The vesicular monoamine transporter-2: an important pharmacological target for the discovery of novel therapeutics to treat methamphetamine abuse

Abstract

Methamphetamine abuse escalates, but no approved therapeutics are available to treat addicted individuals. Methamphetamine increases extracellular dopamine in reward-relevant pathways by interacting at vesicular monoamine transporter-2 (VMAT2) to inhibit dopamine uptake and promote dopamine release from synaptic vesicles, increasing cytosolic dopamine available for reverse transport by the dopamine transporter (DAT). VMAT2 is the target of our iterative drug discovery efforts to identify pharmacotherapeutics for methamphetamine addiction. Lobeline, the major alkaloid in Lobelia inflata, potently inhibited VMAT2, methamphetamine-evoked striatal dopamine release, and methamphetamine self-administration in rats but exhibited high affinity for nicotinic acetylcholine receptors (nAChRs). Defunctionalized, unsaturated lobeline analog, meso-transdiene (MTD), exhibited lobeline-like in vitro pharmacology, lacked nAChR affinity, but exhibited high affinity for DAT, suggesting potential abuse liability. The 2,4-dicholorophenyl MTD analog, UKMH-106, exhibited selectivity for VMAT2 over DAT, inhibited methamphetamine-evoked dopamine release, but required a difficult synthetic approach. Lobelane, a saturated, defunctionalized lobeline analog, inhibited the neurochemical and behavioral effects of methamphetamine; tolerance developed to the lobelane-induced decrease in methamphetamine self-administration. Improved drug-likeness was afforded by the incorporation of a chiral N-1,2-dihydroxypropyl moiety into lobelane to afford GZ-793A, which inhibited the neurochemical and behavioral effects of methamphetamine, without tolerance. From a series of 2,5-disubstituted pyrrolidine analogs, AV-2-192 emerged as a lead, exhibiting high affinity for VMAT2 and inhibiting methamphetamine-evoked dopamine release. Current results support the hypothesis that potent, selective VMAT2 inhibitors provide the requisite preclinical behavioral profile for evaluation as pharmacotherapeutics for methamphetamine abuse and emphasize selectivity for VMAT2 relative to DAT as a criterion for reducing abuse liability of the therapeutic.

Keywords: AV-2-192; GZ-793A; Lobelane; Lobeline; Methamphetamine; VMAT2.

Figure 2.1. Chemical structures

Tetrabenazine (TBZ) is a benzoquinolizine compound that reversibly inhibits VMAT2 function. Lobeline is a lipophilic, nonpyridino alkaloid present in Lobelia inflata. Lobelane is a defunctionalized, saturated meso-analog of lobe-line. MTD is a lobelane analog bearing unsaturated phenethyl side chain linkers. GZ-793A is a lobelane analog bearing a dihydroxypropyl moiety on the central nitrogen atom and methoxy substituents in the para-position on the phenyl rings. AV-1-229 bears a high degree of structural similarity to lobelane, with substitution of a pyrrolidine ring for the piperidine ring. UKCP-110, also known as AV-1-228, is the demethylated nor-analog of AV-1-229.

Figure 2.2

Model of the DA presynaptic terminal in the presence of methamphetamine (left) and in the presence of lobeline analog plus methamphetamine.

Figure 2.3. Chemical structures of the novel 2,5-disubstituted pyrrolidine analogs

Analogs are grouped according to structural similarity: Group 1: analogs with either lengthened or shortened side chain linkers in comparison with AV-1-229; Group 2: analogs incorporating methoxy and hydroxyl moieties on the phenyl rings; and Group 3: analogs incorporating phenylethanol and phenylmethanol substituents on the nitrogen atom on the central pyrrolidine ring.

Figure 2.4. Positive correlation between 2,5-disubstituted pyrrolidine analog affinity for the DTBZ binding site and the DA uptake site

Data are K_i values obtained from concentration–response curves for analog-induced inhibition of [³H]DTBZ binding and [³H]DA uptake (data not shown). Spearman analysis revealed a positive correlation (Spearman r =0.72; p <0.001) between pyrrolidine analog affinity for the DTBZ binding site and the DA uptake site on VMAT2.

Figure 2.5. Kinetic analysis of the inhibition of [³H]DA uptake at VMAT2 by AV-2-192 and AV-2-197

The nor-pyrrolidine analog (AV-2-192) containing single methylene unit side chain linkers in the cis conformation and its N-methyl counterpart (AV-2-197) were evaluated to determine mechanism of inhibition of [³H]DA uptake. Concentrations of analog utilized (in parentheses) for the kinetic analysis were the respective K_i concentrations from the inhibition curves (data not shown). V_max and K_m values (±SEM) are provided in Table 2.2 (n =4–6 rats/analog).

Figure 2.6. AV-2-192 and AV-2-197 stimulate fractional DOPAC release from rat striatal slice preparations

Top and bottom panels illustrate the time course for AV-2-192 and AV-2-197, respectively, to stimulate fractional DOPAC release. Each striatal slice was superfused with either a single concentration (0.3–30 μM) of AV-2-192 or AV-2-197 alone during the 30 min time period prior to the inclusion of 5 μM methamphetamine (METH) in the buffer. Arrow denotes time point at which analog was added to the buffer. METH was added to the buffer after 30 min and remained in the buffer for 15 min. Buffer control represents slices superfused in the absence of analog and METH. Data are mean (±SEM) pg/min/ml slice weight. ^*p <0.05 different from buffer control condition.

Figure 2.7. Time course of AV-2-192 and AV-2-197 inhibition of methamphetamine (METH)-evoked fractional DA release

Top and bottom panels illustrate the time course for AV-2-192 and AV-2-197, respectively, to inhibit METH-evoked fractional DA release. Each striatal slice was superfused with either a single concentration (0.3–30 μM) of AV-2-192 or AV-2-197 alone during the 30-min time period prior to the inclusion of 5 μM METH in the buffer. Arrow denotes time point at which analog was added to the buffer. METH was added to the buffer after 30 min and remained in the buffer for 15 min. METH control represents slices superfused with METH in the absence of analog. Data are mean (±SEM) pg/min/ml slice weight. ^*p <0.05 different from the METH control condition.

Figure 2.8. AV-2-192 and AV-2-197 inhibit methamphetamine-evoked total DA overflow from rat striatal slices

Data are total DA overflow following methamphetamine administration as a function of concentration of AV-2-192 (top panel) and AV-2-197 (bottom panel). CON denotes slice superfused with 5 μM methamphetamine in absence of analog. Data are mean (±SEM) pg/ml/mg slice weight. ^*p <0.05 and ^**p <0.01 different from the methamphetamine control condition.