Phenyltropanes at Monoamine Transporters

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Drug and Alcohol Dependence 56 (1999) 915

Studies of selected phenyltropanes at monoamine transporters

Michael J. Kuhar a,*, Kathleen M. McGirr a, Richard G. Hunter a, Philip D. Lambert a,Bridgette E. Garrett a, F. Ivy Carroll b

a Diision of Neuroscience, Yerkes Regional Primate Research Center of Emory Uniersity, Atlanta, GA 30322, USAb Research Triangle Institute, Research Triangle Park, NC, USA

Received 9 February 1998; accepted 5 January 1999

Abstract

3-Phenyltropane analogues of cocaine are useful neurobiologic tools for examining mechanisms of neurotransmitter trans-

porters and psychostimulant drugs. They are also potential substitute medications for psychostimulant abuse. In this study, 183-phenyltropane analogues were characterized in uptake and binding studies at dopamine (DAT), norepinephrine (NET) and

serotonin (SERT) transporters from the rat, and in binding at DAT in rat, rhesus monkey, and human brain tissue. In rat brain

tissue, potency in inhibiting uptake generally correlated with the potency in inhibiting binding at all three transporters suggesting

that none of these compounds have antagonist properties. At the DAT, there was a significant correlation of inhibitory potencies

between the rat and monkey, the monkey and human, and the rat and human transporters although some compounds showed

some species difference. These findings suggest that with regard to the 3-phenyltropane series, there is generally little pharmaco-

logic difference between DATs from the three species examined, although binding data from rat may not be a perfect predictor

of uptake inhibition in human. 1999 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Selected phenyltropanes; Monoamine transporters; 3-Phenyltropane analogues; Cocaine; Medication

1. Introduction

Monoamine neurotransmitter transporters are key

targets for a variety of drugs including psychostimu-

lants. Cocaine and its analogues are well known in-

hibitors of monoamine transporters, and hundreds of

analogs have been produced with varying selectivities

(Clarke et al., 1973; Kline et al., 1990; Carroll et al.,

1992a; Kozikowski et al., 1992; Meltzer et al., 1992;

Davies et al., 1993; Simoni et al., 1993; Wang et al.,

1993; Goodman et al., 1994; Meltzer et al., 1994;

Bennett et al., 1995; Carroll et al., 1997). Some are

highly selective for dopamine transporters (DATs; Boja

et al., 1992; Carroll et al., 1992a,b, 1993, 1997) orserotonin transporters (SERTs; Blough et al., 1997) but

most affect multiple transporters. Inhibitory com-

pounds from other structural classes have also been

produced as well (van der Zee et al., 1980; Rothman et

al., 1993; Newman et al., 1994; Deutsch et al., 1996;

Carroll et al., 1997; Froimowitz et al., 1997; Matecka etal., 1997).

Many uptake inhibitors are possibly useful, at least

in principle, as substitute medications for psychostimu-

lant abuse. Reasonable properties of such compounds

would be potency, selectivity, long duration of action

and slow entry into brain (Kuhar, 1997), and many

compounds exhibit these properties (Rothman, 1990;

Stathis et al., 1995; Nader et al., 1997). It would also be

useful to identify a cocaine antagonist, i.e. a compound

that blocked cocaines binding to transporters while

allowing neurotransmitter uptake. In this investigation,

in order to characterize further some phenyltropanes,

we tested selected compounds in synaptosomal uptake

assays and in binding assays with rat, monkey and

human tissue. The selection included compounds highly

selective for DAT, as well as compounds with little

selectivity and varying affinities for all three trans-

porters. This study provides an assessment of func-

tional efficacy as well as the pharmacological properties

of transporters from different species.

* Corresponding author. Tel.: +1-404-727-1737; fax: +1-404-727-

3278.

E-mail address:[email protected] (M.J. Kuhar)

0376-8716/99/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0 3 7 6 - 8 7 1 6 ( 9 9 ) 0 0 0 0 5 - 8


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M.J. Kuhar et al./Drug and Alcohol Dependence 56 (1999) 91510

2. Methods

2.1. Tissue

Striatum, frontal cortex and hindbrain was dissected

from male Sprague Dawley rats (200300 g) brain.

Monkey caudate and putamen was obtained from male

rhesus monkeys (12 years old). Human caudate and

putamen was obtained from three men and one wo-

man (2070 years old). The post-mortem times elapsedbefore the human tissue was frozen ranged from 4

to 16 h.

2.2. Binding assays

Dopamine transporter (DAT) binding was per-

formed in a 0.32-M sucrose, 200 mM phosphate buffer

at pH 7.4 at 4C. Cocaine (30 M) was used as the

displacer and 0.5 nM [3H]WIN-35428 was used as the

radioactive ligand. Frozen rat striatum was homoge-

nized in cold 0.32M sucrose phosphate buffer and

centrifuged at 20 000

g for 10 min. The supernatantwas discarded, the pellet was resuspended and spun

again at 20000g for 10 min. Tissue (1 mg/ml

(final concentration)) was allowed to incubate with the

drug on ice for 120 min after which it was filtered and

counted. This assay was performed identically with

monkey and human tissue, however a 3-mg/ml final

concentration was used for the human tissue.

2.3. Uptake assays

2.3.1. Buffer

For all uptake assays the incubation buffer consisted

of 147 mM NaCl, 240 mM KCl, 65 mM CaCl2, 200

mM sodium phosphate pH 7.4, 70 mM MgSO4, 2

mg/ml dextrose and 0.2 mg/ml ascorbic acid were com-

bined at a final volume of 250 ml.

2.3.2. Dopamine

[3H]Dopamine uptake was performed in the above

assay buffer. Cocaine (100 M) was used as a blank

and 0.5 nM [3H]dopamine was added per assay tube.

Pargyline (1 M) was used to inhibit MAO. Striatal

synaptosomes were prepared by homogenizing fresh rat

striatal tissue in 20 volumes of ice cold 0.32 M sucrose

with a glass and Teflon homogenizer. The tissue was

centrifuged at 800g for 10 min. The pellet was dis-

carded and the supernatant was centrifuged at

20 000g for 10 min. The supernatant was discarded

and the pellet was resuspended in ice cold 0.32 Msucrose at a concentration of 15 mg/ml. The tissue was

allowed to sit on ice for 15 min and was then incu-

bated with the drug for 10 min in a 30C water bath.

The reaction was then started by adding [3H]dopamine.

The reaction was stopped by filtration after 3 min and

the filters subjected to scintillation spectrometry.

Table 1

Uptake and binding inhibition values (IC50, nM) in rat tissue at DAT, NET and SERT for selected 3-phenyltropanesa

Uptake (IC50, nM)Compound Binding (IC50, nM)

SERTNETDAT SERTNETDAT

10401 330089260221310Cocaine

233029502 2.0RTI-113 229313.0

1063 556RTI-120 3.3 5830 24 4005.0

RTI-121 1.5 3.2 12.6 0.4 284 66.84

RTI-141 1.8 7.6 24.0 3.4 834 3365

202047403.71946 3511RTI-150

3.97 412RTI-171 0.9 254 38202.8

RTI-177 1.9 3.88 235 1.3 504 2420

9 51 4003645 9002.9RTI-199

47803.9287 3770916.6RTI-20410

11 RTI-219 4.1 295 1250 5.7 8560 10 300

0.812 1.2RTI-51 1.7 37 10.60.9

RTI-55 1.1 1.013 0.4 1.3 36 4.2

RTI-108 2.8 3.314 8.5 2.6 126 97.6

RTI-112 1.1 0.815 1.4 0.8 36 10.5

1203419RTI-11616 123096733RTI-126 9417 3820253 78701002690

18 RTI-139 1.92.4 14.5 841.7 56.9

1491.022 83416WIN-3542819 809

0.3Paroxetine 0.320

a Compounds 211 were selected because binding data suggested they are selective for DAT, whereas compounds 1219 are not. Data shown

are averages of at least three determinations where standard errors were less than 20% of the mean. Some of these data are from previous studies;

see text for details.


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M.J. Kuhar et al./Drug and Alcohol Dependence 56 (1999) 9-15 11

Fig. 1. Structures of selected 3-phenyltropanes, cocaine, WIN 35428

and paroxetine.

[3H]norepinephrine was used as the substrate. Cortical

synaptosomes were prepared according to the above

procedure using fresh rat frontal cortex at a concentra-

tion of 100 mg/ml.

2.3.4. Serotonin

The procedure was the same as above except 1 M

citalopram was used to generate blanks and 5 nM

[3H]serotonin was used as the radioactive substrate.

Hindbrain synaptosomes were prepared according to

the above procedures using fresh rat hindbrain at aconcentration of 10 mg/ml.

2.4. Data analysis

IC50 values were determined using the EBDA Radlig

program. Graphs and correlation data were generated

using Graphpad Prism.

3. Results

3-Phenyltropane compounds with two general char-

acteristics were selected on the basis of previously ob-

tained binding data in the rat brain (Carroll et al.,

1992a,b, 1993, 1995, 1997): compounds that were fairly

selective for the dopamine transporter (compounds 2

11 in Table 1), and compounds that had varying de-

grees of selectivity for the three monoamine

transporters (compounds 1219 in Table 1, Fig. 1). The

relative potencies of the compounds for inhibiting neu-

rotransmitter uptake were assessed using synaptosomal

transport assays as described in Section 2. As expected,

potencies varied considerably among the different com-

pounds, but binding potencies and uptake potencies

varied similarly and were correlated for the compounds

as a group (Table 1 and Figs. 2 4). However, some

compounds seemed to be less potent in inhibiting up

Fig. 2. [3H]Dopamine (DA) uptake versus [3H]-WIN35428 binding

inhibition at the dopamine transporter (DAT) by selected 3-phenyl-

tropanes in rat striatal tissue. Dotted lines show 95% confidence

limits. See Table 1 for number index. P0.0001, r2=0.8760.

Fig. 3. [3H]Norepinephrine (NE) uptake versus [3H]-nisoxetine bind-

ing inhibition at the norepinephrine transporter (NET) by selected

3-phenyltropanes in rat cortical tissue. Dotted lines show 95% confi-

dence limits. See Table 1 for number index. P0.0001, r2=0.9120.

2.3.3. Norepinephrine

The procedure was the same as above except 1 M

desipramine was used to generate blanks and 5 nM


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Fig. 6. DAT binding inhibition of selected 3-phenyltropanes in human caudate and putamen and rat striatal tissue. Dotted lines show 95%

confidence limits. See Table 1 for data. P0.0001, r2=0.7059.

Fig. 7. DAT binding inhibition of selected 3-phenyltropanes in rhesus caudate and putamen and human caudate and putamen. Dotted lines show

95% confidence limits. See Table 1 for data. P0.0001, r2=0.812.

cocaine antagonist. Some compounds do indeed seem

to inhibit cocaine analog binding to a relatively higherdegree than neurotransmitter uptake (Simoni et al.,

1993; Slusher et al., 1997) although a complete demon-

stration of cocaine antagonism in vivo has not been

carried out. These compounds include some tricyclic

antidepressants, antipsychotic agents and other com-

pounds (Slusher et al., 1997). In the 3-phenyltropane

series, while some compounds may show slightly higher

potency in inhibiting binding compared to uptake (Si-

moni et al., 1993), large differences in potency have

never been found. In this study, potencies in inhibiting

binding as well as uptake seem similar to that for

cocaine and WIN 35428 as evidenced by the high

correlations between binding and uptake. Thus it

seems highly unlikely that any of the compounds tested

here can be cocaine antagonists at any of the trans-

porters.


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M.J. Kuhar et al./Drug and Alcohol Dependence 56 (1999) 91514

The data obtained in this study suggest that the

dopamine transporters from different species have simi-

lar pharmacologic properties. There were strong corre-

lations between inhibitory potencies in rat, monkey and

human caudate or putamen with a few compounds

showing some differences. There was a slight tendency

for several DAT selective compounds to become

weaker from rat to monkey to human, and for some

compounds to vary from expected potency in uptake

inhibitions based on binding data. This suggests that

binding data in rat may not precisely predict uptakeinhibition in human, although it appears to be a rea-

sonable general predictor. A high degree of, but not

perfect, homology between the rat and human DAT

amino acid sequence has been shown (Giros and

Caron, 1993) which supports the notion of similar

pharmacology but allows for some differences. Pharma-

cological characterization of expressed rat and human

DAT revealed a similar but not identical pharmacology

(Giros and Caron, 1993). Thus, rat brain transporters

are likely to be useful substitutes or models for human

brain transporters, although perhaps some compounds

may show differences in affinity between the two

transporters.

Acknowledgements

The authors acknowledge the support of: NIH grants

RR00165, DA11178, R37DA05477, and of ONDCP

contract OND6069; E. Nadler for preparing the

manuscript; and Drs J. Kleinman, D. Rye, A. Levey

and B. Wainer for providing human tissue.

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