U.S. patent application number 11/595006 was filed with the patent office on 2007-03-08 for radioligands for the trp-m8 receptor and methods therewith.
Invention is credited to Edward T. Wei.
Application Number | 20070053834 11/595006 |
Document ID | / |
Family ID | 34520890 |
Filed Date | 2007-03-08 |
United States Patent
Application |
20070053834 |
Kind Code |
A1 |
Wei; Edward T. |
March 8, 2007 |
Radioligands for the TRP-M8 receptor and methods therewith
Abstract
One embodiment of the invention is a composition that comprises
a radioactive [.sup.18F], [.sup.76Br]-, [.sup.77Br]-,
[.sup.211At]-, [.sup.123I], [.sup.125I], or
[.sup.131I]-N-radioistope-labeled-aryl-alkyl-alkylcarboxamide
molecule. The composition binds to the transient receptor
potential-M8 (TRP-M8) receptor of cells. The TRP-M8 receptor is
selectively expressed in sensory neurons and in malignant tissues
such as prostate cancer cells. The [.sup.18F], [.sup.76Br]-,
[.sup.77Br]-, [.sup.211At]-, [.sup.123I], [.sup.125I], or
[.sup.131I]-N-radioistope-labeled-aryl-alkyl-alkylcarboxamide
ligand may be used for radioreceptor binding studies, for
diagnostic studies, and for radiotherapy of cancerous tissues.
Affinity of the N-radioistope-labeled-aryl-alkyl-alkylcarboxamide
ligand for the TRP-M8 receptor confers selectivity and specificity
in delivering lethal radiation to the diseased cells.
Inventors: |
Wei; Edward T.; (Berkeley,
CA) |
Correspondence
Address: |
Edward T. Wei
480 Grizzly Peak Blvd.
Berkeley
CA
94708
US
|
Family ID: |
34520890 |
Appl. No.: |
11/595006 |
Filed: |
November 8, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10687188 |
Oct 15, 2003 |
|
|
|
11595006 |
Nov 8, 2006 |
|
|
|
Current U.S.
Class: |
424/1.11 ;
534/11 |
Current CPC
Class: |
A61K 51/0406 20130101;
A61P 35/00 20180101; A61K 51/0476 20130101 |
Class at
Publication: |
424/001.11 ;
534/011 |
International
Class: |
A61K 51/00 20060101
A61K051/00; C07F 5/00 20060101 C07F005/00 |
Claims
1. A N-radioisotope-labeled-aryl-alkyl-alkylcarboxamide ligand
wherein the alkyl moiety of the alkylcarboxamide is a cycloalkane
radical having from 7 to about 14 carbons and with one to three
C.sub.1 to C.sub.5 normal or branched alkyl substituents, the
radioligand having a high affinity to TRP-M8 receptors in cells and
tissues and having a specific activity of at least 20 Ci/mmol or
greater, wherein the TRP-M8 affinity is characterized by a Kd of
about 1.times.10.sup.-5 or less.
2. The radioligand as in claim 1 wherein the radioisotope label is
covalently bound in the molecule.
3. The radioligand as in claim 2 wherein the radioisotope label is
selected from astatine, bromine, fluorine, iodine, astatide,
bromide, fluoride, or iodide nadionuclides.
4. The radioligand as in claim 1 wherein the specific activity is
about 20 Ci/mmol or greater and the radioisotope moiety emits
alpha, beta or gamma radiation.
5. A composition comprising a radioligand, the radioligand being a
N-radioistope-labeled-aryl-alkyl-alkylcarboxamide of Formula 1:
R--(C.dbd.O)--N(H or CH.sub.3)--R'--Y Formula 1 where (a) R is a
saturated or monoethylenically unsaturated alkyl-substituted cyclic
alkyl radical containing a total of 7 to about 14 carbon atoms and
is selected from the group consisting of cyclopentanes,
cyclohexanes, cycloheptanes, and cyclooctanes, each cyclic alkyl
radical containing from 1 to 3 C.sub.1-C.sub.5 normal or branched
alkyl substituents, (b) R' is a normal or branched C.sub.1-C.sub.3
carbon bridge with an optional hydroxy, and (c) Y is an aromatic
radical containing one to four substituents of R.sub.1 or R.sub.2,
and one to three substituents of X, wherein R.sub.1 is selected
from the group hydrogen, hydroxyl, C.sub.1-C.sub.5 alkyl,
C.sub.1-C.sub.3 alkoxy, C.sub.1-C.sub.3 carboxyalkyl,
C.sub.1-C.sub.4 carbonylalkylester, C.sub.1-C.sub.3
oxycarbonylalkyl, C.sub.1-C.sub.3 hydroxyalkyl, R.sub.2 is selected
from the group --SO.sub.2NH-pyrimidine, --SO.sub.3--(H, Me or Et),
or --CH.sub.2--SO.sub.3--(H, Me or Et), acetyl, C.sub.1-C.sub.3
hydroxyalkyl, trifluoromethyl, nitro, cyano, halo, and X is
selected from the group [.sup.18F]--, [.sup.123I]--, [.sup.125I]--,
[.sup.131I]-- [.sup.76Br]-- [.sup.77Br]-- and [.sup.211At]--.
6. The composition as in claim 5 wherein the cycloalkyl radical of
the radioligand is
((1R,2S,5R)-2-isopropyl-5-methyl-cyclohexyl)-.
7. The composition as in claim 5 wherein the radioligand is a
single enantiomer with its chiral center in R'.
8. The composition as in claim 5 wherein the radioligand has a
specific activity of about 20 Ci/mmol or greater and emits alpha,
beta or gamma radiation.
9. The composition as in claim 5 wherein the radioligand is a
ligand for the TRP-M8 receptor.
10. A diagnostic method, comprising: providing a radioligand, the
radioligand being a
N-radioistope-labeled-aryl-alkyl-alkylcarboxamide of Formula 1:
R--(C.dbd.O)--N(H or CH.sub.3)--R'--Y Formula 1 where (a) R is a
saturated or monoethylenically unsaturated alkyl-substituted cyclic
alkyl radical containing a total of 7 to about 14 carbon atoms and
is selected from the group consisting of cyclopentanes,
cyclohexanes, cycloheptanes, and cyclooctanes, each cyclic alkyl
radical containing from 1 to 3 C.sub.1-C.sub.5 normal or branched
alkyl substituents, (b) R' is a normal or branched C.sub.1-C.sub.3
carbon bridge with an optional hydroxy, and (c) Y is an aromatic
radical containing one to four substituents of R.sub.1 or R.sub.2,
and one to three substituents of a radio label X, wherein R.sub.1
is selected from the group hydrogen, hydroxyl, C.sub.1-C.sub.5
alkyl, C.sub.1-C.sub.3 alkoxy, C.sub.1-C.sub.3 carboxyalkyl,
C.sub.1-C.sub.4 carbonylalkylester, C.sub.1-C.sub.3
oxycarbonylalkyl, C.sub.1-C.sub.3 hydroxyalkyl, R.sub.2 is selected
from the group --SO.sub.2NH-pyrimidine, --SO.sub.3--(H, Me or Et),
or --CH.sub.2--SO.sub.3--(H, Me or Et), acetyl, C.sub.1-C.sub.3
hydroxyalkyl, trifluoromethyl, nitro, cyano, halo, and X is
selected from the group [.sup.18F]--, [.sup.123I]--, [.sup.125I]--,
[.sup.131I]-- [.sup.76Br]-- [.sup.77Br]-- and [.sup.211At]--;
contacting the radioligand with cells or tissues under conditions
sufficient to permit specific binding between the radioligand and
TRP-M8 receptors if said receptors are carried by the cells or
tissues; and, examining the cells or tissues for presence of the
radio lable X.
Description
[0001] This application is a continuation-in-part of Ser. No.
10/687,188, filed Oct. 15, 2003, Inventor Wei, entitled
"Radioligands for the TRP-M8 Receptor and Methods Therewith",
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention generally relates to chemicals that bind to
receptors in the TRP (transient receptor potential) ion channel
family, more particularly to the subgroup of long TRP (or TRPM)
channels, and most particularly to those that specifically bind to
the TRP channel called TRP-M8 (trp-p8, CMR.sub.1); TRP-M8 receptors
are present in sensory nerves and activation of these receptors is
associated with cool and cold sensations. These receptors are also
at elevated levels in the tissues of certain cancers, such as
prostate and breast cancer. This invention more particularly
relates to TRP binding compositions containing radioisotopes such
as radioactive fluorine and iodine .sup.18F, .sup.123I, .sup.125I,
or .sup.131I, within the molecular structure, said compositions
being useful, for example, in radioreceptor, diagnostic imaging,
and radiotherapeutic applications.
[0004] 2. Description of Related Art
[0005] About two decades ago a group of scientists discovered novel
compounds that have a physiological cooling action on the skin.
These were described in U.S. Pat. No. 4,193,936 (Watson et al.,
Mar. 18, 1980), U.S. Pat. No. 4,248,859 (Rowsell et al, Feb. 3,
1981) and U.S. Pat No. 4,318,900 (Rowsell, Mar. 9, 1982). Much more
recently a new physiological receptor was discovered. This
1104-amino acid protein, deciphered from the cDNA sequence, was
named trp-p8 because of its structural homology to receptors of the
transient receptor potential (TRP) family. The mRNA for the
synthesis of this specific protein was also detected in samples of
malignant prostate, mammary gland cells, melanoma, and colorectal
cancer cells. The functional role, if any, of TRP-M8 receptors on
malignant cells is not known.
[0006] The TRP-M8 sequence of the gene/protein was published in
Cancer Research (vol. 61, pg. 3760-3769, May 1, 2001. L. Tsavaler,
M. H. Shapero, S. Morkowski, and R. Laus: "Trp-p8, a novel
prostate-specific gene, is up-regulated in prostate cancer and
other malignancies and shares high homology with transient receptor
potential calcium channel proteins"). Soon afterwards it was
discovered that this receptor was present in sensory neurons and
transduced the sensations of cold temperatures (McKemy et al.
"Identification of a cold receptor reveals a general role for TRP
channels in thermosensation". Nature 416: 52-58, March 2002).
Chemicals that elicit sensations of cold, such as menthol and
icilin, bind to and activate the cold receptor, as measured by
binding constants and by calcium influxes into the cells.
[0007] A nomenclature panel composed of experts in the field has
recommended the TRP-M8 designation for the cold/prostate receptor
because of its structural homology to other protein receptors in
this family. However, some still call this receptor trp-p8 or
CMR.sub.1 (cold-menthol receptor). The tags for the TRP-M8
sequences in the NicePro TrEMBL Database are Q8R405 (mouse TRP-M8),
Q8R444 (rat TRP-M8 or CMR.sub.1) and Q8TAC3 (human TRP-M8, or
trp-p8). The corresponding identity tags in the GenBank are
AF4811480 and AY095352 (mouse), AY072788 (rat) and AY090109
(humans).
[0008] Various radioactive fluorine and iodine compounds are used
in clinical oncology. For example, .sup.18F and .sup.123I are used
in positron emission tomography (PET) and single-photon emission
computed tomography (SPECT), respectively, for the imaging,
diagnosis and staging of neoplastic disease. .sup.125I and
.sup.131I are used for the treatment of cancer, especially thyroid
cancer. Radioiodine compounds in thyroid therapy are remarkably
effective because iodine is incorporated specifically into the
thyroid hormones (thyroxin and tri-iodothyronine). Hence, the
malignant cells are selectively and specifically targeted, with
minimal damage to normal cells and adverse side effects.
[0009] Prostate cancer is the most common cancer among men in the
United States. There is no universally agreed-upon strategic plan
for its diagnosis and management. Brachytherapy, a treatment well
known in the art, involves the implantation of radioactive seeds
directly into the prostate gland. The radioactive seeds used in
brachytherapy may include iodine-125, iodine-131, palladium,
radium, iridium, or cesium. Another common cancer is bladder
malignancy, which will be diagnosed in an estimated 44,640 men and
15,600 women in the United State in 2004, with about 13,000 deaths
from this disease.
[0010] The pharmacological strategy, to bring radio-labeled
compounds to specific targets in malignant cells, to improve
diagnosis, or to treat certain cancers, is called targeted
radiodiagnostics and targeted radiotherapy. New radiofluorinated
and radioiodinated compounds useful for these applications are
being sought.
BRIEF SUMMARY OF THE INVENTION
[0011] The present discovery provides carboxamide ligands that are
usefully labeled with various radionuclides, and which have a high
affinity to TRP-M8 receptors in cells and tissues. Formula 1
illustrates carboxamide ligands of this discovery.
R--(C.dbd.O)--N(H or CH.sub.3)--R'--Y Formula 1 [0012] where (a) R
is a branched hydrophobic carbon unit with 5 to about 14 carbon
atoms, and is preferably a cycloalkane radical with one to three
C.sub.1 to C.sub.5 normal or branched alkyl substituents, [0013]
(b) R' is an optional carbon bridge having C.sub.1-C.sub.3 carbons
which may include a hydroxy group, and [0014] (c) Y is an aromatic
radical containing at least one substituent selected from R.sub.1
and R.sub.2, and at least one substituent X, wherein [0015]
R.sub.1, is selected from the group hydrogen, hydroxyl,
C.sub.1-C.sub.5 alkyl, C.sub.1-C.sub.3 alkoxy, C.sub.1-C.sub.3
carboxyalkyl, C.sub.1-C.sub.4 carbonylalkylester, C.sub.1-C.sub.3
oxycarbonylalkyl, C.sub.1-C.sub.3 hydroxyalkyl, [0016] R.sub.2 is
selected from the group --SO.sub.2NH-pyrimidine, --SO.sub.3--(H, Me
or Et), or --CH.sub.2--SO.sub.3--(H, Me or Et), acetyl,
C.sub.1-C.sub.3 hydroxyalkyl, trifluoromethyl, nitro, cyano, halo,
and [0017] X is selected from the group [.sup.18F]--,
[.sup.123I]--, [.sup.125I]--,
[.sup.131I]--[.sup.76Br]--[.sup.77Br]-- and [.sup.211At]--.
[0018] In one aspect of the present invention,
N-radiohaloaryl-alkylcarboxamide radioligand embodiments with
specific affinity for the TRP-M8 receptor are provided (where there
is no carbon bridge, R', of Formula 1). These radioactive ligands
are useful to study receptor binding (and to identify new drugs
that activate the TRP-M8 receptor), to conduct radioimaging and
radiodiagnostics, and should be useful as radioligands for therapy.
The radionuclides preferred are .sup.18F, .sup.123I 125I, or
.sup.131I. The inventive [.sup.125I]-compounds are useful for
laboratory tests, called radioreceptor assays. The inventive
[.sup.18F], [.sup.123I], [.sup.131I]-compounds are useful for
imaging of the tumor cells in vivo bearing this receptor marker,
and the [.sup.125I] or [.sup.131I]-compounds are further believed
potentially useful for targeted radiotherapy.
[0019] Among particularly preferred compositions of this first
embodiment are those including [.sup.123, 125, or
.sup.131I]--N-(4'-iodo-2'-methylphenyl)-2-isopropyl-5-methylcyclohexaneca-
rboxamide illustrated below as Structure 1. ##STR1##
[0020] Also among particularly preferred compositions of the first
embodiment are those that include
[.sup.18F]--N-(4'-fluoro-2'-hydroxyphenyl)-2-isopropyl-2,3-dimethylbutyra-
mide illustrated as Structure 2. ##STR2##
[0021] In another aspect of this invention, N-radioisotope-labelled
aryl-alkyl-alkylcarboxamide radioligands with specific affinity for
the TRP-M8 receptor are provided (where the carbon bridge of R' has
one, two or three carbons). Among particularly preferred
compositions of the second embodiment are those that include the
compound illustrated by Structure 3. ##STR3##
[0022] In this Structure 3 compound, R' is hydroxyethyl, R.sub.1 is
acetyl or --SO.sub.2NH-pyrimidine, and R.sub.2 is hydroxyl or
hydroxymethyl, and X is selected from the group [.sup.18F]--,
[.sup.123I]--, [.sup.125I]--, [.sup.131I]-- [.sup.76Br]--
[.sup.77Br]-- and [.sup.211At]--
[0023] In yet another aspect of the present invention, methods are
provided in which a Formula 1 compound having a determinable
binding for the TRP-M8 receptor and having a specific activity of
about 20 Ci/mmol or greater is exposed to or contacted with a
plurality of TRP-M8 receptors under conditions sufficient to permit
specific binding therebetween. These methods include radioreceptor
assays, diagnostic imaging and radiotherapy, particularly for the
diagnosis, monitoring and potential therapy of prostate cancer.
[0024] Other advantages and aspects of the present invention will
be understood by reading the following detailed description and the
accompanying claims.
DETAILED DESCRIPTION OF THE INVENTION
[0025] With reference generally to Formula 1 below, radioligands of
the invention have a) a hydrogen bonding site, as exemplified by
the CO and NH groups of a carboxamide, b) a hydrophobic group, as
exemplified by cycloalkyl or branched aliphatic groups, and c) an
aryl group that can be halogenated with radioisotopes. The hydrogen
bond/hydrophobic carbon units optimize docking into the TRP-M8
binding site, and the aryl ring permits delivery of the
isotope.
[0026] The selected isotopes, preferably from the group .sup.18F,
.sup.123I, .sup.125I, and .sup.131I, serve to either mark the
location or quantity of the TRP-M8 receptor or to deliver radiation
to the TRP-M8 bearing cell.
[0027] Embodiments of the present invention can function as ligands
for the TRP-M8 receptor and preferably have high affinity to TRP-M8
sites in cells and tissues and a specific activity of about 20
Ci/mmol or greater.
[0028] Compositions including the inventive radioligands of the
invention have the following applications: [0029] use as ligands
for TRP-M8 radioreceptor assays in the laboratory; [0030] use as
ligands for diagnosis and imaging of TRP-M8 receptors in prostate
tissues and cells; and [0031] use as radiotherapeutics (alone or
co-administered with local anesthetic amidase inhibitors as
potentiators) for prostate disorders such as cancer or benign
hyperplasia.
[0032] Formula 1 illustrates carboxamide ligands of this discovery.
R--(C.dbd.O)--N(H or CH.sub.3)--R'--Y Formula 1 [0033] where (a) R
is a hydrophobic alkyl radical, more particularly a branched
hydrophobic carbon unit with 5 to about 14 carbon atoms, and more
preferably is a cycloalkane radical with one to three C.sub.1 to
C.sub.5 normal or branched alkyl substituents, [0034] (b) R' is an
optional carbon bridge having C.sub.1-C.sub.3 carbons and which may
include a hydroxy group, and [0035] (c) Y is an aromatic radical
containing at least one substituent selected from R.sub.1 and
R.sub.2, and at least one substituent X, wherein [0036] R.sub.1 is
selected from the group hydrogen, hydroxyl, C.sub.1-C.sub.5 alkyl,
C.sub.1-C.sub.3 alkoxy, C.sub.1-C.sub.3 carboxyalkyl,
C.sub.1-C.sub.4 carbonylalkylester, C.sub.1-C.sub.3
oxycarbonylalkyl, C.sub.1-C.sub.3 hydroxyalkyl, [0037] R.sub.2 is
selected from the group --SO.sub.2NH-pyrimidine, --SO.sub.3--(H, Me
or Et), or --CH.sub.2--SO.sub.3--(H, Me or Et), acetyl,
C.sub.1-C.sub.3 hydroxyalkyl, trifluoromethyl, nitro, cyano, halo,
and [0038] X is selected from the group [.sup.18F]--,
[.sup.123I]--, [.sup.125I]--, [.sup.131I] [.sup.76Br]--
[.sup.77Br]-- and [.sup.211At]--.
[0039] Where R' has no carbons (that is, R' is not present), then
preferred embodiment compositions are those comprising a
radioactive compound having the structure [.sup.18F], [.sup.123I],
[.sup.125I], or [.sup.131I]-N-radiohaloaryl-alkylcarboxamide, for
example: [.sup.123, 125 or
131I]--N-(4'-iodo-2'-methylphenyl)-2-isopropyl-5-methylcyclohexanecarb-
oxamide, and
[.sup.18F]--N-(4'-fluoro-2'-hydroxyphenyl)-2-isopropyl-2,3-dimethylbutyra-
mide, illustrated below as Structures 1 and 2 respectively.
##STR4## ##STR5##
[0040] Where R' is present, then among particularly preferred
compositions of the second embodiment are those that include the
compound illustrated by Structure 3. ##STR6##
[0041] In Structure 3, R' is hydroxyethyl, R.sub.1 is acetyl or
--SO.sub.2 NH-pyrimidine, and R.sub.2 is hydroxyl or hydroxymethyl,
and X is selected from the group [.sup.18F]--, [.sup.123I]--,
[.sup.125I]--, [.sup.131I]--, [.sup.76Br]--, [.sup.77Br]--, and
[.sup.211At]--.
[0042] The term "alkyl" used throughout this description in the
context of the R group of Formula 1 is as a generic term to include
both acyclic alkyl groups and cycloalkanes. That is, the
hydrophobic group is provided by a branched hydrophobic carbon
unit, which can be supplied by cycloalkyl or branched aliphatic
groups. However, cycloalkyl groups, particularly those where R is a
cycloalkane derivative of cyclopentanes, cyclohexanes,
cycloheptanes, and cyclooctanes, are preferred.
[0043] Radioactive compounds of the present invention preferably
have a specific activity of at least about 20 Ci/mmol, more
preferably have a specific activity of at least about 250 Ci/mmol.
Radioactive compounds of the invention can function as ligands for
the TRP-M8 receptor, and preferably have a Kd for the receptor of
about 1.times.10.sup.-12 to 1.times.10.sup.-5 molar.
[0044] An aspect of the present invention is that the radionuclide
(preferably .sup.18F, .sup.123I, .sup.125I or .sup.131I) is
incorporated (i.e. covalently bound) within the molecular structure
of the ligand for the receptor. One advantage of this is that the
radiation emitted can readily be detected with radioactivity
counters or imaging systems and is directly correlated to high
affinity binding to TRP-M8 receptors. Such specific direct
radioactive label incorporation into the binding molecule is
uncommon and provides excellent results in radioreceptor
applications as contemplated in the present invention.
[0045] By contrast, a laboratory procedure, for example, the
labeling of a binding protein such as a monoclonal antibody by
.sup.125I, carries the risk that the protein will be denatured by
the iodine and degraded by enzymes, thereby reducing or destroying
its high affinity binding to the receptor target. Moreover, the
points of attachment of iodine to the binding molecule are
non-specific (see Griffiths et al,. Radioactive iodine labeled
proteins for targeted radiotherapy, U.S. Pat. No. 5,976,492, Nov.
2, 1999, herein incorporated by reference).
[0046] Incorporating .sup.18F, .sup.123I, .sup.125I or .sup.131I by
covalent binding into molecules of the present invention avoids the
drawbacks referred to above with radioactive iodine with respect to
denaturation, degradation, and potential loss of activity, since
incorporation of the radioisotope into the molecule does not
significantly change the physical-chemical properties of the
molecule. The chemical features of the molecule that determine
specificity of binding affinity are retained, with the added
property of radiation. Compounds of the invention are sometimes
herein collectively termed "N-radiohaloaryl-carboxamides".
Criteria for Bloactivity on TRP-M8
[0047] In Vivo Assays for TRP-M8 Activation. The endogenous TRP-M8
receptor is a physiological receptor designed to detect temperature
changes in its environment and to transmit this signal to the
central nervous system so that appropriate regulatory responses can
be initiated (e.g. vasoconstriction to reduce heat loss, putting on
warmer clothing, avoiding the cold environment). This receptor also
responds to drug ligands (e.g., menthol, icilin, certain
N-alkylcarboxyl esters and N-alkylcarboxamides) which activate its
message transmission system and elicit sensations of cold.
[0048] The TRP-M8 receptor on cold sensory nerve endings and on
malignant cells, for example in the prostate, are biochemically
identical proteins. Thus, the potency of a molecule to elicit cold
sensations, for example on the tongue or skin, was used as
surrogate index of the binding affinity of the molecule for the
TRP-M8 receptor. A number of potent "N-radiohaloaryl-carboxamides"
were synthesized and tested for use as precursors of the inventive
compounds and the results are shown in Table 1. TABLE-US-00001
TABLE 1 Cold Sensation Threshold on CHEMICAL Tongue*, .mu.g
N-(3'-hydroxy-4'-methylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide
0.1 N-(4'-methoxyphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide
0.1
N-(2',4'-dimethylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide
0.1
N-(4'-methoxy-2'-methylphenyl)-1-isopropylcycloheptanecarboxamide
0.2 N-(4'-methylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide
0.3 N-(4'-nitrophenyl)-2-isopropyl-5-methylcyclohexanecarboxamide
0.3 N-(2'-hydroxyphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide
0.5 N-(4'-fluorophenyl)-2-isopropyl-5-methylcyclohexanecarboxamide
0.5 N-(4'-methoxyphenyl)-2-isopropyl-2,3-dimethylbutyramide 0.5
N-(3'-hydroxy-4'-methylphenyl)-1-isopropylcycloheptanecarboxamide 1
N-(4'-hydroxyphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 1
N-(2',4'-dimethylphenyl)-2-isopropyl-2,3-dimethylbutyramide 1
N-(4'-acetylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 2
N-(4'-methoxyphenyl)-2-isopropyl-2,4-dimethylpentanamide 2
N-(4'-methylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 3
N-(3',4'-dimethylphenyl)-2-isopropyl-2,3-dimethylbutyramide 3
N-(3',4'-dimethoxyphenyl)-1-isopropylcycloheptanecarboxamide 5
N-(4'-ethoxycarbonylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide
5 N-(4'-methoxyphenyl)-1-ethyl-2-methylcycloheptanecarboxamide 6
N-(4'-chlorophenyl)-2-isopropyl-5-methylcyclohexanecarboxamide 8
N-(2',4'-dimethylphenyl)-1-isopropylcycloheptanecarboxamide 15
N-(3',4'-dimethylphenyl)-1-isopropylcycloheptanecarboxamide 15
N-phenyl-2-isopropyl-5-methylcyclohexanecarboxamide 20
N-phenylmethyl-2-isopropyl-5-methylcyclohexanecarboxamide 20
*Filter paper (1 .times. 1 cm) was impregnated with a known amount
of compound and placed on the tongue of the test subject. After 30
sec, the subject was required only to report presence or absence of
a cooling effect. Individual sensitivity varied over a considerable
range; for example, for 23 subjects, chosen at random, the
threshold for a standard such as menthol ranged from 0.02 to 10
.mu.g. Ethoxycarbonyl is COOCH.sub.2H.sub.5.
Cooling Actions on Skin. CPS-195
[2-Isopropyl-5-methyl-cyclohexanecaboxylic acid
[2-hydroxy-2-(3-hydroxy-phenyl)-ethyl]-methyl-amide], CPS-140
[2-Isopropyl-5-methyl-cyclohexanecaboxylic acid
[4-acetylphenyl]-amide] and CPS-125
[2-Isopropyl-5-methyl-cyclohexanecarboxylic acid
[4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide], dissolved 0.5 to 2%
wt/vol in Aquaphor.RTM. ointment and then applied to the surface of
the philtrum of volunteer human subjects produced cooling
sensations lasting from 1.5 to 2.5 hr. These results show that
compounds of this application can activate the TRP-M8 receptors. In
Vitro Assays for TRP-M8 Activation. The methods for the TRP-M8
receptor studies, using methods of calcium ion imaging or
intracellular voltage changes, are described in Behrendt et al.
[Characterization of the mouse cold-menthol receptor TRPM8 and
vanilloid receptor type-1 VR1 using a fluorometric imaging plate
reader (FLIPR) assay. Brit. J Pharmacol. 2004 Feb; 141(4):737-45],
and by A. K. Vogt-Eisele, D. Bura, H. Hatt, and E. T. Wei.
[N-Alkylcarboxamide Cooling Agents: Activities on Skin and Cells
with TRPM8 and TRPA1 Receptors. Acta Dermato-Venereol. 85: 468,
2005.] The data here were collected for the applicant by Dr.
Afrodite Lourbakos of Unilever Research and Development, the
Netherlands, using similar transfection methods and a FLIPR assay
system. For the patch-clamp studies, using measurements of
intracellular voltage changes, some of the data reported here were
collected by Dr. Matthias Bodding of the University of the
Saarland, and by Dr. Angela Vogt-Eisele of the Ruhr University at
Bochum, Germany. FLIPR assay Human embryonic kidney (HEK) cells
were permanently trausfected with plasmids containging the cDNA
coding for the gene for the human TRP-M8 receptor. These cells were
then incubated with a calcium fluorescence indicator (Fura-2) and
incubated at either 29 or 37.degree. C. These cells were then
distributed into a 96-well fluorescence-plate image reader with
automated drug dilution and computerized software for dose-response
analysis. Cacium ion influx into cells after stimulation with
compounds was quantified in fluorescence units. Compounds were
dissolved in DMSO by ultrasonication to a 0.1 M solution. 5 .mu.l
of this stock was added to 5 mg of cyclodextrin and 5 ml of 140
Na-Tyrode, to achieve various final test concentrations. Icilin and
menthol, standard TRP-M8 agonists, were used as positive controls
and gave median effective concentration activities of 0.8 and 25
.mu.M (EC50) activities. The EC50 of various test substances are
shown in Table 2. Further analysis of the dose-response
relationship showed the .DELTA.Fmax (the maximum fluorescence
increase induced by a compound at the maximum concentration tested)
for various compounds to be at the maximum of 14,000 units which
was similar to that seen with icilin and menthol, confirming full
activation of the TRP-M8 receptor.
[0049] Patch-Clamp Electrophysiological Recordings. HEK cells were
prepared as above. Membrane currents were recorded in the
whole-cell configuration using an EPC-9 amplifier (HEKA Elektronik,
Lambrecht, Germany) as described previously (Bodding et al. 2002).
Patch pipettes pulled from borosilicate glass (Kimax.RTM.) had
resistances between 2 and 3 M.OMEGA. when filled with the standard
internal solution (in mM): 145 Cs-glutamate, 10 HEPES, 8 NaCl, 1
MgCl.sub.2, 2 Mg-ATP, 0.1 mM EGTA adjusted to pH 7.2 with CsOH. The
EGTA concentration was 10 mM for the experiments shown in FIG. 6.
Extracellular solution contained (in mM): 145 NaCl, 2 CaCl.sub.2,
2.8 KCl, 2 MgCl.sub.2, 11 glucose, 10 HEPES, adjusted to pH 7.2
with NaOH. Drugs were applied in the bath solution by a custom-made
local perfusion system. The series resistance was compensated for
80 % and ranged between 5 and 10 M.OMEGA.. Currents were filtered
using an 8-pole Bessel filter at 2.9 kHz and digitised at 100
.mu.s. Voltage ramps (-110 mV to 90 mV in 50 ms) were applied at
0.5 Hz from a holding potential of -10 mV using PULSE software
(HEKA Electronics). Several parameters such as capacitance, series
resistance and holding current were monitored simultaneously at a
slower rate (2 Hz) using the X-Chart display (HEKA Electronics). A
liquid junction potential of 10 mV was applied to all voltages. All
experiments were carried out at room temperature (20-23.degree.
C.). Internal solutions were kept on ice to minimize hydrolysis of
ATP. TABLE-US-00002 TABLE 2 EC50 .mu.M EC50 .mu.M Code Names
Structures FLIPR patch-clamp R-phenyl-X CPS-128 4-OEt-- 0.5 NA
CPS-112 (WS-12) 4-OMe-- 0.6 0.2 CPS-113 3-F, 4-OMe-- 1.3 1.2
CPS-124 4-fluoro- 1.3 1.2 CPS-129 2-iodo, 4-methoxy NA 0.3 CPS-123
4-bromo- 6 NA CPS-120 4-iodo- 10 NA CPS-125 4-sulfadiazinyl- 6 3.0
CPS-131 4-sulfadimethoxinyl- 60 NA CPS-141 3-OMe-- 6 NA CPS-127
4-OCF.sub.3-- NR NA CPS-138 3-CF.sub.3, 4-NO.sub.2-- NR NA CPS-132
4-sulfisoxazolyl- NR NA Other Structures CPS-116 3-OMe,
4-OH-benzyl-R 8 NA CPS-195 3-OH, 2-hydroxybenzyl- NA 0.1
N-methylated Comparison Cmpd menthol 25 10.4 Comparison Cmpd icilin
0.8 1.4 NR = no responses, NA = not available
Structure-Activity Relationships for Active Compounds. Precursor
compounds having the desired affinity for the TRP-M8 receptor (such
as listed in Tables 1 and 2) are radiohalogenated according to
standard procedures so as to form inventive comounds for uses in
the present invention. The preferred [.sup.18F], [.sup.123I],
[.sup.125I], or [.sup.131I]--N-halo-aryl-alkyl-alkylcarboxamides of
the invention as illustrated by Formula 1. In addition to
cycloalkanes, such as where the R of Formula 1 is
(1R,2S,5R)-2-Isopropyl-5-methyl-cyclohexyl, R may also be a
branched chain
N-radioistope-labeled-aryl-alkyl-alkylcarboxamide.
[0050] Examples of such branched chains for R are propyl,
isopropyl, butyl, isobutyl, sec-butyl, tert-butyl and pentyl,
isopentyl, neo-pentyl etc. For branched aliphatics, the carboxamide
is attached, for example, to the "3" position of
2,3,4-trimethyl-pentane and 2,4-dimethyl-hexane, and to the "4"
position of 3,5-dimethyl-heptane.
[0051] The structural features of TRP-M8 binding for N-fluoro- or
iodo-aryl-alkyl-alkylcarboxamides side principally in the hydrogen
bonding --C(.dbd.O)--NH-- moiety and the branched chain hydrophobic
carbon unit. The N-substituent can be quite varied; for example,
N-ethyl or N-methyl p-menthane-3-carboxamides have oral cooling
thresholds as low as 0.2 to 1.1 .mu.g, respectively. In receptor
terminology, the N-substituent portion of the molecule is
"promiscuous" and many alternatives are permissible to fit the
TRP-M8 receptor pocket. Thus, the "Y" of Formulas 1 and 2 can be a
substituted aromatic radical, selected from the group phenyl,
benzyl, 1-naphthyl, 2-naphthyl, 1-anthracenyl, 2-anthracenyl,
9-anthracenyl, as well as other polyaromatic aromatic rings such as
indene, azulene, heptalene, indacene, acenapthlene, fluroene,
phenanthrene, and further as well as heterocyclic aromatic rings
such as pyridine, dihydropyridine, pyridazine, pyrimidine,
pyrazine, indole, purine, indolizine, quinoline, isoquinoline,
quinazoline, carbazole, phenazine, phenothiazine, and
phenathridine. A polyaromatic ring will also permit multiple
halogenation which increases the specific activity of the TRP-M8
ligand and enhances measurement of binding, imaging, and delivery
of radiation.
[0052] Radiohalogenation of aromatic rings to generate ligands for
receptor binding, for radioimaging and for radiotherapy is a
chemical technique that is familiar to many practioners of the art.
The preferred isotopes, .sup.18F, .sup.123I, .sup.125I, and
.sup.131I, are most commonly used but it should be noted that
alternative therapeutic radionuclides are also contemplated. For
example, for targeted radiotherapy for small tumors other halogens
such as .sup.76Br and .sup.77Br, and low-energy electron emitters
such as .sup.58mCo, .sup.103mRh, .sup.119Sb, .sup.161Ho, and
.sup.189mOs are also feasible (Bernhardt et al. Low-energy electron
emitters for targeted radiotherapy for small tumours. Acta
Oncologica 40: 602-608, 2001). Radionuclides, such as .sup.212Bi,
.sup.213Bi and .sup.211At, a halogen, which decay by the emission
of alpha-particles, can also be incorporated into the N-aryl-alkyl
moiety and are attractive for applications of targeted radiotherapy
in accordance with the present invention. The halogens, such as Br
and At, may be attached using trialkyl tin reagents and the metal
isotopes, such as Sb and Os, may be attached to the ring using
metal chelating agents.
[0053] Precursor compounds of highest activity and especially
preferred for use in the present invention (after modification to
incorporate radiohalides) are described in Table 1 and further
illustrated by the exemplary preparation of Example 1.
[0054] The particular N-substituent portion of the molecule, namely
the --R'--Y of Formula 1, would vary depend upon the particular
laboratory, radiodiagnostic, or radiotherapeutic applications. For
example, in laboratory applications, the ideal isotope will be
.sup.125I and selection of the ideal probe for the receptor will be
based on potency of activation or binding affinity. In
radiodiagnostic applications, for example, in PET scanning, the
isotope will be .sup.18F, but here the pharmacolinetic behavior of
the prototype needs to be considered because it has to be
distributed in the vascular compartiment and access the target
tissues such as prostate or breast cancer cells. In such molecules,
insertion of oxygenated functions, such as hydroxy ethyl will lower
octanol/water partition coeffcients and facilitate access of the
designed molecule to reach target.
[0055] If the target is designed for killing cancer cells in the
bladder urothelium, the delivery would be via urine of a drug
molecule that has a) has high affinity binding to the TRP-M8
receptor, on the order of a Kd of 10.sup.-9 M or less, b) contains
lethal radiation in the form of an alpha, beta or gamma emission
from a radioisotope within its molecular structure, and c) is
water-soluble, because urine is an aqueous media. The drug may
given by oral or by parenteral administration, e.g. by intravenous
injection or the may also be delivered directly into the bladder
lumen with a transurethral catheter or by intravesical
injection.
[0056] For the urothelium, if delivered by oral or by parenteral
administration, the "letter bomb" drug preferably enters the
bloodstream and then is cleared and concentrated by the kidneys
into the urine. The creation of such a drug requires ingenuity in
design. Ideally, this drug would be: a) relatively polar and
water-soluble and thus filtered and concentrated into the urine, b)
of a molecular weight under 500 daltons so that it readily passes
through the glomeruli and tubules c) of a pKa of 4.5 to 7.5, so
that it is significantly ionized at urinary pH, d) minimally bound
to protein in the blood so that it can be filtered, and e) not be
actively re-absorbed by the renal tubules. Overall, >90% of the
drug should preferably be cleared within 24 hr, to minimize
irradiation of non-cancerous tissues. Finally, the drug must retain
its selective high affinity for the TRP-M8 on the urothelium.
[0057] In Formula 1, the left-hand moiety R--(C.dbd.O)--N (H or
CH.sub.3v) confers structural features for TRP-M8 binding. The
branched chain hydrophobic carbon unit and the amide feature are
essential for receptor fit, with molecules having Kd in the range
of 10.sup.-7 to 10.sup.-6 M. The N-substituent can be quite varied
but a 100 to 1000-fold increase in potency is achieved when a
phenyl or aryl with an oxygen atom containing function (e.g.
para-methoxy or sulfadiazine). With these substituents, the goal of
potencies in the range of 10.sup.-8 to 10.sup.-10 M is achieved.
The N-substituent portion of the molecule is sterically
"promiscuous" and many alternatives are permissible to fit the
TRP-M8 receptor pocket, so that the addition of a radioisotope (X)
in the Formulae, such as .sup.131I, does not substantially change
affinity for the TRP-M8 receptor. The presence of the --R'-- group
permits the addition of polar groups which facilitate the delivery
or pharmacokinetic profile of the radioligand to target. In
addition, the presence of one or more chiral centers on the --R'--
carbon(s) allows the use of selective enantiomers to achieve
greater receptor selectivity and specificity. This stereoisomerism
permits better selection of the radioligand.
Preparation of Inventive Compounds
[0058] The preparation of
N-substituted-aryl-alkyl-alkylcarboxamides is familiar to
practitioners of the art of chemistry and, for example, is
described in U.S. Pat. No. 4,193,936, incorporated by reference.
Starting with the corresponding alkanoyl chloride, a single step
reaction with the appropriate amine yields the desired product. For
example, an alicyclic compound, p-menthane-3-carboxylic acid
(synonym: 2-isopropyl-5-methylcyclohexanecarboxylic acid) is
reacted with thionyl chloride in diethylether to yield the
p-menth-3-oyl chloride which, when stirred with the
substituted-aryl-alkylamine at room temperature for about 4 hr,
generates the corresponding
N-substituted-aryl-alkyl-p-menthane-3-carboxamide. The precipitated
product is readily collected by filtration and may be
recrystallized using solvents such ethyl acetate or purified on
silica gel columns. The final products are solids stable at room
temperature.
[0059] The -aryl-alkylamine may, for example, be
3-methyl4-iodo-phenylamine, or 4-fluorophenylamine, or
4-iodo-1-naphthylamine and the corresponding product after reaction
with p-menth-3-oyl chloride would be
N-(3'-methyl-4'-iodo-phenyl)-2-isopropyl-5-methylcyclohexane-3-carboxamid-
e,
N-(4'-fluorophenyl)-2-isopropyl-5-methylcyclohexane-3-carboxamide,
and
N-(4'-iodo-1'-naphthyl)-2-isopropyl-5-methylcyclohexane-3-carboxamide
respectively.
[0060] Synthesis of non-radioactive
n-substituted-aryl-alkyl-alkylcarboxamides as precursors for the
inventive compounds are depicted in the following schematics I and
2. Schematic 1 shows the synthesis of the desired carboxamide.
Synthesis of radioactive n-substituted-aryl-alkyl-alkylcarboxamides
useful in practicing the present invention to incorporate a halogen
is accomplished with reagents that effect the halogenation process
rapidly (because of the short half-life of the isotopes). A
standard reagent is trimethyl tin that is bonded to the site of
radiohalogenation. Schematic 2 illustrates this halogenation
process with trimethyl tin in forming an embodiment of the
invention in which an analog of Structure 1 is formed with .sup.18
F. In this analog, R.sub.1 is hydrogen, R.sub.2 is para-methoxy,
and X is radioactive fluorine. This analog is active at nanomolar
(10.sup.-9) in promoting calcium entry into TRP-M8 transfected
cells and into LNCaP (lymph node prostate cancer cells)
constitutively expressing these receptors. ##STR7## ##STR8##
Radioactive Ligands for the TRP-M8 Receptor.
[0061] The naturally occurring isotope of iodine has an atomic mass
of 126.9 Daltons. The radioactive isotopes of iodine are .sup.123I,
.sup.125I and .sup.131I with half-lives of 13.2 hr, 60.1 days and
8.0 days, and average energy of radioactive emission of 0.159 Mev,
0.02 Mev and 0.36 Mev, respectively. Preparation of
[.sup.123I]-compounds require special facilities because of the
short half-life of this isotope. By contrast, .sup.125I or
.sup.131I are inexpensive, readily available at high specific
activity of several Ci/matom and obtainable by express mail.
Iodine, being a common constituent of the body, has no inherent
toxicity in its radioactive form, other than the emitted
radiation.
[0062] The natural isotope of fluorine has an atomic mass of 19.0.
The .sup.18F-isotope has a half-life of 1.8 hr and an average
energy of emission of 0.511 Mev and requires special facilities for
preparation. The fluorine compounds used in this invention have no
inherent toxicity at the doses employed, other than the emitted
radiation.
[0063] Amersham Biosciences Corporation (800 Centennial Avenue,
Piscataway, N.J. 08855-1327, USA) is a major supplier of reagents
for the synthesis of radio-labeled chemicals. Starting materials
for [.sup.125I]- or [.sup.131I]-compounds can be obtained from
Amersham at high specific activities. The isotope half-lives and
the average energy of emission dictate the practical use of these
labels: [0064] [.sup.125I]-labeled alkylcarboxamides compounds are
useful for radioreceptor assays in the laboratory; [0065]
[.sup.18F]-, [.sup.123I]- or [.sup.131I]-labeled
N-halo-aryl-alkyl-alkylcarboxamides are useful for scanning or
imaging tissues bearing the TRP-M8 receptor; and [0066]
[.sup.125I]- or [.sup.113I]-labeled
N-halo-aryl-alkyl-alkylcarboxamides may be useful for targeted
radiotherapy, using fractionated dosages to destroy the desired
amount of tissues. Use of [.sup.125I]-labeled
N-halo-aryl-alkyl-alkylcarboxamides for Receptor Assays
[0067] A TRP-M8 receptor has two integral components, an
extracellular ligand binding domain that detects the ligand signal
and an intracellular domain that is involved in signal
transmission. The ligand detection mechanism is structurally
specific and analogous to the lock and key model of classical
pharmacology. The key is the drug ligand and the lock is the
receptor. Signal-transducing receptors are present in small
numbers, on the order of a few thousand receptors per cell.
Nevertheless, the receptors are designed to regulate crucial
cellular functions and therefore become specific targets for drug
discovery and development. Although not precisely understood, the
amino acid residues on the TRP-M8 protein that correspond to ligand
binding domains have recently been identified. For example,
aspartic acid on residue 802 and glycine on residue 805 of rat
TRP-M8 are critical for icilin-induced activation of TRP-M8.
Similarly, tyrosine on residue 845, tyrosine on residue 1005, and
leucine on residue 1009, influence menthol-induced activation of
mouse TRP-M8.
[0068] To measure drug occupancy of the receptor, pharmacologists
use the term "Kd (dissociation constant)" to represent the affinity
of the drug to its receptor. The Kd is based on the molar
concentration of the drug occupying 50% of the receptor population,
so the lower the Kd, the higher the "affinity" or stickiness of the
ligand for its receptor. A drug receptor agonist, that is, a drug
that elicits a biological response, generally has Kd values in the
sub-micromolar (10.sup.-6), nanomolar (10.sup.-9) to picomolar
(10.sup.-12) concentration and represents a "high affinity" binding
site. Similarly, a drug that binds with high affinity to the
receptor, but which does not activate the receptor, may be a high
affinity antagonist, preventing the actions of an agonist. To
measure Kd for different chemicals, it is necessary to have a
primary radioligand that is chemically pure and stable and known to
elicit the desired receptor response (for TRP-M8, it can be the
sensation of cold, or cation influx into transfected cells that
express the receptor). The specific activity of the radioactive
ligand must be high enough to detect high affinity binding of the
receptor in the tissue being studied. This usually means a specific
radioactivity of 30 Ci/mmol or higher.
[0069] For example, a synthetic
[.sup.125I]-N-iodo-aryl-alkyl-alkylcarboxamide ligand, such as
[.sup.125I]-N-(4'-iodophenylethyl)-p-menthane-3-carboxamide
(synonym:
[.sup.125I]-N-(4'-iodophenylethyl)-2-isopropyl-5-methylcyclohexanecarboxa-
mide) is an excellent receptor-assay radioligand. Such a
radioligand can be synthesized, for example, at 30 Ci/mmole, which
is considerably below the theoretical maximum of 2000 Ci/mmole for
[.sup.125I]-labeled compounds. This ligand can then be used for
radioreceptor assays of TRP-M8 agonists, as illustrated by Example
2. A TRP-M8 receptor assay based on
[.sup.125I]-N-iodo-aryl-alkyl-alkylcarboxamide ligand has several
applications, as described infra.
Utility of Radio-Labeled -Alkylcarboxamides on TRP-M8 Receptor
[0070] An agonist, in pharmacological terminology, is a chemical
that activates biological events. The agonist, almost by
definition, acts on a specific biological receptor to initiate
cellular events. The purpose of a radioreceptor assay is to have
methods to identify and measure ligands with low Kd value, and
hence high affinity for the desired receptor. Thus, in practice,
the first step is the characterization of a prototype
[.sup.125I]-agonist of the TRP-M8 receptor. Once, a prototype has
been identified, additional assays of in vitro and in vivo agonist
activity are conducted to demonstrate that the binding is
functional. These bioassays may also be conducted with
non-radioactive alkylcarboxamides to measure the median effective
concentrations (EC50). These methods are standard tools in drug
screening.
[0071] An antagonist, in pharmacological terminology, is a chemical
that binds with high affinity to a receptor, occupies it, and
prevents the actions of an agonist; but the antagonist itself does
not activate biological events. A prototype [.sup.125]-agonist of
the TRP-M8 receptor can be utilized in screening for a TRP-M8
receptor antagonist. Unknowns can be tested for their ability to
displace the [.sup.125I]-agonist from its binding site. A high
affinity antagonist would be a chemical that displaces the
[.sup.125I]-agonist at sub-micromolar concentrations, but by itself
does not produce cold sensations or activate TRP-M8 ion
channels.
Carboxamide Radioligands for Laboratory and Diagnostic
Applications.
[0072] The TRP-M8 receptor is exceptional in that its mRNA
transcript is found in abundance in biopsy samples of human
malignant tissues such as breast cancer, colorectal cancer,
melanoma and especially prostate cancer, but not in normal tissues
with the exception of prostate epithelial cells (Tsavaler et al.,
supra). The standard method used for detecting TRP-M8 MRNA
transcript in human tissues is to use in situ hybridization
techniques with special riboprobes designed to detect the TRP-M8
cDNA. Serial sections of tissues are made, then stained, which
enable histopathologists to visually observe any TRP-M8 receptors
in the stained tissue. Such methods, however, require advanced
laboratory skills and training. More recently, TRP-M8 antibodies
have been developed that allow for the detection of TRP-M8 receptor
protein on the surface of hyperplastic and malignant in prostate
tissues. The use of said antibodies (that is "TRP-M8
immunocytochemistry") has confirmed the presence of TRP-M8 on the
surface of human prostate cancer cells.
[0073] A [.sup.125]-radioreceptor assay in accordance with the
present invention, designed to measure the amount and the presence
of the TRP-M8 receptor protein in biopsy samples, is potentially a
less costly and a more convenient and a more direct alternative
than the aforedescribed techniques of in situ hybridization and
TRP-M8 immunocytochemistry.
[0074] The radioreceptor assay technique has diagnostic
applications for patients having cancers that express the TRP-M8
receptor. For example, a biopsy sample of about 10 mg tissue may be
homogenized and incubated with a
[.sup.125I]-N-iodo-aryl-alkyl-alkylcarboxamide ligand for 30 min,
centrifuged or filtered, dissolved in a solvent, and the
beta-emissions counted on a Geiger, scintillation, or other
radioactive counter. Based on the findings of Tsavaler et al,
supra, one would expect a sharp increase in the amount of TRP-M8
specific binding (Bmax) in malignant tissues, and the relative
lower abundance of binding in normal tissues. Such measurements,
with small amounts of tissue, because of the sensitivity of a
radioreceptor method using a radioligand with high specific
activity, can be used, for example, to detect the presence of
diseased tissues, to track disease progression, and to measure
metastases.
[0075] In the laboratory, .sup.125I is widely used in a technique
called autoradiography. Traditionally, .sup.125I, is used to label
peptides or proteins in a non-specific location, such as the ring
structure of tyrosine and on the .epsilon.-amino group of lysine.
This technique permits the detection and visualization of receptors
or antigens that bind to the labeled agonist or antibody. By the
same principles, the [.sup.125I]-N-iodo-aryl-alkyl-alkylcarboxamide
ligand may also be used in accordance with this invention for
autoradiographic studies of the TRP-M8 receptor and for discerning
its role in hyperplastic and neoplastic processes.
[0076] For example, sections of prostate tissues may be incubated
with the radioligand, rinsed, and then placed on X-ray film, and
the precise sites of TRP-M8 localization mapped by autoradiography.
The availability of the
[.sup.125I]-N-iodo-aryl-alkyl-alkylcarboxamide compositions of the
present invention should facilitate the study of TRP-M8 expression
in hyperplastic and malignant cells and aid in elucidating the role
of TRP-M8 in tumor initiation, transformation, invasiveness and
metastatic activity.
Radioimaging/Radiodiagnostic Uses of [.sup.18F], [.sup.123I],
[.sup.124I] or [.sup.131I]-N-iodo-aryl-alkyl-alkylcarboxamides with
High Affinity for TRP-M8.
[0077] Various radioactive fluorine and iodine compounds are used
in clinical oncology. For example, .sup.18F and .sup.124I are used
in positron emission tomography (PET), and .sup.123I and .sup.131I
in single-photon emission computed tomography (SPECT),
respectively, for the imaging, diagnosis and staging of neoplastic
disease. The emission of coincident or single high energy photons
permits computerized tomography imaging that yields useful
information about receptor marker binding, localization, and
clearance rates. A useful isotope for PET imaging is .sup.18F, an
isotope with a 110 min half life that generates coincident 511 KeV
photons which is measured by PET at a resolution of 0.5 to 1.8 mm
at marker concentrations of 10.sup.-9 to 10.sup.-12 M in tissues. A
useful isotope for SPECT imaging is .sup.123I an gamma-emitter with
a 13.3 hour half life. Eighty-five percent of the isotope's
emissions are 159 KeV photons, which is readily measured by SPECT
instrumentation currently in use.
[0078] The use of [.sup.18F]-deoxyglucose PET methods for
monitoring the progress of prostate cancer has limited success in
part because such prostate cells have limited metabolic activity. A
[.sup.18F]-TRP-M8 ligand, using PET imaging, will have utility as a
non-invasive method in staging this disease. .sup.124I, having a
longer half-life of 4.11 days than .sub.18F can also be used, but
it has limited availability. The high resolution of PET can also
allow the surgeon to detect metastases, to stage the disease, to
assess hormonal sensitivity to androgens, and to gauge the
feasibility of tissue removal. Similarly, a [.sup.123I] and
[.sup.131I]-TRP-M8 ligand can be used for SPECT applications in
prostate diseases.
Radiotherapeutic Use of [.sup.125I] or
[.sup.131I]-N-iodo-aryl-alkyl-alkylcarboxamides with High Affinity
for TRP-M8.
[0079] The expression of TRP-M8 receptor in tissues of the prostate
and bladder and its expression in hyperplastic and neoplastic
conditions [Tsavaler et al. supra] makes this receptor a potential
target for cancer radiotherapy. The TRP-M8 drug design strategy for
this target must be selective and specific: selective in the sense
that hyperplastic or cancer cells express this target more than
normal cells, and specific in the sense that the molecular target
will have structural features that bind the drug with high
affinity. Standard pharmacological strategies for targeting
receptors expressed in hyperplastic and neoplastic cells are to:
[0080] a) make a monoclonal antibody against the target. The
binding of the monoclonal antibody to the receptor leads to cell
death, for example, by triggering apoptosis; [0081] b) make a small
molecule agonist of the receptor to reactions that cause cell
death; and [0082] c) devise an epitope based on the receptor
structure such that the body will develop an antibody response and
the immune system attack against the receptor may reduce cancer
growth.
[0083] I contemplate a fourth alternative. An isotopically-labeled
TRP-M8 receptor agonist or antagonist, for example, [.sup.125I] or
[.sup.131I]-N-iodo-aryl-alkyl-alkylcarboxamide, with high affinity
binding for this receptor can be a "letter bomb" for killing cancer
cells bearing this receptor. Here, the binding affinity (that is,
the address to the receptor) is an innate part of the molecular
framework and the radiation from .sup.125I or .sup.131I is the
lethal message. Unlike current brachytherapy technique, compounds
and compositions of the present invention possess selectivity and
specificity to deliver a sophisticated lethal message to a specific
target address. The targeted TRP-M8 receptor may, for example, be
in the prostate or bladder epithelia.
[0084] The high specific radioactivity that may be attained with
.sup.125I or .sup.131I offers tremendous therapeutic advantage if
the radiation can be focused on a localized target. Standard doses
of oral or intravenous [.sup.131I]-sodium iodide for the treatment
of thyroid malignancy can range from 0.75 to 100 milliCi. As noted
earlier, [125I] or [.sup.131I]-alkylcarboxamides may easily be
synthesized at a specific activity of 250 Ci/mmol or higher to give
a compound with a specific activity of greater than 1 Ci/mg.
Injection or oral intake of 0.1 mg of such compounds will yield
therapeutic dose of .gtoreq.100 milliCi. Because this radiation is
selectively localized to hyperplastic or malignant cells, normal
cells are spared and, I believe desirable therapeutic effects may
be achieved.
[0085] To carry out such therapeutic applications, the following
procedures are contemplated. The anti-tumor activity of a given
[.sup.125I] or [.sup.131I]-N-iodo-aryl-alkyl-alkylcarboxamide
agonist/antagonist of the TRP-M8 receptor is first measured by its
cell-inhibiting or anti-proliferative actions (versus the
non-radioactive isotope) on cell lines expressing the TRP-M8
receptor. If activity is found with EC50 ranges of between
nanomolar to low micromolar concentrations, then the radioactive
compound will be tested in mice bearing transplanted tumor cell
lines expressing the TRP-M8 receptor. Tumor volume, rate of growth,
distant metastases, and histological features of the cancer cells
in nude mice will be evaluated using standard techniques that are
well known in the art. Pre-clinical in vivo test results from the
nude mouse model and other animal models of cancer are the final
prelude to clinical evaluation of the drug candidate in humans.
[0086] Before a [.sup.123I] or
[.sup.131I]-N-iodo-aryl-alkyl-alkylcarboxamide agonist/antagonist
of TRP-M8 is administered to human cancer patients, the level of
TRP-M8 expression in the target tissues preferably is determined. A
standard polymerase-chain reaction of the mRNA for the receptor may
be used on biopsied tissues. Alternatively,
[.sup.125I]-N-iodo-aryl-alkyl-alkylcarboxamide radioreceptor
binding to the biopsied tissues may be measured.
[0087] The N-radioiodo-aryl-alkyl-alkylcarboxamide if administered
intravenously is subject to rapid degradation by liver amidases.
One way to circumvent this rapid biostransformation is to
administer an alternative amide substrate concurrently or just
before the radioactive carboxamide. Such substrates may be
lidocaine (Xylocaine.RTM.) which can be infused at a bolus dose of
50-100 mg over 2 to 3 min and this procedure repeated twice for a
total dose of 300 mg in one hr. Another drug in this category is
procainamide (Procan.RTM.). Procainamide can be given up to 1.5 gm
in a 6 hr period. These anti-arrhythmic cardiac drugs are
considered relatively non-toxic at these doses and may be ideal for
co-administration with radiodiagnostic procedures using [.sup.18F]
or [.sup.123I]-labeled TRP-M8 drugs or for radiotherapeutic doses
of [.sup.125I] or [.sup.131I]-labeled TRP-M8 drugs.
[0088] Another consideration in administering radiotherapeutic
drugs to human cancer patients is that of toxicity. To avoid
irradiation of the TRP-M8 receptor in normal tissues, the drug can
be delivered locally into the tumor (e.g. directly into the
bladder) or into the regional circulation of the malignant tissues.
If the radioactive drug is to be administered by oral intake or by
intravenous injection, it may be possible to protect the TRP-M8 in
normal tissues from the radiation by topical or oral administration
of the non-radioactive drug. For example, the non-radioactive
ligand or a surrogate such as menthol may be administered as a
lozenge, in chewing gum, or as a capsule or pill, to protect the
mucous lining of the gastrointestinal tract against the
radionuclide. Eye-drops and nose-drops containing the
non-radioactive ligand may also be administered to protect the
TRP-M8 receptors in these tissues. In addition, radioprotective
drugs such as thiols may be co-administered if the TRP-M8 receptor
is present in tissues such as the liver or kidney.
[0089] Tests for Bioactivitiy and Anti-Neoplastic Actions on
Bladder Cells. The compounds of this invention bind with high
affinity (.mu.g/mL or nanoM) to the TRP-M8 receptor on biological
membranes. Such affinities can be measured using radioreceptor
assays and are familiar to pharmacologists skilled in the art.
Alternatively, certain cancer cell lines, such as human urothelial
UROtsa cells (Master J. W. et al. Tissue culture model of
transitional cell carcinoma: characterization of twenty-two
urothelial cell lines. Cancer Res 1986: 46:3630-3636, and Rossi, MR
et al. The immortalized UROtsa cell line as a potential cell
culture model of human urothelium. Environ Health Perspect. 2001:
109:801-808), and prostate lymph node LNCaP cells, constitutively
express functional TRP-M8 binding sites on their membrane surfaces.
The EC50 of candidate compounds on calcum fluxes, cell growth, and
proliferative activity in such cells may be measured and Kd
estimated. Potency and pharmacokinetics may then be optimized for
lead candidates to take to animal models of bladder cancer.
[0090] The conventional methods for evaluating the effect of a
therapeutic agent for bladder cancer in animal models have been
time and labor consuming. First, the rodents have to be sacrificed,
the bladders need to be inspected under the dissecting microscope,
and a large number of bladder tissue sections must be made
throughout the entire bladder to histologically evaluate the amount
of tumor present. Moreover, only one time point can be studied per
rodent. Finally, to achieve any statistically significant results,
a large number of animals are needed and one could always question
whether or not the tumor burden was similar in treated and
untreated animals.
[0091] Zhou et al. (Visualizing superficial human bladder cancer
cell growth in vivo by green fluorescent protein expression Cancer
Gene Therapy 2002: 9, 681-686) have developed an elegant bioassay.
Human bladder tumor cells (KU-7 cell line) stably expressing high
levels of green fluorescent protein (GFP) are transplanted into
athymic mice by intravesical instillation. After about 1 week a
small incision is made to expose the bladder and its green
fluorescence pixel area, which represents the tumor burden, is
quantified relative to the total area of the bladder. The incision
is closed, but may be re-opened in each mouse and tumor progression
over time can be quantified. It is possible to follow in a given
mouse the effectiveness of therapy. This is one method for assay of
the contemplated invention. The candidate compound can be tested in
animals by oral administration, by parenteral injection
(subeutaenous or intraperitoneal), or by intravesical instillation.
The amount of green fluorescence then predicts the amount of tumor
growth in the bladder.
[0092] Practice of the Invention. Preferred candidate compounds,
when administered to humans, for example, by intravenous injection,
are cleared rapidly from the bloodstream and concentrate in the
urine. Such a compound will then bind selectively to the TRP-M8
receptor on bladder cancer cells, deliver a lethal dose radiation
to the cancer, and then be excreted via the urine. For certain
non-cancerous bladder conditions, such as interstitial cystitis,
this destruction of the urothelium may also be desirable. To
achieve optimal dose and safety, various parameters affecting urine
concentration of the candidate drug may be selected. For example,
fluid intake can be regulated so that a known volume of urine is
present in the bladder. After dosing, the drug may be flushed out
by increased fluid intake or by using a diuretic such as
furosemide. An inhibitor of renal tubule re-uptake of the drug,
such as sulfinpyrazone, may be used to ensure that the radioactive
drug is not re-absorbed once it is excreted into the urine. The
presence of a soluble radiotherapeutic ensures that the bladder
wall is uniformly exposed to the drug (to help destroy
microstatses) and avoids the need for transurethral
catheterization. It is well understood in the art of cancer
chemotherapy that a single agent may not be sufficient to control
the growth and spread of disease. Thus, other agents may be used in
combination with the present invention. Also, the precise dosage
and duration of treatment is a function of the tissue being treated
and can be determined empirically using known testing protocols or
by extrapolation from in vivo or in vitro test data. It is to be
noted that concentrations and dosage values may also vary with the
age of the individual treated. It is to be further understood that
for any particular subject, specific dosage regimens should be
adjusted over time according to the individual need and the
professional judgment of the person administering or supervising
the administration of the formulations, and that the concentration
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed formulations.
EXPERIMENTAL
Example 1
Synthesis of N-haloarylcycloalkyl-alkylcarboxamide Agonists for
TRP-M8 Receptors (CPS-195) and Synthesis of Exemplary Radioactive
N-halo-aryl-alkyl-alkylcarboxamides
[0093] The acid chloride derivative of menthol required for
preparation of the amides is available from optically pure menthol.
Recent reports have suggested that bi- and tricyclic amide
derivatives of commercially available .alpha.-aminoalcohols may
have optimal hydrophobicity and activity, and these amines will be
coupled to the acid chloride. Halogen exchange assisted by
tetrakis(triphenylphosphine)palladium (O) will afford the iodinated
aryl amide. The iodinated compound will be converted to the key
trimethylstannane intermediate, again on treatment with palladium
(O) catalyst. The purified stannylated amide will be used to
prepare both .sup.125I-labelled and .sup.18F-labelled reagents for
radioreceptor assays and PET imaging, respectively. Generation of
electrophilic iodine by treatment of radiolabelled sodium iodide
with chloramines-T will allow preparation of the .sup.125I-labelled
material required for binding assays. Radioactive fluorine gas will
be used to oxidize the carbon-tin bond to give the fluoroaromatic
compound to be used in the PET imaging experiments. Once we have
information about first-generation ligands from radioreceptor
assays (vide infra), their structures will be
systematically-modified to enhance binding, by changing
substituents at the 4'-position of the aryl ring.
[0094] Synthesis of 2-Isopropyl-5-methyl-cyclohexanecaboxylic acid
[2-hydroxy-2-(3-hydroxy-phenyl)-ethyl]-methyl-amide. Phenyephrine
HCl [(R)-(-)-3-(1-Hydroxy-2-methylamino-ethyl)-phenol.
hydrochloride] was purchased from Aldrich Chemicals, Co.,
Milwaukee, Wis. 1.0 g was dissolved in 28 ml diethyether and 1 ml
double-distilled water and cooled to 0.degree. C. A pinch of the
catalyst diaminopyrimidine was added. 1.90 ml of p-menthoyl
chloride was then added dropwise, followed by 2 ml of
triethylamine. White precipitates appeared in the mixture, which
was stirred overnight at room temperature. The precipitate was
dissolved with ethylacetate, washed with double-distilled water and
dried over sodium sulfate. The organic phase was then evaporated
under reduced pressure to yield the final product (1.8 g), which
crystallized at room temperature. The expected molecular mass was
then confirmed by mass spectroscopy and the absorption spectrum by
nuclear magnetic resonance. This compound was given the code of
CPS-140.
[0095] [.sup.18F], [.sup.123I], [.sup.125I], and
[.sup.131I]-N-halo-aryl-alkyl-alkylcarboxamide radioligands of the
invention were synthesized, for example, at 25 Ci/mmole. The
non-radioactive forms of these chemicals are known to be potent and
active on the TRP-M8 receptor. For example, the fluorinated analog
is active at nanomolar (10.sup.-9) in promoting calcium entry into
TRP-M8 transfected cells and into LNCaP (lymph node prostate cancer
cells) constitutively expressing these receptors.
[0096] A particularly preferred TRP-M8 receptor ligand embodiment
of the present invention, sometimes designated CP-129, is
illustrated by Structure 1 where the radioisotope is .sup.125I. The
precursor of this compound is
N-(2'-iodo-4'methoxyphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide.
One can readily replace the non-radioactive iodine atom with one of
the isotopes .sup.18F, .sup.123I, .sup.125I, or .sup.131I, the
choice of which depends on the intended use.
[0097] CP-129 or its .sup.125I, .sup.131I analogs are prepared from
the nonradiolabeled trimethyl tin precursor by oxidation with
labeled sodium iodide and chloramine-T. The precursor is made from
the parent by replacing the iodo group with a trimethyl tin group
in the presence of tetrakis (triphenyl phosphine) palladium and
bis(trimethyl)tin. The initial nonradiolabeled compound is prepared
by reacting 2-isopropyl-5-methylcyclohexane carbonyl chloride with
2-iodo4-methoxylphenylamine (2-iodo-p-anisidine). It should be
noted the corresponding .sup.18F compound may be made by the same
technique.
[0098] A mixture of
N-(2'-iodo-4'methoxyphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide
(500 mg, 1.25 mmol), tetrakis(triphenylphosphine)-palladium (150
mg, 0.13 mmol, 10% molar equivalent), bis(trimethylstannyl) (510
mg, 1.5 mmol), triethylamine (50 ml), and THF (50 ml) was heated at
reflux for 12 hr. The reaction mixture was evaporated to dryness in
vacuum. The residue was dissolved in ethylacetate and crystallized
by the addition of methanol.
[0099] Radiochemical synthesis to produce CP-129 used the following
method. A THF solution of
N-(2'-trimethylstannyl-4'-methoxyphenyl)-2-isopropyl-5-methylcyclohexanec-
arboxamide (1 mg/ml) was prepared. To 5 ml of this solution was
added Na.sup.125I (0.5 to 1.0 mCi, 3 to 5 ml) in 0.1 N NaOH,
followed by the addition of 0.05 N HCl (10 ml) to adjust to pH 4.0
to 5.5. A freshly prepared solution (1 ml) of chloramine-T (1
mg/ml) was added to the above mixture, and the solution was
incubated at room temperature for 15 min. After this time, 20 ml of
sodium metabisulfite (3 mg/ml) were added to terminate the
reaction, and the solution was incubated for an additional 5 min.
Finally, a saturated solution of sodium bicarbonate (50 ml) was
added to the reaction vial, and the radioactivity was extracted
with chloroform (5 ml). The final product is obtained by HPLC
chromatography and used without carrier. For intravenous injection,
the trimethyl tin compound is supplied as a sterile ethanolic
solution for reaction with radiolabeled NaI and chloramine-T in
sterile saline. Unreacted materials are removed simply using a
C.sub.18 Sep Pak cartridge, yielding CP-129 of more than 98 percent
radiochemical purity. This is illustrated by Schematic 3.
##STR9##
[0100] The incorporation efficiency of radioactivity is nearly
quantitative. It should be noted that, in the above synthesis,
alkyl or cycloalkyl substituents can be singly added onto the
-aryl-alkyl ring using methods well known to the art. The use of
trimethyl tin reagents for radio-labeling is only one example of
such technology for single addition of halogens, and alternative
organometallic reagents are available.
Example 2
Radioreceptor Assay
[0101] The synthesized radioligand of the invention, such as the
[.sup.125I]-CP-129 prepared as in Example 1, at a specific activity
of 25 Ci/mmole, is now used for a radioreceptor assay. In a
standard test-tube method for competitive receptor binding, a
tissue known to contain the TRP-M8 receptor, such as dorsal root
ganglia neuronal cultures or a human prostate cancer cell line, is
incubated with [.sup.125I]-CP-129 until steady-state conditions are
reached (usually 30 to 60 minutes). The bound radioactive ligand is
then separated from the free radioactive ligand by methods well
known in the art such as filtration, centrifugation, dialysis, or
size exclusion chromatography. To differentiate between specific
(receptor) binding from non-specific binding, a non-radioactive
N-halo-aryl-alkyl-alkylcarboxamide, such as N-(3'-fluoro,
4'-methoxyphenyl)-2-isopropyl-5-methyl-cyclohexanecarboxamide, may
be used. After these parameters are established, the next procedure
is to conduct a saturation experiment that will establish the Kd
and the Bmax (which is the density of receptors in a given tissue
and is a pharmacological technique well known in the art). Various
concentrations of radioactive ligand are incubated with the
receptor preparation and the ratio of the bound and free levels of
radioactive ligand is measured. The standard Rosenthal plot or
Scatchard analysis of the binding data yields the constants Kd and
Bmax.
[0102] These and other uses of the present invention will become
readily apparent to the skilled artisan once he or she has read the
disclosure in this application. It is to be understood that while
the invention has been described above in conjunction with
preferred specific embodiments, the description and examples are
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims.
* * * * *