Title: Vitro assay for testing gabapentinoid activity
Abstract: An assay and method for selecting analogs and derivatives of gabapentin based on the compounds inhibitory activity toward MAP kinase and MAP kinase mediated reporter gene expression. The method includes the steps of activating the MAP kinase signaling pathway, detecting the MAP kinase signal, and screening the gabapentin analogs and derivatives for inhibitory activity against the MAP kinase signal.
Patent Number: 6,984,496 Issued on 01/10/2006 to Chung, et al.
| Inventors:
|
Chung; Fu-Zon (Ann Arbor, MI);
Hong; Yulong (Painted Post, NY)
|
| Assignee:
|
Warner-Lambert Company (Morris Plains, NJ)
|
| Appl. No.:
|
057099 |
| Filed:
|
January 24, 2002 |
| Current U.S. Class: |
435/7.2; 435/7.1; 562/507 |
| Current Intern'l Class: |
G01N 33/53 (20060101); G01N 33/56.7 (20060101); C07C 229/00 (20060101); C07C 315/00 (20060101) |
| Field of Search: |
562/507
435/71,72,691,252.3,471
|
References Cited [Referenced By]
U.S. Patent Documents
| 6001581 | Dec., 1999 | Johnson et al.
| |
| Foreign Patent Documents |
| 9216547 | Jan., 1992 | WO.
| |
Other References
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International Journal of Oncology, vol. 14, 1999, pp. 327-335.
Fiore et al., "Activation of p42 Mitogen-Activated Protein Kinase by Glutamate
Receptor Stimulation in Rat Primary Cortical Cultures", Journal of Neurochemistry,
vol. 61, No. 5, 1993, pp. 1626-1633.
Sharif, "Mitogenic signaling by substance P and bombesin-like neuropeptide receptors
in astrocytic/glial brain tumor-derived cell lines (Review)", International
Journal of Oncology, vol. 12, 1998, pp. 273-286.
Lewis, et al., "Signal Transduction Through Map Kinase Cascades", Advances
In Cancer Research, vol. 74, 1998. pp 49-139.
Pin and Duvoisin, "Review†: Neurotransmitter receptors I The Metabotropic
Glutamate Receptors: Structure and Functions", Neuropharmacology, vol. 34,
No. 1, 1995, pp. 1-26.
Morris, "Gabapentin", Epilepsia, vol. 40 (Suppl 5), 1999, pp S63-S70.
Ferrier, "Lamotrigine and Gabapentin: Alternatives in the Treatment of Bipolar
Disorder", Neuropsychobiology vol. 38, 1998, pp. 192-197.
Bryans, et al., "Identification of Novel Ligands for the Gabapentin Binding Site
on the α2δ Subunit of a Calcium Channel and Their Evaluation
as Anticonvulsant Agents", Journal of Medicinal Chemistry, vol. 41, No.
11. 1998, pp. 1838-1845.
Purpura, et al., "Structure-Activity Determinants of Pharmcological Effects of
Amino Acids and Related Compounds on Central Synapses", Neurochemistry,
vol. 3, 1959, pp 238-268.
Spokes, "Gaba in Huntington's Chorea, Parkinsonism and Schizophrenia", Adv
Exp Med Biol, vol. 123, 1978. pp. 461-473.
Wu, et al., "Abnormalities of Neurotransmitter Enzymes in Huntington's Chorea",
Neurochemical Research, vol. 4, No. 5, 1979, pp. 575-586.
Tian, et al., "Structural Motifs Encoded by Individual Exons of the Human Neurokinin-1
Receptor Gene Interact Differentially with Selective Agonists and Antagonists",
vol. 67, 1996, pp 1191-1199.
Bryans, et al., "3-Substituted GABA Analogs with Central Nervous System Activity:
A Review", Medical Research Review, vol. 19, 1999, pp 149-177.
Hoekstra, et al., "Chemical Development of CI-1008, an Enantiomerically Pure
Anticonvulsant", Organic Process Research & Development, vol. 1,1997, pp 26-38.
|
Primary Examiner: Landsman; Robert
Attorney, Agent or Firm: Ashbrook; Charles W., Ganjeizadeh; Mehdi
Parent Case Text
This application is a utility application which claims benefit of U.S. Provisional
Application 60/266,358 filed Feb. 2, 2001; the entire contents of which is hereby
incorporated herein by reference.
Claims
What is claimed is:
1. A method for detecting gabapentinoid activity in a test compound comprising
the steps of:
(a) introducing into host cells that express an NK receptor a heterologous DNA
sequence that encodes a reporter polypeptide which responds to Erk-2 activation;
(b) separating the host cells into at least two groups, a first group and a second
group;
(c) treating the first group of host cells with a test compound that binds to
the α
2δ subunit of a calcium channel;
(d) treating the first group and second group of host cells with an NK receptor
agonist;
(e) determining reporter polypeptide activity in the first group and in the second
group; and
(f) comparing reporter polypeptide activity from the first group to the second
group; and
(g) identifying as a gabapentinoid, a test compound that shows greater inhibition
of said reporter polypeptide activity in said first group of step f) than said
reporter polypeptide activity of said second group in step f).
2. The method of claim 1, wherein the host cells are Chinese hamster ovary (CHO) cells.
3. The method of claim 1, wherein the heterologous DNA sequence encodes luciferase.
4. The method of claim 1, wherein in the NK receptor agonist is substance P.
5. The method of claim 1, wherein step (b) comprises the step of separating the
host cells into a plurality of groups, and step (c) comprises treating each separate
group with a compound having a final concentration of between 1 μM and 1 mM.
6. The method of claim 1, wherein step (d) occurs prior to step (c).
7. A method for detecting gabapentinoid activity in a test compound comprising
the steps of:
(a) introducing into host cells that express the an NK1 receptor a heterologous
DNA sequence that encodes a reporter polypeptide which responds to Erk-2 activation;
(b) separating the host cells into at least two groups, a first group and a second
group;
(c) treating the first group of host cells with a test compound that binds to
the α
2δ subunit of a calcium channel;
(d) treating the first group and second group of host cells with an NK receptor
agonist;
(e) determining reporter polypeptide activity in the first group and in the second
group;
(f) comparing reporter polypeptide activity from the first group to the second
group; and
(g) identifying as a gabapentinoid, a test compound that greater inhibition of
said reporter polypeptide activity in said first group of step f) than said reporter
polypeptide activity of said second group in step f).
8. A method for detecting gabapentinoid activity in a target compound comprising
the step of:
(a) introducing into host cells that express an NK receptor a heterologous DNA
sequence chat encodes a reporter polypeptide which responds to Erk-2 activation;
(b) separating the host cells into at least two groups, a first group and a second
group;
(c) treating the first group of host cells with a target compound;
(d) treating the first group and second group of host cells with an NK receptor
agonist;
(e) determining reporter polypeptide activity in the first group and in the second
group;
(f) comparing reporter polypeptide activity from the first group to the second
group; and
(g) identifying as a gabapentinoid, a target compound that shows greater inhibition
of said reporter polypeptide activity in said first group of step f) than said
reporter polypeptide activity of said second group in step f); wherein
said target compound is a compound of formula
##STR2##
or R5 a2 1 1A
or a pharmaceutically acceptable salt thereof wherein:
R is hydrogen or a lower alkyl;
(h) R1 to R14 are each independently selected from hydrogen, straight or branched
alkyl of from 1 to 6 carbons, phenyl, benzyl, fluorine, chlorine, bromine, hydroxy,
hydroxymethyl, amino, aminomethyl, trifluoromethyl, —CO2H, —CO2R15,
—CH2CO2H, —CH2CO2R15, —OR15 wherein R15 is a straight or branched
alkyl of from 1 to 6 carbons, phenyl, or benzyl, and R1 to R8 are simultaneously
hydrogen.
9. The method of claim 1 wherein said test compound of step g) shows, at 500
μM, at least 20% greater inhibition of said reporter polypeptide activity
in said first group of step f) than said reporter polypeptide activity of said
second group in step f).
10. The method of claim 1 wherein said test compound of step g) show, at 500
μM, at least 25% greater inhibition of said reporter polypeptide activity
in said first group of step f) than said reporter polypeptide activity of said
second group in step f).
11. The method of claim 1 wherein said test compound of step g) shows, at 500
μM, at least 30% greater inhibition of said reporter polypeptide activity
in said first group of step f) than said reporter polypeptide activity of said
second group in step f).
Description
FIELD OF THE INVENTION
The present invention relates generally to assays that test analogs and derivatives
of γ-aminobutyric acid (GABA) for their ability to modulate mitogen-activated
protein kinase (MAP Kinase) activity, and more particularly, but not by way of
limitation, to an in vitro screening assay for selecting analogs and derivatives
of 1-(aminomethyl) cyclohexaneacetic acid (gabapentin) based on the analogs' inhibitory
activity toward Elk-1.
BACKGROUND OF THE INVENTION
GABA is a neurotransmitter involved in normal regulation of the mammalian central
nervous system. An imbalance in GABA concentrations in the central nervous system
has been implicated in several disease states, including; seizures, Huntington's
chorea, Parkinson's disease, spasticity, and neuropathic pain. Treatment of these
disease states has generally centered on increasing GABA concentrations in the
afflicted patient's central nervous system. Purpura et al., Neurochem, 1959;3:238-268;
Spokes.,
Adv. Exp. Med. Biol., 1978:123:461-473; Wu et al.,
Neurochem.
Res., 1979;14:575-586. Unfortunately, direct treatment of afflicted patients
with GABA has proven ineffective as GABA has physiochemical properties that prohibit
it from crossing the blood-brain barrier.
Structurally related compounds to GABA are being pursued as possible
treatments for GABA mediated disorders. Gabapentin (1-(aminomethyl) cyclohexaneacetic
acid) is one such structurally related compound which is known to readily cross
the blood-brain barrier and bind throughout the central nervous system. Gabapentin
is currently used in the treatment of seizures and epilepsy and has been implicated
as a possible treatment in other GABA mediated central nervous system disorders.
There is a need in the relevant art to have additional compounds that have
gabapentin like activity. Other gabapentin related drugs may be more effective
than gabapentin in treating seizures and other central nervous system disorders,
or may be used in patients that have become refractory to gabapentin over time
or have unwanted side effects. Against this backdrop the present invention has
been developed.
SUMMARY OF THE INVENTION
The present invention provides an in vitro assay for screening compounds that
are structurally related to gabapentin for MAP kinase inhibitory activity. It is
envisioned that the present invention could be used as a general screening procedure
in obtaining novel gabapentinoids for use in treatment of disorders of the central
nervous system and neuropathic pain.
In one aspect, the present invention is a method for detecting MAP kinase inhibitory
activity by compounds that are structurally related to gabapentin. In one preferred
embodiment, NK expressing cells are stimulated with substance P which results in
the phosphorylation of the MAP kinase member, Erk-2. In another preferred embodiment,
mGluR expressing cells are stimulated with quisquolate which results in the phosphorylation
of Erk-2. To screen for gabapentinoid activity, test substances are individually
incubated with the activated cells and Erk-2 phosphorylation determined. In preferred
embodiments, Erk-2 phosphorylation after treatment with a test compound is compared
to activated cells treated with a similar concentration of gabapentin.
In another aspect, the present invention is a method for testing a compound for
gabapentinoid activity by determining its effects on a MAP kinase inducible reporter
gene. In one preferred embodiment, cells are engineered to incorporate reporter
constructs under the control of a MAP kinase inducible transcription factor. In
one preferred embodiment, NK expressing cells are engineered to incorporate a reporter
gene under the transcriptional control of an Ets family member, for example Elk-1,
where the Ets family member is activated by activated Erk-2. In another embodiment
of the present invention, mGluR expressing cells are engineered to incorporate
reporter construct under the transcriptional control of an Ets family member, for
example Elk-1.
In another aspect, the present invention is a treatment for neuropathic pain
where
compounds structurally related to gabapentin are screened for Elk-1 inhibitory
activity in the assays discussed above, and compounds having inhibitory activity
administered to a subject.
Finally, in another aspect, the present invention is a treatment for central
nervous system disorders compounds structurally related to gabapentin are screened
for Elk-1 inhibitory activity in the assays discussed above, and compounds having
inhibitory activity administered to a subject.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the inhibitory effects of gabapentin on NK1 mediated Erk-2 phosphorylation.
Chinese Hamster Ovary (CHO) cells expressing rat mGluR5 receptors were pretreated
with gabapentin (100 μM) for 30 minutes and than stimulated with quisquolate
(100 μM) for 10 minutes. Products from treated cells were analyzed by Western
blotting with anti-pErk antibody.
FIG. 2 shows the dose-dependent effects of gabapentin on PMA mediated Erk-2
phosphorylation in differentiated IMR-32 human neuroblastoma cells. Differentiated
IMR-32 cells were pretreated with indicated concentrations of gabapentin for 30
minutes and then stimulated with PMA for 10 minutes. Products from treated cells
were analyzed by Western blotting with anti-pErk antibody.
FIGS. 3A and 3B are graphs showing the dose-dependent inhibitory effects of
gabapentin (A) and pregabalin (B) on NK1 mediated Elk-1 transcription. Relative
light units (Y axis) indicate luciferase activity in the presence of increasing
concentrations of gabapentin or pregabalin (X axis).
FIG. 4 are graphs showing the dose-dependent effects of PD 200390 and PD 201698
on NK1 mediated Elk-1 transcription. Relative light units (Y axis) indicate luciferase
activity in the presence of increasing concentrations of PD 200390 or PD 201698
(Y axis). PD 184352, a potent MEK inhibitor, was also included in the assay as
a positive control.
FIG. 5 is a graph showing the dose-dependent inhibitory effects of gabapentin
on PMA mediated Elk-1 transcription in IMR-32 cells. Relative light units (Y axis)
indicate luciferase activity in the presence of increasing concentrations of gabapentin
or pregabalin (X axis).
FIG. 6 is a graph showing the dose-dependent inhibitory effects of gabapentin
on PMA mediated Elk-1 transcription in CHO cells. Relative light units (Y axis)
indicate luciferase activity in the presence of increasing concentrations of gabapentin
or pregabalin (X axis).
FIG. 7 is a graph showing the dose-dependent inhibitory effects of gabapentin
on NK1 mediated SRE activation. Relative light units (Y axis) indicate luciferase
activity in the presence of increasing concentrations of gabapentin or pregabalin
(X axis).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The following definitions are provided to facilitate understanding of certain
terms used frequently herein and are not meant to limit the scope of the present disclosure.
As used herein "amino acids" mean any of the 20 gene encoded amino acids as well
as any modified amino acid sequences. Modifications may include natural processes
such as posttranslational processing, or may include chemical modifications which
are known in the art. Modifications include but are not limited to: ubiquitination,
acetylation, amidation, covalent attachment of flavin, ADP-ribosylation, cross
linking, iodination, methylation, etc.
As used herein, "control cell" refers to a cell that has been cultured in parallel
with a cell treated under the specified experimental condition; but unlike the
treated cell, the control cell has not undergone the specified experimental condition.
Control cells represent a baseline from which comparisons are made.
The term "Elk-1" as used herein is meant to refer to the p62
TCF transcription
factor shown to be activated by Erk, JNK/SAPK, or p38 MAPK. Elk-1, among other
things, regulates serum response element transcription through an interaction with
serum response factor. For purposes of this disclosure, the term Elk-1 also includes
transcription factors that are substantially the same as Elk-1 but that have minor
substitutions, deletions, or additions to the Elk-1 polypeptide sequence.
The term eukaryotic cell line is used to refer to cells established in ex vivo
culture. It is a characteristic of the eukaryotic cell line of the present invention
that it be capable of supporting MAP kinase signaling. Examples of suitable eukaryotic
cells within the context of the present invention include SF9 insect cells (Summers
and Smith., Texas Agricultural Experiment Station Bulletin, 1987;1555), CHO cells
(Puck et al.,
J. Exp. Med., 1958;108:945-955) including CHO K1, human cervical
carcinoma (Hela) cells (ATCC CCL 2), monkey kidney CV1 line transformed by SV40
(COS-7) cells (ATCC CRL 1651), human embryonic kidney (HEK293) cells (Graham et
al.,
J. Gen. Virol., 1977;36:59), human neuroblstoma (IMR-32) cells (ATCC
CCL-127), etc.
The term "expression" refers to transcription and translation occurring within
a host cell. The level of expression of a DNA molecule in a host cell may be determined
on the basis of either the amount of corresponding mRNA that is present within
the cell or the amount of DNA molecule encoded protein produced by the host cell
(Sambrook et al., Molecular cloning: A laboratory manual, 1989;18.1-18.88).
The term "gabapentin" refers to 1-(aminomethyl) cyclohexaneacetic acid as described
in U.S. Pat. No. 4,024,175.
The term "gabapentinoid" or "gabapentinoids" refers to analogs and derivatives
of gabapentin as well as to compounds that show gabapentin-like inhibitory activity
within the context of embodiments of the present invention.
The term "mGluR" as used herein is meant to refer to the family of G-protein-coupled
receptors known as the metabotropic glutamate receptors. This family includes Group
I mGluRs, mGluR1 and mGluR5, Group II mGluRs, mGluR2 and mGluR3, and Group III
mGluRs, all others. (Pin et al., Neuropharmacology, 1995;34:1-26.)
The term "modulation" as used herein is meant to refer to either upregulating
or downregulating the activity of a protein.
The term "nucleic acid sequence" refers to the order or sequence of deoxyribonucleotides
along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides
determines the order of amino acids along a polypeptide chain. The deoxyribonucleotide
sequence thus codes for the amino acid sequence.
The term "polynucleotide" refers to a linear sequence of nucleotides. The nucleotides
are either a linear sequence of polyribonucleotides or polydeoxyribonucleotides,
or a mixture of both. Examples of polynucleotides in the context of the present
invention include: single- and double-stranded DNA, single- and double-stranded
RNA, and hybrid molecules that have both mixtures of single- and double-stranded
DNA and RNA. Further, the polynucleotides of the present invention may have one
or more modified nucleotides.
As used herein, "protein," "peptide," and "polypeptide" are used interchangeably
to denote an amino acid polymer or a set of two or more interacting or bound amino
acid polymers.
The term "substance P" refers to a tackykinin neuropeptide that acts through
the NK
1, NK
2, and NK
3 receptors.
The term "vector," "extra-chromosomal vector," or "expression vector" refers
to a first piece of DNA, usually double-stranded, which may have inserted into
it a second piece of DNA. Foreign DNA is defined as heterologous DNA, which is
DNA that may or may not be naturally found in the host cell and includes additional
copies of nucleic acid sequences naturally present in the host genome. The vector
transports the foreign DNA into a suitable host cell. Once in the host cell, the
vector may be capable of integrating into the host cell chromosomes. The vector
may also contain the necessary elements to select cells containing the integrated
DNA as well as elements to promote transcription of mRNA from the transfected DNA.
Examples of vectors within the scope of the present invention include, but are
not limited to, plasmids, bacteriophages, cosmids, retroviruses, and artificial chromosomes.
Modes for Carrying out the Invention
The present invention is based upon, among other things, the discovery of a novel
in vitro assay for testing compounds for gabapentinoid activity. In particular,
the invention relates to the discovery that under certain conditions cells respond
to gabapentin, and compounds having gabapentin like activity, through the inhibition
of the MAPK signaling pathway. In preferred embodiments, cells are engineered to
express components of the MAPK signaling pathway, for example neurotransmitters
and G-protein coupled receptors, so that stimulation of the component leads to
activation of target MAK kinases, for example activation of Erk-2. In preferred
embodiments, cells used in the in vitro assay are engineered to express both components
of the MAPK signaling pathway as well as reporter constructs under the inducible
control of activated Erk-2, for example, a luciferase construct under the control
of the Elk-1 transcription factor. The engineered cells are used in testing compounds
for gabapentinoid activity by detecting the compound's inhibitory effects on the
activated MAPK signaling pathway.
The in vitro analysis of gabapentinoid activity is of importance in detecting
novel gabapentinoids in an inexpensive in vitro assay. Compounds that have gabapentinoid
activity in the present invention may then be further tested for activity in in
vivo studies and eventually patient studies.
Analysis of gabapentinoid activity is not limited to gabapentinoid activity
on Erk-2 phosphorylation or Elk-1 mediated reporter gene expression, it may also
be determined using other direct and indirect analysis of Elk-1, as well as through
direct or indirect analysis of the MAPK signaling pathway.
Note that within this application, unless otherwise stated, the techniques utilized
may be found in any of several well-known references such as: Sambrook et al.,
Molecular cloning: A Laboratory Manual, 1989; Goeddel D, eds., Gene Expression
Technology, Methods in Enzymology Academic Press: San Diego, Calif., 1991;185;
Deutshcer M. P., Guide to Protein Purification. In: Methods in Enzymology. 3
rd
ed. Academic Press, Inc., 1990; Innis et al., PCR protocols: A guide to methods
and applications Academic Press: San Diego, Calif., 1990; Freshney R. I., Culture
of animal cells: A manual of basic technique. 2
nd ed., New York: Liss,
Inc., 1987; and Murray E. J., ed., Gene Transfer and Expression Protocols. Clifton,
N.J.: The Humana Press Inc., 109-128.
Vectors and Host Cells
In general, embodiments of the present invention may be implemented through the
transient expression of the foreign DNA or through the stable integration of the
foreign DNA into target cells. Techniques required for this aspect of the invention
are well-known in the art (Sambrook et al. Molecular cloning: A laboratory manual.
2
nd ed., Cold Spring Harbor Press, 1989) and can include calcium phosphate
transfection, dextran sulfate transfection, electroporation, lipofection, and viral
infection (Graham and van der Eb. Virology, 1978;52;456-457; Chisholm et al., DNA
cloning IV: A practical approach, mammalian systems, Glover and Hanes, eds., 1995;141;
Andreason.,
J. Tisss. Cult. Meth., 1993;15:56-62).
In an aspect of the present invention, novel polynucleotides substantially similar
to the Group I mGluRs are subcloned into an extra-chromosomal vector. The subcloned
polynucleotide(s) may be joined to a vector having a cis-acting or regulatory element
for increased propagation in a host cell (note that the transacting factors involved
are supplied to the host, supplied by a second vector or supplied by the vector
itself upon introduction into the host). This aspect of the invention allows for
the in vivo and in vitro expression of either mGluR1 or mGluR5.
It is further envisioned that novel polynucleotides substantially similar to
the
NK1, NK2, or NK3 receptor are subcloned into an extra-chromosomal vector. The subcloned
polynucleotides may be joined to a vector having a cis-acting or regulatory element
for increased propagation in a host cell (note that the transacting factors involved
are supplied to the host, supplied by a second vector or supplied by the vector
itself upon introduction into the host). This aspect of the invention allows for
the in vivo and in vitro expression of either NK1, NK2, or NK3.
It is further envisioned that novel polypeptides substantially similar to Elk-1
or GAL4-Elk-1 are subcloned into an extra-chromosomal vector. The subcloned polynucleotides
may be joined to a vector having a cis-acting or regulatory element for increased
propagation in a host cell (note that the transacting factors involved are supplied
to the host, supplied by a second vector or supplied by the vector itself upon
introduction into the host). This aspect of the invention allows for the in vivo
and in vitro expression of either Elk-1 or the fusion protein GAL4-Elk-1.
Several vectors can be used in the context of this invention, including:
PcDNA3 vector (Invitrogen), vectors having the T3 and T7 polymerase promoters,
vectors having the SV40 promoter, or the CMV promoter, pTRE2 vector used in the
Tet-on™ inducible expression system (Clontech labs), the pFA2-Elk-1 plasmid
(Stratagene), pFR-Luc plasmid (stratagene), pCDNA3.1/Hygro (Invitrogen), or any
other promoter that either can direct expression of a polypeptide off a polynucleotide,
or that one wishes to test for the ability to direct expression of a polypeptide
off a polynucleotide.
The host cells of the present invention may be of any type, including, but not
limited to, noneukaryotic and eukaryotic cells. Host cells are cultured using standard
tissue culture techniques in conventional media as is well-known in the art. The
level of expression of the DNA introduced into a host cell of the invention depends
on multiple factors, including gene copy number, efficiency of transcription, messenger
RNA a desired polypeptide according to the present invention will typically involve
optimizing one or more of those factors.
In Vitro Gabapentinoid Activity Assay and Method
In one aspect, the present invention provides an in vitro assay by which target
compounds are tested or screened for gabapentinoid activity by determining the
compounds inhibitory effects on Elk-1 mediated reporter gene expression.
NK Receptor
The in vitro assay is based on the principle that activated NK-1, NK-2, or NK-3
(or any combination of the three receptors) stimulate Erk-2 activity. Activated
Erk-2, among other things, activates any number of proteins belonging to the Ets
family of helix-turn-helix transcription factors, for example Elk-1. In the present
invention, NK expressing cells are engineered to incorporate reporter constructs,
for example vectors having the luciferase gene, under the transcriptional control
of an Ets family member, preferably Elk-1. Compounds having gabapentinoid activity
are ultimately detected and quantitated through inhibition of Elk-1 mediated reporter
gene expression.
In a preferred embodiment of the present invention, the NK receptor is the NK-1
receptor, and the reporter construct has one or more serum response elements or
CREB (cAMP response element binding protein) elements that are recognized by Elk-1.
Stimulation of the NK-1 receptor with agonist, for example substance P, results
in the activation of Erk-2, and hence the activation of Elk-1. Activated Elk-1
binds to the response elements and induces reporter gene expression. Typical agonist
concentrations are from 0.1 to 100 nM, and preferably from 1 to 10 nM. Suitable
reporter genes for use with the present invention include, but are not limited
to, the luciferase gene, chloramphenicol acetyltransrerase gene, β-galactosidase
gene, and β-lactamase gene.
A preferred NK1 expressing cell line for use in the present invention is one
that
is stably transfected with recombinant DNA comprising the NK1 receptor (or the
NK2 or NK3 receptors or multiples of NK1, NK2, or NK3 receptors) (see Tian Y. et
al.,
J. Neurochem., 1996;67:1191-1199). Recombinant cell lines are quantitated
for NK1 receptor expression by employing assays for NK1 biological activity or
by employing assays that are independent of such activity, such as Western blotting
or immunoassay using antibodies that are capable of reacting with NK1. (Sambrook
et al., Molecular Cloning: A Laboratory Manual, 1989;18.1-18.88)
Another preferred cell line for use with the present invention is a NK expressing
cell line stably transfected with recombinant DNA substantially comprising the
Gal4/Elk-1 fusion transcription factor, and that further has copies of a Gal4-controlled
reporter construct. In these cases, activation of the Elk-1 portion of the fusion
protein through the MAP kinase signaling pathway leads to Gal4 controlled expression
of the reporter gene. MAP kinase activation of the Elk-1 fusion protein causes
the protein to bind to the Gal-4 binding sites on the reporter gene and induce
reporter construct expression. Note that other Elk-1 fusion protein/reporter gene
construct pairs may be used in the context of the present invention as long as
NK mediated activation of the MAP kinase pathway induces the Elk-1 fusion protein
to express the reporter construct.
Target compounds to be screened in the above described in vitro assay are
synthesized as described in international patent application WO 9921824, which
is herein incorporated by reference. Compounds may be incubated with cells prior
to, at the same time, or after stimulation of the NK receptor with a NK receptor
agonist. In a preferred embodiment, incubation of the compound(s) with the above
described cells occurs prior to NK receptor activation, suitable compound incubation
times prior to NK activation are variable, but 30 to 45 minutes is typical. Total
incubation times of target compounds on the host cells is variable and set for
the user's convenience as well as for the level of reporter construct expression
needed for detection in the users laboratory; however, suitable times are typically
about 3 to 6 hours, and more preferably about 3.5 to 5 hours.
WO 9921824, is now issued as U.S. Pat. No. 6,635,673. Both WO 9921824, and U.S.
Pat. No. 6,635,673 disclose, in the first paragraph under DETAILED DESCRIPTION
OF THE INVENTION, that "[t]he compounds of the instant invention and their pharmaceutically
acceptable salts are as defined by formulas 1 and 1A
##STR1##
or a pharmaceutically acceptable salt thereof wherein:
R is hydrogen or a lower alkyl; R1 to R14 are each
independently selected from hydrogen, straight or branched alkyl of from
1 to 6 carbons, phenyl, benzyl, fluorine, chlorine, bromine, hydroxy, hydroxymethyl,
amino, aminomethyl, trifluoromethyl, —CO2H, —CO2R15,
—CH2CO2H, —CH2CO2R15,
—OR15 wherein
R15 is a straight or branched alkyl of from 1 to 6 carbons, phenyl,
or benzyl, and R1 to R8 are not simultaneously hydrogen".
Both WO 9921824, and U.S. Pat. No. 6,635,673 disclose that "[t]he compounds of
the invention show good binding affinity to the α2δ subunit.
Gabapentin (Neurontin®) is about 0.10 to 0.12 μM in this assay. Since
the compounds of the instant invention also bind to the subunit, they are expected
to exhibit pharmacological properties comparable to gabapentin." See WO 9921824,
lines 5 to 9, page 18; and U.S. Pat. No. 6,635,673, column 13, paragraph 2.
In general, each compound is tested for inhibitory activity through a range of
concentrations, for example, a compound may be incubated on a series of independent
groups of cells, where each group of cells receives a varying concentration of
compound, from 25 to 1500 μM, and preferably from 50 to 1000 μM. In
some embodiments of the present invention, the host cells are incubated with a
single dose of target compound, for example, a single dose between 25 and 1500
μM, and preferably a single dose of about 450 to 550 μM.
With respect to the detection of reporter expression in the presence and absence
of the tested compound, cells are harvested and reporter gene expression determined.
Luciferase assays are performed according to the procedures suggested by the manufacture
(Promega, Wis.), and procedures for other common reporter gene assays are well-known
in the art. Percent reporter gene inhibition is correlated with gabapentinoid activity,
where the stronger the gabapentinoid activity the greater the percent reporter
inhibition. Although compounds showing any level of inhibition, above a known negative
control, are considered positive for gabapentinoid activity, compounds that show
at least 20%, preferably 25%, and most preferably 30% inhibition at 500 μM
are considered hits and will be identified as having gabapentinoid activity. Note
also that comparisons may be made between the target compound and similarly treated
gabapentin, where a hit is a compound having an inhibitory activity similar to
or greater than gabapentin activity in the assay. Note also, where a compound is
tested over a concentration range, a preferable response is one that is dose responsive.
mGluR
In another aspect, the in vitro assay is based on the principle that activated
Group I mGluR receptors stimulate Erk-2 activity. Activated Erk-2, among other
things, phosphorylates and activates any number of proteins belonging to the Ets
family of helix-turn-helix transcription factors, for example Elk-1. Activated
Elk-1 induces reporter gene expression which can be followed in the presence and
absence of compounds, where compounds having gabapentinoid activity inhibit Elk-1
inducible reporter expression.
In a preferred embodiment of the present invention, the reporter construct has
one or more serum response elements or CREB (cAMP response element binding protein)
binding sites. Stimulation of mGluR1 or mGluR5 (or a combination of the two) with
agonist, for example quisqualic acid, results in activation of Erk-2, and hence
activation of Elk-1. Suitable quisqualic acid concentrations for use with the present
invention are from 10 to 500 μM, and are preferably from 50 to 200 μM.
Activated Elk-1 binds to the serum response elements or CREB elements on the reporter
construct and induces reporter gene expression. Suitable reporter genes for use
with the present invention include, but are not limited to, the luciferase gene,
chloramphenicol acetyltransrerase gene, β-galactosidase gene, and β-lactamase gene.
In another preferred embodiment of the present invention, host cells are constructed
to stably express a Gal4-Elk-1 fusion protein, and the reporter construct has one
or more Gal4 binding sites that bind the GAL4-Elk-1 fusion protein. In these cases,
as above, activation of the Elk-1 portion of the fusion protein through the MAP
kinase signaling pathway leads to Gal4 controlled expression of the reporter gene.
MAP kinase activation of the Elk-1 fusion protein causes the protein to bind to
the Gal-4 binding sites on the reporter gene and induce reporter construct expression.
Note that other Elk-1 fusion protein/reporter gene construct pairs may be used
in the context of the present invention as long as mGluR mediated activation of
the MAP kinase pathway induces the Elk-1 fusion protein to express the reporter construct.
Recombinant cell lines are quantitated for mGluR expression by employing
assays for mGluR biological activity or by employing assays that are independent
of such activity, such as Western blotting or immunoassay using antibodies that
are capable of reacting with Group I mGluRs. (Sambrook et al., Molecular cloning:
A laboratory manual, 1989;18.1-18.88).
Target compounds to be screened for gabapentinoid activity are synthesized
as described in the NK embodiments. Compounds may be incubated with cells prior
to, at the same time, or after stimulation of the mGluR receptor with mGluR agonist.
In preferred embodiments, compounds are incubated with the above described cells
prior to mGluR activation, suitable times are variable, but 30 to 45 minutes prior
to mGluR activation is typical. Total incubation times of target compounds on the
host cells is variable and set for the user's convenience as well as for the level
of reporter construct expression needed for detection in the assay; however, suitable
times are typically for about 3 to 6 hours and most preferably from 3.5 to 5 hours.
In general, each test compound is tested for its inhibitory activity through a
range of concentrations, for example, a compound may be tested at concentrations
from 25 to 1500 μM, and preferably from 50 to 1000 μM. In some embodiments
of the present invention, the host cells are incubated with a single dose of target
compound, for example a dose between 25 and 1500 μM, and preferably a single
dose of about 450 to 550 μM.
Detection of gabapentinoid activity in a test compound is essentially as
described above in the NK embodiments.
PMA Stimulation
In another aspect, the in vitro assay of the present invention is based on the
discovery that gabapentinoid activity may be screened through PKC mediated Erk-2
activation. PKC competent cells are stimulated with PMA, preferably in the range
of 50 nM, to activate Erk-2. Activated Erk-2, as discussed above, activates Elk-1
which induces reporter gene expression. Compounds tested for gabapentinoid activity
are incubated in PMA stimulated cells and gabapentinoid activity followed through
inhibition of reporter gene expression. As above, cells may be incubated with the
target compound before treatment with PMA. Screening aspects are the same as discussed
above. Note that this embodiment allows for the screening of gabapentinoid activity
in both neuronal-like (IMR-32) cells and nonneuronal (CHO) cells, and does not
require the overexpression of mGluR or NK receptors.
Erk-2 Phosphorylation
It is envisioned that other gabapentinoid detection assays be within the scope
of the present invention. For example, host cells having intact MAPK signaling
pathways may be used to screen compounds for gabapentinoid activity by detecting
a compounds inhibitory effects on the MAP kinase signaling pathway. For example,
IMR-32 cells may be treated with PMA and total Erk-2 phosphorylation determined
through Western blotting. Target compounds are incubated with the PMA stimulated
cells to determine the compounds inhibitory effect on Erk-2 phosphorylation and
compared to a nontreated control and a gabapentin treated control. Alternatively,
Erk-2 phosphorylation may be followed in cells that have intact MAPK signaling
pathways where either NK or mGluR receptors are overexpressed. Erk-2 phosphorylation
is achieved by treatment with substance P or quisqualate, respectively, and a target
compound's inhibitory effects on Erk-2 phosphorylation correlated to gabapentinoid
activity. Phosphorylation of Erk-2 may be detected using a Western blot assay,
immunoprecipitation assay, etc. Note that the cell lines and methods used in this
embodiment are essentially as described above.
Method of Screening a Compound for Gabapentinoid Activity
Analogs and derivatives of gabapentin are screened for gabapentinoid activity
in accordance with one method of the invention. Host cells are engineered to express
either NK receptor or Group I mGluR. Host cells are optionally transfected to also
contain copies of an Erk-2 inducible reporter gene, for example a reporter gene
responsive to the Elk-1 transcription factor. In preferred embodiments of the method,
host cells having the Elk-1 inducible reporter gene are further engineered to overexpress
the Elk-1 transcription factor. In other embodiments, host cells having the Elk-1
inducible reporter gene are engineered to overexpress a fusion protein having a
functional Elk-1 portion and a reporter gene element DNA binding portion. Note
that other Erk-2 responsive Ets family members may be substituted into the above
described method.
Next, cells are separated into groups for treatment with a target analog or
derivative of gabapentin (note that a negative and positive control group may be
prepared in parallel with the cells used for target compound testing). Each individual
group has a substantially equal number of cells for treatment with target compound.
Each separate group of cells is treated with a similar concentration of target
compound and allowed to incubate for 30 to 45 minutes. Optionally, a single target
compound may be tested at increasing concentrations on a series of separate groups.
Cells are then treated with either substance P or quisqualate, depending on whether
the host cells express the NK receptor or mGluR. Cells are allowed to incubate
for 3 to 6 hours. Each group of cells is harvested and reporter gene expression
determined as is well-known in the art. Comparisons are made between each compound
for inhibitory effects on reporter gene expression as well as against the negative
and positive control.
In another embodiment of the method, NK or mGluR expressing cells and treated
with compounds and agonist as above, cells are then harvested and Erk-2 phosphorylation
determined (via Western blot or other well known technique). The inhibitory effects
of each compound on Erk-2 phosphorylation is determined and compared to known controls.
Gabapentinoid Administration Methods
Gabapentinoids showing inhibitory activity in the in vitro assays
of the present invention can be formulated as pharmaceutical compositions and administered
to a mammalian host, including a human patient, in a variety of forms adapted to
the chosen route of administration. The compounds are preferably administered in
combination with a pharmaceutically acceptable carrier, and may be combined with
or conjugated to specific delivery agents, including targeting antibodies and/or cytokines.
Gabapentinoids can be administered by known techniques, such as orally,
parentally (including subcutaneous injection, intravenous, intramuscular, intrasternal,
or infusion techniques), by inhalation spray, topically, by absorption through
a mucous membrane, or rectally, in dosage unit formulations containing conventional
nontoxic pharmaceutically acceptable carriers, adjuvants, or vehicles. Pharmaceutical
compositions of the invention can be in the form of suspensions or tablets suitable
for oral administration, nasal sprays, creams, sterile injectable preparations,
such as sterile injectable aqueous or oleagenous suspensions or suppositories.
For oral administration as a suspension, the compositions can be prepared according
to techniques well-known in the art of pharmaceutical formulation. The compositions
can contain microcrystalline cellulose for imparting bulk, alginic acid or sodium
alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners
or flavoring agents. As immediate release tablets, the compositions can contain
microcrystalline cellulose, starch, magnesium stearate and lactose or other excipients,
binders, extenders, disintegrants, diluents, and lubricants known in the art.
For administration by inhalation or aerosol, the compositions can be prepared
according to techniques well-known in the art of pharmaceutical formulation. The
compositions can be prepared as solutions in saline, using benzyl alcohol or other
suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons,
or other solubilizing or dispersing agents known in the art.
For administration as injectable solutions or suspensions, the compositions can
be formulated according to techniques well-known in the art, using suitable dispersing
or wetting and suspending agents, such as sterile oils, including synthetic mono-
or diglycerides, and fatty acids, including oleic acid.
For rectal administration as suppositories, the compositions can be prepared
by mixing with a suitable nonirritating excipient, such as cocoa butter, synthetic
glyceride esters, or polyethylene glycols, which are solid at ambient temperatures,
but liquefy or dissolve in the rectal cavity to release the drug.
Preferred administration routes include orally, parenterally, as well as
intravenous, intramuscular, or subcutaneous routes. More preferably, the compounds
of the present invention are administered parenterally, i.e., intravenously or
intraperitoneally, by infusion or injection. In one embodiment of the invention,
the compounds may be administered directly to a tumor by tumor injection; or by
systemic delivery by intravenous injection.
Solutions or suspensions of the compounds can be prepared in water, isotonic
saline (PBS) and optionally mixed with a nontoxic surfactant. Dispersions may also
be prepared in glycerol, liquid polyethylene, glycols, DNA, vegetable oils, triacetin,
and mixtures thereof. Under ordinary conditions of storage and use, these preparations
may contain a preservative to prevent the growth of microorganisms.
The pharmaceutical dosage form suitable for injection or infusion use can include
sterile, aqueous solutions or dispersions, or sterile powders comprising an active
ingredient which are adapted for the extemporaneous preparation of sterile injectable
or infusible solutions or dispersions. In all cases, the ultimate dosage form should
be sterile, fluid, and stable under the conditions of manufacture and storage.
The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising,
for example, water, ethanol, a polyol such as glycerol, propylene glycol, or liquid
polyethylene glycols and the like, vegetable oils, nontoxic glyceryl esters, and
suitable mixtures thereof. The proper fluidity can be maintained, for example,
by the formation of liposomes, by the maintenance of the required particle size,
in the case of dispersion, or by the use of nontoxic surfactants. The prevention
of the action of microorganisms can be accomplished by various antibacterial and
antifungal agents, for example: parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be desirable to include isotonic agents, for
example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the inclusion in the composition of agents
delaying absorption, for example, aluminum monosterate hydrogels and gelatin.
Sterile injectable solutions are prepared by incorporating the compounds
in the required amount in the appropriate solvent with various other ingredients
as enumerated above and, as required, followed by filter sterilization. In the
case of sterile powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and freeze-drying techniques,
which yield a powder of the active ingredient plus any additional desired ingredient
present in the previously sterile-filtered solutions.
The present invention may be better understood with reference to the accompanying
examples that are intended for purposes of illustration only and should not be
construed to limit the scope of the invention. All literature citations herein
are expressly incorporated by reference.
EXAMPLE 1
Gabapentin Inhibits Rat mGluR5 Mediated ERK Phosphorylation in CHO Cells
CHO cells engineered to express mGluR5 (CHO-mGluR5 cells) were plated in 12-well
tissue culture plates at a density of 20,000 cells/well and grown for 24 hours,
followed by further culturing in serum free medium for 16 hours. CHO-mGluR5 cells
were incubated in Krebs buffer for 30 minutes at which time 1 mM sodium pyruvate
and 10 μ/mL glutamic-pyruric transaminase was added. CHO-mGluR5 cells were
then stimulated with quisqualic acid for 5 minutes, washed with cold PBS buffer
containing 1 mM sodium orthovanadate and 1 mM phenylmethylsulfonyl fluoride, then
solubilized in lysis buffer (10 mM Hepes, pH 7.4, 70 mM NaCl, 50 mM b-glycerol
phosphate, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl
fluoride, and 1× protease inhibitor cocktail). CHO-mGluR5 cells treated with
gabapentin were pretreated with 100 μM gabapentin for 30 minutes prior to
quisqualic acid stimulation. Gabapentin was prepared as outlined in Bryans et al.,
Medicinal Research Reviews, 1999;19:149-177. Proteins in cell extracts were
separated by SDS-PAGE and transferred to nitrocellulose membrane. The membrane
was blocked overnight at 4° C. with 4% BSA in Tris-buffered saline containing
0.2% (v/v) Tween 20 (TBST). The membrane was then incubated for 1 hour at room
temperature (RT) with antiphospho MAP kinase antibody (1:1000 dilution) in 1% BSA
and TBST, then washed with TBST followed by incubation with antirabbit IgG HRP
antibody. After a final wash with TBST, the membrane was visualized using enhanced
chemiluminescence reagents.
Results
Activation of Erk-1 and -2 by stimulating CHO-mGluR5 cells with 100 μM
quisqualate was examined by using an antibody specific to phosphorylated Erk-1
and -2. As shown in FIG. 1 (Lane 1), there was little phosphorylated Erk-1 and
-2 in unstimulated CHO cells. Addition of 100 μM quisqualate for 5 minutes
quickly increased the level of phosphorylated Erk-2, but had little effect on changing
the level of phosphorylated Erk-1 (Lane 2). Preincubation of the mGluR5/CHO cells
with 100 μM gabapentin for 30 minutes reduced the quisqualate activated phosphorylated
Erk-2 by 50% (Lane 4). Gabapentin treated CHO-mGluR5 cells, in the absence of quisqualate,
showed little or no phosphorylated Erk-1 and -2 (Lane 3). As a positive control,
PD 184352 (known MEK inhibitor) block the effect of quisquolate (data not shown).
The data from this study illustrates the utility of the present invention for
screening compounds for gabapentinoid activity.
Since P38 and Jun kinases are closely related to MAP kinase, the effects of
substance-P on these kinases was examined. Addition of 10 nM substance-P to cells
overexpressing NK1 (CHO-NK-1 cells), significantly activated MAP kinase, but had
no effect on either P38 or Jun kinase activation as determined by Western blot
using activation-specific antibodies (data not shown). Also unlike CX3C receptor,
NK1 has no effect on activation of AKT, indicating that AKT signaling is not involved
in the gabapentin-sensitive MAP kinase pathway (data not shown). This data indicates
that NK1 elicited MAP kinase signaling is a fairly specific target for screening
compounds for gabapentinoid activity through the compounds ability to inhibit NK-1
elicited MAP kinase signaling.
EXAMPLE 2
Gabapentin Inhibits PMA Mediated ERK Phosphorylation in Differentiated IMR-32 Cells
IMR-32 cells were grown in MEM media (Gibco BRL, 11095-080), supplemented
with 10% fetal bovine serum (Gibco BRL, 26140-087), 1% antibiotic-antimycotic (Gibco
BRL, 15240-096), and 1% L-glutamine (Gibco BRL, 25030-032). Cells, grown to confluency,
were differentiated for a period of 7 to 10 days, or longer, by adding 1 mM dibutyryl
cAMP (Sigma D-0627) and 2.5 μM 5-bromo-2-deoxyuridine (Sigma B-9285) to the
media. The differentiation media was fed continuously to cells until the cells
were used in an experiment. Cells were divided into groups and preincubated for
30 minutes either in the presence or absence of indicated concentration of gabapentin
then subsequently challenged with 50 nM PMA for 5 minutes. Cells were washed with
cold PBS buffer containing 1 mM sodium orthovanadate and 1 mM phenylmethylsulfonyl
fluoride, then solubilized in lysis buffer (10 mM Hepes, pH 7.4, 70 mM NaCl, 50
mM b-glycerol phosphate, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl
fluoride, and 1× protease inhibitor cocktail). Proteins in cell extracts were
separated by SDS-PAGE and transferred to nitrocellulose membrane. The nitrocellulose
membrane was blocked overnight at 4° C. with 4% BSA in Tris-buffered saline
containing 0.2% (v/v) Tween 20 (TBST). The membrane was then incubated for 1 hour
at RT with antiphospho MAP kinase antibody (1:1000 dilution) in 1% BSA and TBST,
then washed with TBST followed by incubation with antirabbit IgG HRP antibody.
After a final wash with TBST, the membrane was visualized using enhanced chemiluminescence reagents.
Results
To determine the biological relevance of gabapentin effects on the MAP kinase
pathway, differentiated IMR-32 cells treated with PMA were examined. As shown in
FIG. 2, treatment with PMA (50 μM) increased Erk-2 phosphorylation (upper
panel) in differentiated IMR-32 cells, and the effects were dose-dependently blocked
by gabapentin. As controls, the lower panel showed that the total amount of Erk-1
and -2 in all samples are the same. Since differentiated IMR-32 cells have many
characteristics of a neuronal cell type, these data indicate that gabapentin effects
on the MAP kinase pathway are a biologically relevant phenomena. These data also
indicate that mGluR5 is not the direct target of gabapentin, since gabapentin showed
similar inhibitory effects when MAP kinase was activated by PKC instead of through
mGluR5 (not shown).
The data from this Example illustrates the utility of the present invention in
that inhibitory gabapentinoid activity on MAP kinase is dose dependent and can
be tested on multiple cell types that have MAP kinase activity.
EXAMPLE 3
In Vitro Screen for Gabapentinoid Activity
CHO-NK-1 cells were seeded to plates and grown to confluency overnight.
Lipofectamine transfection was performed on the confluent cells to insert Elk-1
and luciferase vectors necessary for a MAP kinase dependent reporter system. Prior
to transfection, the cells were washed in OPTI media (Gibco). To each confluent
well in a 24-well plate, 20 ng of Elk-1 and 200 ng of luciferase DNA vectors were
added. In addition to the vectors, 1.5 μL of Lipofectamine Reagent (Gibco)
and OPTI media (Gibco) were also added for a total transfection volume of 200 μL
(a master mix was made prior to use). After a 3-hour incubation, the media was
changed to F-12 growing media (500 μL/well) for an additional 3-hour incubation.
After the second incubation, the media was changed again to DMEM (500 μL/well)
for serum starvation and incubated overnight (all incubations at 37° C.).
In addition, lipofectamine transfection was performed on CHO-NK-1 confluent cells
to insert a GAL4-Elk-1 fusion protein and luciferase vector using the methods described above.
Luciferase Assay
The DMEM media was decanted off the plate, and the cells were washed with 500
μL of Kreb buffer. After the wash, 460 μL of Kreb buffer containing
2 mM sodium pyruvate was added to each well and incubated for 30 minutes at 37°
C. Next, 20 μL of compound was added to each well at the desired concentration
and incubated for another 30 minutes. Note that gabapentin was prepared as discussed
in Bryans et al., Medicinal Research Reviews, 1999; 19:149-177, pregabalin was
prepared as outlined in Hoekstra et al.,
Org. Process Res. Dev., 1997;1(1):26-38
and PD 201698 and PD 200390 were prepared as outlined in International Patent Application
WO 9921828. Finally, the cells were stimulated with Substance-P (final concentration=20
nM), and the cells were incubated for 4 hours at 37° C. After the 4-hour incubation,
the buffer was decanted off the plate, and the plate was washed twice with Dulbecco's-PBS.
Cells were lysed with 100 μL per well of cell Lysis Buffer (Promega), and
the plate was vortexed for 10 minutes. Finally, 10 μL of the cell lysate
was transferred to a 96-well reader plate, and each well's luminescence was measured
using a luminometer (the Promega Luciferase Reporter system was used).
Note that the 24-well assay format may be modified into a 96-well assay format
for higher throughput capabilities. In the 96-well format the assay is modified
by scaling down the 24-well format 4-fold. Therefore, each well in the 96-well
format has one fourth the amount of reagents as the 24-well assay format. A permanent
CHO cell line has also been created by electroporation of the NK-1, Elk-1, and
luciferase vectors, thus circumventing the need for lipofectamine transfection.
Results
A major downstream target for MAP kinase is the activation of the Elk-1 transcription
factor. Therefore, the effects of gabapentin and pregabalin were examined on Elk-1
mediated reporter gene activation.
Using the luciferase assay system discussed above, both gabapentin and pregabalin
showed dose dependent inhibition of the Elk-1 mediated reporter gene (elicited
by substance P) (FIG. 3). In particular, substance P treated CHO/NK-1 cells
showed significant Elk-1 mediated luciferase activity. However, treatment of the
CHO/NK-1 cells with both substance P and gabapentin (FIG. 3A) or pregabalin (FIG.
3B) showed dose dependent inhibition of the Elk-1 mediated luciferase activity.
Although the Elk-1 mediated luciferase assay is not a direct measure of
gabapentin activity, as was measuring for phosphorylated Erk-1 and -2 (Examples
1 and 2), the Elk-1 reporter gene assay is highly quantitative and provides a high-throughput
assay for screening numerous compounds for gabapentinoid acitivy.
To determine whether there is a correlation between the inhibitory effects of
gabapentinoids on Elk-1 mediated reporter gene activation and the gabapentinoids
biological efficacy in vivo, two pharmacologically well-characterized gabapentinoids
were tested for inhibitory effects on Elk-1 mediated luciferase activity. As shown
in FIG. 4 (left panel), PD 200390, a potent gabapentinoid in in vivo animal pain
models, showed dose-dependent inhibition on Elk-1 mediated luciferase activity.
In contrast, PD 201698, a biologically
