alpha-(diphenylmethoxy)tropane (3; benztropine). However, 5 remained nonselective over muscarinic m(1) receptors (dopamine transporter, K(i) = 11.8 nM; m(1), K(i) = 11.6 nM) which could potentially confound the interpretation of behavioral data, for this compound and other members of this series. Thus, significant effort has been directed toward developing analogues that retain high affinity at the dopamine transporter but have decreased affinity at muscarinic sites. Recently, it was discovered that by replacing the N-methyl group of 5 with the phenyl-n-butyl substituent (6) retention of high binding affinity at the dopamine transporter (K(i) = 8.51 nM) while decreasing affinity at muscarinic receptors (K(i) = 576 nM) was achieved, resulting in 68-fold selectivity. In the present series, a further improvement in the selectivity for the dopamine transporter was accomplished, with the chiral analogue (S)-N-(2-amino-3-methyl-n-butyl)-3alpha-[bis(4'-fluorophenyl)metho xy] tropane (10b) showing a 136-fold selectivity for the dopamine transporter versus muscarinic m(1) receptors (K(i) = 29.5 nM versus K(i) = 4020 nM, respectively). In addition, a comparative molecular field analysis (CoMFA) model was derived to correlate the binding affinities of all the N-substituted 3alpha-[bis(4'-fluorophenyl)methoxy]tropane analogues that we have prepared with their 3D-structural features. The best model (q(2) = 0. 746) was used to accurately predict binding affinities of compounds in the training set and in a test set. The CoMFA coefficient contour plot for this model, which provides a visual representation of the chemical environment of the binding domain of the dopamine transporter, can now be used to design and/or predict the binding affinities of novel drugs within this class of dopamine uptake inhibitors

Structure-activity relationships at monoamine transporters and muscarinic receptors for N-substituted-3alpha-(3'-chloro-, 4'-chloro-, and 4',4''-dichloro-substituted-diphenyl)methoxytropanes

The design, synthesis, and evaluation of 3alpha-(diphenylmethoxy)tropane (benztropine) analogues have provided potent and selective probes for the dopamine transporter. Structure-activity relationships (SARs) have been developed that contrast with those described for cocaine, despite significant structural similarity. Furthermore, behavioral evaluation of many of the benztropine analogues in animal models of cocaine abuse has suggested that these two classes of tropane-based dopamine uptake inhibitors have distinct pharmacological profiles. In general, the benztropine analogues do not demonstrate efficacious locomotor stimulation in mice, do not fully substitute for a cocaine discriminative stimulus, and are not appreciably self-administered in rhesus monkeys. These compounds are generally more potent than cocaine as dopamine uptake inhibitors in vitro, although their actions in vivo are not consistent with this action. These observations suggest that differing binding profiles at the serotonin and norepinephrine transporters as well as at muscarinic receptors might have significant impact on the pharmacological actions of these compounds. In addition, by varying the structures of the parent compounds and thereby modifying their physical properties, pharmacokinetics as well as pharmacodynamics will be directly affected. Therefore, in an attempt to systematically evaluate the impact of chemical modification on these actions, a series of N-substituted (H, CH3, allyl, benzyl, propylphenyl, and butylphenyl) analogues of 3'-chloro-, 4'-chloro-, and 4,4''-dichloro-3alpha-(diphenylmethoxy)tropanes were synthesized. These compounds were evaluated for displacement, in rat tissue, of [3H]WIN 35,428 from the dopamine transporter, [3H]citalopram from the serotonin transporter, [3H]nisoxetine from the norepinephrine transporter, and [3H]pirenzepine from muscarinic m1 receptors. SARs were developed and compared to a series of N-substituted-3alpha-(bis-4'-fluorophenyl)methoxytropanes. The present SARs followed previously reported studies with the single exception of the N-butylphenyl substituent, which did not provide the high affinity binding in any of these three sets of analogues, as it did in the 4',4''-difluoro series. X-ray crystallographic analyses of the three parent ligands (1a, 2a, and 3a) were compared to that of 3alpha-(bis-4'-fluorophenyl)methoxytropane which provided supportive evidence toward the proposal that the combination of steric bulk in both the 3-position and the N-substituent, in this class of compounds, is not optimal for binding at the dopamine transporter. These studies provide binding profile data that can now be used to correlate with future behavioral analyses of these compounds and may provide insight into the kind of binding profile that might be targeted as a potential treatment for cocaine abuse

JPET #185025 Decreases in Cocaine Self Administration with Dual Inhibition of the Dopamine Transporter and σ Receptors

Abstract: 241 words Introduction: 678 word

Radiometric studies on the oxidation of (1-14c) fatty acids by drug-susceptible and drug-resistant mycobacteria

A radiometric assay system has been used to study oxidation patterns of (1-14C) fatty acids by drug-susceptible and drug-resistant organisms of the genus Mycobacterium. Two strains of M. tuberculosis susceptible to all drugs, H37Rv and Erdman, were used. Drug-resistant organisms included in this investigation were M. tuberculosis H37Rv resistant to 5 ug/ml isoniazid, M. bovis, M. avium, M. intracellular, M. kansasii and M. chelonei. The organisms were inoculated in sterile reaction vials containing liquid 7H9 medium, 10% ADC enrichment and 1.0 uCi of one of the (1-14C) fatty acids (butyric, hexánoic, octanoic, decanoic, lauric, myristic, palmitic, stearic, oleic, linoleic, linolenic). Vials were incubated at 37°C and the 14CO2 envolved was measured daily for 3 days with a Bactec R-301 instrument. Although each individual organism displayed a different pattern of fatty oxidation, these patterns were not distinctive enough for identification of the organism. No combination of fatty acids nor preferential oxidation of long chain or of short chain fatty acids were able to separate susceptible from resistant organisms. Further investigation with a larger number of drug susceptible mycobacteria including assimilation studies and oxidation of other substrates may be required to achieve a distinction between drug-susceptible and drug-resistant mycobacteria