Radioligands for the TRP-M8 receptor and methods therewith

Abstract

One embodiment of the invention is a composition that comprises a radioactive [18F], [76Br]-, [77Br]-, [211At]-, [123I], [125I], or [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 [18F], [76Br]-, [77Br]-, [211At]-, [123I], [125I], or [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.

Description

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

1. Field of the Invention

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, CMR₁); 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 ¹⁸F, ¹²³I, ¹²⁵I, or ¹³¹I, within the molecular structure, said compositions being useful, for example, in radioreceptor, diagnostic imaging, and radiotherapeutic applications.

2. Description of Related Art

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.

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.

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 CMR₁ (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 CMR₁) 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).

Various radioactive fluorine and iodine compounds are used in clinical oncology. For example, ¹⁸F and 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. ¹²⁵I and 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.

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.

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

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═O)—N(H or CH₃)—R′—Y  Formula 1

• • 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 C₁ to C₅ normal or branched alkyl substituents,
    • (b) R′ is an optional carbon bridge having C₁-C₃ carbons which may include a hydroxy group, and
    • (c) Y is an aromatic radical containing at least one substituent selected from R₁ and R₂, and at least one substituent X, wherein
      • R₁, is selected from the group hydrogen, hydroxyl, C₁-C₅ alkyl, C₁-C₃ alkoxy, C₁-C₃ carboxyalkyl, C₁-C₄ carbonylalkylester, C₁-C₃ oxycarbonylalkyl, C₁-C₃ hydroxyalkyl,
      • R₂ is selected from the group —SO₂NH-pyrimidine, —SO₃—(H, Me or Et), or —CH₂—SO₃—(H, Me or Et), acetyl, C₁-C₃ hydroxyalkyl, trifluoromethyl, nitro, cyano, halo, and
      • X is selected from the group [¹⁸F]—, [¹²³I]—, [¹²⁵I]—, [¹³¹I]—[⁷⁶Br]—[⁷⁷Br]— and [²¹¹At]—.

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 ¹⁸F, ¹²³I 125I, or ¹³¹I. The inventive [¹²⁵I]-compounds are useful for laboratory tests, called radioreceptor assays. The inventive [¹⁸F], [¹²³I], [¹³¹I]-compounds are useful for imaging of the tumor cells in vivo bearing this receptor marker, and the [¹²⁵I] or [¹³¹I]-compounds are further believed potentially useful for targeted radiotherapy.

Among particularly preferred compositions of this first embodiment are those including [^{123, 125}, or ¹³¹I]—N-(4′-iodo-2′-methylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide illustrated below as Structure 1.

Also among particularly preferred compositions of the first embodiment are those that include [¹⁸F]—N-(4′-fluoro-2′-hydroxyphenyl)-2-isopropyl-2,3-dimethylbutyramide illustrated as Structure 2.

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.

In this Structure 3 compound, R′ is hydroxyethyl, R₁ is acetyl or —SO₂NH-pyrimidine, and R₂ is hydroxyl or hydroxymethyl, and X is selected from the group [¹⁸F]—, [¹²³I]—, [¹²⁵I]—, [¹³¹I]— [⁷⁶Br]— [⁷⁷Br]— and [²¹¹At]—

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.

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

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.

The selected isotopes, preferably from the group ¹⁸F, ¹²³I, ¹²⁵I, and ¹³¹I, serve to either mark the location or quantity of the TRP-M8 receptor or to deliver radiation to the TRP-M8 bearing cell.

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.

Compositions including the inventive radioligands of the invention have the following applications:

• • use as ligands for TRP-M8 radioreceptor assays in the laboratory;
  • use as ligands for diagnosis and imaging of TRP-M8 receptors in prostate tissues and cells; and
  • use as radiotherapeutics (alone or co-administered with local anesthetic amidase inhibitors as potentiators) for prostate disorders such as cancer or benign hyperplasia.

Formula 1 illustrates carboxamide ligands of this discovery.
R—(C═O)—N(H or CH₃)—R′—Y  Formula 1

• • 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 C₁ to C₅ normal or branched alkyl substituents,
    • (b) R′ is an optional carbon bridge having C₁-C₃ carbons and which may include a hydroxy group, and
    • (c) Y is an aromatic radical containing at least one substituent selected from R₁ and R₂, and at least one substituent X, wherein
      • R₁ is selected from the group hydrogen, hydroxyl, C₁-C₅ alkyl, C₁-C₃ alkoxy, C₁-C₃ carboxyalkyl, C₁-C₄ carbonylalkylester, C₁-C₃ oxycarbonylalkyl, C₁-C₃ hydroxyalkyl,
      • R₂ is selected from the group —SO₂NH-pyrimidine, —SO₃—(H, Me or Et), or —CH₂—SO₃—(H, Me or Et), acetyl, C₁-C₃ hydroxyalkyl, trifluoromethyl, nitro, cyano, halo, and
      • X is selected from the group [¹⁸F]—, [¹²³I]—, [¹²⁵I]—, [¹³¹I] [⁷⁶Br]— [⁷⁷Br]— and [²¹¹At]—.

Where R′ has no carbons (that is, R′ is not present), then preferred embodiment compositions are those comprising a radioactive compound having the structure [¹⁸F], [¹²³I], [¹²⁵I], or [¹³¹I]-N-radiohaloaryl-alkylcarboxamide, for example: [^{123, 125 or 131}I]—N-(4′-iodo-2′-methylphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide, and [¹⁸F]—N-(4′-fluoro-2′-hydroxyphenyl)-2-isopropyl-2,3-dimethylbutyramide, illustrated below as Structures 1 and 2 respectively.

Where R′ is present, then among particularly preferred compositions of the second embodiment are those that include the compound illustrated by Structure 3.

In Structure 3, R′ is hydroxyethyl, R₁ is acetyl or —SO₂ NH-pyrimidine, and R₂ is hydroxyl or hydroxymethyl, and X is selected from the group [¹⁸F]—, [¹²³I]—, [¹²⁵I]—, [¹³¹I]—, [⁷⁶Br]—, [⁷⁷Br]—, and [²¹¹At]—.

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.

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 1×10⁻¹² to 1×10⁻⁵ molar.

An aspect of the present invention is that the radionuclide (preferably ¹⁸F, ¹²³I, ¹²⁵I or 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.

By contrast, a laboratory procedure, for example, the labeling of a binding protein such as a monoclonal antibody by 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).

Incorporating ¹⁸F, ¹²³I, ¹²⁵I or 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

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.

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 1
                                 Cold Sensation
                                 Threshold on
CHEMICAL                         Tongue*, μg
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 × 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 μg. Ethoxycarbonyl is COOCH2H5.

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® 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 Bödding 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° 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 μl 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 μM (EC50) activities. The EC50 of various test substances are shown in Table 2. Further analysis of the dose-response relationship showed the ΔFmax (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.

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 (Bödding et al. 2002). Patch pipettes pulled from borosilicate glass (Kimax®) had resistances between 2 and 3 MΩ when filled with the standard internal solution (in mM): 145 Cs-glutamate, 10 HEPES, 8 NaCl, 1 MgCl₂, 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 CaCl₂, 2.8 KCl, 2 MgCl₂, 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 MΩ. Currents were filtered using an 8-pole Bessel filter at 2.9 kHz and digitised at 100 μs. 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° C.). Internal solutions were kept on ice to minimize hydrolysis of ATP.

TABLE 2
		EC50 μM	EC50 μM
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-OCF3—	NR	NA
CPS-138	3-CF3, 4-NO2—	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 [¹⁸F], [¹²³I], [¹²⁵I], or [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.

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.

The structural features of TRP-M8 binding for N-fluoro- or iodo-aryl-alkyl-alkylcarboxamides side principally in the hydrogen bonding —C(═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 μg, 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.

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, ¹⁸F, ¹²³I, ¹²⁵I, and 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 ⁷⁶Br and ⁷⁷Br, and low-energy electron emitters such as ^58mCo, ^103mRh, ¹¹⁹Sb, ¹⁶¹Ho, and ^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 ²¹²Bi, ²¹³Bi and 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.

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.

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 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 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.

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 10⁻⁹ 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.

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.

In Formula 1, the left-hand moiety R—(C═O)—N (H or CH_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 10⁻⁷ to 10⁻⁶ 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 10⁻⁸ to 10⁻¹⁰ 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 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

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.

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-carboxamide, N-(4′-fluorophenyl)-2-isopropyl-5-methylcyclohexane-3-carboxamide, and N-(4′-iodo-1′-naphthyl)-2-isopropyl-5-methylcyclohexane-3-carboxamide respectively.

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 ¹⁸ F. In this analog, R₁ is hydrogen, R₂ is para-methoxy, and X is radioactive fluorine. This analog is active at nanomolar (10⁻⁹) in promoting calcium entry into TRP-M8 transfected cells and into LNCaP (lymph node prostate cancer cells) constitutively expressing these receptors.
Radioactive Ligands for the TRP-M8 Receptor.

The naturally occurring isotope of iodine has an atomic mass of 126.9 Daltons. The radioactive isotopes of iodine are ¹²³I, ¹²⁵I and 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 [¹²³I]-compounds require special facilities because of the short half-life of this isotope. By contrast, ¹²⁵I or 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.

The natural isotope of fluorine has an atomic mass of 19.0. The 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.

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 [¹²⁵I]- or [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:

• • [¹²⁵I]-labeled alkylcarboxamides compounds are useful for radioreceptor assays in the laboratory;
  • [¹⁸F]-, [¹²³I]- or [¹³¹I]-labeled N-halo-aryl-alkyl-alkylcarboxamides are useful for scanning or imaging tissues bearing the TRP-M8 receptor; and
  • [¹²⁵I]- or [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 [¹²⁵I]-labeled N-halo-aryl-alkyl-alkylcarboxamides for Receptor Assays

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.

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 (10⁻⁶), nanomolar (10⁻⁹) to picomolar (10⁻¹²) 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.

For example, a synthetic [¹²⁵I]-N-iodo-aryl-alkyl-alkylcarboxamide ligand, such as [¹²⁵I]-N-(4′-iodophenylethyl)-p-menthane-3-carboxamide (synonym: [¹²⁵I]-N-(4′-iodophenylethyl)-2-isopropyl-5-methylcyclohexanecarboxamide) 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 [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 [¹²⁵I]-N-iodo-aryl-alkyl-alkylcarboxamide ligand has several applications, as described infra.

Utility of Radio-Labeled -Alkylcarboxamides on TRP-M8 Receptor

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 [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.

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 [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 [¹²⁵I]-agonist from its binding site. A high affinity antagonist would be a chemical that displaces the [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.

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.

A [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.

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 [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.

In the laboratory, ¹²⁵I is widely used in a technique called autoradiography. Traditionally, ¹²⁵I, is used to label peptides or proteins in a non-specific location, such as the ring structure of tyrosine and on the ε-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 [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.

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 [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 [¹⁸F], [¹²³I], [¹²⁴I] or [¹³¹I]-N-iodo-aryl-alkyl-alkylcarboxamides with High Affinity for TRP-M8.

Various radioactive fluorine and iodine compounds are used in clinical oncology. For example, ¹⁸F and ¹²⁴I are used in positron emission tomography (PET), and ¹²³I and 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 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 10⁻⁹ to 10⁻¹² M in tissues. A useful isotope for SPECT imaging is 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.

The use of [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 [¹⁸F]-TRP-M8 ligand, using PET imaging, will have utility as a non-invasive method in staging this disease. ¹²⁴I, having a longer half-life of 4.11 days than 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 [¹²³I] and [¹³¹I]-TRP-M8 ligand can be used for SPECT applications in prostate diseases.

Radiotherapeutic Use of [¹²⁵I] or [¹³¹I]-N-iodo-aryl-alkyl-alkylcarboxamides with High Affinity for TRP-M8.

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:

• 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;
• b) make a small molecule agonist of the receptor to reactions that cause cell death; and
• 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.

I contemplate a fourth alternative. An isotopically-labeled TRP-M8 receptor agonist or antagonist, for example, [¹²⁵I] or [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 ¹²⁵I or 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.

The high specific radioactivity that may be attained with ¹²⁵I or ¹³¹I offers tremendous therapeutic advantage if the radiation can be focused on a localized target. Standard doses of oral or intravenous [¹³¹I]-sodium iodide for the treatment of thyroid malignancy can range from 0.75 to 100 milliCi. As noted earlier, [125I] or [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 ≧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.

To carry out such therapeutic applications, the following procedures are contemplated. The anti-tumor activity of a given [¹²⁵I] or [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.

Before a [¹²³I] or [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, [¹²⁵I]-N-iodo-aryl-alkyl-alkylcarboxamide radioreceptor binding to the biopsied tissues may be measured.

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®) 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®). 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 [¹⁸F] or [¹²³I]-labeled TRP-M8 drugs or for radiotherapeutic doses of [¹²⁵I] or [¹³¹I]-labeled TRP-M8 drugs.

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.

Tests for Bioactivitiy and Anti-Neoplastic Actions on Bladder Cells. The compounds of this invention bind with high affinity (μg/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.

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.

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.

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

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 α-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 ¹²⁵I-labelled and 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 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.

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° 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.

[¹⁸F], [¹²³I], [¹²⁵I], and [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 (10⁻⁹) in promoting calcium entry into TRP-M8 transfected cells and into LNCaP (lymph node prostate cancer cells) constitutively expressing these receptors.

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 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 ¹⁸F, ¹²³I, ¹²⁵I, or ¹³¹I, the choice of which depends on the intended use.

CP-129 or its ¹²⁵I, 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 ¹⁸F compound may be made by the same technique.

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.

Radiochemical synthesis to produce CP-129 used the following method. A THF solution of N-(2′-trimethylstannyl-4′-methoxyphenyl)-2-isopropyl-5-methylcyclohexanecarboxamide (1 mg/ml) was prepared. To 5 ml of this solution was added Na¹²⁵I (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 C₁₈ Sep Pak cartridge, yielding CP-129 of more than 98 percent radiochemical purity. This is illustrated by Schematic 3.

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

The synthesized radioligand of the invention, such as the [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 [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.

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.

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 C1 to C5 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 1×10−5 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═O)—N(H or CH3)—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 C1-C5 normal or branched alkyl substituents,

(b) R′ is a normal or branched C1-C3 carbon bridge with an optional hydroxy, and

(c) Y is an aromatic radical containing one to four substituents of R1 or R2, and one to three substituents of X, wherein R1 is selected from the group hydrogen, hydroxyl, C1-C5 alkyl, C1-C3 alkoxy, C1-C3 carboxyalkyl, C1-C4 carbonylalkylester, C1-C3 oxycarbonylalkyl, C1-C3 hydroxyalkyl, R2 is selected from the group —SO2NH-pyrimidine, —SO3—(H, Me or Et), or —CH2—SO3—(H, Me or Et), acetyl, C1-C3 hydroxyalkyl, trifluoromethyl, nitro, cyano, halo, and X is selected from the group [18F]—, [123I]—, [125I]—, [131I]— [76Br]— [77Br]— and [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═O)—N(H or CH3)—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 C1-C5 normal or branched alkyl substituents,

(b) R′ is a normal or branched C1-C3 carbon bridge with an optional hydroxy, and

(c) Y is an aromatic radical containing one to four substituents of R1 or R2, and one to three substituents of a radio label X, wherein R1 is selected from the group hydrogen, hydroxyl, C1-C5 alkyl, C1-C3 alkoxy, C1-C3 carboxyalkyl, C1-C4 carbonylalkylester, C1-C3 oxycarbonylalkyl, C1-C3 hydroxyalkyl, R2 is selected from the group —SO2NH-pyrimidine, —SO3—(H, Me or Et), or —CH2—SO3—(H, Me or Et), acetyl, C1-C3 hydroxyalkyl, trifluoromethyl, nitro, cyano, halo, and X is selected from the group [18F]—, [123I]—, [125I]—, [131I]— [76Br]— [77Br]— and [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.