Type III deiodinase inhibitors and uses thereof

Abstract

The present invention relates to compounds that inhibit the activity of Type III deiodinase (DIO3). The present invention further relates to methods for treating or preventing depression, depression associated with other psychiatric or general medical diseases or conditions, condition amenable to treatment with known anti-depressants and cancer, particularly by using the compounds of the invention.

Claims

1. A method for treating ovarian cancer, comprising the step of administering to a subject in need thereof a therapeutically effective amount of at least one compound selected from the group consisting of: ##STR00030## and salts thereof, or a pharmaceutical composition comprising said compound and a pharmaceutically acceptable carrier or excipient.

2. The method according to claim 1, wherein the compound is represented by the structure of formula 4: ##STR00031## or a salt thereof.

3. The method according to claim 1, wherein the compound is represented by the structure of formula 5: ##STR00032## or a salt thereof.

4. The method according to claim 1, wherein the compound is represented by the structure of formula 6: ##STR00033## or a salt thereof.

5. The method according to claim 1, wherein the compound is represented by the structure of formula 7: ##STR00034## or a salt thereof.

6. The method according to claim 1, wherein the compound is represented by the structure of formula 8: ##STR00035## or a salt thereof.

7. The method according to claim 1, wherein the ovarian cancer is associated with aberrant Type III deiodinase (DIO3) expression or activity.

8. The method according to claim 7, wherein the compound inhibits the activity of Type III deiodinase (DIO3).

9. The method according to claim 8, wherein inhibition of the DIO3 activity results in increased amount of T3 in the subject.

10. The method according to claim 1, wherein the ovarian cancer is a high grade ovarian cancer.

11. A method of attenuating Type III deiodinase (DIO3) expression and/or activity, comprising the step of contacting the enzyme with an effective amount of at least one compound selected from the group consisting of: ##STR00036## and salts thereof, or a pharmaceutical composition comprising said compound and a pharmaceutically acceptable carrier or excipient.

12. The method according to claim 11, wherein attenuating DIO3 expression and/or activity results in treatment of ovarian cancer in a subject.

13. The method according to claim 11, wherein the compound is represented by the structure of formula 4: ##STR00037## or a salt thereof.

14. The method according to claim 11, wherein the compound is represented by the structure of formula 5: ##STR00038## or a salt thereof.

15. The method according to claim 11, wherein the compound is represented by the structure of formula 6: ##STR00039## or a salt thereof.

16. The method according to claim 11, wherein the compound is represented by the structure of formula 7: ##STR00040## or a salt thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 demonstrate activation and inactivation of thyroid hormones by the three iodothyronine deiodinases DIO1 (ID-1), DIO2 (ID-2) and DIO3 (ID-3).

(2) FIG. 2 shows inner ring monodeiodination of T4 to rT3 or of T3 to 3,3′-T2 by DIO3 mimic (compound A).

(3) FIG. 3 demonstrates deiodination of thyroxine by compound A, a DIO3 mimic.

(4) FIG. 4 shows Inhibition of DIO3 mimic by Compound C (Dibromo-N-methylmaleimide).

(5) FIG. 5 shows Inhibition of DIO3 mimic by Compound 3 (2,3-dibromo-N-3-(4-hydroxyphenyl)propanoic acid) maleimide; TYR-DBRMD).

(6) FIG. 6 shows .sup.1H NMR spectra of compound 3 in DMSO-d.sub.6.

(7) FIG. 7 shows .sup.13C NMR spectra of compound 3 in DMSO-d.sub.6.

(8) FIG. 8 shows .sup.1H NMR spectra of compound 4 in DMSO-d.sub.6.

(9) FIG. 9 shows .sup.13C NMR spectra of compound 4 in DMSO-d.sub.6.

(10) FIG. 10 is a bar graph showing brain T3 levels (pg/ml) following intracerebroventricular (ICV) administration of IOP-DIBRMD (2,3-dibromo-N-2,4,6-triiodobenzyl)butanoic acid)maleimide; IOP, compound 6) by osmotic minipumps for 3 weeks (*Significant; One sided, non-paired t test).

(11) FIG. 11 is a bar graph showing the effect of IOP-DIBRMD (IOP, compound 6) on latency to feed in the novelty suppressed feeding test. Marginal means derived from the ANCOVA are shown. The effect of T3 in the model is significant (F=6.21; p=0.026).

(12) FIG. 12 is a bar graph showing the effect of ITYR-DIBRMD (2,3-dibromo-N-3-(4-hydroxy-3,5-diiodophenyl)propanoic acid) maleimide; ITYR, compound 5) on latency to feed in the novelty suppressed feeding test. Marginal means derived from the ANCOVA are shown. Animals treated with IOP show a reduced latency to feed. Model shows significance (F=7.23; p=0.006). Effect of T3 in the model is significant (F=7.62; p=0.01)

(13) FIG. 13A shows DIO3 expression in ovarian cancer cells (A2780, SKOV3 and OVCAR3 cell lines) as measured by FACS analysis.

(14) FIG. 13B demonstrated the effect of compounds C1-C6 on the density of the ovarian cancer cell line OVCAR3. Cells were treated with 0.5 μM DIO3 inhibitors for 48 h and imaged by light microscopy.

(15) FIG. 13C demonstrated the effect of compounds C1-C6 on the viability of the ovarian cancer cell line OVCAR3. Cells were treated with 0.5 μM DIO3 inhibitors for 48 h and analyzed by WST-1 viability assay.

DETAILED DESCRIPTION OF THE INVENTION

(16) The present invention provides novel compounds that specifically inhibit the activity of Type III deiodinase (DIO3). DIO3 is particularly active in the brain, and DIO3 inhibition may thus result in an increased amount of the hormone 3,3′,5,-triiodothyronine (T3) in brain. DIO3 has also been indicated to be associated with hyperproliferative states and with human solid tumors. Specifically, previous studies reported elevated expression and activity of DIO3 in a number of human cancer diseases (Dentice et al. 2013, ibid).

(17) The present invention discloses for the first time that inhibition of DIO3 activity is useful for treating and/or preventing depression. Without wishing to be bound by certain specific theory or mechanism of action, inhibition of DIO3 by the compounds of the invention leads to elevated content of T3 in the brain. The novel compounds of the present invention are also useful for treating and/or preventing cancer and other diseases or disorders associated with elevated expression or activity of DIO3.

(18) Definitions

(19) The term “C.sub.1-C.sub.6 alkyl” as used herein alone or as part of another group refers to any saturated aliphatic hydrocarbon, including straight-chain, and branched-chain which contains 1 to 6 carbon atoms. Examples of C1-C6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, 1-penyl, 2-pentyl, 3-pentyl, 1-hexyl, 2-hexyl, and 3-hexyl. According to the principles of the present invention, the C1-C6 alkyl group is substituted at any location with a COOH moiety.

(20) The term “tyrosine side chain” refers to the following moiety:

(21) ##STR00012##

(22) The term “iodotyrosine side chain” refers to a tyrosine side chain in which one or more of the phenyl hydrogens have been substituted by iodine. One non-limiting example of an iodotyrosine side chain is a 3-5-diiosotyrosine group represented by the moiety:

(23) ##STR00013##

(24) The term “hydroxytyrosine side chain” refers to a tyrosine side chain in which one or more of the phenyl hydrogens have been substituted by a hydroxy. One non-limiting example of a hydroxytyrosine side chain is 3-hydroxytyrosine group (alternatively designated 3,4-dihydroxyphenylalanine), which is represented by the moiety:

(25) ##STR00014##
also known as “DOPA”.

(26) The term “aspartic acid side chain” refers to the group —CH.sub.2—COOH.

(27) The term “ethyl diiodophenol” refers to an ethylphenol group in which two of the phenol hydrogens have been substituted by iodine. One non-limiting example of an ethyl diiodophenol is represented by the moiety:

(28) ##STR00015##

(29) Several of the compounds of the present invention contain side chains of naturally occurring alpha-amino acids. The naturally occurring amino acids are, e.g., glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine and lysine. Each possibility represents a separate embodiment of the present invention.

(30) The term “depression” as used herein includes, but is not limited to, major depressive episodes in the context of major depressive disorder or bipolar disorder, schizoaffective disorder and other psychiatric states that are characterized by depressed mood and/or feelings of sadness, despair, discouragement, “blues”, melancholy, feelings of low self esteem, guilt and self reproach, withdrawal from interpersonal contact, and somatic symptoms such as eating and sleep disturbances.

(31) It is to be explicitly understood that the present invention encompasses treating depression that is associated with other psychiatric disorders, with general medical conditions or with alcohol or drug abuse.

(32) Examples of other psychiatric disorders that may be associated with depression include, but are not limited to, dysthymia, posttraumatic stress disorder, schizophrenia, schizoaffective disorder, post-partum depression, eating disorders including anorexia nervosa and bulimia, anxiety disorders, Parkinson's disease, Alzheimer's disease, fibromyalgia and chronic fatigue syndrome.

(33) Anxiety disorders include, but are not limited to, obsessive compulsive disorder, generalized anxiety disorder, panic disorder and social phobia.

(34) Examples of general medical conditions associated with depression include, but are not limited to, heart diseases, cancer, diabetes, HIV/AIDS and neurological conditions such as stroke and multiple sclerosis.

(35) The present invention also encompasses treating disease and conditions known to be amenable to treatment with anti-depressants other than the compounds of the present invention. According to certain embodiments, such diseases or conditions include, but are not limited to, obsessive compulsive disorder, panic disorder, generalized anxiety disorder, pre-menstrual syndrome (PMS), posttraumatic stress disorder, social phobia, agoraphobia, fibromyalgia, chronic fatigue syndrome chronic pain, bulimia, anorexia nervosa, obesity, alcohol abuse, smoking cessation and nicotine withdrawal syndrome symptoms.

(36) The term “cancer” refers to a medical condition characterized by abnormal cell growth in a tissue. The term encompasses a wide range of cancers, classified according to cell type origin, including, without limitation, carcinomas, sarcomas, myelomas, leukemias and lymphomas. Particular types of cancers, classified according to the affected tissue, include, without limitation, brain cancer, lymphoproliferative disorders, breast cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, bone cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, cancer of the thyroid, head and neck cancer, cancer of the central nervous system, cancer of the peripheral nervous system, skin cancer, and kidney cancer. Further cancer types, include, without limitation, hepatocellular carcinoma, hematoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, invasive ductal carcinoma, papillary adenocarcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (well differentiated, moderately differentiated, poorly differentiated or undifferentiated), renal cell carcinoma, hypernephroma, hypernephroid adenocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinoma including small cell, non-small and large cell lung carcinoma, bladder carcinoma, glioma, astrocyoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, retinoblastoma, neuroblastoma, colon carcinoma, rectal carcinoma, hematopoietic malignancies including all types of leukemia and lymphoma including: acute myelogenous leukemia, acute myelocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma.

(37) According to some embodiments, the cancer is associated with aberrant DIO3 expression. A cancer associated with aberrant expression of DIO3 is one in which DIO3 expression or activity results in a level of active DIO3 that is greater than that observed in corresponding (similar cell types) non-cancerous cells. In other embodiments, it is a cancer associated with low levels of active T3. A cancer associated with low levels of T3 is one in which functional or active T3 is lower than in a corresponding non cancerous cell.

(38) According to certain embodiments, the compounds of the invention are for treating solid tumors.

(39) As used herein, the terms “type III deiodinase”, “type III iodothyronine deiodinase”, type 3 deiodinase”, “type 3 iodothyronine deiodinase”, DIO3 and D3 are used herein interchangeably and refer to a protein belonging to the iodothyronine deiodinase family that catalyzes the inactivation of thyroid hormone by inner ring deiodination of the prohormone thyroxine (T4) and the bioactive hormone 3,3′,5-triiodothyronine (T3) to inactive metabolites, 3,3′,5′-triiodothyronine (rT3) and 3,3′-diiodothyronine (3,3′-T2), respectively.

(40) As used herein, the term “inhibition” with regard to inhibiting the activity of DIO3 refers to reducing the activity of DIO3 compared to its activity under the same conditions without the addition of an inhibitory compound. According to certain embodiments, the DIO3 activity is reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% compared to its activity under the same conditions without the addition of an inhibitory compound. Each possibility represents a separate embodiment of the present invention.

(41) The term “specific inhibition” with regard to the inhibition of DIO3 by the compounds of the present invention refers to compounds which inhibit DIO3 to a greater extent (e.g., a lower K.sub.i) than it inhibits DIO2 or DIO1 enzymes. A specific DIO3 inhibitor also includes agents that inhibit DIO3, but fail to inhibit DIO2 or DIO1 at comparable concentrations.

(42) The term “therapeutically effective amount” refers to the amount of a compound of the present invention that, when administered to a subject in need thereof, results in inhibition of DIO3 activity. According to certain embodiments, inhibition of DIO3 activity results in increased T3 amounts in the brain compared to the amounts observed in other organs.

(43) The term “preventing” as used herein means causing the clinical symptoms of the disease state not to develop in a subject that may be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state.

(44) The term “treating: as used herein refers to inhibiting the disease state, i.e., arresting the development of the disease state or its clinical symptoms, or relieving the disease state, i.e., causing temporary or permanent regression of the disease state or its clinical symptoms. The term is interchangeable with any one or more of the following: abrogating, ameliorating, inhibiting, attenuating, blocking, suppressing, reducing, halting, alleviating or preventing symptoms associated with the disease.

(45) According to one aspect, the present invention provides a compound having the general formula I:

(46) ##STR00016## wherein A is

(47) ##STR00017## R′ is independently at each occurrence a halogen, OH, NO.sub.2 or N(R).sub.2 wherein each R is independently H or a C.sub.1-C.sub.6 alkyl; R.sup.2 is COOH, OH, NO.sub.2, N(R).sub.2 or a linear or branched C.sub.1-C.sub.6 alkyl substituted with at least one group selected from the group consisting of COOH, halogen, OH, NO.sub.2, and N(R)2; R.sup.3 is H or COOH; R.sup.4 is iodophenyl, heterocyclyl, heteroaryl, or the side chain of an amino acid selected from the group consisting of tyrosine, 3,4-dihydroxyphenylalanine (DOPA), iodotyrosine, aspartic acid, valine, leucine, isoleucine, methionine, tryptophan, threonine, asparagine, glutamine, cysteine, proline, arginine, histidine, lysine, glutamic acid; or R.sup.4 is represented by the structure:

(48) ##STR00018##

(49) wherein R.sup.5 is independently at each occurrence OH, halogen, NO.sub.2 or N(R).sub.2 and m is 0, 1, 2, 3 or 4; L.sup.1 and L.sup.2 are each a leaving group selected from a halogen and a sulfonate; n is 1, 2, 3 or 4; and salts thereof.

(50) In one embodiment, the compound of formula (I) is a phenylcarboxylic acid derivative (e.g., p-benzoic acid derivative or iopanoic acid derivative), represented by the structure of formula (II):

(51) ##STR00019##

(52) In another embodiment, the compound of formula (I) is represented by the structure of formula (III):

(53) ##STR00020##

(54) Compounds useful in treating and/or preventing any of the diseases or conditions described herein are compounds of formula (I), for example compounds of formulae (II) or (III). Additional compounds useful in treating and/or preventing any of the diseases or conditions described herein are compounds of formula (I-A):

(55) ##STR00021## wherein A is

(56) ##STR00022## R.sup.1 is independently at each occurrence a halogen, OH, NO.sub.2 or N(R).sub.2 wherein each R is independently H or a C.sub.1-C.sub.6 alkyl; R.sup.2 is COOH, OH, NO.sub.2, N(R).sub.2 or a linear or branched C.sub.1-C.sub.6 alkyl substituted with at least one group selected from the group consisting of COOH, halogen, OH, NO.sub.2, and N(R).sub.2; R.sup.3 is H or COOH; R.sup.4 is iodophenyl, heterocyclyl, heteroaryl, the side chain of an amino acid selected from the group consisting of a naturally occurring alpha-amino acid, 3,4-dihydroxyphenylalanine (DOPA) and iodotyrosine; or R.sup.4 is represented by the structure:

(57) ##STR00023## wherein R.sup.5 is independently at each occurrence OH, halogen, NO.sub.2 or N(R).sub.2 and m is 0, 1, 2, 3 or 4; L.sup.1 and L.sup.2 are each a leaving group selected from a halogen and a sulfonate; n is 0, 1, 2, 3 or 4; and salts thereof.

(58) Preferred embodiments of formula I, I-A and II are compounds of formula 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, as described herein, and salt thereof.

(59) One or more of the compounds of the invention, may be present as a salt. The term “salt” encompasses salts formed by standard acid-base reactions between an acidic moiety and an organic or inorganic cation. The term “organic or inorganic cation” refers to counter-ions for the carboxylate anion of a carboxylate salt or the counter ion for the phenoxide moiety. The counter-ions are chosen from the alkali and alkaline earth metals (such as lithium, sodium, potassium, barium, aluminum and calcium); ammonium and mono-, di- and tri-alkyl amines such as trimethylamine, cyclohexylamine; and the organic cations, such as dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, bis(2-hydroxyethyl) ammonium, phenylethylbenzylammonium, dibenzylethylenediammonium, and like cations.

(60) The present invention also includes solvates of any of compounds described herein. “Solvate” means a physical association of a compound of the invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation. “Solvate” encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates and the like. “Hydrate” is a solvate wherein the solvent molecule is water.

(61) According to another aspect, the present invention provides a pharmaceutical composition comprising at least one compound of the present invention and a pharmaceutically acceptable diluents or carrier.

(62) As sued herein, the term “pharmaceutically acceptable carrier or excipient” means an additive, carrier, excipient or diluent which is useful in preparing a pharmaceutical composition that is generally safe non-toxic, and neither abrogates nor causes otherwise undesirable effect to the administered compound. The term includes such additives that are acceptable for human as well as veterinary pharmaceutical use.

(63) The term “subject” includes humans and animals amenable to therapy with a DIO3 inhibitor including animals afflicted with cancer or depression or depression associated disease or disorder. According to some embodiments, the subject is a human subject. Each possibility represents a separate embodiment of the invention.

(64) The term “subject” as used herein, includes, for example, a subject who has been diagnosed to be afflicted with cancer or depression or depression associated disease or disorder or a subject who has been treated to ameliorate cancer or depression or depression associated disease or disorder, including subjects that have been refractory to the previous treatment. Also encompassed within the present invention is a healthy subject having a risk of being affected with cancer or depression or depression associated disease or disorder. According to some embodiments, the subject is afflicted with cancer and has been identified to express DIO3 by the tumor. According to some embodiments, the expression is overexpression and the overexpression is determined following analysis and comparison with control or reference. According to some embodiments, the control is selected from the group consisting of: a predetermined cutoff value, a value obtained from a healthy individual, a panel of values from a set of healthy individuals and a value or a set of values obtained from a group of individuals afflicted with defined severities of cancer. Each possibility represents a separate embodiment of the invention. According to yet another embodiment, the predetermined cutoff value is obtained from the subject to be treated at at-least one prior-referenced time point.

(65) According to some embodiments, the expression of DIO3 may be determined by any method known in the art, for example by an immunoassay.

(66) The pharmaceutical compositions of the invention can be prepared by methods and contain carriers which are well-known in the art, for example as described in Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams & Wilkins: Philadelphia, Pa., 2000.

(67) In general, the compounds of the invention are administered in a therapeutically effective amount by any of the accepted modes of administration for agents that serve similar utilities. Suitable dosage ranges are depending upon numerous factors such as the severity of the symptoms to be treated, the age and relative health of the subject, the potency of the compound used; the route and form of administration; and the specific indication towards which the administration is directed. One of ordinary skill in the art of treating such conditions will be able, without undue experimentation and in reliance upon personal knowledge and the disclosure of this Application, to ascertain a therapeutically effective amount of the compounds of the present invention for a given condition.

(68) Compounds of the invention may be administered as pharmaceutical formulations including those suitable for parenteral administration (including intramuscular, subcutaneous and intravenous), oral administration (including buccal and sub-lingual), nasal, topical, pulmonary, intrathecal administration, or administration by inhalation. A preferred manner of administration is generally oral using a convenient daily dosage regimen which can be adjusted according to the degree of affliction. According to one embodiment, the DIO3 inhibitor is administered locally into the tissue to be treated. According to one embodiment, the tissue is a brain tissue, and the administration route is directly into the brain tissue.

(69) According to some embodiments, the composition comprising a DIO3 inhibitor may further comprise an additional agent. The additional agent may be any treatment for cancer, depression or depression associated disease or condition. According to some embodiments, treatment comprises administering to a subject in need thereof a plurality of DIO3 inhibitors.

(70) A compound or compounds of the invention, together with one or more conventional adjuvants, carriers, or diluents, may be placed into the form of pharmaceutical compositions and unit dosages. The pharmaceutical compositions and unit dosage forms may be comprised of conventional ingredients in conventional proportions, with or without additional active compounds or principles, and the unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. The pharmaceutical compositions may be employed as solids, such as tablets or filled capsules, semisolids, powders, sustained release formulations, or liquids such as solutions, suspensions, emulsions, elixirs, or filled capsules for oral use; or in the form of sterile injectable solutions for parenteral use.

(71) According to yet additional aspect, the present invention provides a method for treating or preventing depression or a depression-associated disease or condition, the method comprises administering to a subject in need thereof a therapeutically effective amount of a compound that inhibit the activity of Type III deiodinase (DIO3) thereby treating or preventing depression a depression-associated disease or condition.

(72) According to certain embodiments, inhibition of the DIO3 activity results in increased amount of T3 in the subject. According to currently certain exemplary embodiments, the amount of T3 is increased in the brain of the subject.

(73) In some embodiments, the compounds of the present invention are designed to have specificity or some selectivity to inhibition of DIO3, such that the other deiodinases (DIO1 and DI02) are not inhibited or inhibited to a lesser extent. DIO3 is known to be found primarily in the brain, and, more importantly, it is not found in heart or bone tissue. Thus, the specific inhibition of DIO3 results in increased amount of T3 in the brain, where it is most required to assert its anti-depressant activity, while having none or negligible of the deleterious side effects associated with non-specific elevation of T3 concentration, including enhancement of bone depletion, muscle weakness and tachycardia (Ocasio and Scanlan, Current Opinion in Endocrinology and Diabetes 2005; 12, 363-370; Yoshihara and Scanlan, Curr Top Med Chem 2003; 3, 1601-1616).

(74) In some embodiments, the compounds of the invention also inhibit DIO1 and DIO2, in addition to DIO3, but they are designed to selectively target the brain, or they are formulated specifically for brain delivery, such that they target DIO3 selectively due to preferential localization to the brain.

(75) According to yet another aspect, the present invention provides a method for treating or preventing cancer, the method comprises administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention.

(76) DIO3 expression and activity have been indicated previously to be associated with tumorigenesis. Thus, according to further embodiments the compounds of the present invention, which inhibit DIO3 are useful as treatment for cancer.

(77) According to some embodiments, the cancer is a solid cancer. Non limiting examples of solid cancers which are encompassed within the embodiments of the present invention include pancreatic cancer; bladder cancer; colorectal cancer; breast cancer; prostate cancer; renal cancer; hepatocellular cancer; lung cancer; ovarian cancer; cervical cancer; gastric cancer; esophageal cancer; head and neck cancer; melanoma; neuroendocrine cancer; brain tumors.

(78) According to some embodiment, the cancer is colon cancer or basal cell carcinoma (BCC).

(79) According to certain exemplary embodiments, the cancer is ovarian cancer, endometrial cancer.

(80) According to some embodiments, the compounds of the invention are designed to selectively target overproliferating cells, particularly cancer cells, or they are formulated specifically for delivery into these types of cells such that they target DIO3 selectively due to preferential localization within these cells.

(81) The DIO3 inhibitors of the present invention may be administered by any suitable route of administration. According to some embodiments, the DIO3 inhibitor is prescribed for a chronic administration. Chronic administration may be performed, for example, by a single dose, once daily. According to some embodiments, the DIO3 inhibitor is prescribed for acute administration.

(82) The therapeutically effective amount of a compound of the invention to be administered to a subject depends on the condition to be treated and subject parameters including, but not limited to, gender, age, weight type and severity of the condition. The therapeutically effective amount can be initially assessed from in vitro assays. Target concentrations will be those concentrations of active compound(s) that are capable of inhibiting DIO3 activity by at least about 5%-95% in comparison to DIO3 activity in untreated cell, either normal or overproliferating cells. Target concentrations of active compound(s) that are capable of inhibiting DIO3 by at least about 60-70% or even 90% or higher in in vitro assays are preferred.

(83) Therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a circulating concentration that has been found to be effective in animals. Animal models of cancer and for depression assessment are known in the art. For treating cancer, the dosage in humans can be adjusted by monitoring inhibition of cell proliferation and/or tumor growth and adjusting the dosage upwards or downwards, to achieve the desired percentage of inhibition. For treating depression, the dosage in humans can be adjusted in view of the effective dosage that alleviated or abolishes the depression associated symptoms. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.

(84) It is to be explicitly understood that any mechanism by which inhibition of DIO3 activity overcomes depression or depression associated disease or disorder is encompassed within the scope of the present invention. It has been previously acknowledged that the phenomena attributed to T3 may be mediated by T3 activation of gene transcription. Accordingly, most of the reports regarding the use of T3 as antidepressant describe positive influence of T3 administration after a lag time of about a week. However, other antidepressants are known to have immediate effect. For example, ketamine has been reported to have anti-depression activity after acute administration to humans (Aan Het Rot et al. Biological Psychiatry, 2012; 72(7), 537-547), and to affect the behavioral performance of rats in forced swim test (FST) (Yang C. et al., 2012. Journal of Biomedicine and Biotechnology, Epub 2012 Apr. 8).

(85) It has been also described that in addition to being active by induction of gene transcription the thyroid gland hormones show immediate physiological effects. Thus, without wishing to be bound by any specific theory or mechanism of action, administration of DIO3 inhibitors can assert immediate antidepressant effects.

(86) The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

EXAMPLES

Example 1: Design and Synthesis of DIO3 Mimics

(87) To date, pure DIO3 enzyme is not available. An inventor of the present invention and co-workers have designed and synthesized molecules that functionally mimic DIO3 in terms of deiodinase activity (Manna et al., 2010, ibid; Manna et al., 2012, ibid). Such DIO3-mimetic molecules have now been utilized for design and synthesis of compounds capable of inhibiting DIO3 enzyme. The present invention discloses such compounds that functionally inhibit the activity of the DI03-mimetic molecule and the wild-type (wt) DIO3 enzyme.

(88) The deiodination of thyroxine by compound A (a DIO3 mimetic; FIGS. 2 and 3) in the presence of various thiourea derivatives was studied. When the commonly used antithyroid drugs such as thiourea derivatives (e.g., 6-n-propyl-2-thiouracil (PTU), 6-methyl-2-thiouracil (MTU) and 1-methyl-3H-imidazole-2-thione or methimazole (MMI)) were used, no inhibition of the deiodinase activity was observed. Although PTU and MTU have been shown to inhibit DIO1, these compounds do not inhibit DIO3. Specifically, thiourea compounds (e.g., PTU) have been shown to react with the selenenyl iodide intermediate of DIO1. Those results suggest that compound A not only deiodinates T4 in the inner-ring, but also behaves similarly to DIO3 with respect to inhibition. These studies suggest that thiourea-based compounds are not suitable as inhibitors for DIO3.

(89) Another known inhibitor of DIO1, i.e. iodoacetic acid (IAA), may react with the selenol group of the enzyme. This compound is specific for DIO1 and cannot inhibit DIO2 and DIO3. Although IAA can effectively inhibit the activity of several Cys peptidases (i.e., the Sec-containing glutathione peroxidase (GPx), and thioredoxin reductase (TrxR)) by forming the corresponding alkylated Cys or Sec derivatives, the reason for the insensitivity of DIO3 toward IAA is not clear. Treatment of compound A with IAA resulted in a deiodination instead of the formation of Se-carboxymethylated derivative. In contrast, a compound that lacks the second selenol moiety underwent facile carboxymethylation by IAA. These observations suggest that IAA may undergo rapid deiodination in the presence of DIO3, which may account for the insensitivity of this enzyme toward IAA.

(90) A careful analysis of the amino acid residues at the active site of DIO3 indicates that there is a cysteine (Cys) residue in the enzyme that may assist the selenocysteine (Sec) in the deiodination reaction. In the mimic, the second selenol moiety in the 8-position of the naphthalene ring is expected to perform a similar function. The current approach aims at targeting both the Sec and Cys instead of targeting the Sec alone. Previous attempts to develop inhibitors for DIO3 based on Sec reactivity were unsuccessful.

(91) As described above, it is contemplated that any compound that can react with the both selenol moieties simultaneously in the synthetic DIO3 mimic of compound A may inhibit the deiodinase activity of this compound.

(92) ##STR00024##

(93) Without wishing to be bound by any particular mechanism or theory, it was hypothesized that the dibromomaleimide derivatives of the present invention may inhibit the activity of the DIO3 mimic by reacting with the two selenol groups to form a stable adduct.

(94) In view of the above, the following compounds (compound B and C) were synthesized. The two simultaneous nucleophilic attacks of the selenol moieties at the carbons adjacent to the carbonyl groups are expected to block the selenium centers. The reactions of compound A with compounds B (2,3-dibromo-N-benzylmaleimide) and C (2,3-dibromo-N-methylmaleimide) were studied. The reaction of compound A with compounds B and C produced the adducts D (1,8-bis(2,3-diseleno-N-benzylmaleimide)naphthalene) and E (1,8-bis(2,3-diseleno-N-methylmaleimide)naphthalene).

(95) ##STR00025##

(96) Subsequently, the deiodination of T4 by compound A in the presence of compounds B and C was studied. The results demonstrate that compounds B and C do inhibit the deiodination of T4 (exemplified in FIG. 4 for compound C).

(97) Another compound, a novel tyrosine derivative 3 (TYR-DBRMD) was synthesized. The following description demonstrates the route employed for the synthesis of TYR-DBRMD.

(98) Synthesis of TYR-DBRMD (Compound 3)

(99) The novel tyrosine derivative 3 was obtained from dibromomaleic anhydride (1) and L-tyrosine. As the commercially available starting material (compound 2) is expensive, it was prepared from maleic anhydride (1) in a sealed Teflon tube by using aluminum chloride as catalyst. For the synthesis of compound 2, the procedure reported in Dubernet et al. (Dubernet, M. et al., Tetrahedron, 2005; 61, 4585-4593) was followed:

(100) ##STR00026##

(101) Synthesis of 3,4-Dibromomaleic anhydride: A mixture of maleic anhydride (2.0 g, 20.39 mmol), aluminum chloride (40.8 mg, 0.3 mmol), and bromine (2.1 ml, 40.78 mmol), was heated at 120° C. for 16 h in sealed tube. After that, it was allowed to cool to room temperature and the reaction mixture was added to ethyl acetate. The ethyl acetate solution was filtered and the filtrate was evaporated. To the resulting reddish solid, 100 ml CHCl.sub.3 was added and filtered. The filtrate was evaporated to get off-white (yellow) solid in 87% yield. 13C NMR(CDCl.sub.3) δ(ppm): 131.8, 159.0.

(102) ##STR00027##

(103) To a solution of 3,4-dibromomaleic anhydride (500 mg, 1.95 mmol) in acetic acid (10 ml), tyrosine (425 mg, 2.35 mmol) was added. The mixture was heated at reflux condition for 12 h. The solvent was removed under reduced pressure. The crude mixture was purified by column chromatography over silica gel using petroleum ether and ethyl acetate as mobile phase to afford the desired product (485 mg, 59%) as off-white solid.

(104) .sup.1H NMR(DMSO-d.sub.6) δ (ppm): 3.02-3.08 (1H, m), 3.25-3.30 (1H, m), 4.90-4.94 (1H, dd, J=8.0 Hz) 6.60 (2H, d, J=8.0 Hz), 6.89 (2H, d, J=8.0 Hz) (FIG. 6).

(105) .sup.13C NMR(CDCl.sub.3) δ (ppm): 33.9, 55.8, 116.1, 127.7, 130.1, 130.6, 156.8, 168.4, 170.5 (FIG. 7).

(106) m.p. 175-177° C.

(107) Synthesis of PBENZ-DBRMD (Compound 4):

(108) ##STR00028##

(109) To a solution of 3,4-dibromomaleic anhydride (500 mg, 1.95 mmol) in acetic acid (10 ml), p-amino benzoic acid (322 mg, 2.35 mmol) was added. The mixture was heated at reflux condition for 12 h. The solvent was removed under reduced pressure. The crude mixture was purified by column chromatography over silica gel using petroleum ether and ethyl acetate as mobile phase to afford the desired product (550 mg, 82%) as yellow solid.

(110) .sup.1H NMR(DMSO-d.sub.6) δ (ppm): 7.50 (2H, d, J=8.0 Hz), 8.04 (2H, d, J=8.0 Hz) (FIG. 8).

(111) .sup.13C NMR(DMSO-d.sub.6) δ (ppm):127.5, 130.6, 130.9, 131.1, 136.0, 163.9, 167.6 (FIG. 9).

Example 2: Deiodination Assay

(112) The deiodination reactions were carried out in 100 mM phosphate buffer (pH 7.5) with 10 mM dithiothreitol (DTT) at 37° C. Diselenol (Compound A) was freshly prepared by reducing the corresponding diselenide, with NaBH4 prior to use. The reaction products were analyzed by reverse-phase HPLC (Lichrospher C18 column, 4.6 μm, 150 mm×5 mm) with gradient elution using acetonitrile/ammonium acetate (pH 4.0) as the mobile phase. The formation of rT3 was monitored at λ=275 nm and the amounts of deiodinated products formed in the reactions in the presence or absence of test compounds were calculated by comparing the peak areas.

(113) Inhibition of the DIO3 mimic by TYR-DBRMD (Compound 3)

(114) Compounds 3 and 4 were selected based on their water solubility. The TYR (tyrosine derivative, compound 3) is more soluble in water (due to the presence of —OH and —COOH groups) than PBENZ (benzoic acid derivative, compound 4), but the PBENZ is expected to be more lipophilic. The reactivity of the two compounds toward the DIO3 mimic is was found to be similar, although they may exhibit different binding behavior with the enzyme. However, the affinity data with the mimic may not correlate with that of the enzyme, as there will be additional interactions (hydrogen bonding etc) with the inhibitors at the active site of the enzyme, which are absent with the mimetic molecule.

(115) The two compounds (3 and 4) react stoichiometrically with the mimic. The IC.sub.50 values for inhibiting the DIO3 mimic were found to be in the micromolar range (FIG. 5) as the mimics used were in the micromolar concentrations for the deiodination of thyroxine. Assuming that in vivo, the enzymes are present in sub-nanomolar concentrations and the inhibitors are expected to form a covalent bond with the enzyme, it is expected the IC.sub.50 values will be in the nanomolar concentration range.

(116) Additional inhibitors of DIO3 contemplated by the present invention are compounds 5, 6, 7, 8, 9, 10, 11 and 12 presented below. Those compounds comprise several modifications such as iodine ions (in ITYR-DIBRMD—compound 5, and IOP-DIBRMD—compound 6). These compounds can be stabilized by halogen bonding at the enzyme active sites, caused by the iodine atoms that are attached to maleimide moiety, thus greater deiodinase isoenzyme specificity is supposedly achieved. As DIO3 readily accepts iodinated substrates, the introduction of iodine may help the compounds to bind strongly to the active site. For example, the commercially available iodine-containing compound, Iopanoic acid (IOP) has been shown to be a substrate for DIO3 (Huang MP et al., Thyroid, 2011, 21, 1263).

(117) ##STR00029##

Example 3: High Throughput Assays for Determining Deiodinase 3 Inhibitors Effects on Enzyme Activity

(118) Rapid evaluation of the effects of novel putative deiodinase 3 inhibitors on activity levels of DIO3 is established by a high output system employing two main lines of in vitro assays:

(119) 1) Mouse brain-derived microsome assay: Mouse brain tissue is homogenized and centrifuged to obtain microsomal fractions. Protein concentration in each fraction pellet is determined by Bradford Assay. Deiodinase 3 activity determination, in the presence of putative deiodinase inhibitors, is carried out by incubation of a given amount of the fraction with the radioligand [3,5-.sup.125]T3, each incubation done with blanks (no protein) to correct for non enzymatic degradation of radioligand. Reactions are stopped and radioiodide .sup.125I emission is measured by precipitation of the [.sup.125I]iodothyronines and isolation of the emitted (by the inner ring deiodination reaction) .sup.125I radioiodide on microcolumns. Radioactivity in the eluate is monitored using a scintillation detector.

(120) 2) Cell based assay: Astrocytes from neonate mice are harvested and a cell suspension is prepared, plated on Petri dishes and maintained in a cell incubator. After 7 days, cells are disrupted and homogenates are prepared. In the presence of the putative deiodinase 3 inhibitor compound, the homogenates is incubated with [.sup.125I]T3. Reactions are stopped by the addition of NH.sub.4OH containing 10 micro molar T3. The [.sup.125I]3,3′-T2 produced by deiodinase 3 action is separated from [.sup.125I]T3 by chromatography. Then the radioactive products of the reaction are counted for determination of D3 activity, expressed as fentomoles of 3,3′-T.sub.2 per minute per milligram protein.

Example 4: Effects of Intracerebroventricular (ICV) Administration of ITYR-DIBRMD (ITYR; Compound 5) and IOP-DIBRMD (IOP; Compound 6) on Depression

(121) Materials and Methods:

(122) Chronic Intra-cerebroventricular (ICV) administration via osmotic minipumps

(123) The intra-cerebroventricular injection was performed via a cannula inserted as follows: first, for the chronic administration of compounds or vehicle, osmotic minipumps (Alzet model 1004 for up to 28 days administration) were employed. Empty minipumps were filled under sterile conditions with 0.1 ml of PBS or with PBS containing either one of the DIO3 inhibitors and then connected to brain infusion kits. The filled minipumps connected to the brain infusion kits were than primed in an incubator at 37° C. before being implanted into the animals. At the surgery itself, each mouse was anaesthetized by intraparenteral (i.p.) injection of anesthesia. The anesthesia solution was prepared as follows: for each 2 ml of solution, 900 μl ketamine plus 100 μl xylazine 2% was supplemented with 1000 μl of 0.9% NaCl saline solution. 70 μl of the anesthesia solution was administered i.p to a 20 gram (about 100 μl for 30 gram) weighing animal. After being anesthetized the animal was hooked to a stereotactic table. A longitudinal cut was made in the head skin, the upper part of the skull was exposed and a hole was produced using 0.5 mm diameter drill and the cannula was inserted. The hole was located at the following coordinates relative to the bregma: Anterior-ventral-dorsal=2.5 mm; Medial-lateral=1.0 mm and anterior-posterior=0.0 mm Thereafter each minipump—was inserted subcutaneously to the interscapular space and the base of the infusion kit cannula was glued, using specialized Loctite adhesive, to the skull base so the cannula shaft itself will protrude through the drilled hole to the lateral ventricle, and through a catheter linked to ALZET brain infusion kits number 3, the compounds or vehicle were intracerebroventricullary through the drilled hole.

(124) The wound formed in the skull was sown with silk sutures, and after it awoke, each animal was transferred to the home cage for recovery, typically for 48 h, after which the experiment may be started.

(125) 39 male BALB/c male mice aged 8 weeks were implanted with ALZET model 1004 osmotic minipumps for intracerebroventricular (ICV) administration of compounds in 3 treatment groups (13 mice in each group): (1) the putative DIO3 inhibitor IOP-DIBRMD (IOP, compound 6) dissolved in phosphate buffered solution (PBS) vehicle; (2) the putative DIO3 inhibitor ITYR-DIBRMD (ITYR, compound 5) dissolved in PBS; (3) PBS vehicle. Minipumps contained 0.1 ml of PBS into which the putative DIO3 inhibitors ITYR (5 mg/kg) or IOP (2 mg/kg) were dissolved, or PBS vehicle. 8, 13 and 9 mice survived the experiment in the PBS, ITYR and IOP groups, respectively. Deaths were related to the surgical procedure. After recovery from minipump implantation surgery, animals were returned to their home cages for continuous ICV compound administration for 3 weeks, before behavioral testing commenced.

(126) Assessment of Antidepressant-like Activity and Serum and Brain Thyroid Hormone Levels

(127) Novelty Suppressed Feeding Test (NSFT):

(128) After the last treatment administration, all food was removed from the cage for 24 hours (water was available ad libitum). At the end of this period, each animal was introduced into a 50×50×30 (height) cm plastic arena located in a specialized behavioral room in which all behavioral tests were tracked by cameras linked to a computer installed with EthoVision behavioral tracking software of the latest edition (XT 10). A pellet of food was placed on an elevated surface in the center of the arena. Time elapsing from the introduction of the animal into the arena until it commences eating (latency to feed), total distance move, velocity and time spent in the arena periphery (lateral 10 cm on each side) and center were recorded. The animal was then removed from the arena immediately after it begins to eat or after not doing so for 5 min. After the test, the animal was immediately transferred to its home cage and left to consume a previously weighed amount of food for 10 minutes. On completion of this period the food was weighed again to calculate the home cage food consumption. The rating of the animals' behavior was conducted by two experimenters who were blind to the treatment received by each mouse. The mean of the two ratings was calculated and used for the statistical analysis.

(129) Serum and Brain Thyroid Hormone Levels

(130) At the end of the tests, animals were anesthetized with veterinary Pental (13.333 mg/kg) and cardiac blood was drawn for peripheral T3 (triiodothyronine) and T4 (thyroxine) levels. Brains were harvested for evaluating brain T3, T4 levels. Hormone levels in the sera and brains were measured by SunLong Biotech Mouse Ultrasensitivity total and free T3 and T4 ELISA Kits, employing producing a standard curve followed by actual measurement, in which OD levels obtained by spectrophotometer were converted, using the standard curve, to pg/ml values.

(131) Results

(132) i) Effect of IOP and ITYR on Brain and Serum Hormone Levels

(133) As shown in Table 1 and FIG. 10, IOP treatment for 3 weeks via ICV administration resulted in a significant increase in brain free T3 levels (about 46%). Brain free T3 level was not significantly increased when ITYR was administered. Neither treatment significantly increased brain free T4 levels or the levels of either free hormone in the serum.

(134) TABLE-US-00001 TABLE 1 Brain and serum hormone levels after treatment with IOP and ITYR Free T3 (pg/ml) Brain Serum No. of Signifi- Signifi- Treatment mice Mean SD cance* Mean SD cance* Vehicle  8  72.74  9.63 14.45 2.83 (PBS) IOP 10  84.30 15.31 *0.041 12.23 1.64 NS ITYR 12  81.98 12.24 NS 12.33 1.30 NS Free T4 (ng/ml) Brain Serum No. of Signifi- Signifi- Treatment mice Mean SD cance* Mean SD cance* Vehicle  8 250.54 53.51 36.05 8.53 (PBS) IOP 10 244.11 44.72 NS 35.75 9.79 NS ITYR 12 262.86 32.70 NS 37.00 8.92 NS *One sided, non-paired t test; active compound vs. vehicle

(135) ii) Effect of IOP and ITYR on Latency to Feed in the Novelty Suppressed Feeding Test

(136) The time taken for a rodent to start eating a pellet of food located in the center of an open field arena (latency to feed) is a reflection of anxiety level and is also seen as reflecting level of anhedonic behavior. In the current experiment, treatment with both IOP and ITYR resulted in a reduced latency to feed compared to mice treated with vehicle

(137) (FIGS. 11 and 12, respectively). Data were analyzed by analysis of covariance with brain T3 level entered as the covariate. The effect of T3 in mice treated with both IOP and ITYR was found to be significant.

Example 5: Effect of D103 Inhibitors on Ovarian Cancer Cells

(138) Cell lines: Human ovarian adenocarcinoma cells employed in the study were OVCAR-3 (ATCC HTB-161), A2780 (Sigma Aldrich) and SKOV-3 (ATCC HTB-77). Human normal embryonic kidney cells, HEK 293 (ATCC CRL1573) were used. All cells were cultured in RPMI1640 supplemented with 10% heat-inactivated FBS and antibiotics. Before conducting an experiment, cells were cultured for 24 h in RPMI1640 supplemented with 1.5% heat-inactivated charcoal stripped FBS and antibiotics.

(139) Flow cytometry (MACSQuant, Miltenyi): The cells were treated with 70% ethanol at −20° C. for 1 hour, stained with rabbit anti Human DIO3 monoclonal antibody (Alexa Fluor (R) 647) from Novus Biologicals and analyzed for endogenous DIO3 level by FACS.

(140) Viability: WST-1 (Roche; 10% final concentration) was incubated with cells at 37° C. for 2 h and read with a MicroELISA reader at 440 nm.

(141) Microscopy: The cells were visualized using a microscope equipped with a camera (model IX71; Olympus) with a 20_/0.50 objective lens and Cell{circumflex over ( )}A (version 3.1) Olympus software imaging.

(142) Compounds

(143) The following DIO3 inhibitory compounds were examined: PBENZ (compound 4); TYR (compound 3); IOP (compound 6); ITYR (Compound 5): DOP (compound 7) and ASP (compound 8).

(144) The anti cancerous (anti-proliferative) effect of the DIO3 inhibitors described above was examined using the above-described human ovarian cancer cells (OVCAR3, SKOV3 and A2780). The cells were cultured in RPMI 1640/10% FBS/antibiotics.

(145) T3 influences cellular growth, differentiation and apoptosis, and thus its level within cells is strictly regulated. Elevated DIO3 levels and thus reduced T3 levels are favorable to the neoplastic process. Therefore, cancer cells that show elevated DIO3 are a good model for testing the potential anti-neoplastic efficacy of DIO3 inhibitors. Accordingly, the level of endogenous DIO3 protein in the different cancer cells was assessed by FACS analysis using monoclonal antibody against the human DIO3. Results indicate that OVCAR3 cells exhibited the highest DIO3 protein level (FIG. 13A) and this cell type was used as the experimental model.

(146) OVCAR3 cells were collected and seeded in 96 well plates (10,000/well). The six compounds were then dissolved in DMSO and added to a final 0.5 μM concentration for 48 h of incubation. After incubation the cells were imaged by light microscopy (FIG. 13B). A marked reduction in cell density and deformed cell morphology was observed with three agents, ITYR, DOP and ASP. These cells were then examined for cell viability by WST-1 assay (FIG. 13C). No effect was evident using PBENZ, a low reduction in viability was observed using TYR and IOP and a significant reduction in cell viability by the same 3 agents described above, ITYR, DOP and ASP was shown.

(147) The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.