The AAPS Journal 2006; 8 (4) Article 78 (http://www.aapsj.org).
`Themed Issue: NIDA/AAPS Symposium on Drugs of Abuse: Mechanisms of Toxicity, Toxicokinetics and Medical Consequences,
`November 4-5, 2005
`Guest Editors - Rao S. Rapaka and Jagitsing H. Khalsa
` Vesicular Monoamine Transporter 2: Role as a Novel Target for
`Drug Development
` Submitted: June 30 , 2006 ; Accepted: July 18 , 2006; Published: November 10, 2006
` Guangrong Zheng , 1 Linda P. Dwoskin , 1 and Peter A. Crooks 1
` 1 College of Pharmacy, University of Kentucky, Department of Pharmaceutical Sciences, Lexington, KY 40536-0082
`
` A BSTRACT
` In the central nervous system, vesicular monoamine trans-
`porter 2 (VMAT2) is the only transporter that moves cyto-
`plasmic dopamine (DA) into synaptic vesicles for storage
`and subsequent exocytotic release. Pharmacologically
`enhancing DA sequestration by VMAT2, and thus prevent-
`ing the oxidation of DA in the cytoplasm, may be a strategy
`for treating diseases such as Parkinson ’ s disease. VMAT2
`may also be a novel target for the development of treat-
`ments for psychostimulant abuse. This review summarizes
`the possible role of VMAT2 as a therapeutic target, VMAT2
`ligands reported in the literature, and the structure-activity
`relationship of these ligands, including tetrabenazine ana-
`logs, ketanserin analogs, lobeline analogs, and 3-amine-2-
`phenylpropene analogs. The molecular structure of VMAT2
`and its relevance to ligand binding are briefl y discussed.
`
`intestinal tract. 5-7 VMAT2 is also expressed in at least 2
`endocrine cell populations and in neurons. 6 Both VMAT1
`and VMAT2 are more widely expressed during embryonic
`development. 8 Substrate recognition and inhibitor sensitivi-
`ties for differences between VMAT1 and VMAT2 have been
`studied using membrane vesicles prepared from stable trans-
`formed cell lines from Chinese hamster ovaries (CHO) that
`express the respective proteins. 9 VMAT2 has a consistently
`higher affi nity for all of the monoamine substrates tested,
`particularly histamine, and has a greater sensitivity than
`VMAT1 to the inhibitor tetrabenazine (TBZ).
` The natural alkaloid reserpine and TBZ are considered 2 clas-
`sical VMAT inhibitors. 10 Reserpine inhibits the transport of
`amines into chromaffi n granules and synaptic storage vesi-
`cles 11 , 12 by binding with high affi nity to VMAT, presumably at
`the amine recognition site. It has been suggested that TBZ, on
`the other hand, binds to a site on VMAT that is different from
`the substrate binding site at which reserpine interacts. 12-14
`
` K EYWORDS: vesicular monoamine transporter 2 , Parkinson ’ s
`disease , psychostimulant abuse , tetrabenazine , ketanserin ,
` lobeline
`
` INTRODUCTION
` The vesicular monoamine transporter (VMAT), a member of
`the vesicular neurotransmitter transporter family, is respon-
`sible for the translocation of monoamines (serotonin, dopa-
`mine, norepinephrine, and histamine) from the cytoplasm
`into synaptic vesicles via a proton electrochemical gradient
`generated by the vacuolar type H + -adenosine triphospha-
`tase. 1 Two pharmacologically distinct VMAT isoforms,
`VMAT1 and VMAT2, have been cloned and described. 2-4
`Adult human and rodent monoaminergic neurons of the cen-
`tral nervous system (CNS) and sympathetic postganglionic
`neurons express only VMAT2, 5-7 while VMAT1 is predomi-
`nantly expressed in neuroendocrine cells such as chromaffi n
`cells of the adrenal medulla and enterochromaffi n cells of the
`
` Corresponding Author: Peter A. Crooks, College of
`Pharmacy, University of Kentucky, 907 Rose Street, Room
`501B, Lexington, KY 40503 . Tel: (859) 257-1718 ; Fax:
` (859) 257-7585 ; E-mail: pcrooks@email.uky.edu
`
` VMAT2 AND NEUROPROTECTION
` Oxidative deamination of monoamines by monoamine oxi-
`dase is accompanied by the reduction of molecular oxygen
`to a toxic product, hydrogen peroxide. 15 Therefore, mainte-
`nance of low cytoplasmic concentrations of neurotransmit-
`ters by their reuptake into synaptic vesicles for storage is
`important to minimize their inherent toxicity. 16 Further-
`more, storage of neurotransmitters in synaptic vesicles pre-
`cludes their metabolism in the cytoplasmic compartment
`and reduces the synthetic demands on the cell. 16 In the cen-
`tral nervous system, VMAT2 is the only transporter that
`moves cytoplasmic dopamine (DA) into synaptic vesicles
`for storage and subsequent exocytotic release. 1
` Parkinson ’ s disease is a degenerative, progressive disorder
`that dramatically affects neurons of the substantia nigra and
`the basal ganglia. The etiology of Parkinson ’ s disease has
`not been elucidated, but exposure to endogenous or envi-
`ronmental toxins may contribute to the development of the
`disease. 17-21 In this regard, DA may play a role as an endog-
`enous toxin, since the normal metabolism of DA produces
`hydrogen peroxide as a byproduct, and the formation of
`DA-associated reactive oxygen species may contribute to
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`the loss of nigrostriatal DA neurons. 22 Accordingly, phar-
`neurotransmitter transporters that regulate synaptic DA con-
`macologically enhancing DA sequestration by VMAT2, and
`centrations. 43-45 Recent studies have demonstrated that psy-
`thus preventing the oxidation of DA in the cytoplasm, may
`chostimulants alter VMAT2 function. 46 , 47 Cocaine inhibits
`be a strategy for treatment of Parkinson ’ s disease.
`DA transporter function, induces a rapid and reversible
`increase in vesicular DA uptake and dihydrotetrabenazine
` Exposure to the neurotoxin N -methyl-4-phenyltetrahydropyri-
`(DTBZ) binding, and causes a shift in the ratio of cytoplas-
`dine (MPTP) results in clinical symptoms closely approximat-
`mic to vesicular DA, all of which suggests that VMAT2
` N -Methyl-4-phenylpyridinium
`ing Parkinson ’ s disease. 17
`may be a novel target for the development of treatments for
`(MPP + ), the active toxic metabolite of MPTP, is a substrate for
`cocaine abuse. 48 Amphetamine and its analogs, such as
`VMAT2. 23-27 VMAT2 sequesters MPP + in synaptic vesicles
`methamphetamine, decrease vesicular DA sequestration by
`and thereby protects catecholamine-containing neurons from
`inhibiting vesicular uptake and promoting release from the
`MPP + -induced toxicity and degeneration. 3 , 28-32 CHO cells,
`vesicles. 49 , 50 Amphetamine diffuses across the vesicular
`which are normally sensitive to MPP + toxicity, because they
`membrane, decreasing the pH gradient, which results in the
`lack a plasma membrane amine transporter, can be made rela-
`loss of free energy needed for monoamine sequestration. 49-52
`tively insensitive to MPP + toxicity by transfection with VMAT
`Also, amphetamine that accumulates in the vesicles com petes
`complementary DNA. 3 In addition, when the transfected CHO
`with monoamines for protons, resulting in an increase in the
`cells are treated with reserpine, which inhibits VMAT2 func-
`diffusion of uncharged monoamines out of the vesicle. 52
`tion, the cells then become sensitive to MPP + toxicity. 3 Other
`High-dose methamphetamine treatment decreases vesicular
`studies using heterozygous VMAT2 knockout mice show that
`DA uptake and DTBZ binding, suggesting that there is a
`the knockouts are more susceptible to the neurotoxic effects of
`signifi cant alteration in VMAT2 function and localization at
`MPTP compared with the wild-type mice. 28 , 30 , 33 Furthermore,
`the vesicular membrane. 53 VMAT2 heterologous knockout
`heterozygous VMAT2 knockout mice are more sensitive to
`mice exhibit reduced amphetamine-conditioned place pref-
`methamphetamine-induced neurotoxicity and are more vul-
`erence (reward) and enhanced sensitivity to the locomotor
`nerable to the toxic effects of L-3,4-dihydroxyphenylalanine
`effects of apomorphine, ethanol, cocaine, and amphet-
`(L-DOPA, a DA precursor used to treat Parkinson ’ s disease)
`amine. 28 , 54 VMAT2 knockout studies also indicate that
`compared with wild-type mice. 34 , 35 The latter results suggest
`VMAT2 plays an important role in mediating the behavioral
`that reduction in VMAT2 activity might attenuate the effi cacy
`effects of psychostimulants. Taken together, these results
`of L-DOPA therapy in Parkinson ’ s patients. Finally, increased
`support the idea that VMAT2 should be considered as a valid
`sequestration of DA in synaptic vesicles by VMAT2 has been
`target for the development of pharmacotherapies to treat
`suggested to be protective in Parkinson ’ s disease. 36
`psychostimulant abuse. Other evidence supporting the role
` Recently, studies have suggested that pharmacological agents
`of VMAT2 in psychostimulant pharmacology is the fi nding
`that increase VMAT2 activity are neuroprotective. For exam-
`that benzoquinolizine derivatives, such as TBZ, which have
`ple, methylphenidate increases vesicular DA uptake in rats
`high affi nity for VMAT2, decrease locomotor activity and agg-
`and prevents persistent dopaminergic defi cits induced by high-
`ressiveness in monkeys 55 and decrease methamphetamine-
`dose methamphetamine administration. 37 , 38 Pramipexole, a
`induced hyperactivity in rodent animal models. 55
`DA D2/D3 agonist used as a therapy for Parkinson ’ s disease,
`increases vesicular DA uptake and protects against the loss of
`nigrostriatal DA neurons in methamphetamine-, 3-acetylpyri-
`dine-, and ischemia-induced neurotoxicity. 39-41 Additionally,
`apomorphine, a DA D2/D3 agonist used in Europe as a treat-
`ment for Parkinson ’ s disease and for impotence, increases
`vesicular DA uptake, and this mechanism has been suggested
`to be important for its associated neuroprotection. 42
` Taken together, the results of the above studies indicate that
`VMAT2 expression and function are important in counter-
`acting the neurotoxicity of MPP + and perhaps of other envi-
`ronmental and endogenous neurotoxins that play an etiologic
`role in neurodegenerative disease. 21
`
` VMAT2 LIGANDS
` TBZ and Its Analogs
` TBZ ( 1 , Figure 1 ), a benzoquinolizine compound, has been
`shown to deplete cerebral monoamines in rat brain by revers-
`ibly inhibiting VMAT2. 56 First introduced in 1956 as an anti-
`psychotic drug, 57 TBZ is currently used to treat hyperkinetic
`movement disorders, such as chorea associated with
`Huntington ’ s disease, tics in Tourette ’ s syndrome, and
`movement stereotypes in tardive dyskinesia. 58-60 The side
`effects associated with TBZ include sedation, depression,
`akathisia, and parkinsonism. 58 TBZ inhibits catecholamine
`uptake by VMAT2 with a K i of 3 nM 14 and acts as an inhibitor
`of both presynaptic and postsynaptic DA receptors in rat
` VMAT2 AND PSYCHOSTIMULANT ABUSE
`brain. 61 [ 11 C]TBZ (label on the 9- O -methyl group) has been
` Psychostimulant-induced behavioral activation and rein-
`synthesized 62 and used as an in vivo radioligand for positron
`forcement are mediated, at least in part, via interaction with
`emission tomography (PET) imaging of VMAT2. 63-66
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` Figure 1. Structures of tetrabenazine and its analogs ( 1-4 ).
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`The AAPS Journal 2006; 8 (4) Article 78 (http://www.aapsj.org).
`phy (HPLC). The (+)-isomer (2R,3R,11bR, 3a ) 74 shows
`high affi nity in vitro ( K i = 0.97 nM) for rat VMAT2, whereas
`the ( – )-isomer shows very low affi nity for VMAT2 ( K i = 2.2
`μM). Thus the binding of a -DTBZ to VMAT2 is enantiose-
`lective, with the (+)-isomer having higher affi nity. 75 , 76
` Another 4 possible DTBZ isomers (2S,3S,11bR, 3e ; 2R,3R,11bS,
` 3f ; 2R,3S,11bR, 3g ; 2S,3R,11bS, 3h , Figure 3 ) have been synthe-
`sized and tested for inhibition of VMAT2 binding using rat vesicu-
`lar membranes. Isomer 3g showed the highest affi nity ( K i = 28
`nM) in the [ 3 H]DTBZ binding assay. 77 , 78
` Methoxytetrabenazine (MTBZ) ( 4 , Figure 1 ) is another
`TBZ analog with high affi nity ( K d = 3.9 nM) for VMAT2. 79
`Similar to DTBZ, [ 3 H] and [ 11 C]MTBZ have also been syn-
`thesized 73 and used in in vitro and in vivo studies. 79-81
` Nucleophilic addition of organometallic reagents to the C-2
`keto group of TBZ generated a series of 2-alkylated DTBZ
`analogs, such as the 2-Me, 2-Et, 2-Pr, 2-iso-Pr, and 2-iso-Bu
`derivatives (all racemic mixtures, Figure 4 ). 82-85 These com-
`pounds have been evaluated for inhibition of [ 3 H]MTBZ
`binding to VMAT2 in rat striatum. 85 The b -methyl com-
`pound 5a showed the highest affi nity ( K i = 2.6 nM) in this
`series, with a nearly 5-fold higher affi nity than its diastereo-
`mer 5b ( K i = 12 nM), which is consistent with the fi nding
`that a -DTBZ exhibits higher affi nity for VMAT2 than does
` b -DTBZ. 73 Compound 5b and compounds 6 to 9 all contain
`a b -hydroxyl group and showed a general decrease in bind-
`ing affi nity upon either lengthening or branching of the
`alkyl group at C-2. 85 These results indicate that analogs
`containing considerable steric bulk at position 2 can be tol-
`erated. Thus, compound 10 ( Figure 4 ), in which an 125 I atom
`has been introduced for autoradiographic studies of VMAT2,
`has been synthesized. 86 ( ± )-Compound 10 can be separated
`by chiral HPLC into its optical isomers, and the fi rst eluted
`enantiomer binds to VMAT2 with a K d of 0.22 nM. 87
` Structure-activity relationship (SAR) studies involving TBZ
`analogs have shown that quaternization of the amine nitro-
`gen at position 5, aromatization of ring C, and elimination
`of the carbonyl group afforded compounds that were devoid
`
` TBZ analogs have been synthesized with different alkyl
`groups at the C-3 position in the molecule, such as com-
`pound Ro 4-1632 ( 2 , Figure 1 ). These analogs retain good
`amine-depleting activity. 55
` In vivo, TBZ is rapidly and extensively metabolized to its
`reduced form, DTBZ ( 3 , Figure 1 ). 67 [ 3 H]DTBZ (label on
`the C-2 hydrogen) has been used as a selective radioligand
`in in vitro brain homogenate binding studies and in autora-
`diographic studies, and is reported to have a K d value of 3.0
`nM. 13 , 14 , 68-70 [ 11 C]DTBZ (label on the 9- O -methyl group)
`has also been synthesized 71 and used for in vivo PET imag-
`ing of VMAT2. 66 , 72
` TBZ contains 2 chiral carbon centers at C-3 and C-11b;
`thus, theoretically, TBZ can exist as 4 possible stereoiso-
`mers (3R,11bR; 3S,11bS; 3R,11bS; and 3S,11bR). TBZ
`usually refers to the racemic compound, that is, a 1:1 mix-
`ture of the 3R,11bR and 3S,11bS isomers. Synthetic DTBZ,
`the product of hydride reduction of the 2-keto group of TBZ,
`can exist in 2 a -DTBZ forms (2R,3R,11bR, 3a ; and
`2S,3S,11bS, 3b , Figure 2 ) and 2 b -DTBZ forms (2S,3R,11bR,
` 3c ; and 2R,3S,11bS, 3d , Figure 2 ). a -DTBZ and b -DTBZ
`can be separated by column chromatography, and the
` a -DTBZ isomer ( K i = 6 nM) shows slightly higher binding
`affi nity in vitro for rat brain VMAT2 than does b -DTBZ ( K i =
`20 nM). 73 The 2 enantiomers of a -DTBZ have been sepa-
`rated using chiral High Performance Liquid Chromatogra-
`
` Figure 2. Stereoisomers of dihydrotetrabenazine ( 3a-d ).
`
` Figure 3. Stereoisomers of dihydrotetrabenazine ( 3e-h ).
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` Figure 4. Structures of tetrabenazine analogs ( 5a-b and 6-10 ).
`
`of monoamine-depleting activity. 55 , 88 Thus, a basic amine
`nitrogen at position 5 is a prerequisite for TBZ-like activ-
`ity. 89 Also, methoxy groups at positions 9 and 10 appear to
`be essential for TBZ-like activity; the methylenedioxy com-
`pound 11 ( Figure 5 ) was 3 orders of magnitude less potent
`than Ro 4-1284 ( 6 ). 90
` Replacing the carbonyl oxygen in TBZ with a bis -methylthio
`group (compound 12 , Figure 5 ) affords a compound with
`similar activity to TBZ. 91 Olefi nation of the carbonyl group
`to afford compound 13 ( Figure 5 ) (EC 50 = 14 nM) resulted
`in potent inhibition of [ 3 H]DTBZ binding. 92
` Based upon a limited number of TBZ analogs ( 14 - 17 , Fig-
`ure 6 ), a correlation between the lipophilicity of the analogs
`and their affi nity for the DTBZ binding site has been estab-
`lished. 93 Compounds shown to have higher partition coeffi -
`cients (octanol/buffer) generally exhibited a greater ability
`to inhibit the specifi c binding of [ 3 H]DTBZ (IC 50 = 6 nM
`for 14 , 47 nM for 17 , 110 nM for 16 , and 2500 nM for 15 )
`to VMAT2. 93 Accordingly, compound 20 ( Figure 6 ), an
`iodinated and photosensitive derivative of TBZ, has been
`synthesized and exhibited an IC 50 of 428 nM to inhibit
`[ 3 H]DTBZ binding. 92 However, both its precursor (com-
`pound 18 , IC 50 = 8.1 nM) and the non-iodinated analog ( 19 ,
`IC 50 = 53 nM) of compound 20 showed higher affi nity at
`VMAT2 than did compound 20 . 92
` Several derivatives of compound 16 (ie, compounds 21 - 24 ,
` Figure 7 ) have been synthesized; of these, the amino com-
`pounds 21 and 22 retained affi nity for VMAT2 ( K i = 7.6 nM
`and 72.2 nM, respectively, in the [ 125 I]iodovinyl-TBZ bind-
`ing assay), whereas the amido compounds 23 and 24 exhib-
`ited diminished affi nity for VMAT2 ( K i = 730 nM and >10
`000 nM, respectively, in the [ 125 I]iodovinyl-TBZ binding
`assay). 94
`
` Ketanserin and Its Analogs
` Ketanserin ( 25 , Figure 8 ), a well-known serotonin 5-HT2
`receptor antagonist, 95 also binds to VMAT on chromaffi n
`
` Figure 6. Structures of tetrabenazine analogs ( 14-20 ).
`
`granules and synaptic vesicles. 96-98 In the studies by Darchen
`et al, 96 Henry et al, 97 and Leysen et al, 98 ketanserin competi-
`tively inhibited the binding of [ 3 H]DTBZ to VMAT2, and
`[ 3 H]ketanserin binding.
`conversely, TBZ displaced
`[ 3 H]Ketanserin binds to the TBZ binding site with a K d of
`45 nM at 30ºC and a K d of 6 nM at 0ºC. 96
` A ketanserin derivative, 7-azidoketanserin ( 26 , Figure 8 ),
`also binds to the TBZ binding site of bovine chromaffi n
`granule membranes with a K i of 23 nM (inhibition of
`[ 3 H]DTBZ binding). 99 An iodinated azido derivative of ket-
`anserin, 7-azido-8-iodoketanserin ( 27 , Figure 8 ), binds to
`the same specifi c TBZ binding site as ketanserin with a K d
`of 5.5 nM at 0ºC 99 ; 7-azido-8-[ 125 I]iodoketanserin has been
`successfully used for photoaffi nity labeling of TBZ binding
`sites of different tissues, including rat striatum, rabbit
` platelets, human pheochromocytoma, and human adrenal
`medulla. 99
` Lengthening the distance between the piperidine and the
`benzoyleneurea moieties of the ketanserin molecule by
`addition of 2 methylene groups results in a compound ( 28 ,
` Figure 9 ) that exhibits a 20-fold decrease in affi nity ( K i =
`950 nM) for the [ 3 H]DTBZ binding site. 96 Reducing the
`keto group of ketanserin (compound 29 , Figure 9 ) also
`decreases affi nity ( K i = 350 nM) for this site. Additionally,
`replacing the benzoyleneurea moiety with other heterocy-
`cles (eg, compounds 30-32 , Figure 9 ) also decreases affi nity
`( K i = 950, 814, and 3600 nM, respectively) for the [ 3 H]DTBZ
`binding site. However, minor structural changes to the
`
` Figure 5. Structures of tetrabenazine analogs ( 11-13 ).
`
` Figure 7. Structures of tetrabenazine analogs ( 21-24 ).
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` Figure 10. Structure of lobeline ( 35 ).
`
` Figure 8. Structures of ketanserin and its analogs ( 25-27 ).
`
`neurochemical studies, suggesting that they do not act via a
`common mechanism. Nevertheless, lobeline has often been
`considered to be a nicotinic receptor agonist. Conversely,
`we and others have established that lobeline acts as a potent,
`but nonselective, nicotinic receptor antagonist. 104 , 114-117
`Lobeline inhibits nicotine-evoked [ 3 H]DA overfl ow from
`rat striatal slices with an IC 50 of 1 μM, suggesting that lobe-
`line acts as an antagonist at nicotinic receptors mediating
`nicotine-evoked DA release (ie, a 6 b 2 b 3* subtype). 116 Lobeline
`also inhibits nicotine-evoked 86 Rb + effl ux from rat thalamic
`synaptosomes with an IC 50 of 0.7 μM, indicating that lobe-
`line is also an antagonist at a 4 b 2* nicotinic receptors. 116
`Moreover, lobeline also inhibits [ 3 H]methyllycaconitine
`binding to rat brain membranes with a K i of 6.26 μM, indi-
`cating that there is an interaction with the a 7* nicotinic
`receptor subtype. 117 Lobeline has also been reported to
`be an antagonist (IC 50 of 8.5 μM) at human a 7* nicotinic
`receptors expressed in Xenopus oocytes. 118
` In addition to interacting with nicotinic acetylcholine recep-
`tors (nAChRs), lobeline inhibits [ 3 H]DTBZ binding to
`VMAT2 with an IC 50 of 0.90 μM and inhibits [ 3 H]DA
`uptake into rat striatal vesicle preparations with an IC 50 of
`0.88 μM. 119 , 120 Therefore, lobeline is a nonselective nAChR
`antagonist that also inhibits VMAT2 function. Importantly,
`lobeline has been shown to inhibit both the neurochemical
`and the behavioral effects of amphetamine in rodents. 111 , 121-123
`The mechanism underlying the lobeline-induced inhibition
`of these effects has been suggested to be noncompetitive
`inhibition of VMAT2 function. 114 The observation that lobeline
`is not self-administered is consistent with fi ndings that lobe-
`line does not evoke DA release. 111 , 114 , 119 Furthermore, the
`observation that lobeline inhibits methamphetamine-evoked
`DA release from superfused rat striatal slices 116 is consis-
`tent with its ability to decrease methamphetamine self-
`administration in rats. 123 These studies clearly implicate
`VMAT2 as a potential target for the development of
`agents to treat methamphetamine abuse. Regardless,
`lobeline is a novel prototypical molecule from which
`subtype-selective nAChR ligands and selective VMAT2
`inhibitors may be developed following appropriate struc-
`tural modifi cation.
` Systematic structural modifi cation of the lobeline molecule
`provided 2 non-oxygen-containing
`lobeline analogs:
` N -methyl-2,6-di-( cis -phenylethenyl)piperidine ( meso -trans-
`diene [MTD], 36a , Figure 11 ) and N -methyl-2,6-di-( cis -
`phenylethyl)piperidine (lobelane, 37a , Figure 11 ). The latter
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`benzoyleneurea moiety, such as introducing a hydroxyl
`group into the ring (compound 33 , Figure 9 ) or replacing 1 of
`the oxygen atoms with a sulfur atom (compound 34 , Figure
`9 ), retains the affi nity ( K i = 14 and 40 nM, respectively). 96
`
` Lobeline and Its Analogs
` A lipophilic alkaloid from Lobelia infl ata , ( – )-lobeline
`(lobeline, 2R,6S,10S-, 35 , Figure 10 ), displaces [ 3 H]nicotine
`binding from native nicotinic receptors in the CNS with
`high affi nity ( K i = 4-30 nM). 100-104 Although lobeline has
`no structural resemblance to nicotine, and SARs do not sug-
`gest a common pharmacophore, 105 it has many nicotinelike
`effects, such as tachycardia and hypertension, 106 brady-
`cardia and hypotension in anesthetized rats, 107 anxiolytic
`activity, 108 and improvement of learning and memory. 109 In
`contrast to nicotine, lobeline only marginally supports self-
`administration in mice 110 and does not support self-admin-
`istration in rats. 111 Additionally, chronic lobeline treatment
`does not increase locomotor activity in rats and does not
`produce conditioned place preference. 112 , 113 Thus, lobe-
`line and nicotine have different effects in behavioral and
`
` Figure 9. Structures of ketanserin analogs ( 28-34 ).
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` Figure 11. Structures of lobeline analogs ( 36a-c and 37a-c ).
`
`2 analogs showed affi nity for VMAT2 at the TBZ binding
`site ( K i of 9.88 μM for 36a , and 0.97 μM for 37a ), with
`negligible affi nity for the ligand binding sites on a 4 b 2* and
` a 7* nAChRs. 117 , 124 Compounds 36b and 36c ( Figure 11 )
`are 2 stereoisomers of 36a ; 36c was equipotent with its
` meso -isomer, MTD, but 36b was slightly less potent (2- to
`3-fold) than MTD at VMAT2. Within the lobelane series of
`compounds (ie, compounds 37a , 37b , and 37c , Figure 11 ), a
`change in C2, C6 stereochemistry from cis to trans afforded
`a modest reduction (5- to 6-fold) in affi nity at VMAT2. The
` trans enantiomers 37b and 37c exhibited comparable affi ni-
`ties at VMAT2. These data indicate that the VMAT2 bind-
`ing site is not sensitive to major stereochemical changes to
`the MTD and lobelane molecules at the C2 and C6 piperid-
`ino ring carbons.
` Interestingly, 2 conformationally fl exible, ring-opened com-
`pounds, 38a and 38b ( Figure 12 ) ( K i = 5.21 and 3.96 μM,
`respectively) and 2 acyclic compounds, 39 and 40 ( Figure
`12 ) ( K i = 2.37 and 3.07 μM, respectively), exhibited lower,
`but comparable, affi nity for VMAT2 compared with lobel-
`ane. Thus, ring opening or complete removal of the piperi-
`dine ring results in only a modest reduction in affi nity at
`VMAT2 compared with lobelane ( 37a ). The presence of a
`basic amine functionality is likely a prerequisite for VMAT2
`recognition, as is evidenced by the fact that quaternized
`compounds 41 ( K i > 100 μM) and 42 ( K i = 16.5 μM) ( Fig-
`ure 12 ) show signifi cant loss in affi nity for VMAT2. 124
` The entire lobelane structure appears to be critical for high-
`affi nity binding at VMAT2, since fragments of lobelane or
`MTD, such as compounds 43 and 44 ( Figure 13 ) (both K i >
`100 μM), exhibited no affi nity for VMAT2. 125
`
` Figure 13. Structures of lobeline analogs ( 43 and 44 ).
`
` Isomerized lobelane analogs, such as compound 45 ( Figure
`14 ) ( K i = 1.36 m M), retained affi nity for VMAT2, indicating
`that the position of the piperidine N atom relative to the C2
`and C6 side chains does not appear to be critical for VMAT2
`interaction, and that the VMAT2 binding site can tolerate
`changes in distance between the piperidine nitrogen and the
`2 phenyl rings. 125 In the lobelane structure, changes in the
` N -substituent can also be tolerated. Nor -lobelane ( 46 , K i =
`2.31 m M), nor - N -ethyl lobelane ( 47 , K i = 3.41 m M), and
` nor - N -n-propyl lobelane ( 48 , K i = 1.87 m M) ( Figure 14 )
`exhibit only a slight decrease in affi nity for VMAT2 com-
`pared with lobelane. 125 Replacing the phenyl rings of lobel-
`ane with naphthalene rings (compound 49 , K i = 0.63 m M) or
`introducing substituents into the phenyl rings (eg, in com-
`pounds 50 , K i = 0.57 m M; 51 , K i = 0.43 m M; and 52 , K i =
`0.52 m M) ( Figure 14 ) retains or somewhat improves affi nity
`at VMAT2.
` To increase the rigidity of the lobelane molecule, analogs
`were prepared in which the piperidine ring has been replaced
`with a tropene ring. The resulting compounds ( 53 , K i = 1.30
` m M; 54 , K i = 1.38 m M; and 55 , K i = 4.80 m M) ( Figure 15 )
`exhibited affi nity at VMAT2 comparable with lobelane. 126
`
` 3-Amino-2-Phenylpropene Derivatives
` Recently, a series of 3-amino-2-phenylpropene derivatives
`( Figure 16 ) have been reported as novel competitive inhibi-
`tors of the bovine chromaffi n granule membrane mono-
`amine transporter (bVMAT2). 127 With a K i of 40.3 m M,
`3-amino-2-phenylpropene (APP, 56 ) inhibits DA uptake
`into bVMAT2. Introduction of a hydroxyl group into the 3 or 4
`
` Figure 12. Structures of lobeline analogs ( 38a-b and 39-42 ).
`
` Figure 14. Structures of lobeline analogs ( 45-52 ).
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`to form an ion pair and appear to provide a structural frame-
`work for substrate recognition. 129 Experiments employing a
`chimera of VMAT1 and VMAT2 indicate that 2 domains,
`TMD5 through TMD8 and TMD9 through TMD12, cooper-
`ate to confer the high-affi nity interaction of VMAT2 with
`TBZ and histamine. 130 In addition, the domain encompass-
`ing TMD3 and TMD4 infl uences serotonin affi nity but not
`histamine affi nity or TBZ sensitivity. 130 The domain encom-
`passing TMD5 through TMD7 of VMAT2 in the context of
`N-terminal VMAT2 sequences reduces the apparent affi nity
`for serotonin but not histamine or the sensitivity to TBZ. 130
`Tyrosine 434 and aspartate 461 in TMD9 through TMD12
`are identifi ed as being responsible for the high-affi nity inter-
`action of TBZ, histamine, and serotonin, but not for DA. 131
`Photoaffi nity labeling of purifi ed rat VMAT2 indicates that
`TMD1 and TMD10/11 are possibly juxtaposed and may
`interact in a functionally signifi cant manner. 132 Cysteine
`mutagenesis and derivatization of human VMAT2 revealed
`that cysteines 439, 476, and/or 497, and possibly cysteines
`126 and/or 333, are important for [ 3 H]TBZOH binding, and
`cysteines 176, 207, and 439 together are important for
`[ 3 H]serotonin transport. 133 Furthermore, a disulfi de bond
`between lumenal cysteine 126 in loop 1/2 and cysteine 333
`in loop 7/8 has been identifi ed. 134
`
` Figure 15. Structures of lobeline analogs ( 53-55 ).
`
`position of APP affords compounds 57 ( K i = 16.7 m M) and
` 58 ( K i = 15.5 m M), respectively, equally improved potency
`for bVMAT2. However, compound 59 ( K i = 103 m M), which
`has a methoxyl group at the 4 position of the phenyl ring of
`APP, led to a decrease in potency. However, compound 60 ,
`in which a methoxyl group is at the 3 position of the phenyl
`ring, led to a slight improvement in inhibitory potency with
`respect to APP. Methyl ( 61 , K i = 55.9 m M) or fl uoro ( 62 , K i =
`42.3 m M) substitution at the 4 position of the phenyl ring
`had no effect on the inhibitory potency, while chloro ( 63 ),
`bromo ( 64 ), and iodo ( 65 ) substitution led to a modest
`increase in inhibitory potency ( K i = 18.0, 17.7, and 12.9
` m M, respectively).
`
` VMAT2 STRUCTURE AND MOLECULAR BASIS
`FOR BINDING
` Predictions regarding the molecular structure of VMAT2
`from its protein sequence are that it comprises 12 putative
`transmembrane domains (TMDs) with both N- and C- ter-
`mini in the cytoplasm and a large, hydrophobic, N -glycosyl-
`ated loop between TMDs 1 and 2 facing the vesicle lumen. 1
`Structural biology studies have identifi ed important resi-
`dues that may contribute to ligand binding and monoamine
`transport. Mutagenesis studies indicate that aspartate 33,
`which contains a negative charge, in TMD1 and serines 180
`to 182 in TMD3 of VMAT2 play a critical role in substrate
`recognition, presumably by interacting with the protonated
`amino group of the ligand and hydroxyl groups on the cate-
`chol or indole ring, respectively. 128 In addition, lysine 139
`in TMD2 and aspartate 427 in TMD11 of VMAT2 interact
`
` CONCLUSION
` Signifi cant progress has been made over the last 20 years in
`elucidating the role of VMAT2 in monoamine transport and
`its potential as a therapeutic target. VMAT2 sequesters cyto-
`plasmic DA and thus prevents the oxidation of DA in the
`cytoplasm; VMAT2 also sequesters neurotoxins within ves-
`icles. These data indicate that VMAT2 may play a role in
`neuroprotection and that molecules that interact with
`VMAT2 may have value as treatments for diseases such as
`Parkinson ’ s disease. VMAT2 may also be a novel target for
`the development of treatments for psychostimulant abuse,
`and the discovery of molecules that modulate VMAT2 func-
`tion may afford useful tools for examining the pivotal role
`of this transporter in the neurochemical and behavioral
`effects of psychostimulant drugs, thus providing potential
`pharmacotherapies.
`
` ACKNOWLEDGMENTS
` This study was supported by National Institutes of Health
`grant DA 13519.
`
` Figure 16. Structures of 3-amino-2-phenylpropene and its
`analogs ( 56-65 ).
`
` REFERENCES
` 1 . Yelin R , Schuldiner S . Vesicular neurotransmitter transporters:
`pharmacology, biochemistry, and molecular analysis. In: Reith MEA, ed.
` Neurotransmitter Transporters; Structure, Function, and Regulation.
`
` 2nd. Totowa, NJ : Humana Press ; 2002 :313- 354.
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` 2 . Erickson JD , Eiden LE , Hoffman BJ . Expression cl