Is the inhibition of nicotinic acetylcholine receptors by bupropion involved in its clinical actions?

The International Journal of Biochemistry & Cell Biology 41 (2009) 2098–2108

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Medicine in focus

Is the inhibition of nicotinic acetylcholine receptors by bupropion involved in its clinical actions? Hugo R. Arias ∗ Department of Pharmaceutical Sciences, College of Pharmacy, Midwestern University, 19555 N. 59th Avenue, Glendale, AZ 85308, USA

a r t i c l e

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Article history: Received 21 April 2009 Received in revised form 23 May 2009 Accepted 26 May 2009 Available online 2 June 2009 Keywords: Depression Nicotine addiction Antidepressants Bupropion Neurotransmitter transporters Nicotinic acetylcholine receptors

a b s t r a c t In this mini review we will focus on those molecular and cellular mechanisms exerted by bupropion (BP), ultimately leading to the antidepressant and anti-nicotinic properties described for this molecule. The main pharmacological mechanism is based on the fact that BP induces the release as well as inhibits the reuptake of neurotransmitters such as a dopamine (DA) and norepinephrine (NE). Additional mechanisms of action have been also determined. For example, BP is a noncompetitive antagonist (NCA) of several nicotinic acetylcholine receptors (AChRs). Based on this evidence, the dual antidepressant and anti-nicotinic activity of BP is currently considered to be mediated by its stimulatory action on the DA and NE systems as well as its inhibitory action on AChRs. Considering the results obtained in the archetypical mouse muscle AChR, a sequential mechanism can be hypothesized to explain the inhibitory action of BP on neuronal AChRs: (1) BP first binds to AChRs in the resting state, decreasing the probability of ion channel opening, (2) the remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process, and (3), BP interacts with a binding domain located between the serine (position 6 ) and valine (position 13 ) rings that is shared with the NCA phencyclidine and other tricyclic antidepressants. This new evidence paves the way for further investigations using AChRs as targets for the action of safer antidepressants and novel anti-addictive compounds. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Clinical depression is a chronic illness that affects approximately 5–8% of the population of American adults or about 15 million people each year (NIMH, 2008). Those suffering from depression experience symptoms such as persistent feelings of sadness, hopelessness, worthlessness, and loss of interest in typical daily activities. If this disorder is left untreated, the patient may eventually develop thoughts of suicide or engage in suicidal behavior. This common mental health disorder is the leading cause of disability in the USA and other developing countries. People from all ethnic, socio-economic, sex, and age groups are susceptible to depression. Although we do not have a clear view of the causes underlying mental depression, genetic and/or epigenetic factors might be

Abbreviations: Bupropion (BP), (±)-2-(tert-butylamino)-1-(3-chlorophenyl) propan-1-one; DA, dopamine; DAT, dopamine transporter; NE, norepinephrine; NET, norepinephrine transporter; NDRI, norepinephrine-dopamine reuptake inhibitor; 5-HT, 5-hydroxytryptamine (serotonin); ACh, acetylcholine; NCA, noncompetitive antagonist; AChR, nicotinic acetylcholine receptor; MAO, monoamine oxidase; TCAs, tricyclic antidepressants; VTA, ventral tegmental area; [3 H]TCP, [piperidyl34-3 H(N)]-N-(1-(2 thienyl)cyclohexyl)-3,4-piperidine; PCP, phencyclidine. ∗ Tel.: +1 623 572 3589; fax: +1 623 572 3550. E-mail address: [email protected]. 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.05.015

involved (Levinson, 2006; Caspi et al., 2003). For example, the latest evidence from brain imaging studies supports the view that the familial component is very important in the development of this disease (Peterson et al., 2009). Fig. 1 shows that the cortical thickness of right brain hemispheres from nondepressed persons with family history of depression (high-risk group) is thinner than that from persons without family history of depression (low-risk group). However, a familial trait is not necessary of genetic origin, and might also be consequence of changes in the environment when children are growing up with parents or grandparents who are depressed. Fortunately, depression is a CNS disorder that when properly treated, the symptoms will diminish or disappear completely. This opens up the possibility for a patient diagnosed with depression to lead a healthy, typical lifestyle. Antidepressants have been used therapeutically to treat depression for many years. However, it is still unclear exactly how these drugs work. Several antidepressants prevent the reuptake of specific neurotransmitters from the synaptic cleft, leaving them available for interaction with receptors on the postsynaptic neuron. Some other antidepressants inhibit the enzymes (i.e., monoamine oxidase, MAO) involved in monoamine degradation, increasing the concentration of biogenic amines including serotonin (5-HT), norepinephrine (NE), and dopamine (DA). Depending on their mechanisms of action and on their structural features, there are currently nine main cate-

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Fig. 1. Side views of the right and left hemispheres of the brain (modified from Peterson et al., 2009). The colors represent the differences in cortical thickness between the groups with (high-risk group) and without (low-risk group) family history of depression. Blue and purple represent the thinning of the cortex, with purple regions having the greatest thinning. Green areas show no significant differences between the two groups.

gories of antidepressants (reviewed in Baldessarini, 2001; Arias et al., 2006a): (1) MAO inhibitors (e.g., phenelzine), the oldest used antidepressants, (2) selective 5-HT reuptake inhibitors (e.g., fluoxetine), (3) selective NE reuptake inhibitors (e.g., reboxetine), (4) dual NE-5-HT reuptake inhibitors (e.g., venlafaxine), (5) tricyclic antidepressants (TCAs), which are classified based on their common structure, and although they are old antidepressants some of them inhibits both the 5-HT and NE transporters (e.g., amitriptyline, imipramine, doxepin, and clomipramine), whereas some others are considered as specific NE reuptake inhibitors (e.g., desipramine and nortriptyline), (6) 5-HT type 2 receptor inhibitors (e.g., trazodone), (7) ␣2 -adrenergic antagonist and 5-HT type 2 and 3 receptor inhibitors (e.g., mirtazapine), and (8) natural antidepressants (e.g., hyperforin), whose mechanisms of action are still unclear. Finally, (9) bupropion (BP) [(±)-2-(tert-butylamino)-1-(3chlorophenyl)propan-1-one] (see its molecular structure in Fig. 2) is a unique antidepressant whose aminoketone structure differs from that for other antidepressants in the market and functionally is classified as a dual NE-DA reuptake inhibitor (NDRI). In this mini review we will focus on those molecular and cellular mechanisms exerted by BP regarding its antidepressant and anti-nicotinic activities. The most accepted mechanism of action for BP is that this antidepressant inhibits the catecholamine reuptake in presynaptic neurons, modulating the concentrations of the neurotransmitters DA and NE in the synaptic cleft. Fig. 3 shows the most accepted mechanism of action for BP as a NDRI. However, the affinity of BP for these neurotransmitter transporters is only moderate (see Table 1), and there is not clear-cut evidence explaining the dual antidepressant and anti-nicotinic modes of action elicited by BP. In this regard, the combined inhibition of nicotinic acetylcholine receptors (AChRs) and neurotransmitter transporters produced by BP might account for its clinical efficacy in smoking cessation therapy and as an antidepressant. Moreover, the contribution of the BP-induced AChR inhibition to its clinical action could be two-fold important: as part of the side effects (i.e., dry mouth, nausea, and insomnia) elicited by BP and/or as part of its clinical outcome. Thus, a better understanding of the interaction of BP with the AChR in different conformational states to determine its noncompetitive inhibitory mechanism is crucial to develop safer and more efficient antidepressants and/or anti-nicotinic drugs. In this regard, the interaction of BP with AChRs in different conformational states was recently determined by functional and structural approaches (Arias et al., 2009).

2. Bupropion is a catecholamine transporter inhibitor Bupropion has been used for long time as an antidepressant (Wellbutrin® ) as well as in the pharmacotherapy for smoking cessation (Zyban® ) (Wilkes, 2006; Dwoskin et al., 2006). BP, as well as other more specific antidepressants, can be used for the treatment of atypical depression, which is associated with interpersonal deficits such as rejection sensitivity and social avoidance (reviewed in Levitan, 2007). It has also recently been used “off-label” for the treatment of attention deficit hyperactivity disorder (ADHD) (reviewed in Slatkoff and Greenfield, 2006; Covey et al., 2008), and it is the only antidepressant with high efficacy to prevent depressive relapse for seasonal affective disorder (SAD) (reviewed in Stahl et al., 2004). Finally, BP has showed positive effects as an adjunct for weight loss in nondepressed, obese individuals (Anderson et al., 2002). From the clinical point of view, BP only produces minor side effects including dry mouth, nausea, and insomnia, lacking major events produced by other antidepressants such as sexual dysfunction, weight gain, and sedation (reviewed in Stahl et al., 2004). These therapeutic properties have been discovered and adopted on the basis of the many pharmacological properties that this molecule exhibits. For instance, BP has been considered to be a dual NDRI (Fig. 3), a vesicular monoamine transport enhancer (Rau et al., 2005), an anti-inflammatory agent against the actions of cytokines such as tumor necrosis factor-␣, a cytochrome P450 CYT2D6 inhibitor (Wilkes, 2006), and a noncompetitive antagonist (NCA) of several AChRs (see Section 5). Currently, BP constitutes an important pharmacological tool for biomedical research given its proved capacity to inhibit DA and NE reuptakes. The first neurochemical studies on the central mechanisms of BP suggested that the antidepressant activity of BP is not merely due to MAO inhibition or biogenic amines release from nerve endings (Ferris et al., 1982). In this study, the authors showed that BP was a weak inhibitor of catecholamine transporters, but its selective blockade of the DA transporter (DAT) in vivo could be correlated with mild stimulation of the CNS in rodents. These and other properties, such as the lack of desensitization of ␤-adrenergic receptors in rat cerebral cortex, served to postulate BP as an “atypical” antidepressant with different modes of action of those for MAO inhibitors and TCAs. Shortly thereafter, new studies reinforced these observations by discriminating the antidepressant activity of BP from MAO inhibition (Ferris et al., 1983). Although in this study it was clear that DA neurons have to be present for BP to exert its effects on the CNS, they found that at antidepressant doses of BP, the DA turnover

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Fig. 2. Molecular structure of bupropion [(±)-2-(tert-butylamino)-1-(3chlorophenyl)propan-1-one] and of its endogenous metabolites.

is decreased. Based on these findings, the authors concluded that the antidepressant properties of BP cannot be merely explained on the basis of alterations in either presynaptic or receptor-mediated postsynaptic events in cholinergic or serotonergic pathways. The acute effect of BP on the extracellular concentration of DA and its metabolites by means of microdialysis in freely moving rats was previously studied (Nomikos et al., 1989). The doseand time-dependent increase in DA release (peaking at 20 min) and the changes in metabolites produced by BP in the striatum and in the nucleus accumbens lasted for 2 h. Interestingly, the increase in extracellular DA levels correlated with stereotyped behavior only during the first hour. The authors concluded that BPinduced DA release contributed to the observed behavioral effects through complex mechanisms still unknown, and is likely involved in the antidepressant actions of this molecule. In this study, BP was assumed to act as a DA reuptake inhibitor. Indeed, further studies simply accepted the DA reuptake inhibition effect of BP as its main feature (Beauregard et al., 1991). Furthermore, when BP was

given to rats before and after the iontophoretical DA application, it resulted in a reduced duration of the inhibitory responses evoked by DA on spontaneous neuronal firing in anterior cingulate, while lengthening them in prefrontal cortex, neostriatum and nucleus accumbens. The dopaminergic component of BP actions was reinforced in a model of canine cataflexy (Mignot et al., 1993). This event is typically controlled by adrenergic mechanisms, and therefore the BP-induced DA reuptake inhibition was non-sensitive. Acute BP administration, in addition to increasing DA concentration, enhances NE in the same mesocorticolimbic areas (Li et al., 2002). Microdialysis studies indicated that that acute administration of BP increases NE concentrations and reduces firing of NE neurons in the brain steams in a dose-dependent manner (reviewed in Ascher et al., 1995). Interestingly, DA and NE blocking drugs inhibited the effect elicited by BP in animal models of depression (Cooper et al., 1980). Recently, BP has been also shown to increase brain and core temperatures in freely moving rats in a microdialysis trial (Hasegawa et al., 2005). The study demonstrated that the acute inhibition of DA and NE reuptake, but not of 5-HT reuptake, is responsible for the elevation of brain and core temperatures. Taken together, these results support the existence of a presynaptic, positive-feedback mechanism triggered by DA and/or NE which in turn favors DA and NE release, respectively, upon its reuptake in nerve terminals. Another relevant contribution to the understanding of BP’s mechanisms of action in humans was made evaluating the in vivo activity of BP in human brains by positron emission tomography (PET) (Learned-Coughlin et al., 2003). This technique determines the extent of duration of DAT occupancy by BP and its metabolites in human volunteers under sustained-release oral treatment. As expected, BP and its metabolites inhibited the striatal reuptake of the specific DAT radioligand [11 C]-␤CIT-FE {[11 C]-N-␻-fluoroalkyl2␤-carboxy-3-␤-(4-iodophenyl) nortropane ester} thus, producing an occupancy of ∼26%. These results were confirmed by using single photon emission computed tomography (SPECT), where ∼24% DAT occupancy was obtained (reviewed in Stahl et al., 2004). These results contrast with that shown by Meyer et al. (2002), where occupancy of only ∼14% was obtained in depressed patients. Additional SPECT studies indicated that although ∼21% DAT is occupied during BP treatment, there is a lack of correlation between the efficacy of therapy and DAT occupancy (Árgyelán et al., 2005). This evidence supports the hypothesis that DA reuptake inhibition by BP is only partially responsible for its therapeutic effects in human subjects. From the molecular point of view, BP binds to the DAT, which has twelve transmembrane segments (TM1–TM12), initially to S359, located in the middle of TM7, whereas A279, located close to the extracellular end of TM5, is responsible for the subsequent steps in the blockade of translocation in DAT (Mortensen and Amara, 2006). These two amino acids, and probably F367, forming a cluster of residues that contribute to BP sensitivity, are located far from the substrate binding pocket located between TM1 and TM6. The homologous residues V276 and I356 in NET also interact specifically with BP. The combination of findings from the molecular to the animal behavior level has served to recognize BP, in regards of its neuropharmacological activities, as a dual NDRI.

3. Pharmacokinetics and metabolism of bupropion Studies on BP’s pharmacokinetics represent an issue deserving further attention, especially in the brain, since they establish the basis for the understanding of the many interactions of this drug with receptors and transporters in the CNS. One of the first reports describing BP kinetics and metabolism was performed using rats, dogs, and normal volunteers (Schroeder, 1983). The

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Table 1 Interaction of bupropion and its endogenous metabolites with different neurotransmitter transporters. Used technique

Tissue or cell

DAT IC50 or Ki (␮M)

NET IC50 (␮M)

[3 H]DA uptake

DAT or NET expressed in COS-7 cells DAT or NET expressed in COS-7 cells Human NET expressed in C6 glial cells Rat caudate putamen membranes Mouse striatum

0.95 ± 0.25

4.63 ± 0.58

1.51 ± 0.32

3.42 ± 0.48

[3 H]Nisoxetine competition binding [3 H]NE uptake [3 H]WIN 35,428 competition binding In vivo [3 H]WIN 35,428 competition binding [3 H]WIN 35,428 competition binding [3 H]Mazindol competition binding [3 H]DA uptake [3 H]DA uptake [3 H]DA uptake [3 H]DA uptake [3 H]NE uptake [3 H]NE uptake [3 H]5-HT uptake [3 H]5-HT uptake

SERT IC50 (␮M)

Reference Mortensen and Amara (2006) Mortensen and Amara (2006) Foley and Cozzi (2002)

1.37 ± 0.14 0.373

Katz et al. (2000)

0.199

Stathis et al. (1994)

Rabbit caudate membranes

0.055 ± 0.004

Aloyo et al. (1995)

Rat striatal synaptosomes

0.178 ± 0.038

Dersch et al. (1994)

0.52

Giros et al. (1992)

0.33

Giros et al. (1992)

2.0 ± 0.6; 23 ± 8a ; 47 ± 2b 2.0 ± 0.8; 17 ± 9a ; 23 ± 13b

Ascher et al. (1995) Ascher et al. (1995)

−

Rat DAT expressed in Ltk cells Human DAT expressed in Ltk− cells Rat striatal synaptosomes Mouse striatal synaptosomes Rat hypothalamic synaptosomes Mouse hypothalamic synaptosomes Rat hypothalamic synaptosomes Mouse hypothalamic synaptosomes

5 ± 1; 7 ± 3a ; 16 ± 7b

Ascher et al. (1995)

4 ± 1; 4 ± 1a ; 10 ± 13b

Ascher et al. (1995) 58 ± 15; 105 ± 11a ; 67 ± 2b

Ascher et al. (1995)

36 ± 21 100 ± 11a ; 92 ± 15b

Ascher et al. (1995)

DAT, NET, and SERT, are dopamine, norepinephrine, and serotonin transporters, respectively. IC50 , concentration of bupropion to produce 50% inhibition of binding or uptake at the neurotransmitter transporter. Ki , inhibition constant. [3 H]WIN 35,428, (−)-2-␤-carbomethoxy-3-␤-(4-fluorophenyl)tropane. Values for a hydroxybupropion and b threo-hydrobupropion, respectively.

results demonstrated that BP was rapidly and completely absorbed, widely distributed in tissues, and metabolized extensively prior to its excretion. BP metabolism in rats and dogs was produced predominantly by side chain oxidative cleavage, while reduction of the intact parent aminoketone to an aminoalcohol was a major pathway in men. Hydroxylation of the tert-butyl group of BP to hydroxybupropion (see both hydroxybupropion isomers in Fig. 2), one of the major metabolites, is produced by the isoenzyme CYP2B6. Thus, CYP2B6 polymorphism and drug-drug interactions might be risk factors increasing potential adverse events like seizures, which normally is very rear (∼0.1%). In addition, the carbonyl group of BP is reduced to the amino-alcohol isomers threo- and erythrohydrobupropion (see molecular structures in Fig. 2), which are

further metabolized to inactive metabolites and finally excreted in the urine. The results also showed that BP was concentrated in many tissues, with a brain to plasma ratio of about 25:1. In this regards, BP reaches the brain in considerable amounts, attaining those concentrations needed to exert its therapeutic effects at the central level. Interestingly, no pharmacokinetics significant differences between smokers and nonsmokers or between male and female volunteers were observed for BP or its metabolites (Hsyu et al., 1997). Hydroxybupropion has almost the same affinity as BP for NET (Ascher et al., 1995) (see Table 1) and ∼50% the antidepressant activity of BP. However, during clinical treatment hydroxybupropion reaches concentrations ∼10-fold higher than that of the parent drug (Ascher et al., 1995; Hsyu et al., 1997). This increase in con-

Fig. 3. Most probable mechanism of action for bupropion as a dual norepinephrine-dopamine reuptake inhibitor (NDRI) (modified from Stahl et al., 2004). The portions of the NDRI molecule, namely BP, interacting with either the NE reuptake or the DA reuptake pump is colored in yellow and red, respectively. After BP blocks both NE and DA pumps, NE (purple) and DA (blue) concentrations are increased in the synaptic cleft, and finally each neurotransmitter interacts with its respective NE (blue) and DA (green) receptor in the postsynaptic membrane.

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centration would counteract for the observed low affinity for NET compared to that for DAT (see Table 1), causing meaningful inhibition of NE reuptake, even at therapeutic dosages. Finally, both (S)and (R)-BP isomers have the same affinity for monoamine transporters, indicating lack of enantioselectivity (Musso et al., 1993) (see Table 1). These aspects should be considered for future design of therapeutic strategies. 4. Anti-nicotinic action of bupropion Tobacco use causes about four million deaths per year worldwide and billions of dollars are spent for the treatment of tobacco-related diseases. The most important active (addictive) component in tobacco is the alkaloid (−)-nicotine. Nicotine is so powerful that adolescent smokers already present the first symptoms of nicotine dependence such as withdrawal, craving, and relapse, within the first weeks of smoking (reviewed in DiFranza, 2008). The combination of several sources of information, but mainly from human twin studies, suggest that an important genetic component underlying nicotine addiction exist (reviewed in Mineur and Picciotto, 2008; Benowitz, 2009). Nevertheless, environmental and emotional factors also contribute to other aspects of nicotine addiction including reward, craving, and relapse. The most prevalent hypothesis of nicotine addiction includes a scenario where nicotine first stimulates DA release in neurons from the ventral tegmental area (VTA), promoting long-term potentiation of inputs onto dopaminergic neurons (Mansvelder et al., 2006, 2007). The DA-producing neurons originated in the VTA extend to DA-sensitive cells located in the nucleus accumbens. This dopaminergic pathway is neuroanatomically part of the mesocorticolimbic system which is functionally considered as the “brain reward circuitry”. Drugs of abuse block DA reuptake (e.g., cocaine) and/or increase DA release (e.g., nicotine) in VTA terminals, enhancing DA signaling in the nucleus accumbens and thereby promoting drug addiction behavior. Fig. 4 depicts the mechanisms of action of nicotine in the VTA neurons. VTA neurons express several AChR subunits which can form ␣7 (i.e., homomeric) and non-␣7 (i.e., heteromeric) AChRs (reviewed in Mansvelder et al., 2006). Nicotine

Fig. 4. Nicotine-induced stimulation in ventral tegmental area (VTA) and the inhibition elicited by bupropion (modified from Mansvelder et al., 2007). (A) Nicotine directly increases dopamine excitability in VTA neurons through activation of non␣7 AChRs. Nicotine activation of presynaptic ␣7 AChRs in glutamatergic neurons enhance glutamate inputs to VTA dopamine neurons. Non-␣7 AChRs at the preterminal, somatic and dendritic regions of GABAergic neurons maintain the basal inhibitory tone to DA neurons via cholinergic input from the pontomesencephalic tegmental nuclei (PMT). (B) Nicotine effects on these AChRs are inhibited by bupropion. Bupropion-induced inhibition of AChRs on GABAergic neurons may enhance dopamine release and contribute to the antidepressant effects of the drug.

directly increases DA excitability in VTA neurons through activation of non-␣7 AChRs, and stimulates presynaptic ␣7 AChRs in glutamatergic neurons enhancing glutamate inputs to VTA neurons. In addition, nicotine activates transiently non-␣7 AChRs at the preterminal, somatic and dendritic regions of GABAergic neurons increasing inhibitory GABAergic inputs to DA neurons. However, this effect is decreased after nicotine-induced AChR desensitization. Although we do not know with certainty which non-␣7 AChRs are the most importants in this process, the combination of several studies indicate that AChR subunits ␤2 (Picciotto et al., 1998) and ␣4 (Tapper et al., 2004) are implicated in the reinforcing properties mediated by nicotine, whereas subunit ␤4 is involved in the withdrawal effects of the alkaloid (Salas et al., 2003) (reviewed in Mineur and Picciotto, 2008). Additional studies using genetically modified mice support the idea that particular AChR subunit combinations, but not any subunit combination, are involved in nicotine reward, dependence, and withdrawal (reviewed in Fowler et al., 2008). Studies in rat hippocampal slices showed that ␣4␤2 and ␣3␤4 AChRs are more sensitive to the desensitizing and upregulating effects of nicotine at concentrations found in the venous blood of cigarette smokers (Alkondon and Albuquerque, 2005), suggesting that these AChR types could be relevant in early cue-related learning associated with nicotine addiction. Bupropion is recommended as a first aid for smoking cessation, and its efficacy can be attributed to two combined activities: BP produces stimulatory effects in the CNS similar to that by nicotine and noncompetitively inhibits several AChRs (see Table 2). Although BP is considered a psychomotor stimulant (Wilkinson and Bevins, 2007; Sidhpura et al., 2007b), this drug has been shown to attenuate the expression of nicotine withdrawal symptoms in both animal models and human subjects. Considering these results, we can speculate that BP effectiveness in the treatment of people who are motivated to quit smoking is based on the nicotine-like effects produced by low doses of BP, finally serving as a suitable substitute for nicotine. This hypothesis is supported by the current evidence showing that BP attenuates nicotine-induced reinstatement in rats, and improves the quitting success rate in humans, even after one year (Jorenby et al., 1999). BP also shows a positive response in different animal models (reviewed in Dwoskin et al., 2006). For example, BP attenuates nicotine-induced unconditioned behaviors, sharing or enhancing discriminative stimulus properties of nicotine, and affects nicotine self-administration in a complex manner, i.e., low doses augments whereas high doses attenuates self-administration. Current studies show that BP facilitates the acquisition of nicotine conditioned place preference in rats, further suggesting that BP enhances the rewarding properties of nicotine. And vice versa, that nicotine pretreatment enhances the locomotor activity elicited by BP (Sidhpura et al., 2007a; Wilkinson et al., 2006). BP is responsible for increased locomotor activity in freely moving rats through mechanisms typically involving DA reuptake inhibition and blockade of AChRs, potentiating the behavioral effects of nicotine (Sidhpura et al., 2007a). The effects of BP on AChR-mediated DA release were compared in rat striatal synaptosomes and slices (Sidhpura et al., 2007b). The contribution of this study is based on the proposal that a modest blockade of DAT by low concentrations of BP generates a feedback inhibition via D2 DA presynaptic autoreceptors, and that this is overcome at higher concentrations of BP. However, at this higher concentration, BP does not directly inhibit AChRs. Therefore, the BP-induced inhibition of DAT and AChRs are two events clearly dependent on BP’s concentration. Other interesting findings have been recently described that may account for the anti-nicotinic activity of BP (Mansvelder et al., 2007). For instance, it has been shown that BP inhibits the cellular effects of nicotine in the rat VTA, which in turn contributes to its anti-nicotinic effects (see Fig. 4). In addition, BP-induced

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Table 2 Interaction of bupropion and its endogenous metabolites with several AChRs. AChR type

Used technique

Human ␣1␤1␥␦ Human ␣1␤1␥␦ Torpedo Torpedo Mouse ␣1␤1␧␦

Ca2+ influx fluorimetry Rb+ efflux [3 H]TCP competition bindingb [3 H]Imipramine competition bindingb

Human ␣3␤4e Human ␣3␤4e Rat ␣4␤2 Rat ␣3␤2 Rat ␣7 Human ␣3␤4* Human ␣4␤2 Human ␣4␤4 Human ␣1␤1␥␦ Human ␣3␤4* Rat ␣3␤4* Rat ␣3␤2* Rat ␣3␤4 Rat ␣3␤4 Rat ␣3␤4 Rat ␣3␤4 Human ␣4␤2 Human ␣3␤4* Human ␣4␤4 Human ␣1␤1␥␦ a b c d e f g h

Resting IC50 a or Ki b (␮M)

Desensitized Ki b (␮M)

20.5 ± 3.9c ; 10.5 ± 2.1c 10.5 ± 1.0

86

Patch-clamp Ca2+ influx fluorimetry 86 Rb+ efflux Voltage-clamp Voltage-clamp Voltage-clamp 86 Rb+ efflux 86 Rb+ efflux 86 Rb+ efflux 86 Rb+ efflux Ca2+ influx fluorimetry Nicotine-evoked [3 H]NE overflow in hippocampal slices Nicotine-evoked [3 H]DA overflow in striatal slices 86 Rb+ efflux 86 Rb+ efflux 86 Rb+ efflux HPLC-immobilized ␣3␤4 AChRs 86 Rb+ efflux 86 Rb+ efflux 86 Rb+ efflux 86 Rb+ efflux

Activated IC50 a (␮M)

5.1 ± 0.3b 21.4 ± 3.4b 0.40 ± 0.04d

8.0 ± 2.6d 1.3 ± 0.6d ∼60d

2.0 ± 0.1b 11.6 ± 1.3b 40.1 ± 5.1 0.82 ± 0.10 1.51 ± 0.32 55

1.8 ± 1.1 12.0 ± 1.1 14.0 ± 1.1 7.9 ± 1.1 0.82 ± 0.10 0.32 1.27 7 14f 2g ; 18h 0.18 31.0 ± 1.g ; 3.3 ± 1.1h 6.5 ± 1.g ; 10.0 ± 1.5h 41.0 ± 1.g ; 30.0 ± 1.1h 7.6 ± 1.g ; 28.0 ± 1.4h

Reference Arias et al. (2009) Fryer and Lukas (1999) Arias et al. (2009) Arias et al. (2009) Arias et al. (2009) Sidhpura et al. (2007a,b) Fryer and Lukas (1999) Slemmer et al. (2000) Slemmer et al. (2000) Slemmer et al. (2000) Damaj et al. (2004) Damaj et al. (2004) Damaj et al. (2004) Damaj et al. (2004) Sidhpura et al. (2007a,b) Miller et al. (2002) Miller et al. (2002) Bondarev et al. (2003) Bondarev et al. (2003) Bondarev et al. (2003) Jozwiak et al. (2004) Damaj et al. (2004) Damaj et al. (2004) Damaj et al. (2004) Damaj et al. (2004)

IC50 is the required drug concentration to produce 50% inhibition of agonist-activated AChRs. Ki values were obtained in the presence of ␣-bungarotoxin (resting state) or carbamylcholine (desensitized state), respectively. These values were obtained by pre-incubating the cells with BP for 5 min or by co-injecting BP and epibatidine for several seconds, respectively. These values were obtained by a brief pre-incubation with BP before agonist activity was measured. The native AChR can have additional subunits. These values correspond to either (–)- or (+)-threo-hydrobupropion. These values correspond to (2R,3R)-hydroxybupropion. These values correspond to (2S,3S)-hydroxybupropion.

blockade of non-␣7 AChRs on GABAergic neurons may diminish tonic inhibition of VTA neurons (see Fig. 4), subsequently enhancing DA release, which may contribute to its antidepressant action. This disinhibition of the DA system may also reduce the negative symptoms produced during smoke quitting, diminishing relapse, and contributing to the overall effectiveness of BP. Recent studies explored the hypothesis that BP ameliorates, at least partially, nicotine withdrawal by a DA-dependent mechanism (Paterson et al., 2007). For this purpose, the authors investigated the effects of a chronic infusion of BP on behavioral aspects of nicotine withdrawal including brain reward thresholds and somatic signs of withdrawal. The authors concluded, based on the findings that BP lowers reward thresholds, increases K+ -evoked DA release, and blocks withdrawal-associated somatic signs, that the BP-induced increase in DA extracellular levels in the nucleus accumbens shell decreases the anhedonic component of nicotine withdrawal, an issue clearly in favor of smoking cessation facilitation. However, it is not clear how the extent of BP-induced DA neurons disinhibition might contribute to the observed mechanisms described in Fig. 4 (Mansvelder et al., 2007). Pre-clinical studies indicate that the combination of low doses of BP with other anti-addictive drugs such as 18-methoxycoronaridine (a novel ibogaine analog and a NCA with relatively high specificity for the ␣3␤4 AChR; Maissonueve and Glick, 2003), mecamylamine (an unspecific NCA of AChRs with antidepressant-like activity; Rabenstein et al., 2006), or dextromethorphan (a NCA of both AChRs and NMDA-type of glutamate receptors; Hernandez et al., 2000) decreases nicotine self-administration in rats (Glick et al., 2002). This anti-addictive result was attributable to the additive effects elicited by the different drugs, without producing the side effects (e.g., decrease in water intake) generated by each particular drug

at higher concentrations. Since all these drugs produce inhibition of the ␣3␤4 AChR (Glick et al., 2002; Maissonueve and Glick, 2003; Hernandez et al., 2000), and nicotine induces ␣3␤4 AChR up-regulation (Alkondon and Albuquerque, 2005), this AChR type might be implicated in the process of nicotine addiction and in the beneficial effects elicited by BP. In fact, ␣3␤4 AChRs are expressed in relatively high amounts in the habenulo-interpeduncular pathway (reviewed in Maissonueve and Glick, 2003). This cholinergic circuitry is considered a second brain reward system that modulates the mesocorticolimbic pathway, and vice versa (Glick et al., 2002; Maissonueve and Glick, 2003; Taraschenko et al., 2005, 2007). BP has also been found to decrease methamphetamine effects and craving in humans (Newton et al., 2006). The decrease in craving can be explained by BP-induced NE transporter (NET) inhibition which subsequently increases synaptic NE concentration, finally reducing NE cell firing (Dong and Blier, 2001). These results pave the way for further evaluation of BP as a therapy for different drugs of abuse. In summary, whether the actions of BP are mediated by DA and/or NE reuptake inhibition, by direct action on cholinergic receptors, or all of them, the central actions of this drug remain to be elucidated with priority as it represents a suitable strategy for different depression- and addictive-related disorders. 5. Role of AChRs in depression and in the pharmacological action of antidepressants AChRs are the paradigm of the Cys-loop ligand-gated ion channel superfamily. This genetically linked superfamily includes types A and C GABA, type 3 5-HT, and glycine receptors (reviewed in Arias, 2006). The malfunctioning of these receptors has been con-

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sidered as the origin of several neurological disorders (reviewed in Hogg et al., 2003; Lloyd and Williams, 2000). For instance, the importance of several AChR types in nicotine addiction has been explained in Section 4. The evidence showing a higher rate of smokers in depressed patients than in the general population supports the hypothesis that AChRs may play an important role in depression mechanisms (reviewed in Picciotto et al., 2002). Another piece of information that links AChRs with the functional components involved in depression is provided by studies using knockout mice lacking the DAT, where the density of several AChR types is modified in an area-dependent manner (Weiss et al., 2007). In some dopaminergic areas, the small decrease in the ␤2-containing AChR density contrasted with the higher decrease and increase in the ␣6 and ␣7 subunit densities, respectively. In addition, mutant mice were hypersensitive to locomotor activity produced by low doses of nicotine, probably by enhanced nicotine-induced extracellular DA levels. The mutant animals were also hypersensitive to hypolocomotion induced by high doses of nicotine or by the specific ␣7 AChR agonist, choline. The cholinergic-adrenergic theory is one of the accepted hypotheses used to describe the mechanisms of depression. This theory is based on the fact that hyperactivity or hypersensitivity of the cholinergic system over the adrenergic system can lead to depressed mood states (reviewed in Shytle et al., 2002). The hyperactivity of the cholinergic system might be provoked by excessive neuronal AChR stimulation. In this regard, the therapeutic action of many antidepressants may be mediated in part through inhibition of one or more AChRs. Structurally and functionally different antidepressants behave pharmacologically as NCAs of several AChRs (Gumilar et al., 2003; Sanghvi et al., 2008; reviewed in Arias et al., 2006a). Interestingly, several DAT, NET, and 5-HTT blockers, different from the known antidepressants, also inhibit distinct AChRs (Szasz et al., 2007, and references therein). For the particular case of BP, in vivo and in vitro results indicate that this antidepressant also inhibits several AChRs in a noncompetitive manner (Alkondon and Albuquerque, 2005; Slemmer et al., 2000; Fryer and Lukas, 1999; Arias et al., 2009; Bondarev et al., 2003; Damaj et al., 2004; reviewed in Arias et al., 2006a). Table 2 shows the inhibitory potency and affinity of BP and its endogenous metabolites on several AChR types in different conformational states. Of particular importance is the fact that BP inhibits the ␣3␤4 AChR, since this receptor type has been considered as the main target for the anti-addictive actions mediated by BP and other NCAs (Glick et al., 2002; Maissonueve and Glick, 2003; Taraschenko et al., 2005). An interesting study performed by Miller et al. (2002) showed the capability of BP to inhibits nicotine-evoked [3 H]DA overflow in rat striatal slices preloaded with [3 H]DA and in rat hippocampal slices preloaded with [3 H]NE. It was concluded that, besides of its known ability to inhibit DA and NE transporters, BP also acts as a NCA of ␣3␤2 and ␣3␤4 AChRs in both brain regions.

6. Molecular mechanisms of AChR inhibition mediated by bupropion The inhibitory mechanism produced by bupropion on AChRs was studied by several functional approaches. In a first attempt, the effect of BP on epibatidine-activated Ca2+ influx in TE671 cells expressing the ␣1␤1␧␦ AChR was determined (Arias et al., 2009). The inhibitory potency of BP was in the 10–20 ␮M concentration range as was previously determined by 86 Rb+ efflux experiments using the same cell type (Fryer and Lukas, 1999) (see Table 2). Comparing these values with that found in other AChR types (see Table 2), we can conclude that the BP specificity for different AChRs follows the sequence: ␣3- > ␣4- ∼ ␣1- > ␣7-containing AChRs.

Considering the serum levels of BP attained after its oral administration (∼0.5–1 ␮M; Hsyu et al., 1997), the blockade of non-␣3-containing AChRs is unlikely. However, hydroxybupropion, which has practically the same activity as BP for ␣4␤2 AChRs (Damaj et al., 2004; see Table 2), can attain ∼10-fold the plasma concentration of the parent compound (Ascher et al., 1995; Hsyu et al., 1997), increasing the possibility that ␣4␤2 AChRs contribute to the clinical efficacy of BP. In order to distinguish the effect of BP on a particular conformational state, macroscopic currents of agonist-activated receptors rapidly pre- (mainly in the resting state) or co-incubated (mainly in the open state) with BP were recorded (Fig. 5) (Arias et al., 2009). Macroscopic currents show that the interaction of BP with the open state decreases the decay time constant of currents activated by 300 ␮M ACh, indicating that BP increases the desensitization rate from the open state. The current decay was adequately fitted using a single exponential function, which discards the possibility of a fast open-channel blockade mechanism. However, distinguishing between the occurrence of slow channel blockade and the increase in the desensitization rate would be difficult (Gumilar et al., 2003). Macroscopic current recordings also show a decrease in the peak current when BP interacts with the resting channel. This effect might be due to either an increase in desensitization of the resting state or a direct blockade of unliganded channels. A similar effect is observed with TCAs (Gumilar et al., 2003). The potency of inhibition is greater when BP acts on the resting state than on the open state. A similar finding was reported for the inhibition of different Cys-loop receptors by TCAs (Gumilar and Bouzat, 2008; Choi et al., 2003). The two different effects detected for each different conformational state, i.e. reduced peak current and increased decay rate, are additive when the receptor is exposed continuously to the drug. Thus, the mechanism by which BP inhibits AChR function is selective for each conformational state.

7. Localization of the bupropion binding site in the AChR ion channel To characterize the BP binding sites in different receptor conformational states, a combination of several methods were used on the Torpedo AChR (Arias et al., 2009). The results from the radioligand competition binding experiments indicate that BP binds with ∼2-fold higher affinity to the desensitized AChR compared to the resting AChR. These experiments also indicate that BP inhibits the binding of both [3 H]imipramine and [3 H]TCP [the structural and functional analog of the NCA phencyclidine (PCP)] to both desensitized and resting AChRs (Arias et al., 2009). Based on Schild-type analysis showing that imipramine inhibits [3 H]TCP binding to Torpedo AChRs in a steric fashion (Sullivan et al., 2008), we suggest that the BP binding site overlaps the PCP locus. However, we cannot predict whether the location of the BP binding site is the same or distinct in each conformational state. The location of the PCP binding site depends on the conformational state of the AChR ion channel (reviewed in Arias et al., 2006a). For instance, photoaffinity labeling studies using [3 H]ethidium diazide, which binds with high affinity to the PCP locus, helped to determine the structural components of this site in the desensitized state (Pratt et al., 2000). The results indicated that [3 H]ethidium diazide mainly labeled residues at and close to the leucine ring (position 9 ) from the ␣1-M2 transmembrane segment. In addition, new [125 I]TID photoaffinity labeling results suggest that the PCP binding site is located between the threonine (position 2 ) and serine (position 6 ) rings (Hamouda et al., 2008). Our photoaffinity labeling studies using [3 H]2-azidoimipramine supports the idea

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Fig. 5. Effects of bupropion on agonist-induced macroscopic currents in HEK293 cells expressing mouse ␣1␤1␧␦ (adult) AChRs (modified from Arias et al., 2009). (A) Bupropion effects from the open state. Left: Ensembled mean currents obtained from outside-out patches activated in absence (control) or simultaneous application of ACh and BP, without preincubation with BP (protocol −/+; open state). Each trace represents the average of 4–8 applications of agonist. Curves from right to left correspond to: control and recovery, 50, 100, and 200 ␮M bupropion. The calculated decay time constants ( d ) are 25 ms for control and recovery curves, and 11.3, 6.3, and 4.5 ms, for 50, 100, and 200 ␮M BP, respectively. Membrane potential: −50 mV. Right: Concentration–response curve for the decrease in the decay time constant (n = 5). (B) Effects of bupropion from the resting/activatable state. Left: Effect of BP application protocol +/− (resting/activatable state): 2 min pre-incubation of BP following ACh application. Each trace represents the average of 4–8 applications of agonist. Curves from right to left correspond to: control and recovery, 0.25, 0.5, 1, and 5 ␮M BP, respectively. The peak current decreases with increased BP concentrations. Membrane potential: −50 mV. The calculated IC50 values are summarized in Table 2. Right: Concentration–response curve for the decrease in the peak current on the resting/activatable state. (C) Effects of bupropion application protocol on macroscopic currents. Superimposed currents responses to 300 ␮M ACh and BP concentrations correspond to IC50 for the resting/activatable state (∼0.4 ␮M) and for the open state (∼40 ␮M) using different protocols. From right to left curves correspond to control condition (−/− protocol;  d = 13.4 ms), simultaneous 300 ␮M ACh/40 ␮M BP application without pre-incubation with bupropion (−/+ protocol;  d = 6.5 ms; peak current 99% of the control), ACh application following 2 min pre-incubation with 0.4 ␮M BP (± protocol;  d = 13.9 ms; peak current = 50.4% of the control), and simultaneous 300 ␮M ACh/40 ␮M BP application after pre-incubation with 0.4 ␮M BP (+/+ protocol;  d = 6.3 ms; peak current = 47% of the control). The calculated IC50 values are summarized in Table 2.

that TCAs bind to the PCP locus in the desensitized ion channel (Sanghvi et al., 2008). On the other hand, site-directed mutagenesis studies determined that the PCP binding site in the open ion channel includes residues located between the serine (position 6 ) and leucine (position 9 ) rings (Eaton et al., 2000). Finally, we suggested that the aromatic tertiary amino group from PCP (or TCP) might interact with acidic residues (i.e., ␣1-Glu262 ) located at the outer ring (position 20 ) (Arias et al., 2002, 2003, 2006b; reviewed in Arias et al., 2006a). Considering our previous findings, we suggest that the secondary amino group from the BP molecule can also be responsible for the binding to one of the two Glu262 residues by electrostatic interactions, when the receptor is in the resting state. Proposed mechanisms of BP binding were additionally studied by molecular modeling. Docking simulations followed by molecular dynamics of BP were performed on ␣3␤4 (Jozwiak et al., 2004)

and on Torpedo and mouse muscle AChRs (Arias et al., 2009). Fig. 6 shows the interaction of neutral BP with the Torpedo AChR ion channel. The results from the docking simulations using muscletype AChRs indicate no enantioselectivity for BP. Although there is no experimental evidence of BP enantioselectivity on AChRs, the (2R,3R)-hydroxybupropion isomer inhibits the muscle-type AChR with 3.7-fold higher potency than the (2S,3S)-hydroxybupropion isomer (Damaj et al., 2004) (see Table 2), indicating the possibility of BP enantioselectivity. In this regard, further experiments need to be performed to address this question. The analysis of the obtained molecular complexes clearly indicates that BP in either the neutral or protonated form binds to the middle portion of muscle-type AChR ion channels between the serine (position 6 ) and valine rings (position 13 ) (see Fig. 6). Exactly the same locus was observed for PCP and imipramine in the Torpedo AChR ion channel (Sanghvi et al., 2008). Another distinction

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was observed for BP interacting with the ␣3␤4 ion channel: BP in the protonated state interacted with the polar region of the intermediate ring (position 1 ), whereas the neutral form was positioned between the valine/phenylalanine (position 15 ) and serine (position 8 ) rings (Jozwiak et al., 2004). The pocket formed by the cleft between the phenyl ring provided by ␤4-Phe and the isopropyl moiety from ␣3-Val interacts with the hydrophobic portion of BP, whereas hydrogen bonds are formed between polar residues at the serine ring and the polar region of BP. Considering this difference, distinct BP binding site locations may exist on each AChR ion channel. Nevertheless, we have to take into consideration that the ␣3␤4 AChR ion channel was constructed by homology with the model of the 23 mer peptide imitating the M2 sequence of the Torpedo AChR ␦ subunit (Jozwiak et al., 2004), whereas the Torpedo AChR ion channel model (Arias et al., 2009) was based on the cryo-electron microscopy structure determined at ∼4 Å resolution (PDB ID 2BG9; Unwin, 2005). The evidence obtained using the Torpedo AChR model also supports the [3 H]TCP competition experiments, indicating that there is a binding site for antidepressants that overlaps the PCP binding sites in the resting and desensitized Torpedo AChR ion channels (Sanghvi et al., 2008; Arias et al., 2009). Considering the above results, we envision a dynamic process where BP binds first to the resting AChR, decreasing the probability of ion channel opening. The remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process. Bupropion inhibits AChR function by interacting with a binding domain shared by TCAs and by PCP that is located between the serine and valine rings. 8. Concluding remarks

Fig. 6. Complex formed between (S)-bupropion and the Torpedo AChR ion channel obtained by molecular docking. Computational simulations were performed using the same protocol as recently reported (Sanghvi et al., 2008; Arias et al., 2009). Briefly, BP molecules were docked into the Torpedo ion channel model (PDB ID 2BG9; Unwin, 2005) using the following settings (Molegro Virtual Docker): numbers of runs = 100; maximal number of iterations = 10,000; maximal number of poses = 10. (A) Side view of the lowest energy complex showing four subunits rendered in secondary structure mode, whereas the BP in the neutral form is rendered in element color coded ball mode. (B and C) Side views of the target protein rendered in semitransparent surface with visible secondary structure and explicit CPK atoms of residues forming the valine (position 13 in green) and serine (position 6 in red) rings. BP (B) and PCP (C) in the neutral forms are rendered in stick mode with hydrogen atoms not shown explicitly. On these pictures the ␦ subunit was removed for clarity, and the order of remaining subunits is (from left to right) ␣1, ␥, ␣1, and ␤1.

Different components involved in the process of neurotransmission are important targets for the pharmacological action of BP in the CNS. The current evidence indicates that BP stimulates the release and inhibits the reuptake of NE and DA, as well as blocks different AChRs. This triple action might be the basis of the particular mechanisms by which BP mediates its clinical actions. In this regard, AChRs can be envisioned as targets for the pharmacological action of newer and safer antidepressants as well as for novel anti-addictive compounds. For example, the agonist cytisine, a natural quinolizidine alkaloid obtained from the seeds of Cytisus sp. and several genera of the Faboideae subfamily of plants, is already in the European market under the name Tabex® as an aid for antismoking therapy (Etter et al., 2008; reviewed in Tutka, 2008). Its synthetic derivative, varenicline (named Chantix® in the USA and Chamix® in Europe and other countries), is used as an anti-nicotinic drug (Niaura et al., 2006; reviewed in Kaur et al., 2009; Tutka, 2008), and has shown potential for the treatment of depression (Rollema et al., 2009), and alcohol addiction (Steensland et al., 2007). Newer cytisine derivatives, still in pre-clinical trials, present antidepressant activities (Mineur et al., 2009), and might be lead compounds for depression therapy. Acknowledgements This research was supported by grants from the Science Foundation Arizona and Stardust Foundation and the College of Pharmacy, Midwestern University, AZ, USA. References Alkondon M, Albuquerque EX. Nicotinic receptor subtypes in rat hippocampal slices are differentially sensitive to desensitization and early in vivo functional up-regulation by nicotine and to block by bupropion. J Pharm Exp Ther 2005;313:740–50.

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