r-bPiDI, an α6ß2* Nicotinic Receptor Antagonist, Decreases Nicotine-Evoked Dopamine Release and Nicotine Reinforcement.

α6ß2* nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic neurons mediate nicotine-evoked dopamine (DA) release and nicotine reinforcement. α6ß2* antagonists inhibit these effects of nicotine, such that α6ß2* receptors serve as therapeutic targets for nicotine addiction. The present research assessed the neuropharmacology of 1,10-bis(3-methyl-5,6-dihydropyridin-1(2H)-yl)decane (r-bPiDI), a novel small-molecule, tertiary amino analog of its parent compound, N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI). bPiDI was previously shown to inhibit both nicotine-evoked DA release and the reinforcing effects of nicotine. In the current study, r-bPiDI inhibition of [(3)H]nicotine and [(3)H]methyllycaconitine binding sites was evaluated to assess interaction with the recognition binding sites on α4ß2* and α7* nAChRs, respectively. Further, r-bPiDI inhibition of nicotine-evoked DA release in vitro in the absence and presence of α-conotoxin MII and following chronic in vivo nicotine administration were determined. The ability of r-bPiDI to decrease nicotine self-administration and food-maintained responding was also assessed. Results show that r-bPiDI did not inhibit [(3)H]nicotine or [(3)H]methyllycaconitine binding, but potently (IC50 = 37.5 nM) inhibited nicotine-evoked DA release from superfused striatal slices obtained from either drug naïve rats or from those repeatedly treated with nicotine. r-bPiDI inhibition of nicotine-evoked DA release was not different in the absence or presence of α-conotoxin MII, indicating that r-bPiDI acts as a potent, selective α6ß2* nAChR antagonist. Acute systemic administration of r-bPiDI specifically decreased nicotine self-administration by 75 %, and did not alter food-maintained responding, demonstrating greater specificity relative to bPiDI and bPiDDB, as well as the tertiary amino analog r-bPiDDB. The current work describes the discovery of r-bPiDI, a tertiary amino, α-conotoxin MII-like small molecule that acts as a potent and selective antagonist at α6ß2* nAChRs to specifically decrease nicotine self-administration in rats, thus, establishing r-bPiDI as a lead compound for development as a treatment for nicotine addiction.

GZ-793A, a lobelane analog, interacts with the vesicular monoamine transporter-2 to inhibit the effect of methamphetamine.

(R)-3-[2,6-cis-Di(4-methoxyphenethyl)piperidin-1-yl]propane-1,2-diol (GZ-793A) inhibits methamphetamine-evoked dopamine release from striatal slices and methamphetamine self-administration in rats. GZ-793A potently and selectively inhibits dopamine uptake at the vesicular monoamine transporter-2 (VMAT2). This study determined GZ-793A's ability to evoke [³H]dopamine release and inhibit methamphetamine-evoked [³H]dopamine release from isolated striatal synaptic vesicles. Results show GZ-793A concentration-dependent [³H]dopamine release; nonlinear regression revealed a two-site model of interaction with VMAT2 (High- and Low-EC50 = 15.5 nM and 29.3 µM, respectively). Tetrabenazine and reserpine completely inhibited GZ-793A-evoked [³H]dopamine release, however, only at the High-affinity site. Low concentrations of GZ-793A that interact with the extravesicular dopamine uptake site and the High-affinity intravesicular DA release site also inhibited methamphetamine-evoked [³H]dopamine release from synaptic vesicles. A rightward shift in the methamphetamine concentration-response was evident with increasing concentrations of GZ-793A, and the Schild regression slope was 0.49 ± 0.08, consistent with surmountable allosteric inhibition. These results support a hypothetical model of GZ-793A interaction at more than one site on the VMAT2 protein, which explains its potent inhibition of dopamine uptake, dopamine release via a High-affinity tetrabenazine- and reserpine-sensitive site, dopamine release via a Low-affinity tetrabenazine- and reserpine-insensitive site, and a low-affinity interaction with the dihydrotetrabenazine binding site on VMAT2. GZ-793A inhibition of the effects of methamphetamine supports its potential as a therapeutic agent for the treatment of methamphetamine abuse.

A translational pharmacology approach to understanding the predictive value of abuse potential assessments.

Within the drug development industry the assessment of abuse potential for novel molecules involves the generation and review of data from multiple sources, ranging from in-vitro binding and functional assays through to in-vivo nonclinical models in mammals, as well as collection of information from studies in humans. This breadth of data aligns with current expectations from regulatory agencies in both the USA and Europe. To date, there have been a limited number of reviews on the predictive value of individual models within this sequence, but there has been no systematic review on how each of these models contributes to our overall understanding of abuse potential risk. To address this, we analyzed data from 100 small molecules to compare the predictive validity for drug scheduling status of a number of models that typically contribute to the abuse potential assessment package. These models range from the assessment of in-vitro binding and functional profiles at receptors or transporters typically associated with abuse through in-vivo models including locomotor activity, drug discrimination, and self-administration in rodents. Data from subjective report assessments in humans following acute dosing of compounds were also included. The predictive value of each model was then evaluated relative to the scheduling status of each drug in the USA. In recognition of the fact that drug scheduling can be influenced by factors other than the pharmacology of the drug, we also evaluated the predictive value of each assay for the outcome of the human subjective effects assessment. This approach provides an objective and statistical assessment of the predictive value of many of the models typically applied within the pharmaceutical industry to evaluate abuse potential risk. In addition, the impact of combining information from multiple models was examined. This analysis adds to our understanding of the predictive value of each model, allows us to critically evaluate the benefits and limitations of each model, and provides a method for identifying opportunities for improving our assessment and prediction of abuse liability risk in the future.

Novel N-1,2-dihydroxypropyl analogs of lobelane inhibit vesicular monoamine transporter-2 function and methamphetamine-evoked dopamine release.

Lobelane, a chemically defunctionalized saturated analog of lobeline, has increased selectivity for the vesicular monoamine transporter 2 (VMAT2) compared with the parent compound. Lobelane inhibits methamphetamine-evoked dopamine (DA) release and decreases methamphetamine self-administration. Unfortunately, tolerance develops to the ability of lobelane to decrease these behavioral effects of methamphetamine. Lobelane has low water solubility, which is problematic for drug development. The aim of the current study was to determine the pharmacological effect of replacement of the N-methyl moiety with a chiral N-1,2-dihydroxypropyl (N-1,2-diol) moiety, which enhances water solubility, altering the configuration of the N-1,2-diol moiety and incorporating phenyl ring substituents into the analogs. To determine VMAT2 selectivity, structure-activity relationships also were generated for inhibition of DA and serotonin transporters. Analogs with the highest potency for inhibiting DA uptake at VMAT2 and at least 10-fold selectivity were evaluated further for ability to inhibit methamphetamine-evoked DA release from superfused striatal slices. (R)-3-[2,6-cis-di(4-methoxyphenethyl)piperidin-1-yl]propane-1,2-diol (GZ-793A), the (R)-4-methoxyphenyl-N-1,2-diol analog, and (R)-3-[2,6-cis-di(1-naphthylethyl)piperidin-1-yl]propane-1,2-diol (GZ-794A), the (R)-1-naphthyl-N-1,2-diol analog, exhibited the highest potency (K(i) ∼30 nM) inhibiting VMAT2, and both analogs inhibited methamphetamine-evoked endogenous DA release (IC(50) = 10.6 and 0.4 µM, respectively). Thus, the pharmacophore for VMAT2 inhibition accommodates the N-1,2-diol moiety, which improves drug-likeness and enhances the potential for the development of these clinical candidates as treatments for methamphetamine abuse.

meso-Transdiene analogs inhibit vesicular monoamine transporter-2 function and methamphetamine-evoked dopamine release.

Lobeline, a nicotinic receptor antagonist and neurotransmitter transporter inhibitor, is a candidate pharmacotherapy for methamphetamine abuse. meso-Transdiene (MTD), a lobeline analog, lacks nicotinic receptor affinity, retains affinity for vesicular monoamine transporter 2 (VMAT2), and, surprisingly, has enhanced affinity for dopamine (DA) and serotonin transporters [DA transporter (DAT) and serotonin transporter (SERT), respectively]. In the current study, MTD was evaluated for its ability to decrease methamphetamine self-administration in rats relative to food-maintained responding. MTD specifically decreased methamphetamine self-administration, extending our previous work. Classical structure-activity relationships revealed that more conformationally restricted MTD analogs enhanced VMAT2 selectivity and drug likeness, whereas affinity at the dihydrotetrabenazine binding and DA uptake sites on VMAT2 was not altered. Generally, MTD analogs exhibited 50- to 1000-fold lower affinity for DAT and were equipotent or had 10-fold higher affinity for SERT, compared with MTD. Representative analogs from the series potently and competitively inhibited [(3)H]DA uptake at VMAT2. (3Z,5Z)-3,5-bis(2,4-dichlorobenzylidene)-1-methylpiperidine (UKMH-106), the 3Z,5Z-2,4-dichlorophenyl MTD analog, had improved selectivity for VMAT2 over DAT and importantly inhibited methamphetamine-evoked DA release from striatal slices. In contrast, (3Z,5E)-3,5-bis(2,4-dichlorobenzylidene)-1-methylpiperidine (UKMH-105), the 3Z,5E-geometrical isomer, inhibited DA uptake at VMAT2, but did not inhibit methamphetamine-evoked DA release. Taken together, these results suggest that these geometrical isomers interact at alternate sites on VMAT2, which are associated with distinct pharmacophores. Thus, structural modification of the MTD molecule resulted in analogs exhibiting improved drug likeness and improved selectivity for VMAT2, as well as the ability to decrease methamphetamine-evoked DA release, supporting the further evaluation of these analogs as treatments for methamphetamine abuse.

The novel pyrrolidine nor-lobelane analog UKCP-110 [cis-2,5-di-(2-phenethyl)-pyrrolidine hydrochloride] inhibits VMAT2 function, methamphetamine-evoked dopamine release, and methamphetamine self-administration in rats.

Both lobeline and lobelane attenuate methamphetamine self-administration in rats by decreasing methamphetamine-induced dopamine release via interaction with vesicular monoamine transporter-2 (VMAT2). A novel derivative of nor-lobelane, cis-2,5-di-(2-phenethyl)-pyrrolidine hydrochloride (UKCP-110), and its trans-isomers, (2R,5R)-trans-di-(2-phenethyl)-pyrrolidine hydrochloride (UKCP-111) and (2S,5S)-trans-di-(2-phenethyl)-pyrrolidine hydrochloride (UKCP-112), were evaluated for inhibition of [(3)H]dihydrotetrabenazine binding and [(3)H]dopamine uptake by using a rat synaptic vesicle preparation to assess VMAT2 interaction. Compounds were evaluated for inhibition of [(3)H]nicotine and [(3)H]methyllycaconitine binding to assess interaction with the major nicotinic receptor subtypes. In addition, compounds were evaluated for inhibition of methamphetamine-evoked endogenous dopamine release by using striatal slices. The most promising compound, UKCP-110, was evaluated for its ability to decrease methamphetamine self-administration and methamphetamine discriminative stimulus cues and for its effect on food-maintained operant responding. UKCP-110, UKCP-111, and UKCP-112 inhibited [(3)H]dihydrotetrabenazine binding (K(i) = 2.66 ± 0.37, 1.05 ± 0.10, and 3.80 ± 0.31 µM, respectively) and had high potency inhibiting [(3)H]dopamine uptake (K(i) = 0.028 ± 0.001, 0.046 ± 0.008, 0.043 ± 0.004 µM, respectively), but lacked affinity at nicotinic receptors. Although the trans-isomers did not alter methamphetamine-evoked dopamine release, UKCP-110 inhibited (IC(50) = 1.8 ± 0.2 µM; I(max) = 67.18 ± 6.11 µM) methamphetamine-evoked dopamine release. At high concentrations, UKCP-110 also increased extracellular dihydroxyphenylacetic acid. It is noteworthy that UKCP-110 decreased the number of methamphetamine self-infusions, while having no effect on food-reinforced behavior or the methamphetamine stimulus cue. Thus, UKCP-110 represents a new lead in the development of novel pharmacotherapies for the treatment of methamphetamine abuse.

Lobeline esters as novel ligands for neuronal nicotinic acetylcholine receptors and neurotransmitter transporters.

Vesicular monoamine transporter-2 (VMAT2) is a viable target for development of pharmacotherapies for psychostimulant abuse. Lobeline (1) is a potent antagonist at alpha4beta2 * nicotinic acetylcholine receptors, has moderate affinity (K(i)=5.46microM) for VMAT2, and is being investigated currently as a clinical candidate for treatment of psychostimulant abuse. A series of carboxylic acid and sulfonic acid ester analogs 2-20 of lobeline were synthesized and evaluated for interaction with alpha4beta2 * and alpha7 * neuronal nicotinic acetylcholine receptors (nAChRs), the dopamine transporter (DAT), serotonin transporter (SERT) and VMAT2. Both carboxylic acid and sulfonic acid esters had low affinity at alpha7 * nAChRs. Similar to lobeline (K(i)=4nM), sulfonic acid esters had high affinity at alpha4beta2 * (K(i)=5-17nM). Aromatic carboxylic acid ester analogs of lobeline (2-4) were 100-1000-fold less potent than lobeline at alpha4beta2 * nAChRs, whereas aliphatic carboxylic acid ester analogs were 10-100-fold less potent than lobeline at alpha4beta2 *. Two representative lobeline esters, the 10-O-benzoate (2) and the 10-O-benzenesulfonate (10) were evaluated in the (36)Rb(+) efflux assay using rat thalamic synaptosomes, and were shown to be antagonists with IC(50) values of 0.85microM and 1.60microM, respectively. Both carboxylic and sulfonic acid esters exhibited a range of potencies (equipotent to 13-45-fold greater potency compared to lobeline) for inhibiting DAT and SERT, respectively, and like lobeline, had moderate affinity (K(i)=1.98-10.8microM) for VMAT2. One of the more interesting analogs, p-methoxybenzoic acid ester 4, had low affinity at alpha4beta2 * nAChRs (K(i)=19.3microM) and was equipotent with lobeline, at VMAT2 (K(i)=2.98microM), exhibiting a 6.5-fold selectivity for VMAT2 over alpha4beta2 nAChRs. Thus, esterification of the lobeline molecule may be a useful structural modification for the development of lobeline analogs with improved selectivity at VMAT2.

Regulatory and strategic considerations for addressing immunogenicity and related responses in biopharmaceutical development programs.

The last three decades have seen the biotherapeutic drug market evolve from promising concept to market dominance in a range of clinical indications. This growth has been spurred by the success of established drug classes like monoclonal antibodies, but also by the introduction of biosimilars, and more recently, multiple novel cell and gene therapies. Biotherapeutic drug development presents many unique challenges, but unintended immune responses are among the most common reasons for program attrition. Anti-drug antibodies can impact the safety and efficacy of drug products, and related immune responses, like the cytokine release syndrome that occurred in the infamous TGN-1412 clinical trial, can be challenging to predict with nonclinical models. For this reason, it is important that development programs proceed with a scientifically grounded and measured approach to these responses. This process begins at the discovery stage with the application of "quality by design," continues into the clinic with the development of quality assays and management strategies, and culminates in the effective presentation of this information in regulatory documents. This review provides an overview of some of the key strategic and regulatory considerations for biotherapeutics as they pertain to immunogenicity and related responses.

The promiscuity of the dopamine transporter: implications for the kinetic analysis of [3H]serotonin uptake in rat hippocampal and striatal synaptosomes.

Evidence indicates that monoaminergic neurotransmitter transporters are promiscuous, transporting substrates other than their cognate neurotransmitters. For example, serotonin is transported by the dopamine transporter (DAT) under conditions in which serotonin transporter (SERT) activity is eliminated (e.g., pharmacological inhibition). We performed a kinetic analysis of [(3)H]serotonin uptake in rat striatal synaptosomes (expressing DAT and SERT) and hippocampal synaptosomes (expressing SERT, but not DAT). Nonspecific [(3)H]serotonin uptake was defined as the amount of uptake remaining in the presence of fluoxetine (10microM) or paroxetine (0.05microM). In hippocampal synaptosomes, K(m) and V(max) values for [(3)H]serotonin uptake did not differ whether fluoxetine or paroxetine was used to define nonspecific uptake. However, in striatal synaptosomes, both K(m) and V(max) values for [(3)H]serotonin uptake were greater when fluoxetine, rather than paroxetine, was used to define nonspecific uptake. These data suggest that, at the concentrations employed, fluoxetine inhibits serotonin uptake at both DAT and SERT, whereas paroxetine only inhibits serotonin uptake at SERT. Thus, when DAT is inhibited by GBR 12909, kinetic parameters for serotonin uptake via SERT in striatum are not different from those obtained in hippocampus. These findings have important implications regarding the analysis of monoaminergic reuptake in brain regions exhibiting heterogeneous transporter expression.

GZ-793A inhibits the neurochemical effects of methamphetamine via a selective interaction with the vesicular monoamine transporter-2.

Lobeline and lobelane inhibit the behavioral and neurochemical effects of methamphetamine via an interaction with the vesicular monoamine transporter-2 (VMAT2). However, lobeline has high affinity for nicotinic receptors, and tolerance develops to the behavioral effects of lobelane. A water-soluble analog of lobelane, R-N-(1,2-dihydroxypropyl)-2,6-cis-di-(4-methoxyphenethyl)piperidine hydrochloride (GZ-793A), also interacts selectively with VMAT2 to inhibit the effects of methamphetamine, but does not produce behavioral tolerance. The current study further evaluated the mechanism underlying the GZ-793A-mediated inhibition of the neurochemical effects of methamphetamine. In contrast to lobeline, GZ-793A does not interact with the agonist recognition site on α4ß2* and α7* nicotinic receptors. GZ-793A (0.3-100µM) inhibited methamphetamine (5µM)-evoked fractional dopamine release from rat striatal slices, and did not evoke dopamine release in the absence of methamphetamine. Furthermore, GZ-793A (1-100µM) inhibited neither nicotine (30µM)-evoked nor electrical field-stimulation-evoked (100Hz/1min) fractional dopamine release. Unfortunately, GZ-793A inhibited [3H]dofetilide binding to human-ether-a-go-go related gene channels expressed on human embryonic kidney cells, and further, prolonged action potentials in rabbit cardiac Purkinje fibers, suggesting the potential for GZ-793A to induce ventricular arrhythmias. Thus, GZ-793A selectively inhibits the neurochemical effects of methamphetamine and lacks nicotinic receptor interactions; however, development as a pharmacotherapy for methamphetamine use disorders will not be pursued due to its potential cardiac liabilities.

Angiotensin AT1 and AT2 receptor antagonists modulate nicotine-evoked [³H]dopamine and [³H]norepinephrine release.

Tobacco smoking is the leading preventable cause of death in the United States. A major negative health consequence of chronic smoking is hypertension. Untoward addictive and cardiovascular sequelae associated with chronic smoking are mediated by nicotine-induced activation of nicotinic receptors (nAChRs) within striatal dopaminergic and hypothalamic noradrenergic systems. Hypertension involves both brain and peripheral angiotensin systems. Activation of angiotensin type-1 receptors (AT1) release dopamine and norepinephrine. The current study determined the role of AT1 and angiotensin type-2 (AT2) receptors in mediating nicotine-evoked dopamine and norepinephrine release from striatal and hypothalamic slices, respectively. The potential involvement of nAChRs in mediating effects of AT1 antagonist losartan and AT2 antagonist, 1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid (PD123319) was evaluated by determining their affinities for α4ß2* and α7* nAChRs using [³H]nicotine and [³H]methyllycaconitine binding assays, respectively. Results show that losartan concentration-dependently inhibited nicotine-evoked [³H]dopamine and [³H]norepinephrine release (IC50: 3.9 ± 1.2 and 2.2 ± 0.7 µM; Imax: 82 ± 3 and 89 ± 6%, respectively). In contrast, PD123319 did not alter nicotine-evoked norepinephrine release, and potentiated nicotine-evoked dopamine release. These results indicate that AT1 receptors modulate nicotine-evoked striatal dopamine and hypothalamic norepinephrine release. Furthermore, AT1 receptor activation appears to be counteracted by AT2 receptor activation in striatum. Losartan and PD123319 did not inhibit [³H]nicotine or [³H]methyllycaconitine binding, indicating that these AT1 and AT2 antagonists do not interact with the agonist recognition sites on α4ß2* and α7* nAChRs to mediate these effects of nicotine. Thus, angiotensin receptors contribute to the effects of nicotine on dopamine and norepinephrine release in brain regions involved in nicotine reward and hypertension.

Exploring the effect of N-substitution in nor-lobelane on the interaction with VMAT2: discovery of a potential clinical candidate for treatment of methamphetamine abuse.

A series of N-substituted lobelane analogues was synthesized and evaluated for their [3H]dihydrotetrabenazine binding affinity at the vesicular monoamine transporter and for their inhibition of vesicular [3H]dopamine uptake. Compound 19a, which contains an N-1,2(R)-dihydroxypropyl group, had been identified as a potential clinical candidate for the treatment of methamphetamine abuse.

Design, synthesis and interaction at the vesicular monoamine transporter-2 of lobeline analogs: potential pharmacotherapies for the treatment of psychostimulant abuse.

The vesicular monoamine transporter-2 (VMAT2) is considered as a new target for the development of novel therapeutics to treat psychostimulant abuse. Current information on the structure, function and role of VMAT2 in psychostimulant abuse are presented. Lobeline, the major alkaloidal constituent of Lobelia inflata, interacts with nicotinic receptors and with VMAT2. Numerous studies have shown that lobeline inhibits both the neurochemical and behavioral effects of amphetamine in rodents, and behavioral studies demonstrate that lobeline has potential as a pharmacotherapy for psychostimulant abuse. Systematic structural modification of the lobeline molecule is described with the aim of improving selectivity and affinity for VMAT2 over neuronal nicotinic acetylcholine receptors and other neurotransmitter transporters. This has led to the discovery of more potent and selective ligands for VMAT2. In addition, a computational neural network analysis of the affinity of these lobeline analogs for VMAT2 has been carried out, which provides computational models that have predictive value in the rational design of VMAT2 ligands and is also useful in identifying drug candidates from virtual libraries for subsequent synthesis and evaluation.

Des-keto lobeline analogs with increased potency and selectivity at dopamine and serotonin transporters.

A series of des-keto lobeline analogs has been synthesized and evaluated for their ability to inhibit the dopamine transporter (DAT) and serotonin transporter (SERT) function and for their affinity for the synaptic vesicle monoamine transporter (VMAT2), as well as for alpha4beta2( *) and alpha7( *) neuronal nicotinic acetylcholine receptors (nAChRs). The enantiomers 8R-hydroxylobel-9-ene (3a) and 10S-hydroxylobel-7-ene (3c) exhibited high potency and selectivity at SERT and DAT, respectively.