Patent Application
20120264804
Influx of extracellular calcium is critical for a number of vital cellular processes. Calcium influx is generally mediated by calcium channels, which are grouped into several families one of which is the T type calcium channel family. Pharmacological modulation of the T type calcium channel's function is of importance in the practice of medicine; for example. T type calcium channel inhibitors are in widespread use in the treatment of neurological diseases epilepsy, petit mal seizure, absence seizure, neuropathic pain, and etc.) and cardiovascular diseases (e.g. hypertension, unstable angina, and etc.). For example, mibefradil, a T type calcium inhibitor, was clinically efficacious in treating hypertension and cardiac arrhythmia. Studies also suggest that T-type calcium channels may play an important role in age related macular degeneration.
It has been known for some time that Ca2+ entry is a regulatory component of the cell cycle and such that inhibition of it blocks proliferation. However, the mechanism by which Ca2+ entry occurs continues to be a matter of some debate.
Voltage gated calcium requires rapid changes in membrane potential, called action potentials, for activation of calcium influx. Electrically non-excitable cells, such as the majority of cancer cell types, do not have action potentials. Consequently, voltage gated calcium channels are thought to have no role in electrically non-excitable cells (Venkatachalam, K., Van Rossum, D. B, Patterson, R. L., Ma, H.-T., and Gill, D. L. 2002. The cellular and molecular basis of store-operated calcium entry. Nat. Cell Biol. 4:E263-E272).
Accordingly, one embodiment provides a method for treating a disease or condition in a mammal associated with influx of extracellular calcium via T type calcium channels, which comprises administering to the mammal a therapeutically effective amount of a T type calcium channel inhibitor, a prodrug thereof or a pharmaceutically acceptable salt of said inhibitor or prodrug. Another embodiment provides a method for treating a disease or condition in a mammal associated with influx of extracellular calcium via T type calcium channels, which comprises administering to the mammal a therapeutically effective amount of a T type calcium channel inhibitor that inhibits a splice variant of the α1H isoform of T type calcium channels or a pharmaceutically acceptable salt of said inhibitor. Preferably, the disease or condition is selected from the group consisting of unstable angina, hypertension, epilepsy, neuropathic pain, petit mal seizure, absence seizure, age related macular degeneration, cancer, and pre-cancerous condition.
Preferably, the above-mentioned T type calcium channel inhibitor has a structure represented by Formula (I):
wherein
R1 is selected from the group consisting of C1-C4 alkyl, hydroxy and C1-C4 alkoxy;
X is selected from the group consisting of N and CH;
Z is selected from the group consisting of NH, O, S and CH2;
R2 is selected from the group consisting of H, halo, NH2, C1-C4 alkyl, hydroxy and C1-C4 alkoxy; and
R3 is selected from the group consisting of H, halo, NH2, C1-C4 alkyl, hydroxy and C1-C4 alkoxy. In one embodiment R1 is selected from the group consisting of C1-C4 alkyl, hydroxy and C1-C4 alkoxy, X is N, Z is O or CH2, R2 is H, halo, NH2 or hydroxy and R3 is H.
Another embodiment provides a method for reducing proliferation of electrically non-excitable cells, which comprises administering a T type calcium channel inhibitor, wherein said T type calcium channels inhibitor blocks a splice variant of an α1H isoform of T type calcium channels thereof.
Another embodiment provides a method for inhibiting calcium entry into electrically non-excitable cells, which comprises administering a T type calcium channel inhibitor, wherein said T type calcium channels inhibitor blocks a splice variant of an α1H isoform of T type calcium channels.
One embodiment provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) as described above, a prodrug of said compound or a pharmaceutically acceptable salt of said compound or prodrug; and a pharmaceutically acceptable carrier, vehicle or diluent.
In another embodiment, a method for the treatment of cancer or pre-cancerous condition in a mammal, which comprises administering to the mammal a therapeutically effective amount of a compound of formula (I) as described above, a prodrug thereof, or a pharmaceutically acceptable salt of said compound or prodrug in combination with one or more anti-tumor agent is provided.
One embodiment a pharmaceutical combination composition comprising a therapeutically effective amount of a combination of a compound of formula (I) as described above, a prodrug thereof, or a pharmaceutically acceptable salt of said compound or prodrug; and one or more anti-tumor agent.
In one embodiment, the splice variant is δ25, 512, 513, 544, 577 or a combination thereof. In another embodiment, the splice variant is δ25.
In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.
As used herein, the term “isolated” or “purified” and like terms relate to an enrichment of a molecule or compound relative to other components normally associated with the molecule or compound in a native environment. The term “purified” does not necessarily indicate that complete purity of the particular molecule has been achieved during the process. A “highly purified” compound as used herein refers to a compound that is greater than 90% pure.
As used herein, the term “treating” includes administering therapy to prevent, cure, or alleviate the symptoms associated with, a specific disorder, disease, injury or condition. For example treating cancer includes inhibition or complete growth arrest of a tumor, reduction in the number of tumor cells, reduction in tumor size, inhibition of tumor cell infiltration into peripheral organs/tissues, and/or inhibition of metastasis as well as relief, to some extent, of one or more symptoms associated with the disorder. The treatment of cancer also includes the administration of a therapeutic agent that directly decreases the pathology of tumor cells, or renders the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy. As used herein, the term “treating” includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
As used herein, the term “pharmaceutically acceptable carrier, vehicle or diluent” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.
The term “therapeutically effective amount” means an amount of a compound of the present invention that ameliorates, attenuates or eliminates a particular disease or condition or prevents or delays the onset of a particular disease or condition.
By “mammal” it is meant to refer to all mammals, including, for example, primates such as humans and monkeys. Examples of other mammals included herein are rabbits, dogs, cats, cattle, goats, sheep and horses. Preferably, the mammal is a female or male human.
The phrase “compound(s) of the present invention” or “compound(s) of Formula (I)” or the like, shall at all times be understood to include all active forms of such compounds, including, for example, the free form thereof, e.g., the free acid or base form, and also, all prodrugs, polymorphs, hydrates, solvates, tautomers, and the like, and all pharmaceutically acceptable salts, unless specifically stated otherwise. It will also be appreciated that suitable active metabolites of such compounds are within the scope of the present invention.
The expression “prodrug” refers to compounds that are drug precursors which following administration, release the drug in vivo via some chemical or physiological process (e.g., a prodrug on being brought to the physiological pH or through enzyme action is converted to the desired drug form).
The expression “pro-cancerous condition” refers to a growth that is not malignant, but is likely to become so if not treated, A “pre-cancerous condition” is also known as “pre-malignant condition” by one of ordinary skill in the art.
As used herein the term “anti-tumor agent” relates to agents known in the art that have been demonstrated to have utility for treating neoplastic disease. For example, antitumor agents include, but are not limited to, antibodies, toxins, chemotherapeutics, enzymes, cytokines, radionuclides, photodynamic agents, and angiogenesis inhibitors. Toxins include ricin A chain, mutant Pseudomonas exotoxins, diphtheria toxoid, streptonigrin, boamycin, saporin, gelonin, and pokeweed antiviral protein. Chemotherapeutics include 5-fluorouracil (5-FU) daunorubicin, cisplatinum, bleomycin, melphalan, taxol, tamoxifen, mitomycin-C, and methotrexate as well as any of the compounds described in U.S. Pat. No. 6,372,719 (the disclosure of which is incorporated herein by reference) as being chemotherapeutic agents. Radionuclides include radiometals. Photodynamic agents include porphyrins and their derivatives. Angiogenesis inhibitors are known in the art and include natural and synthetic biomolecules such as paclitaxel, O-(chloroacetyl-carbomyl) fumagillin (“TNP-470” or “AGM 1470”), thrombospondin-1, thrombospondin-2, angiostatin, human chondrocyte-derived inhibitor of angiogenesis (“hCHIAMP”), cartilage-derived angiogenic inhibitor, platelet factor-4, gro-beta, human interferon-inducible protein 10 (“IP 10”), interleukin 12, Ro 318220, tricyclodecan-9-yl xanthate (“D609”), irsogladine, 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide (“GPA 1734”), medroxyprogesterone, a combination of heparin and cortisone, glucosidase inhibitors, genistein, thalidomide, diamino-antraquinone, herbimycin, ursolic acid, and oleanolic acid. Anti-tumor therapy includes the administration of an anti-tumor agent or other therapy, such as radiation treatments, that has been reported as being useful for treating cancer.
As used herein, the term “halogen” or “halo” includes bromo, chloro, fluoro, and iodo.
The term “haloalkyl” as used herein refers to an alkyl radical bearing at least one halogen substituent, for example, chloromethyl, fluoroethyl or trifluoromethyl and the like.
The term “C1-Cn alkyl” wherein n is an integer, as used herein, represents a branched or linear alkyl group having from one to the specified number of carbon atoms. Typically C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.
The term “C2-Cn alkenyl” wherein n is an integer, as used herein, represents an olefinically unsaturated branched or linear group having from 2 to the specified number of carbon atoms and at least one double bond. Examples of such groups include, but are not limited to, 1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl, and the like.
The term “C2-Cn alkynyl” wherein n is an integer refers to an unsaturated branched or linear group having from 2 to the specified number of carbon atoms and at least one triple bond. Examples of such groups include, but are not limited to, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, and the like.
The term “C3-Cn cycloalkyl” wherein n=4-8, represents cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
As used herein, the term “optionally substituted” refers to from zero to four substituents, wherein the substituents are each independently selected. Each of the independently selected substituents may be the same or different than other substituents.
As used herein the term “aryl” refers to a mono or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, benzyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like.
The term “heterocyclic group” refers to a mono or bicyclic carbocyclic ring system containing from one to three heteroatoms wherein the heteroatoms are selected from the group consisting of oxygen, sulfur, and nitrogen.
The term “pharmaceutically acceptable salt” refers to salts which retain the biological effectiveness and properties of the compounds of the present invention and which are not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl group groups or groups similar thereto.
Recently, progress has been made in identifying candidate channels for mediating Ca+ entry (Li, S. W., WestwickJ., and Poll, C T. 2002. Receptor-operated Ca2+ influx channels in leukocytes: a therapeutic target?Trends Pharmacol. Sci. 23:63-70; Mori, Y., Wakamori, M., Miyakawa, T. Hermosucra, M., Hara, Y., Nishida, M., Hirose, K., Mizushima, A., Kurosaki, M., Mori, E. et al. 2002. Transient receptor potential 1 regulates capacitative Ca2+ entry and Ca2+ release from endoplasmic reticulum in B lymphocytes. J Exp. Med. 195:673-681; Tsavaier, L., Shapero, M. H., Morkowski, S., and Laus, R. 2001. 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. Cancer Res. 61:3760-3769; Peng, J. B., Zhuang, L., Berger, U. V., Adam, R. M., Williams, B. J., Brown, E. M., Hediger, M. A., and Freeman, M. R. 2001. CaTI expression correlates with tumor grade in prostate cancer, Biochem. Biophys. Res. Commun. 282:729-734; Benham, C. D., Davis, J. B., and Randall, A. D. 2002. Vanilloid and TRP channels: a family of lipid-gated cation channels. Neuropharmacology 42:873-888). However, these Ca2+ channels do not completely fulfill the criteria for the Ca2+ entry pathway in electrically non-excitable cells (Clapham, D. E. 2002. Sorting out MIC, TRP, and CRAC Ion Channels, J. Gen. Physiol 120:217-220; Densmore, J. J., Haverstick, D. M., Szabo, G., and Gray, L. S. 1996. A voltage operable current is involved in activation-induced Ca2+ entry in human lymphocytes whereas ICRAC has no apparent role. Am J. Physiol. 271:C1494-C1503). This lack of knowledge may be the result of a number of factors. While several of the candidates for mediation of Ca2+ entry in electrically non-excitable cells have been characterized at the molecular level (Putney, J. W., Jr. and McKay, R. R. 1999. Capacitative calcium entry channels. Bioassays 21:38-46), a commonly accepted candidate, ICRAC (Clapham, D. E. 2002. Sorting out MIC, TRP, and CRAC Ion Channels. J. Gen. Physiol 120:217-220; Cahalan, M. D., Wulff, H., and Chandy, K. G. 2001. Molecular properties and physiological roles of ion channels in the immune system. J Clin Immunol. 21:235-252), has not been, although it was first identified about ten years ago (Hoth, M. and Penner, R. 1993. Calcium release-activated calcium current in rat mast cells. J. Physiol. 465:359-386; Hoth, M. and Penner, R. 1992. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 355:353-356). It is also difficult to tie the function of these channels to inhibition of proliferation of cancer cell lines because of the lack of specific Ca2+ entry blockers for electrically non-excitable cells. There is as well the unavoidable disjunction between Ca2+ entry as measured by Ca2+ selective fluorescent dyes and electrophysiological methods. The patch clamp technique is extraordinarily powerful for examining the biophysical details of the function of an ion channel (Neher, E, and Sakmrann, B. 1992. The patch clamp technique. Scientific American March; 44-51), however the level of membrane control it both achieves and requires makes it less suited to identifying a channel's physiological role. Fluorescence techniques are very limited in obtaining biophysical detail, but better able to study physiological roles. This disconnection makes it difficult to determine if the effects of physiologically relevant stimuli (as determined by fluorescence measurements) are reproduced at the electrophysiological level.
In accordance with the present invention, it is believed that the mechanism of Ca2+ entry in electrically non-excitable cells involves a Ca2+ channel sharing characteristics with the T type family of voltage gated Ca2+ channels (Densmore, J. J., Haverstick, D. M., Szabo, G., and Gray, L. S. 1996. A voltage operable current is involved in activation-induced Ca2+ entry in human lymphocytes whereas ICRAC has no apparent role. Am. J. Physiol. 271:C1494-C1503; Densmore, J. J., Szabo, G., and Gray, L. S. 1992. A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes, FEBS Lett. 312:161-164; Haverstick, D, M. and Gray, L. S. Increased intracellular Ca2+ induces Ca2+ influx in human T lymphocytes. Molecular Biology of the Cell 4, 173-184. 1993; Haverstick, D, M., Densmore, J. J., and Gray, L. S. 1998. Calmodulin regulation of Ca2+ entry in Jurkat T cells. Cell Calcium 23:361-368). It could be argued that it is difficult to envision a physiologic role for voltage gated Ca2+ channels in cells that do not have action potentials. This argument is, however, based upon the assumption that a voltage gated Ca2+ channel can be only be activated by an action potential. Such an assumption is false a priori because the means by which a protein can be regulated by imposed experimental conditions is not necessarily identical with, or even similar to, the mechanism by which it is controlled physiologically. Although secondary to regulation by membrane potential, the known biochemical regulation of voltage gated Ca2+ channels in a variety of systems (Heady, T. N., Gomora, J. C., Macdonald, T. L., and Perez-Reyes, E, 2001. Molecular pharmacology of T-type Ca2+ channels. Jpn. J. Pharmacol. 85:339-350; 1-Hockerman, G. H., Peterson, B-Z., Johnson, B. D., and Catterall, W. A. 1997. Molecular determinants of drug binding and action on L-type calcium channels. Annu. Rev. Pharmacol Toxicol. 37:361-396; Slish, D. F., Schultz, D., and Schwartz, A. 1992. Molecular biology of the calcium antagonist receptor. Hypertension 19:19-24) also suggests that the categorical distinction between electrical and biochemical regulation of Ca2+ channels maybe somewhat simplistic.
Described herein is an alternative approach to dissecting the Ca2+ entry pathway in electrically non-excitable cells. First, the advantage of Ca2+ entry blockade by Ni2+ was taken advantage of, as measured by fluorescence techniques (Merritt, J. E. and Rink, T. J. 1987. Regulation of cytosolic free calcium in fura-2-loaded rat parotid acinar cells. J. Biol. Chem. 262:17362-17369; Merritt, J. E., Jacob, R., and Hallam, T. J. 1989. Use of manganese to discriminate between calcium influx and mobilization front internal stores in stimulated human neutrophils. J. Biol. Chem. 264: 1522-1527; Skryma, R., Mariot, P., Bourhis, X. L., Coppenolle, F. V., Shuba, Y., Abeele, F. V., Legrand, G., Humez, S., Boilly, B., and Prevarskaya, N. 2000, Store depletion and store-operated Ca2+ current in human prostate cancer LNCaP cells: involvement in apoptosis, J. Physiol. (Lond.) 527 Pt 1:71-83), to identify compounds in the published literature with a similar ability. The structure/activity relationship of these compounds aided in the synthesis of novel compounds with enhanced potency to block Ca2+ entry into and proliferation of several cancer cell lines. Two representative novel compounds were then shown to block the Ca2+ current through the heterologously expressed α1H isoform of T type Ca2+ channels. Cell lines sensitive to the novel compounds express messages for α1H, its δ25 splice variant, or both. These observations demonstrate the possibility of directed chemical synthesis of compounds that inhibit Ca2+ entry and thereby proliferation of cancer cells.
Three genes encode T-type channels, CACN1G, CACNA1H and CACNAH, expressing Ca,3.1 (α1G), Ca,3.2 (α1H), and Ca,3.3 (αH) subunits, respectively. Calcium channel, voltage-dependent, T type, alpha 1H subunit, also known as CACNA1H, is a protein which in humans is encoded by the CACNA1H gene. This gene encodes, Ca3.2, a T-type member of the α1 subunit family, a protein in the voltage-dependent calcium channel complex. There are two isoforms of CACNA1H-isoform 1 (identifier: O95180-1), also known as: A1H-a and isoform 2 (identifier: O95180-2) also known as: A1H-b. The sequence of isoform 2 differs from isoform 1 in that 1587-1592 are missing. Calcium channels mediate the influx of calcium ions into the cell upon membrane polarization and consist of a complex of α1, α2δ, β, and γ subunits in a 1:1:1:1 ratio. The α1 subunit has 24 transmembrane segments and forms the pore through which ions pass into the cell. There are multiple isoforms of each of the proteins in the complex, either encoded by different genes or the result of alternative splicing of transcripts. Alternate transcriptional splice variants, encoding different isoforms, have been characterized for CACNA1H.
There are several splice variants of the full length, canonical Ca,3.2 that are derivatives of the gene CACNA1H (accession numbers NM—001005407.1, AF051946.34, AJ420779.1, AF073931.1 and NP—001005407.1 (human mRNA and protein) and NM—021415 and NP—067390 (mouse mRNA and protein)). These variants are aggregated in GenBank in a UniGene Cluster, which in the case of Ca,3.2 is designated Hs.459642. For example, The α1H Ca2+ and its δ25 splice variant (accession number AF223563) both are members of the T type Ca2+ channel family by sequence homology and have been assigned to the Hs.122359 (since retired and is now referred to as the Hs.459642) UniGene cluster within the NCBI database. The Hs.459642 UniGene cluster includes accession number AC120498 (195680 bp DNA Homo sapiens chromosome 16 clone RP11-616M22, complete sequence); accession number AE006466 (265786 bp Homo sapiens 16p13.3 sequence section 5 of 8), accession number AF223560 (878 bp DNA Homo sapiens low-voltage-activated calcium channel alpha13.2 subunit (CACNA1H) gene, exons 1, 2 and partial eds), accession number AF223561 (Homo sapiens low-voltage-activated calcium channel alpha13.2 subunit (CACNA1H) gene, exons 7, 8, and partial eds.), accession number AF223562 (Homo sapiens low-voltage-activated calcium channel alpha13.2 subunit (CACNA1H) gene, exon 9 and partial eds.), accession number AF223563 (Homo sapiens low-voltage-activated calcium channel alpha 13.2 subunit delta25B splice variant (CACNA1H) gene, exons 11 through 36), accession number AL031703 (Human DNA sequence from clone LA16e-302G6 on chromosome 16 Contains part of the CACNA1H (calcium channel, voltage-dependent, alpha 1H subunit) gene, ESTs, GSSs and CpG islands, complete sequence), accession number AL031715 (Human DNA sequence from clone LA 16-357D8 on chromosome 16, complete sequence), accession number CH-1471112 (Homo sapiens 211000035837318 genomic scaffold, whole genome shotgun sequence), accession number AF051946 (Homo sapiens T-type calcium channel alpha 1H subunit (CACNA1H) mRNA, complete eds), accession number AF070604 (Homo sapiens clone 24597 mRNA sequence), accession number AF073931 (Homo sapiens low-voltage activated calcium channel alpha 1H mRNA, complete eds.), accession number AJ420779 (Homo sapiens mRNA for calcium channel, voltage-dependent, T type, alpha 1Hb subunit (CACNA1HB gene), accession number AK074965 (Homo sapiens cDNA FLJ90484 fis, clone NT2RP3003000, highly similar to Voltage-dependent. T-type calcium channel subunit alpha-1H), accession number BM55438 (AGENCOURT—6546908 NIH_MGC—119 Homo sapiens cDNA clone IMAGE:57424975-, mRNA sequence), accession number CA335096 (NISC_lt06a11.yl COGENE 8.5 EPT Homo sapiens cDNA clone IMAGE:5605917 5-, mRNA sequence), accession number CD243650 (AGENCOURT—1412077 NIH_MGC—18 Homo sapiens cDNA clone IMAGE:30383454 5-, mRNA sequence), accession number DQ363526 (Homo sapiens low-voltage-activated calcium channel alpha 1H subunit splice variant 512 (CACNA1H) mRNA, partial eds, alternatively spliced), accession number DQ363527 (Homo sapiens low-voltage-activated calcium channel alpha1H subunit splice variant 513 (CACNA1H) mRNA, partial eds, alternatively spliced), accession number DQ363528 (Homo sapiens low-voltage-activated calcium channel alpha1H subunit splice variant 544 (CACNA1H) mRNA, partial eds, alternatively spliced), and accession number DQ363529 (Homo sapiens low-voltage-activated calcium channel alpha1H subunit splice variant 577 (CACNA1H) mRNA, partial eds, alternatively spliced). Accession numbers of proteins coded by the above include AAK61268.1, AAF60160.2, AAF60161.1, AAF60162.1, AAF60163.1, CAC42094.1, EAW85683.1, EAW85684.1, AAC67239.3, AAD176668, CAD12646.1, BAG52041.1, ABC88009.1, ABC88010.1, ABC88011.1 and, ABC88012.1, (See, for example, Zhong et al. Human Molecular Genetics, 2006, Vol. 15, No. 9, 1497-1512, which is incorporated herein by reference).
As used herein, “fragments,” “analogues” or “derivatives” of the polypeptides/nucleotides described include those polypeptides/nucleotides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and which may be natural or unnatural. In one embodiment, variant, derivatives and analogues of polypeptides/nucleotides will have about 70% identity with those sequences described herein. That is, 70% of the residues are the same. In a further embodiment, polypeptides/nucleotides will have greater than 75% identity. In a further embodiment, polypeptides/nucleotides will have greater than 80% identity. In a further embodiment, polypeptides/nucleotides will have greater than 85% identity. In a further embodiment, polypeptides, nucleotides will have greater than 90% identity. In a further embodiment, polypeptides/nucleotides will have greater than 95% identity. In a further embodiment, polypeptides/nucleotides will have greater than 99% identity.
“Sequence Identity” as it is known in the art refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are “identical” at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993): Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the disclosures of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12:387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215:403 (1990)). The BLASTX program is publicly available from NCBI and other sources {BLAST Manual, Altschul, S. et al, NCVI NLM NIH Bethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403 (1990), the disclosures of which are incorporated herein by reference}. These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% “sequence identity” to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 95% identity relative to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 95% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 95% sequence identity with a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence. Preferably, residue positions that are not identical differ by conservative amino acid substitutions.
General methods regarding polynucleotides and polypeptides are described in: Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y. 1989; Current Protocols in Molecular Biology, edited by Ausubel F. M. et al., John Wiley and Sons, Inc. New York; PCR Cloning Protocols, from Molecular Cloning to Genetic Engineering, Edited by White B. A., Humana Press, Totowa, N.J., 1997, 490 pages; Protein Purification, Principles and Practices, Scopes R. K., Springer-Verlag, New York, 3rd Edition, 1993, 380 pages; Current Protocols in Immunology, edited by Coligan J. E. et al., John Wiley & Sons Inc., New York, which are herein incorporated by reference.
In accordance with one embodiment a novel compound that inhibits Ca2+ entry, and thereby proliferation of cancer cells is provided. The compounds have the general structure:
wherein
R1 is selected from the group consisting of C1-C4 alkyl, hydroxy and C1-C1 alkoxy;
X is selected from the group consisting of N and CH;
Z is selected from the group consisting of NH, O, S and C2;
R2 is selected from the group consisting of H, halo, NH2, C1-C4 alkyl, hydroxy and C1-C4 alkoxy; and R3 is selected from the group consisting of H, halo, NH2, C1-C4 alkyl, hydroxy and C1-C4 alkoxy. In one embodiment R1 is selected from the group consisting of C1-C4 alkyl, hydroxy and C1-C4 alkoxy, X is N, Z is O or CH2, R2 is H, halo, NH2 or hydroxy and R3 is H.
The novel compounds of the present invention can be combined with standard pharmaceutically acceptable carriers or other known anti-tumor and chemotherapeutic agents.
TH-1177 was synthesized in three simple steps as described (Haverstick, D. M., Heady, T, N., Macdonald, T. L., and Gray, L. S. 2000. Inhibition of human prostate cancer proliferation in vitro and in a mouse model by a compound synthesized to block Ca2+ entry. Cancer Res 60:1002-1008) L-Proline methyl ester was coupled with 4-methoxyphenylacetic acid using benzotriazol 1-yl-oxytripyrrolidinephosphonium to generate methyl 1-[2-(4 methoxyphenyl)acetyl]pyrrolidine-2-carboxylate, a yellowish oil. The resulting amide was subsequently reduced to the amino alcohol with LiAlH4 and AlCl3 in tetrahydrofuran. The resulting colorless oil was coupled with 4-chlorobenzhydrol under Williamson conditions with catalytic p-toluenesulfonic acid hi refluxing toluene. The final brownish oil was isolated by column chromatography, and its structure was confirmed by nuclear magnetic resonance and mass spectrometry. TH-1177 was dissolved in DMSO for use.
Cancer cell lines were obtained from the American Type Culture Collection (Manassas, Va.). Cell lines were maintained in RPMII 640 supplemented with glutamine and 5% fetal bovine serum containing SerXtend (Irvine Scientific). The fetal bovine serum used for culture was heat-inactivated by maintaining the serum at 56° C. for 1 h.
Cells were incubated in growth media containing 1 uM of the acetoxy-methyl ester of the Ca2+-sensitive fluorescent dye indo-1 (indo-1/AM; Molecular Probes, Eugene, Oreg.) for 1 h at 37° C. Cells were washed three times in buffer A [10 mM HEPES (pH 7.4), 1 mM MgCl2. 3 mM KCl, 1 mM CaCl2, 140 mM NaCl, 0.1% glucose, and 1% fetal bovine serum] and suspended to a final concentration of 106 cells/mi. Before stimulation, cells were warmed to 37° C. Changes in [Ca2+]i were monitored in an SLM 8100C spectrofluorometer (SLM/Aninco; Urbana, Ill.) using previously published methods (Densmore, J. J., Haverstick, D. M., Szabo, G., and Gray, L. S. 1996. A voltage operable current is involved in activation-induced Ca2+ entry in human lymphocytes whereas ICRAC has no apparent role. Am. J. Physiol. 271:C149-C1503; Haverstick, D. M., Densmore, J. J., and Gray, L. S. 1998. Calmnodulin regulation of Ca2+ entry in Jurkat T cells. Cell Calcium 23:361-368).
LNCaP cells at 2.5×104 cells/well or PC-3 cells at 5×104 cells/well, both in a final volume of 100 IJI, were plated in triplicate in standard flat-bottomed 96-well tissue culture plates in the presence of drug or vehicle (DMSO). Unless otherwise indicated, cells were grown for 48 h at 37° C. in a CO2 incubator. Relative cell growth was determined with the CellTiter 96 aqueous cell proliferation assay (Promega, Madison, Wis.) as described by the manufacturer using an automated plate reader. Results were calculated in a blinded fashion and are the means of triplicate determinations.
Extracellular Ni2+ blocks the Ca2+ entry pathway in electrically non-excitable cells (Merritt, J. E., Jacob, R., and Hallam, T. J. 1989. Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils. J. Biol. Chem. 264:1522-1527; Jones, G. R. N. 1985. Cancer therapy: Phenothiazines in an unexpected role. Tumori 71:563-569) as well as the current through T type Ca2+ channels (Lee, J.-H. Gomora, J. C, Cribbs, L. L., and Perez-Reyes, E. 2000. Nickel block of three cloned T-type Ca channels: low concentrations selectively block α1H. Biophys. J. 77:3042). We made use of these facts and conducted a search of the Medline database for compounds that block Ca2+ entry in any system that was also sensitive to inhibition of Ca2+ entry by Ni2+. The identified compounds were then used as the basis for a reiterated search. This strategy was continued until the only citations returned were those that had been retrieved already indicating that the database had been saturated. These agents, some of which are listed in Table 1, were tested for the ability to block proliferation of and Ca2+ entry into the Jurkat human cancer cell line. These compounds were tested in various cancer cell lines (Materials and Methods) with results similar to those obtained with the Jurkat cell line (data not shown). The correlation between these two inhibitory activities in the Jurkat cell line, expressed as ICSO's, is shown in
The Ca2+ ionophore ionomycin partially overcomes the effects of TH-1177. We have used one of our compounds, TH-1177, as the prototype for the others (Haverstick, D. M., Heady, T. N., Macdonald, T. L., and (Gray, L. S. 2000. Inhibition of human prostate cancer prolilferation in vitro and in a mouse model by a compound synthesized to block Ca2+ entry, Cancer Res 60: 1002-1008), If TH-1177 is acting via inhibition of Ca2+ entry, its effects should be at least partially reversed by direct elevation of [Ca2+]i using a Ca2+ ionophore. As shown in
Cancer cell lines sensitive to our agents express message for the α1H Ca2+ channel or its splice variants including its delta25 splice variant.
We have presented data previously suggesting that a member or members of the T type Ca2+ channel family have a role in mediating Ca2+ entry in electrically excitable cells (Densrnore, J. J., Haverstick, D. M., Szabo, G., and Gray, L. S. 1996. A voltage operable current is involved in activation-induced Ca2+ entry in human lymphocytes whereas ICRAC has no apparent role. Am. J. Physiol. 271:C1494-C1503; Densmore, J. J., Szabo, G., and Gray, L. S. 1992. A voltage-gated calcium channel is linked to the antigen receptor in Jurkat T lymphocytes. FEBS Lett. 312:161-164; Haverstick, D. M. and Gray, L. S. Increased intracellular Ca2+ induces Ca2+ influx in human T lymphocytes. Molecular Biology of the Cell 4, 173-184. 1993; Haverstick, D. M., Densmore, J. J., and Gray, L. S. 1998. Calmodulin regulation of Ca2+ entry in Jurkat T cells. Cell Calcium 23:361-368). It has been shown recently that a prostate cancer line expresses the α1H isoform of T type Ca2+ channels at levels that vary with differentiation status (Mariot, P., Vanoverberghe, K., Lalevee, N., Rossier, M. F., and Prevarskaya, N. 2002. Overexpression of an alpha 1H (Cav3.2) T-type calcium channel during neuroendocrine differentiation of human prostate cancer cells. J Biol. Chem 277:10824-10833). Using the same primers, we identified two different amplicons in cancer cell lines (
Diasteromers TH-1177 and TH-1211 inhibit proliferation of PC3 prostate cancer cells and block α1H with the same stereoselectively.
TH-1177 has two chiral centers and TH-1211 is its stereoisomer about one of them (
We have shown here the possibility that the α1H isoform of T type Ca2+ channels or its splice variants including its δ25 splice variant has a role in Ca2+ entry into and proliferation of electrically non-excitable cells. Our data show that novel compounds can be created based upon an SAR generated from compounds that are known to inhibit Ca2+ entry in systems that are also sensitive to Ni2+. importantly, inhibition of proliferation of several cancer cell lines by these novel compounds is most likely via blockade of Ca2+ entry. The same cell lines that are sensitive to our agents express message for α1H Ca2+ channels, its δ25 splice variant, or both. The compounds were shown to inhibit the Ca2+ current mediated by α1H Ca2+ channels. TH-1177 and TH-1211 stereoselectively inhibit Ca2+ entry into and proliferation of cancer cell lines and show the same stereoselective block of canonical α1H. These data strongly suggest that the α1H Ca2+ channel and its splice variants, including its δ25 splice variant participate in Ca2+ entry in the cancer cell lines tested in these studies.
Linking biophysical analysis of Ca2+ channel function to a physiological function such as proliferation can pose challenges. We have demonstrated that our compounds block a heterologously expressed Ca2+ channel and that only those cancer cell lines with message for that channel, or its splice variants, are sensitive to inhibition by the same agents. Furthermore, TH-1177 is more potent at inhibiting Ca2+ entry via expressed α1H as measured by biophysical techniques than the stereoisomer of it, TH-1211. TH-1177 and TH-1211 also show the same rank order of potency at inhibiting proliferation and Ca2+ entry in cancer cell lines when these are assayed by more commonly used biochemical methods. The absolute potencies of the agents as measured by IC50 values are strikingly similar whether measured by biophysical or biochemical methods. Thus, the results from a combination of experimental approaches were synthesized into a picture of the likely mechanism of Ca+ entry in some cancer cells.
Expression of the α1H Ca2+ channel has been demonstrated in LNCaP cells and the expression level correlates with differentiation state (Mariot, J P., Vanoverberghe, K., Lalevee, N., Rossier, M, F., and Prevarskaya, N. 2002, Overexpression of an alpha 1H (Cav3.2) T-type calcium channel during neuroendocrine differentiation of human prostate cancer cells. J Biol. Chem 277:10824-10833). Although the sequence of the δ25 splice variant has been deposited in GenBank (accession number AF223563), its function has not been described to our knowledge. However, both are members of the T type Ca2+ channel family by sequence homology and have been assigned to the Hs.122359 UniGene cluster within the NCBI database. The physiological roles of T type Ca2+ channels are not wholly clear at present although they may playa role as pacemakers in the heart and central nervous system (Chemin, J., Monteil, A., Perez-Reyes, E., Bourinet, E., Nargeot, J., and Lory, P. 2002, Specific contribution of human T-type calcium channel isotypes (α1G, α1H and α1I) to neuronal excitability. J. Physiol. (Lond.) 540:3-14; McDonald, T. F., Pelzer, S., Trautwein, W., and Pelzer, D. J. 1994. Regulation and modulation of calcium channels in cardiac, skeletal, and smooth muscle cells. Physiol Rev 74:365-507. The expression of these Ca2+ channels also appears to be developmentally regulated (Brooks, G., Harper, J. V., Bates, S. E., Haworth, R. S., Cribbs, L. L., Perez-Reyes, E., and Shattock, M J. 1999. Over expression of the voltage-gated T-type calcium channel induces vascular smooth muscle cell proliferation. Circulation 100:1-209 (Abstr.); Clozel, J. P., Ertel, E. A., and Ertel, S. I. 1999. Voltage-gated T-type Ca2+ channels and heart failure. Proc. Assoc. Am Physicians 111:429-437; Harper, J. V., McLatchie, L. Perez-Reyes, E., Cribbs, L. L., Shattock, M J., and Brooks, G. 2000. T-type calcium channel expression is necessary for G1-S progression in vascular smooth muscle, Circulation 102:11-48 (Abstr.); Monteil, A., Chemin. J., Bourinet, E., Mennessier, G., Lory, P., and Nargeot, J. 2000. Molecular and functional properties of the human α1G subunit that forms T-type calcium channels, J Biol. Chem. 275:6090-6100) and the data reported here suggest that both canonical α1H and its splice variants, including its δ25 splice variant are responsible for the Ca2+ entry required for proliferation of some cancer cell lines.
The presently described synthetic compounds may have clinical utility because treatment with TH-1177 of mice bearing xenografted human PC3 prostate cancer cells significantly extended the lifespan of them (Haverstick, D. M., Heady, T. N., Macdonald, T. L., and Gray, L. S. 2000. Inhibition of human prostate cancer proliferation in vitro and in a mouse model by a compound synthesized to block Ca2+ entry. Cancer Res 60: 1002-1008). Thus, Ca2+ channel entry inhibitors will provide clinicians with an addition to their armamentarium for the treatment of cancer.
This application is a continuation-in-part of U.S. Application Ser. No. 12/687,641 filed Jan. 14, 2011 which is a divisional of U.S. application Ser. No. 11/660,401, filed Sep. 11, 2011, now abandoned, which is a U.S. National Stage Application of PCT/US2005/029851, filed Aug. 22, 2005, which claims priority under 35 U.S.C. 119(e) from U.S. Provisional Application Ser. No. 60/603,159 filed Aug. 20, 2004, which applications are herein incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60603159 | Aug 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11660401 | Sep 2007 | US |
| Child | 12687641 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12687641 | Jan 2010 | US |
| Child | 13267609 | US |
