lobelane analogs with various methylene linker lengths and acyclic lobelane analogs as potential

291 Pages·2017·2.86 MB·English

UUnniivveerrssiittyy ooff KKeennttuucckkyy
UUKKnnoowwlleeddggee
Theses and Dissertations--Pharmacy College of Pharmacy
2014
LLOOBBEELLAANNEE AANNAALLOOGGSS WWIITTHH VVAARRIIOOUUSS MMEETTHHYYLLEENNEE LLIINNKKEERR
LLEENNGGTTHHSS AANNDD AACCYYCCLLIICC LLOOBBEELLAANNEE AANNAALLOOGGSS AASS PPOOTTEENNTTIIAALL
PPHHAARRMMAACCOOTTHHEERRAAPPIIEESS TTOO TTRREEAATT MMEETTHHAAMMPPHHEETTAAMMIINNEE AABBUUSSEE
Zheng Cao
University of Kentucky, [email protected]
RRiigghhtt cclliicckk ttoo ooppeenn aa ffeeeeddbbaacckk ffoorrmm iinn aa nneeww ttaabb ttoo lleett uuss kknnooww hhooww tthhiiss ddooccuummeenntt bbeenneefifittss yyoouu..
RReeccoommmmeennddeedd CCiittaattiioonn
Cao, Zheng, "LOBELANE ANALOGS WITH VARIOUS METHYLENE LINKER LENGTHS AND ACYCLIC
LOBELANE ANALOGS AS POTENTIAL PHARMACOTHERAPIES TO TREAT METHAMPHETAMINE ABUSE"
(2014). Theses and Dissertations--Pharmacy. 32.
https://uknowledge.uky.edu/pharmacy_etds/32
This Doctoral Dissertation is brought to you for free and open access by the College of Pharmacy at UKnowledge. It
has been accepted for inclusion in Theses and Dissertations--Pharmacy by an authorized administrator of
UKnowledge. For more information, please contact [email protected].
SSTTUUDDEENNTT AAGGRREEEEMMEENNTT::
I represent that my thesis or dissertation and abstract are my original work. Proper attribution
has been given to all outside sources. I understand that I am solely responsible for obtaining
any needed copyright permissions. I have obtained needed written permission statement(s)
from the owner(s) of each third-party copyrighted matter to be included in my work, allowing
electronic distribution (if such use is not permitted by the fair use doctrine) which will be
submitted to UKnowledge as Additional File.
I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and
royalty-free license to archive and make accessible my work in whole or in part in all forms of
media, now or hereafter known. I agree that the document mentioned above may be made
available immediately for worldwide access unless an embargo applies.
I retain all other ownership rights to the copyright of my work. I also retain the right to use in
future works (such as articles or books) all or part of my work. I understand that I am free to
register the copyright to my work.
RREEVVIIEEWW,, AAPPPPRROOVVAALL AANNDD AACCCCEEPPTTAANNCCEE
The document mentioned above has been reviewed and accepted by the student’s advisor, on
behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of
the program; we verify that this is the final, approved version of the student’s thesis including all
changes required by the advisory committee. The undersigned agree to abide by the statements
above.
Zheng Cao, Student
Dr. Linda P. Dwoskin, Major Professor
Dr. Jim R. Pauly, Director of Graduate Studies
LOBELANE ANALOGS WITH VARIOUS METHYLENE LINKER LENGTHS AND
ACYCLIC LOBELANE ANALOGS AS POTENTIAL PHARMACOTHERAPIES TO
TREAT METHAMPHETAMINE ABUSE

DISSERTATION

A dissertation submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in the College of Pharmacy at the University of Kentucky

By
Zheng Cao
Lexington, Kentucky
Director: Dr. Linda Dwoskin, Professor of Pharmaceutical Sciences
Lexington, Kentucky
2014

Copyright © Zheng Cao 2014
ABSTRACT OF DISSERTATION
LOBELANE ANALOGS WITH VARIOUS METHYLENE LINKER LENGTHS AND
ACYCLIC LOBELANE ANALOGS AS POTENTIAL PHARMACOTHERAPIES TO
TREAT METHAMPHETAMINE ABUSE
Methamphetamine interacts with vesicular monoamine transporter-2
(VMAT2) to inhibit dopamine (DA) uptake and promotes DA release from
presynaptic vesicles, increasing cytosolic DA available for methamphetamine-
induced reverse transport by DA transporters. By inhibiting VMAT2, lobelane, a
defunctionalized, saturated lobeline analog, decreases methamphetamine-
evoked DA release and methamphetamine self-administration in rats. In this
dissertation structure-activity relationships around the lobelane structure were
investigated on racemic lobelane analogs with varying methylene linker lengths
at central piperidine ring. Affinity for dihydrotetrabenazine (DTBZ) sites on
VMAT2 and for inhibition of VMAT2 function was determined to be 0.88-63 and
0.024-4.6 µM, respectively, and positively correlated. The most potent and
selective analog, (±)-cis-2-benzyl-6-(3-phenylpropyl)piperidine [(±)-GZ-730B], for
VMAT2 uptake was identified as the lead. The ability of (±)-GZ-730B to inhibit
methamphetamine-evoked [3H]DA release from striatal synaptic vesicles and
endogenous DA release from striatal slices was determined. The lead analog-
induced inhibition of methamphetamine-evoked vesicular [3H]DA release did not
translate to inhibition of methamphetamine-evoked DA release in the more intact
striatal slices. Moreover, poor water solubility of these lobelane analogs
prohibited further in vivo work. Subsequent work focused on analogs with the C-3
and C-4 carbons in the piperidine ring eliminated to afford racemic acyclic
lobelane analogs. Generally, acyclic analogs exhibited greater water solubility
and less lipophilicity compared to lobelane. Acyclic analogs exhibited affinities (K
i
= 0.096-17 μM) for [3H]DTBZ sites that correlated positively with affinity (K = 3.3-
i
300 nM) for inhibition of [3H]DA uptake. Pure enantiomers of potent racemic
analogs were synthesized, and found to potently, selectively, and competitively
inhibit [3H]DA uptake at VMAT2 and to release vesicular [3H]DA in a biphasic
manner. Lead enantiomer (R)-N-(1-phenylpropan-2-yl)-3-phenylpropan-1-amine
[(R)-GZ-924] inhibited methamphetamine-evoked [3H]DA release from striatal
synaptic vesicles, but not from the more intact striatal slices. Surprisingly, (R)-
GZ-924 inhibited nicotine-evoked [3H]DA overflow from striatal slices, revealing
nonspecific effects. Importantly, (R)-GZ-924 inhibited methamphetamine self-
administration in rats. However, the analog also inhibited food-maintained
responding, revealing a lack of specificity. The lead analog will not be pursued
further as a pharmacotherapy due to the lack of specificity. Further evaluation of
the pharmacophore is needed to discover analogs which specifically inhibit the
neurochemical and behavioral effect of methamphetamine.
KEYWORDS: Lobeline, Lobelane, Methamphetamine, Vesicular Monoamine
Tranporter-2, (R)-GZ-924

Zheng Cao
Student’s Signature

03/15/14
Date
LOBELANE ANALOGS WITH VARIOUS METHYLENE LINKER LENGTHS AND
ACYCLIC LOBELANE ANALOGS AS POTENTIAL PHARMACOTHERAPIES TO
TREAT METHAMPHETAMINE ABUSE
by

Zheng Cao

Dr. Linda P. Dwoskin
Director of Dissertation

Dr. Jim R. Pauly
Director of Graduate Studies
December 4, 2012
Acknowledgement
I would like to thank my advisor Dr. Linda Dwoskin for the support and
guidance that help my scientific career. In addition, I would like to thank my
committee members: Dr. Michael Bardo, Dr. Kimberly Nixon, and Dr. Kyung-Bo
Kim for their time and advice. I would like to thank Dr. Lawrence Gottlob for
agreeing to be my outside examiner. I would like to thank former and current
members in Dr. Dwoskin’s lab: Dr.Justin Nickell, Dr. Kiran Babu Siripurapu,
Agripina Deaciuc, Dr. Andrew Smith, Dr. Mahesh Darna, Dr. David Horton, Dr.
Vidya Narayanaswami, and Dr. Sucharita Sen Somkuwar. Their frequent
discussion with me improved my English speaking and the understanding of their
and my own project. I would like to thank Dr. Peter Crooks and Dr. Guangrong
Zheng for synthesis of the compounds and their expertise on chemistry. I would
like to thank members in Dr. Bardo’s lab: Dr. Josh Beckmann, Dr. Carrie
Wilmouth and Emily Denehy for performing the behavior study and their
expertise. I would like to thank Catina Rossoll and Charolette Garland for their
assistance and coordination. This research was supported by NIH DA13519 and
UL1TR000117. The University of Kentucky holds patents on lobeline and the
analogs described in the current work. A potential royalty stream to LPD, GZ and
PAC may occur consistent with University of Kentucky policy.
I would like to thank my parents, Hongzhi Cao and Weimin Hu, for years
of support and love. You are the best parents. I would like to thank my wife,
iii
Xiaoyan Zhang, for your love and patience and I could never finish this
dissertation without you.

iv
Table of Contents
Acknowledgement .......................................................................................................... iii
List of Tables ................................................................................................................ viii
List of Figures ................................................................................................................. ix
CHAPTER 1 Introduction ............................................................................................ 1
1.1 Methamphetamine ............................................................................................ 1
1.1.1 Physicochemical Characteristics ............................................................... 1
1.1.2 History and Background ............................................................................ 1
1.1.3 Pharmacokinetics ...................................................................................... 5
1.1.4 Clinical Pharmacology ............................................................................... 6
1.2 DA and Reward ................................................................................................ 8
1.2.1 DA Biosynthesis, Metabolism and Storage ................................................ 8
1.2.2 DA Receptors ...........................................................................................10
1.2.3 Dopaminergic Pathways ...........................................................................11
1.2.4 DA Transporter (DAT) ..............................................................................12
1.2.5 Vesicular Monoamine Transporter (VMAT)...............................................17
1.3 Methamphetamine Mechanism of Action .........................................................24
1.3.1 Methamphetamine on DA biosynthesis ....................................................25
1.3.2 Methamphetamine on DA metabolism ......................................................26
1.3.3 Methamphetamine on Plasma Membrane Transporters ...........................27
1.3.4 Effect of Methamphetamine on VMAT2 ....................................................29
1.4 Methamphetamine Neurotoxicity .....................................................................37
1.5 Review of Potential Treatment and Therapeutic Targets for Methamphetamine
Abuse 41
1.5.1 Behavioral Therapy ..................................................................................41
1.5.2 Replacement Therapy ..............................................................................43
1.5.3 5-HT Receptors as a Therapeutic Target .................................................44
1.5.4 Immunotherapy ........................................................................................45
1.5.5 Gamma-aminobutyric Acid (GABA) Receptors as Therapeutic Targets ....46
1.5.6 Sigma Receptors as Therapeutic Targets ................................................47
1.5.7 DA Receptors as Therapeutic Targets ......................................................48
1.5.8 Plasma Membrane Transporters as Therapeutic Target ...........................50
1.5.9 Acetylcholine Neurotransmitter System ....................................................52
1.5.10 Opioid Receptors as Therapeutic Targets ................................................53
1.5.11 Nicotinic Receptors as Therapeutic Targets .............................................54
1.6 VMAT2 as Therapeutic Target .........................................................................56
1.6.1 Lobeline ...................................................................................................57
1.6.2 Lobelane Physicochemical Characteristics and Pharmacology ................63
1.7 Drug-likeness ..................................................................................................63
1.8 Hypothesis and Specific Aims .........................................................................64
CHAPTER 2 Lobelane analogs with varying methylene linker lengths as novel ligands
that interact with vesicular monoamine transporter-2 .....................................................68
2.1 Introduction .....................................................................................................68
2.2 Methods ..........................................................................................................71
2.2.1 Animals ....................................................................................................71
2.2.2 Materials ..................................................................................................71
v
2.2.3 [3H]DTBZ Binding .....................................................................................73
2.2.4 Vesicular [3H]DA Uptake ..........................................................................74
2.2.5 Synaptosomal [3H]DA Uptake ...................................................................75
2.2.6 Vesicular [3H]DA Release .........................................................................76
2.2.7 [3H]Dofetilide Binding Assay to HERG Channels Expressed in HEK-293
Cells Membranes ...................................................................................................77
2.2.8 Inhibition of Methamphetamine-Evoked Endogenous DA Release ...........80
2.2.9 Data analysis ............................................................................................82
2.3 Results ............................................................................................................84
2.3.1 Inhibition of [3H]DTBZ binding at VMAT2 ..................................................84
2.3.2 Inhibition of [3H]DA uptake at VMAT2 .......................................................86
2.3.3 Selectivity of (±)-GZ-729C, and (±)-GZ-730B for VMAT2 over DAT and
hERG channel ........................................................................................................89
2.3.4 Release of [3H]DA from striatal synaptic vesicles .....................................90
2.3.5 (±)-GZ-729C and (±)-GZ-730B inhibited methamphetamine-evoked [3H]DA
release from striatal synaptic vesicles ....................................................................91
2.3.6 Lack of (±)-GZ-729C inhibition of methamphetamine-evoked endogenous
fractional DA release from striatal slices .................................................................93
2.3.7 Lack of (±)-GZ-730B inhibition of methamphetamine-evoked endogenous
fractional DA release from striatal slices .................................................................95
2.4 Discussion .......................................................................................................96
CHAPTER 3 Acyclic Lobelane Analogs Inhibit Vesicular Monoamine Transporter-2
Function and Methamphetamine Self-administration in Rats ....................................... 124
3.1 Introduction ...................................................................................................1 24
3.2 Methods ........................................................................................................1 28
3.2.1 Animals ..................................................................................................1 28
3.2.2 Materials ................................................................................................1 28
3.2.3 [3H]DTBZ Binding ...................................................................................1 31
3.2.4 Vesicular [3H]DA Uptake ........................................................................ 132
3.2.5 Kinetics of Vesicular [3H]DA Uptake .......................................................1 33
3.2.6 Synaptosomal [3H]DA Uptake ................................................................. 134
3.2.7 Vesicular [3H]DA Release ....................................................................... 135
3.2.8 [3H]Dofetilide Binding Assay to HERG Channels Expressed in HEK-293
Cells Membranes ................................................................................................. 140
3.2.9 Inhibition of Methamphetamine-Evoked Endogenous and [3H]DA Release
from Striatal Slices ...............................................................................................1 43
3.2.10 Inhibition of Nicotine-Evoked [3H]DA Overflow Assay ............................. 145
3.2.11 [3H]Nicotine and [3H]MLA Binding Assays .............................................. 147
3.2.12 Methamphetamine Self-Administration ................................................... 148
3.2.13 Food-Maintained Responding................................................................. 150
3.2.14 Data Analysis .........................................................................................1 51
3.3 Results ..........................................................................................................1 55
3.3.1 Inhibition of [3H]DTBZ Binding at VMAT2C ............................................. 155
3.3.2 Inhibition of [3H]DA Uptake at VMAT2C .................................................. 157
3.3.3 Correlation of K Values for the [3H]DTBZ Binding and [3H]DA Uptake at
i
VMAT2C ..............................................................................................................1 60
3.3.4 Mechanism of Inhibition of [3H]DA Uptake at VMAT2C ........................... 161
3.3.5 Inhibition of [3H]Dofetilide Binding to HERG Channels ............................ 161
3.3.6 Inhibition of [3H]DA Uptake at DAT ......................................................... 162
vi