The primary focus of this research is to develop a better understanding of the pharmacological mechanisms underlying the behavioral effects of cocaine that lead to its abuse, and the consequences of that abuse. This better understanding will advance basic knowledge of the pharmacology of cocaine, and drug abuse. A better understanding of the pharmacology of cocaine and drug abuse will lead to advances in our discovery of new treatment modalities for cocaine abuse which will ultimately have a positive public health impact in curtailing drug abuse and the transmission of HIV infection. Unique compounds based on the structure of cocaine and benztropine (BZT) have been synthesized that provide information on the nature of the interaction of cocaine with its binding site on the dopamine transporter (DAT). Several studies have evaluated brain dopamine levels and the pharmacokinetics of novel compounds with regard to their penetration into brain. Previous studies showed that BZT and its analogs have relatively high affinity for the DAT without having cocaine-like behavioral effects. Because BZT also has affinity for histamine receptors, affinities of the analogs at these receptors were assessed to determine if histamine actions could explain the differences between BZT analogs and cocaine. H1 receptor affinity was determined by displacement of [3H]mepyramine (Kd=15.93 nM) in whole rat brain, H2 receptor with [125I]aminopotentidine (Kd=0.34 nM) in guinea pig striatum and H3 receptor with [3H]N- methylhistamine (Kd=0.16 nM) in rat frontal cortex. The affinities of the BZT analogs in displacing these radioligands were then compared to their affinities at the dopamine transporter using [3H]WIN 35,428 (Kd = 29.6 nM). The BZT analogs showed affinity for the H1 receptor ranging from 16 to 2200 nM. Affinities at H2 and H3 receptor ranged from 200-4000 nM and 5-100 uM, respectively. No correlation between affinities at DAT and any of the histamine receptors was observed. There were several compounds that showed affinities at H1 higher than DAT, but none that showed H2 or H3 affinity higher than DAT. Among those with H1 affinity higher than DAT were compounds that had behavioral effects distinct from those of cocaine. However, among those compounds with DAT affinities greater than H1 were others that had behavioral effects distinct from those of cocaine. Currently, these data do not indicate that affinities at histamine receptors alone can account for the differences between BZT analogs and cocaine. In related studies we have found that the histamine antagonists are generally ineffective in modifying the effects of cocaine. This functional study is in agreement with the binding study in indicating that that affinities at histamine receptors alone can account for the differences between BZT analogs and cocaine. A series of 2-substituted-3-phenyltropanes were synthesized as analogs of cocaine and tested in vitro for their abilitv to displace bound [3H]WIN 35,428 (2b) and inhibit dopamine uptake in rat caudate-putamen tissue. The analogs bound with high affinity (Ki values from 11 to 22 nM) to the dopamine transporter. Increased lipophilicity at the -C(2)-position was found to lead to increased binding affinity and increased dopamine uptake potency. The unsaturated ester 7 was found to possess weak dopamine uptake inhibition relative to the high binding affinity (IC50/Ki = 10.2). In vivo measurement of stimulated locomotor activity and drug discrimination against cocaine (10 mg/kg; i.p.) with selected analogs (4, 6 and 7) demonstrated that the behavioral effects of these drugs were approximately equipotent with cocaine. The SAR of this series of cocaine analogs supports a pharmacophore model in which lipophihc interactions between the -C(2)-position of 3-phenyltropanes and the cocaine binding site on the dopamine transporter lead to enhanced potency while electrostatic interactions have a nonspecific effect. Assessing the locomotor-stimulant effects of cocaine in DAT knockout (KO) mice has proven difficult due to significant differences in the baseline activity of KO and wild-type (WT) controls. We developed a procedure to assess the activity of these subjects at when expressed at comparable levels, and to do this in a manner that is not intrusive. Intrusive manipulation of the DAT KO mice can introduce confounding reactions to the stress from handling. Intravenous cocaine injection produced a typicall stimulation of locomotor activity in WT mice, however norepinephrine (NE) transporter KO mice exhibited a reduced response to their WT controls. A comparable sensitization developed in NET-KOs and WTs after several injections of cocaine. The DAT-KO and DAT-heterozygote (HT) mice displayed no locomotor activation following either acute or repeated cocaine administration. Contrary to previous reports, the present studies suggest that the NET may be involved in an acute response to cocaine, but not in the sensitization that develops to repeated injections of cocaine. In contrast, the DAT appears to be necessary for both the acute locomotor response to cocaine and the subsequent development of sensitization. Recent studies have determined that analogs of cocaine with di-phenyl ether substitutions at the 2-position of the tropane ring can compete for cocaine binding sites in brain and inhibit dopamine uptake. These compounds however, do not produce cocaine-like subjective effects in animals. This is the first time that a close cocaine analog has been shown to lack a behavioral profile similar to that of cocaine. Although these compounds do not produce subjective effects like those of cocaine, they retain psychomotor stimulant effects. The separation of these two effects is not normally obtained. Therefore these compounds may be important for a better understanding and differentiation of the biological substrates that underlie these two important behavioral effects of cocaine.
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