Previous research has suggested that lobeline, a plant alkaloid derived from Lobelia inflate, has potential to be an efficacious pharmacotherapy for the treatment of methamphetamine dependence. In addition to attenuating methamphetamineinduced dopaminergic alterations in vitro, lobeline has been shown to decrease the primary rewarding effects and discriminative stimulus properties of methamphetamine in rats. It is of clinical interest to assess the utility of lobeline to decrease methamphetamine conditioned cues as these cues have been shown to significantly contribute to relapse. The current studies assessed the ability of lobeline to block the acquisition and expression of methamphetamine-induced conditioned place preference in rats. Lobeline blocked the acquisition of methamphetamine-induced conditioned place preference when a low dose of methamphetamine was used during conditioning. However, this blockade was surmounted with higher doses of methamphetamine. Furthermore, the expression of methamphetamine-induced conditioned place preference is attenuated following repeated administration, indicating that lobeline not only blocks the primary reinforcing effects of methamphetamine, but it also blocks the environmental cues that become associated with drug administration. These results provide further evidence that lobeline may be an efficacious treatment for methamphetamine dependence. The rewarding properties of psychostimulants are thought to be mediated, at least in part, by the nucleus accumbens shell. The effects of lobeline on methamphetamine-induced alterations in this dopaminergic region were assessed using microdialysis in rats. Acute lobeline did not have an effect on the methamphetamine-induced increases in dopamine, indicating that repeated lobeline administration may be more efficacious. Interestingly, lobeline potentiated the methamphetamine-induced decrease of the dopamine metabolite, DOPAC. These results suggest that acute lobeline may function to redistribute vesicular dopamine pools within the terminal bouton.

This dissertation work investigated the effect of acute and repeated in vivo administration of lobeline on dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) function. The effects of lobeline were then compared to the effects of acute and repeated in vivo administration of methylphenidate and amphetamine to determine if lobeline produced similar effects compared to these Attention Deficit Hyperactivity Disorder (ADHD) medications. These medications are considered the first line of pharmacotherapy for ADHD, although there is a growing concern associated with their potential for abuse and other side effects. This merits the need for novel ADHD treatments that have a safer side effect profile. If lobeline alters DAT and VMAT2 function in the same way as methylphenidate or amphetamine, further investigation may be necessary to evaluate lobeline as a potential treatment for ADHD. Kinetic analysis of [3H]dopamine (DA) was utilized to determine the effect on DAT and VMAT2 function in rat striatum. Results from the DAT experiments, revealed that lobeline as well as amphetamine had no effect on DAT function. However, methylphenidate increased DAT function after acute and 7-day treatment. None of the drug treatment regimens altered Km. To determine if the methylphenidateinduced increase in DAT function was due to DAT trafficking, biotinylation and Western blot analyses were performed. Acute administration of methylphenidate did not alter surface DAT, however repeated administration of methylphenidate for 7 days decreased intracellular DAT, suggesting that methylphenidate redistributes DAT in a time-dependent manner. Similar results were found in the VMAT2 experiments. Lobeline and amphetamine had no effect on VMAT2 function after acute or repeated administration. Amphetamine decreased the Km after repeated administration for 7 days. Methylphenidate increased VMAT2 function after acute and repeated administration for 7 days. The overall results of these experiments suggest that methylphenidate interacts with DAT and VMAT2 in a different manner than amphetamine and lobeline. In addition, since lobeline and amphetamine had no effect on DAT and VMAT2 function, further investigation is warranted to elucidate the underlying mechanisms of the therapeutic actions of these agents. This additional information will aid in the development of novel treatments for ADHD.

Methamphetamine abuse is a serious public health concern affecting millions of people worldwide, and there are currently no viable pharmacotherapies to treat methamphetamine abuse. Methamphetamine increases extracellular dopamine (DA) concentrations through an interaction with the DA transporter (DAT) and the vesicular monoamine transporter-2 (VMAT2), leading to reward and abuse. While numerous studies have focused on DAT as a target for the discovery of pharmacotherapies to treat psychostimulant abuse, these efforts have been met with limited success. Taking into account the fact that methamphetamine interacts with VMAT2 to increase DA extracellular concentrations; the focus of the current work was to develop novel compounds that interact with VMAT2 to inhibit the effects of methamphetamine. Lobeline, the principal alkaloid found in Lobelia inflata, inhibits VMAT2 binding and function. Inhibition of VMAT2 was hypothesized to be responsible for the observed lobeline-induced inhibition of methamphetamine-evoked DA release in striatal slices and decrease in methamphetamine self-administration in rats. Lobeline has recently completed Phase Ib clinical trials demonstrating safety in methamphetamine abusers. Lobeline is also a potent inhibitor of nicotinic acetylcholine receptors (nAChRs), limiting selectivity for VMAT2. Chemical defunctionalization of the lobeline molecule afforded analogs, meso-transdiene (MTD) and lobelane, which exhibited decreased affinity for nAChRs. MTD, an unsaturated analog of lobeline, exhibited similar affinity for VMAT2 and increased affinity for DAT compared to lobeline. Conformationally-restricted MTD analogs exhibited decreased affinity for DAT compared to MTD, while retaining affinity at VMAT2. One analog, UKMH-106 exhibited high affinity and selectivity for VMAT2 and inhibited METH-evoked DA release from striatal slices. Unfortunately, the MTD analogs exhibited poor water solubility which limited further investigation of these promising analogs. Importantly lobelane, a saturated analog of lobeline, exhibited increased affinity and selectivity for VMAT2 compared to lobeline. To improve water solubility, a N-1,2-dihydroxypropyl (diol) moiety was incorporated into the lobelane molecule. GZ-793A, an N-1,2-diol analog, potently and competitively inhibited VMAT2 function, exhibiting over 50-fold selectivity for VMAT2 over DAT, serotonin transporters and nAChRs. GZ-793A released DA from preloaded synaptic vesicles, fitting a two-site model with the high-affinity site inhibited by tetrabenazine and reserpine (classical VMAT2 inhibitors), suggesting a VMAT2-mediated mechanism of release. Further, low concentrations of GZ-793A that selectively interact with high-affinity sites on VMAT2 to evoke DA release, inhibit methamphetamine-evoked DA release from synaptic vesicles. Results showed that increasing concentrations of GZ-793A produced a rightward shift in the METH concentration response; however, the Schild regression revealed a slope different from unity, consistent with surmountable allosteric inhibition. In addition, GZ-793A specifically inhibited methamphetamine-evoked DA release in striatal slices and methamphetamine self-administration in rats. To examine the possibility that GZ-793A produced DA depletion, the effect of a behaviorally active dose of GZ-793A on DA content in striatal tissue and striatal vesicles was determined. GZ-793A administration did not alter DA content in striatal tissue or vesicles and pretreatment with GZ-793A prior to methamphetamine administration did not exacerbate the DA depleting effects of methamphetamine. Importantly, GZ-793A was shown to protect against methamphetamine-induced striatal DA depletions. Thus, GZ-793A represents an exciting new lead in the development of pharmacotherapies to treat methamphetamine abuse.

Methamphetamine interacts with vesicular monoamine transporter-2 (VMAT2) to inhibit dopamine (DA) uptake and promotes DA release from presynaptic vesicles, increasing cytosolic DA available for methamphetamine-induced reverse transport by DA transporters. By inhibiting VMAT2, lobelane, a defunctionalized, saturated lobeline analog, decreases methamphetamine-evoked DA release and methamphetamine self-administration in rats. In this dissertation structure-activity relationships around the lobelane structure were investigated on racemic lobelane analogs with varying methylene linker lengths at central piperidine ring. Affinity for dihydrotetrabenazine (DTBZ) sites on VMAT2 and for inhibition of VMAT2 function was determined to be 0.88-63 and 0.024-4.6 µM, respectively, and positively correlated. The most potent and selective analog, (±)-cis-2-benzyl-6-(3-phenylpropyl)piperidine [(±)-GZ-730B], for VMAT2 uptake was identified as the lead. The ability of (±)-GZ-730B to inhibit methamphetamine-evoked [3H]DA release from striatal synaptic vesicles and endogenous DA release from striatal slices was determined. The lead analog-induced inhibition of methamphetamine-evoked vesicular [3H]DA release did not translate to inhibition of methamphetamine-evoked DA release in the more intact striatal slices. Moreover, poor water solubility of these lobelane analogs prohibited further in vivo work. Subsequent work focused on analogs with the C-3 and C-4 carbons in the piperidine ring eliminated to afford racemic acyclic lobelane analogs. Generally, acyclic analogs exhibited greater water solubility and less lipophilicity compared to lobelane. Acyclic analogs exhibited affinities (Ki = 0.096-17 μM) for [3H]DTBZ sites that correlated positively with affinity (Ki = 3.3-300 nM) for inhibition of [3H]DA uptake. Pure enantiomers of potent racemic analogs were synthesized, and found to potently, selectively, and competitively inhibit [3H]DA uptake at VMAT2 and to release vesicular [3H]DA in a biphasic manner. Lead enantiomer (R)-N-(1-phenylpropan-2-yl)-3-phenylpropan-1-amine [(R)-GZ-924] inhibited methamphetamine-evoked [3H]DA release from striatal synaptic vesicles, but not from the more intact striatal slices. Surprisingly, (R)-GZ-924 inhibited nicotine-evoked [3H]DA overflow from striatal slices, revealing nonspecific effects. Importantly, (R)-GZ-924 inhibited methamphetamine self-administration in rats. However, the analog also inhibited food-maintained responding, revealing a lack of specificity. The lead analog will not be pursued further as a pharmacotherapy due to the lack of specificity. Further evaluation of the pharmacophore is needed to discover analogs which specifically inhibit the neurochemical and behavioral effect of methamphetamine.

Title: Study of pharmacology and function of binding sites of nicotinic acetylcholine receptors Author: Mgr. Martina Kaniaková Department: Institute of Physiology AS CR, v.v.i. Supervisor: RNDr. Jan Krůšek, CSc., Institute of Physiology AS CR, v.v.i. Abstract: Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels. We use the whole-cell patch-clamp technique to study functional and pharmacological properties of muscle and neuronal nicotinic receptors. Rat neuronal receptors were heterologously expressed in COS cells and human embryonic muscle receptors were studied in TE671 cells. Lobeline, a plant alkaloid with a long history of therapeutic use, interacts with the classical agonist-binding site of nAChRs. The final result of this interaction depends on the receptor subtype, lobeline and other agonists concentrations and the time schedule of application. Generally, lobeline is a very weak partial agonist eliciting deep desensitization at several subtypes of nAChRs. In combination with other agonists, lobeline acts as a competitive antagonist or coagonist. Using point mutation procedure we studied the functional role of negatively charged amino acids in the F-loop of β2 and β4 subunits of neuronal receptors. Neutralising mutations in β4 subunit led to up to eighteen-fold increase in the...