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WO2005076958A2 - Procedes de determination du pronostic et du traitement de sujets atteints de troubles lies a pin 1 - Google Patents

Procedes de determination du pronostic et du traitement de sujets atteints de troubles lies a pin 1 Download PDF

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Publication number
WO2005076958A2
WO2005076958A2 PCT/US2005/003703 US2005003703W WO2005076958A2 WO 2005076958 A2 WO2005076958 A2 WO 2005076958A2 US 2005003703 W US2005003703 W US 2005003703W WO 2005076958 A2 WO2005076958 A2 WO 2005076958A2
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Prior art keywords
pinl
cancer
subject
carcinoma
adenocarcinoma
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WO2005076958A3 (fr
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Janusz M. Sowadski
Walter Gilbert
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Pintex Pharmaceuticals Inc
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Pintex Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes

Definitions

  • PPIases peptidyl-prolyl cis-trans isomerases
  • rotamases peptidyl-prolyl cis-trans isomerases
  • PPIases are a family of ubiquitous enzymes that catalyze the cis/trans isomerization of the peptide bond on the n-terminal side of proline residues in proteins (Hunter, Cell 92:141-142, 1998).
  • PPIases are divided into three classes, cyclophilins (Cyps), FK-506 binding proteins (FKBPs) and the Pinl/parvulin class.
  • Pinl is a highly conserved protein that catalyzes the isomerization of only phosphorylated Ser/Thr-Pro bonds (Ranathan, R.
  • Pinl contains anN-terminal WW domain, which functions as a phosphorylated Ser/Thr-Pro binding module (Sudol, M. (1996) Prog. Biophys. Mol. Biol.
  • the instant invention provides methods to determine which subjects in a population would benefit from treatment with a Pinl modulator.
  • the instant invention also provides, in at least one embodiment, methods for determining the prognosis of a subject with a cell proliferative disorder.
  • the invention also provides methods to monitor the efficacy of treatment of individuals with a Pinl associated disorder. Further, kits are provided to perform the methods of the invention.
  • the invention provides a method for treating a subject that has been preselected based on unphosphorylated Pinl levels by administering a Pinl modulator to the Pinl subject such that the treatment of said subject occurs.
  • the subject has high levels of cytoplasmic Pinl .
  • the subject has a Pinl associated state.
  • the Pinl associated state is a cell proliferative disorder.
  • the cell proliferative disorder can be cancer, e.g., a cancer selected from the group consisting of oligodendroglioma, astrocytoma, glioblastomamultiforme, cervical carcinoma, endometriod carcinoma, endometrium serous carcenoma, ovary endometroid cancer, ovary Brenner tumor, ovary mucinous cancer, ovary serous cancer, uterus carcinosarcoma, breast, breast lobular cancer, breast ductal cancer, breast medullary cancer, breast mucinous cancer, breast tubular cancer, thyroid adenocarcinoma, thyroid follicular cancer, thyroid medullary cancer, thyroid papillary carcinoma, parathyroid adenocarcinoma, adrenal gland adenoma, adrenal gland cancer, pheochromocytoma, colon, colon adenoma mild displasia, colon adenoma moderate displasia, colon adenoma severe displasia, colon
  • the cancer is selected from the group consisting of breast, lung, prostate, and colon cancer.
  • the invention provides a method of treating a subject preselected based on their levels of Pinl, wherein said subject is selected based on the levels of phosphorylated Pinl and unphosphorylated Pinl, comprising administering a Pinl modulator to the subject such that the treatment of the subject occurs.
  • the levels of Pinl are determined by measuring the level of phosphorylated and unphosphorylated Pinl using immunohistochemistry.
  • the immunohistochemistry is performed using antibodies specific for phosphorylated and unphosphorylated Pinl .
  • the Pinl associated state is a cell proliferative disorder, e.g., cancer.
  • the cancer is selected from the group consisting of breast, lung, prostate, and colon cancer.
  • the invention provides a method of determining the Pinl status of a subject comprising determining the level and phosphorylation state of Pinl within a sample obtained from said subject.
  • the level of phosphorylated and unphosphorylated Pinl are determined, e.g., by immunohistochemistry.
  • the subject has a Pinl associated state.
  • the Pinl associated state is a cell proliferative disorder, e.g., cancer.
  • the cancer is selected from the group consisting of breast, lung, prostate, and colon cancer.
  • the subject has a neurodegenerative disease, e.g., Alzheimer's disease.
  • the subject is classified into a one of the following groups based on the level, location and phosphorylation state of Pinl : strong nuclear Pinl and negative cytoplasmic Pinl (nPinlStr-cPinlneg), strong nuclear Pinl and positive cytoplasmic Pinl (nPinlStr-cPinlpos), weak nuclear Pinl and negative cytoplasmic Pinl (nPinl Weak-cPinlneg), weak nuclear Pinl and positive cytoplasmic Pinl (nPinl Weak-cPinlpos), negative nuclear Pinl and negative cytoplasmic Pinl(nPinlNeg-cPinlneg), or negative nuclear Pinl and positive cytoplasmic Pinl (nPinlNeg-cPinlpos).
  • the invention provides a method of treating a subject having a Pinl associated state wherein the treatment is determined based on the subject's classification as nPinlStr-cPinlneg, nPinlStr-cPinlpos, nPinlWeak-cPinlneg, nPinl Weak-cPinlpos, nPinlNeg-cPinlneg, or nPinlNeg-cPinlpos.
  • the treatment comprises the administration of a Pinl modulator, and optionally, and one or more other anti-cancer treatments.
  • the invention provides a method of monitoring the efficacy of treatment of a subject by determining the levels of phosphorylated and unphosphorylated Pinl after the administration of a Pinl modulator to the subject; wherein a change in the ratio of phosphorylated to unphosphorylated Pinl is indicative of the efficacy of treatment.
  • the invention provides a kit for selecting a subject that would benefit from treatment with a Pinl modulator including a phosphorylated Pinl specific antibody and/or an unphosphorylated Pinl specific antibody, and directions for use.
  • the invention provides a method for determining the prognosis of a subject having a cell proliferative disorder by determining the levels of pPinl in a biological sample, wherein an elevated level of pPinl in the sample compared to the statistical mean of a population having a cell proliferative disorder is indicative of a good prognosis.
  • the invention provides a method for determining the prognosis of a subject having a cell proliferative disorder by determining the levels of pPinl in a biological sample, wherein an decreased level of pPinl in the sample compared to the statistical mean of a population having a cell proliferative disorder is indicative of a poor prognosis.
  • the biological sample is selected from the group consisting of breast, uterus, ovaries, brain, endometrium, cervix, colon, esophagus, hepatocellular, kidney, mouth, prostate, liver, lung, skin, testicles, endocrine tissue, e.g., thyroid, blood, ascites and brain fluid.
  • the sample is breast tissue.
  • the pPinl levels are determined using an antibody specific for pPinl.
  • the levels of pPinl are determined using a pPinl specific antibody using fluorescence in situ hybridization (FISH).
  • the levels of pPinl are determined using a pPinl specific antibody using immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the invention provides a method of determining the prognosis of a subject having a cell proliferative disorder by obtaining a first biological sample from the subject and determining the level of pPinl in the sample, then obtaining a second biological sample from the subject at a time after collection of the first biological sample and determining the level of pPinl in the sample, wherein an increase in the level of pPinl is indicative of good prognosis.
  • the invention provides a method of determining the prognosis of a subject having a cell proliferative disorder by obtaining a first biological sample from the subject and determining the level of pPinl in the sample, then obtaining a second biological sample from the subject at a time after collection of the first biological sample and determining the level of pPinl in the sample, wherein a decrease in the level of pPinl is indicative of poor prognosis.
  • the invention provides a kit for determining the prognosis of a subject with a cell proliferative disorder comprising an antibody specific for pPinl and instructions for use.
  • the antibody enclosed in the kit is a monoclonal or a polyclonal antibody.
  • the kit can contain a second antibody specific for a second cancer marker.
  • the invention provides a method for treating an individual having a cell proliferative disorder comprising determining the level of pPinl present in a biological sample, wherein the lower the level of pPinl the more aggressive the treatment of said subject with a Pinl inhibitor.
  • Figure 1 depicts the polypeptide sequence of Pinl (SEQ ID NO:l).
  • the serine residue identified in Lu et al. ((2002) J Biol Chem. 277: 2381-4) is underlined and in bold.
  • Figure 2 is a pie chart depicting the percentage of subjects that are positive for unphosphorylated Pinl and the percentage of subjects that are negative for unphosphorylated Pinl. The methods presented in Example 1 were used to determine that 50% of the subjects are positive for unphosphorylated Pinl and 50% of the subjects are negative for unphosphorylated Pinl .
  • Figure 3 is a pie chart depicting the percentage of subjects classified as having strong, weak and negative levels of phosphorylated Pinl.
  • the methods presented in Example 1 were used to determine that 36% of the subjects are classified as having strong levels of phosphorylated Pinl, 33% of the subjects were classified as having weak levels of phosphorylated Pinl, and 31% of the subjects were classified as being negative for phosphorylated Pinl expression.
  • Figure 4 is a pie chart classifying the test population based on the presence and level of phosphorylated and unphosphorylated Pinl .
  • the pie chart depicts six classifications of subjects. The six classifications are Pin lneg-pPinl Strong (9%), Pinl neg-pPinl Weak (15%), Pinlneg-pPinlNeg (26%), Pin lpos-pPinl Strong (27%), Pinlpos-pPinWeak (19%) and Pinlpos-pPinlNeg (4%). Levels of Pinl and pPinl were determined as described in Example 1 using antibodies that preferentially bind unphosphorylated and phosphorylated Pinl, respectively.
  • Figure 5 depicts survival curves based on Pinl nuclear staining determined by immunohistochemistry. The graph depicts fraction of subjects surviving as a function of time (in months). Nuclear Pinl levels are divided into strong, moderate, weak and negative levels.
  • Figure 6 depicts survival curves based on phosphorylated Pinl.
  • the graph depicts the fraction of subjects surviving as a function of time (in months). Nuclear Pinl levels are divided into strong, moderate, weak and negative levels.
  • Figure 7 depicts a bar graph indicating the fraction of pPinl expression as a function of Pinl level.
  • Figure 8 depicts a bar graph indicating the fraction of Her2 positive samples that show Pinl nuclear staining.
  • Figure 9 depicts survival curves based on Her2 status and nuclear Pinl levels. Her2 status was determined by immunohistochemistry. The graph depicts the fraction of subjects surviving as a function of time (in months). Subjects are divided based on nuclear Pinl levels being strong or negative, and Her2 levels being negative and positive.
  • Figure 10 depicts the fraction of Her2 amplified samples as a function of the level of nuclear Pinl staining.
  • Figure 11 depicts survival curves based on Her2 status and nuclear Pinl levels.
  • Her2 status was determined by FISH.
  • the graph depicts the fraction of subjects surviving as a function of time (in months). Subjects are classified based on nuclear Pinl levels being strong or negative, and Her2 levels being negative or positive.
  • Figure 12 depicts the fraction of subjects that are estrogen receptor (ER) positive as a function of nuclear Pinl level.
  • Figure 13 depicts survival curves based on ER status and nuclear Pinl levels. The graph depicts the fraction of subjects surviving as a function of time (in months). Subjects are classified based on nuclear Pinl levels being strong or negative, and ER levels being negative or positive.
  • ER estrogen receptor
  • Figure 14 depicts the fraction of subjects that are progesterone receptor (PR) positive as a function of nuclear Pinl level.
  • PR progesterone receptor
  • Figure 15 depicts survival curves based on PR status and nuclear Pinl levels.
  • the graph depicts the fraction of subjects surviving as a function of time (in months). Subjects are classified based on nuclear Pinl levels being strong or negative, and ER levels being negative or positive.
  • Figure 16 depicts the fraction of subjects that are epidermal growth factor receptor (EGFR) positive as a function of nuclear Pinl level.
  • EGFR epidermal growth factor receptor
  • Figure 77 depicts survival curves based on EGFR status and Pinl levels. The graph depicts the fraction of subjects surviving as a function of time (in months). Subjects are classified based on nuclear Pinl levels being strong or negative, and EGRF levels being negative or positive.
  • Figure 18 depicts the fraction of subjects that are cyclin Dl (CCND1) positive as a function of nuclear Pinl level.
  • Figure 19 depicts survival curves based on CCND1 status and Pinl level.
  • the graph depicts the fraction of subjects surviving as a function of time (in months). Subjects are classified based on nuclear Pinl levels being strong or negative, and CCNDI level being negative or positive.
  • biological sample includes solid and body fluid samples.
  • the biological samples of the present invention may include cells, protein or membrane extracts of cells, blood or biological fluids such as ascites fluid or brain fluid (e.g., cerebrospinal fluid).
  • solid biological samples include samples taken from feces, the rectum, central nervous system, bone, breast tissue, renal tissue, the uterine cervix, the endometrium, the head/neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, and the thymus.
  • body fluid samples include samples taken from the blood, serum, cerebrospinal fluid, semen, prostate fluid, seminal fluid, urine, saliva, sputum, mucus, bone marrow, lymph, and tears. Samples for use in the methods of the invention can be obtained by standard methods including venous puncture and surgical biopsy. In certain embodiments, the biological sample is a breast, lung, colon, or prostate tissue sample obtained by needle biopsy.
  • Pinl status indicates a classification of a subject into one of a defined series of groups based on the presence and level of phosphorylated and unphosphorylated Pinl .
  • Exemplary classifications of Pinl status defined herein are: Pinlneg-pPinl Strong, Pin lneg-pPinl Weak Pinlneg-pPinlNeg, Pinlpos-pPinl Strong, Pinlpos-pPinWeak, and Pinlpos-pPinlNeg.
  • Pinl status can be represented as ratio of phosphorylated Pinl to unphosphorylated Pinl .
  • the presence and level of Pinl can be determined using art recognized techniques, e.g., immunohistochemistry using Pinl specific antibodies. Subjects can be treated for Pinl- associated states based on their Pinl status.
  • nuclear Pinl is intended to include Pinl polypeptide that is localized to the nucleus of a cell. In certain embodiments, nuclear Pinl is predominantly phosphorylated.
  • cytoplasmic Pinl is intended to include Pinl polypeptide that is localized to the cytoplasm of a cell. In certain embodiments, cytoplasmic Pinl is predominantly unphosphorylated.
  • phosphorylation state is intended to denote that the Pinl polypeptide can exist in either a phosphorylated or unphosphorylated state. The phosphorylation state denotes whether the Pinl in a biological sample is phosphorylated or unphosphorylated, or the relative ratios of phosphorylated to unphosphorylated Pinl in a sample.
  • Pinl -associated state or "Pinl associated disorder” includes disorders and states (e.g., a disease state) that are associated with the abnormal activity of Pinl .
  • This abnormal activity can be as a result of the misexpression of Pinl or the misregulation of the production, degradation, or regulation of Pinl, e.g., the phosphorylation/ dephosphorylation of Pinl .
  • Pinl associated disorders that are related to higher than necessary levels of Pinl can be caused by (1) an increase in the level of transcription or translation, or a decrease in the level of degradation, of Pinl such that an abnormally high amount of Pinl polypeptide is present in a cell, or (2) the amount Pinl that is present in the unphosphorylated, i.e., active form, is abnormally high due to either an increase in the dephosphorylation of Pinl or a decrease in the phosphorylation of Pinl .
  • Pinl disorders are often associated with abnormal cell growth, abnormal cell proliferation, or misexpression of Pinl (e.g., Pinl protein or nucleic acid).
  • Pinl -associated states include states resulting from an elevation in the expression of cyclin Dl and/or Pinl.
  • Pinl -associated states also include states resulting from an elevation in the phosphorylation level of c-Jun, particularly phosphorylation of c-Jun on Ser 63/73 -Pro and/or from an elevation in the level of c-Jun amino terminal kinases (JNKs) present in a cell.
  • Pinl -associated states include neoplasia, cancer, undesirable cell growth, and/or tumor growth.
  • Pinl -associated states include states caused by DNA damage, an oncogenic protein (i.e. Ha-Ras), loss of or reduced expression of a tumor suppressor (i.e. Brcal), and/or growth factors.
  • Pinl- associated state is also intended to include diseases or disorders caused by, or associated with, deregulation of genes and/or gene products involved in a biological pathway that includes Pinl and/or cyclin Dl (e.g. beta-catenin, APC or WNT). Beta-catenin, APC and WNT have been linked to cancer development as demonstrated in Biochim Biophys Acta. (2003) 1653: 1-24 and Eur J Surg Oncol. (2003) 29: 107-117.
  • Pinl associated states further include disorders and states associated with regulation or activity of Pinl in the brain, e.g., Alzheimer's disease, wherein the phosphorylation state of tau is influenced by the activity of Pinl .
  • the term "misexpression" includes a non- wild type pattern of gene expression.
  • Expression as used herein includes transcriptional, post transcriptional, e.g., mRNA stability, translational, and post translational stages. Misexpression includes: expression at non- wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the
  • Misexpression includes any expression from a transgenic nucleic acid. Misexpression includes the lack or non-expression of a gene or transgene, e.g., that can be caused by a deletion of all or part of the gene or its control sequences.
  • the term "carcinoma” includes malignancies of epithelial or endocrine tissues, including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostate carcinomas, endocrine system carcinomas, melanomas, choriocarcinoma, and carcinomas of the cervix, lung, head and neck, colon, and ovary.
  • carcinoma also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • adenocarcinoma includes carcinomas derived from glandular tissue or a tumor in which the tumor cells form recognizable glandular structures.
  • sarcoma includes malignant tumors of mesodermal connective tissue, e.g., tumors of bone, fat, and cartilage.
  • leukemia and lymphoma include malignancies of the hematopoietic cells of the bone manow. Leukemias tend to proliferate as single cells, whereas lymphomas tend to proliferate as solid tumor masses.
  • leukemias include acute myeloid leukemia (AML), acute promyelocytic leukemia, chronic myelogenous leukemia, mixed-lineage leukemia, acute monoblastic leukemia, acute lymphoblastic leukemia, acute non-lymphoblastic leukemia, blastic mantle cell leukemia, myelodyplastic syndrome, T cell leukemia, B cell leukemia, and chronic lymphocytic leukemia.
  • AML acute myeloid leukemia
  • AML acute promyelocytic leukemia
  • chronic myelogenous leukemia mixed-lineage leukemia
  • acute monoblastic leukemia acute lymphoblastic leukemia
  • acute non-lymphoblastic leukemia acute non-lymphoblastic leukemia
  • blastic mantle cell leukemia myelodyplastic syndrome
  • T cell leukemia B cell leukemia
  • chronic lymphocytic leukemia chronic lymphocytic leukemia
  • lymphomas examples include Hodgkin's disease, non- Hodgkin's lymphoma, B cell lymphoma, epitheliotropic lymphoma, composite lymphoma, anaplastic large cell lymphoma, gastric and non-gastric mucosa-associated lymphoid tissue lymphoma, lymphoproliferative disease, T cell lymphoma, Burkitt's lymphoma, mantle cell lymphoma, diffuse large cell lymphoma, lymphoplasmacytoid lymphoma, and multiple myeloma.
  • the therapeutic methods of the present invention can be applied to cancerous cells of mesenchymal origin, such as those producing sarcomas (e.g., f ⁇ brosarcoma, myxosarcoma, liosarcoma, chondrosarcoma, osteogenic sarcoma or chordosarcoma, angiosarcoma, endotheliosardcoma, lympangiosarcoma, synoviosarcoma or mesothelisosarcoma); leukemias and lymphomas such as granulocytic leukemia, monocytic leukemia, lymphocytic leukemia, malignant lymphoma, plasmocytoma, reticulum cell sarcoma, or Hodgkin's disease; sarcomas such as leiomysarcoma or rhabdomysarcoma, tumors of epithelial origin such as squamous cell carcinoma, basal cell
  • Additional cell types amenable to treatment according to the methods described herein include those giving rise to mammary carcinomas, gastrointestinal carcinoma, such as colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region.
  • cancers amenable to treatment according to the methods described herein include vaginal, cervical, and breast cancers.
  • the language "inhibiting undesirable cell growth” is intended to include the inhibition of undesirable or inappropriate cell growth.
  • the inhibition is intended to include inhibition of cell proliferation, including rapid proliferation. For example, undesirable cell growth can result in benign masses or malignant tumors.
  • Examples of benign conditions which result from inappropriate cell growth or angiogenesis are diabetic retinopathy, retrolental fibrioplasia, neovascular glaucoma, psoriasis, angiofibromas, rheumatoid arthritis, hemangiomas, Karposi's sarcoma, and other conditions or dysfunctions characterized by dysregulated endothelial cell division.
  • the language "inhibiting tumor growth” or “inhibiting neoplasia” includes the prevention of the growth of a tumor in a subject or a reduction in the growth of a preexisting tumor in a subject, or can be the inhibition of the metastasis of a tumor from one site to another.
  • tumor is intended to encompass both in vitro, e.g., in cell culture, and in vivo tumors that form in any organ or body part of the subject.
  • the tumors preferably are tumors sensitive to the Pinl -modulating compounds of the present invention.
  • Examples of the types of tumors intended to be encompassed by the present invention include those tumors associated with breast cancer, skin cancer, bone cancer, prostate cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, gallbladder, esophagus, pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys.
  • the tumors whose growth rate is inhibited by the present invention include basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small- cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuromas, intestinal ganglloneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion,
  • carcinomas include carcinomas, sarcomas, leukemias, and lymphomas.
  • cancer and “tumor” may be used interchangeably herein.
  • cancer includes primary malignant tumors, e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor, and secondary malignant tumors, e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor.
  • the cancer is one of the numerous forms of breast cancer, e.g., ductal carcinoma, lobular carcinoma, medullary carcinoma, tubular carcinoma, cribriform carcinoma, mucinous carcinoma, papillary carcinoma, apocrine carcinoma, clear cell carcinoma, apocrine metaplasia, intraductal hyperplasia, atypical intraductal hyperplasia, mastopathy, papillloma or sclerosing adenosis.
  • Pinl modulating agents may be used to treat, inhibit, and/or prevent undesirable cell growth, neoplasia, neurodegenerative diseases, and/or cancer in a subject.
  • Pinl modulating compounds may be used to inhibit Pinl activity in a subject.
  • Pinl modulating compounds may be used to inhibit cyclin Dl expression in a subject.
  • Pinl modulating compounds of the invention can be used to treat subjects having Alzheimer's disease.
  • Pinl modulators have a Ki for Pinl of less than 0.2mM, less than O.lmM, less than 750 ⁇ M, less than 500 ⁇ M, less than 250 ⁇ M, less than 100 ⁇ M, less than 50 ⁇ M, less than 500 nM, less than 250nM, less than 50 nM, less than 10 nM, less than 5 nM, or less than 2 nM.
  • Pinl modulating compound refers to compounds that modulate, e.g., inhibit, promote, or otherwise alter, the activity of Pinl.
  • Pinl modulating compounds include both Pinl agonists and antagonists.
  • the Pinl modulating compounds include compounds that interact with the peptidyl prolyl isomerase domain (PPI) and/or the WW domain of Pinl .
  • the Pinl modulating compound is substantially specific to Pinl.
  • Pinl substantially specific for Pinl
  • inhibitors of the invention that have a Kj or K d that is at least 2, 3, 4, 5, 10, 15, or 20 times less than the Kj or K d for other peptidyl prolyl isomerases, e.g., hCyP-A, hCyP-B, hCyP-C, NKCA, hFKBP-12, hFKBP-13, and hFKBP-25.
  • Examples of Pinl modulating compounds include compounds described in U.S. Provisional Application No. 60/537,171, filed January 16, 2004, entitled "Pinl- Modulating Compounds and Methods of Use Thereof; U.S. Application No.
  • the Pinl inhibiting compounds include compounds that interact with the PPI and/or the WW domain of Pinl .
  • preselected is intended to mean that a subject has been identified based on their level and/or phosphorylation state of Pinl to be likely to benefit from treatment with a Pinl modulator. In certain embodiments, a subject is preselected based on the levels of unphosphorylated Pinl, phosphorylated Pinl, or the relative amounts of phosphorylated and unphosphorylated Pinl .
  • prognosis is intended to mean the probable course and outcome of a disease, e.g., a Pinl associated disease. In certain embodiments, the term is intended to mean the likelihood that a subject will live longer than the average length of time that a population of subjects with a similar disease will live. In related embodiments, a subject's prognosis is indicative of the aggressiveness and course of treatment that a subject will receive.
  • Pinl is a highly conserved protein (SEQ ID NO: 1) that catalyzes the isomerization of only phosphorylated Ser/Thr-Pro bonds (Rananathan, R. et al. (1997) Cell 89:875-86; Yaffe, et al. 1997, Science 278:1957- 1960; Shen, et al. 1998,Genes Dev. 12:706-720; Lu, et al. 1999, Science 283:1325- 1328; Crenshaw, et al. 1998, Embo J. 17:1315-1327; Lu, et al.
  • SEQ ID NO: 1 highly conserved protein
  • Pinl contains an N-terminal WW domain, which functions as a phosphorylated Ser/Thr-Pro binding module (Sudol, M. (1996) Prog. Biophys. Mol. Biol. 65:113-32). This phosphorylation- dependent interaction targets Pinl to a subset of phosphorylated substrates, including Cdc25, Wee 1, Mytl, Tau-Rad4, and the C-terminal domain of RNA polymerase II large domain (Crenshaw, D.G., et al. (1998) Embo. J. 17:1315-27; Shen, M. (1998) Genes Dev.
  • Pinl activity is essential for cell growth; depletion or mutations of Pinl cause growth anest, affect cell cycle checkpoints and induce premature mitotic entry, mitotic anest and apoptosis in human tumor cells, yeast or Xenopus extracts (Lu, et al. 1996, Nature 380:544-547; Winkler, et al. 200, Science 287:1644-1647; Hani, et al. 1999. J. Biol. Chem. 274:108-116).
  • Pinl is dramatically misexpressed in human cancer samples and the total level or concentration of Pinl is conelated with the aggressiveness of tumors.
  • inhibition of Pinl by various approaches including Pinl antisense polynucleotides or genetic depletion, kills human and yeast dividing cells by inducing premature mitotic entry and apoptosis.
  • Pinl -catalyzed prolyl isomerization regulates the conformation and function of these phosphoprotein substrates and facilitates dephosphorylation because of the conformational specificity of some phosphatases.
  • Pinl -dependent peptide bond isomerization is an important post-phosphorylation regulatory mechanism, allowing cells to turn phosphoprotein function on or off with high efficiency and specificity during temporally regulated events, including the cell cycle (Lu et al., supra).
  • Other cancer known cancer marker polypeptides can be used in the methods of the invention.
  • estrogen receptor Accession No.: P03372
  • progersterone receptor Accession No.: NP_000917
  • epidermal growth factor receptor accesion No.: AAG43243
  • cyclin Dl accesion No.: NP_444284
  • the invention provides a method of detecting the presence and amount of unphosphorylated Pinl and/or phosphorylated Pinl in a biological sample.
  • the invention also provides a method of determining the amount of phosphorylated Pinl relative to the amount of unphosphorylated Pinl in a sample.
  • the methods of the invention may use antibodies that recognize phosphorylated Pinl and unphosphorylated Pinl, antibodies that are specific for phosphorylated Pinl, and antibodies that are specific for unphosphorylated Pinl.
  • Antibodies that are specific for Pinl are described in US patent 6,596,848, the entire contents of which are incorporated herein by reference.
  • Phosphorylation specific Pinl antibodies are commercially available from, for example, Cell Signaling Technology (Beverly, MA).
  • the invention also provides methods of detecting levels of Pinl in combination with other known cancer markers, e.g., Her2, ER, PR, EGFR, or CCND1.
  • levels of known cancer markers can be determined using antibodies specific for these markers and methods standard to one of skill in the art.
  • the phrase "antibodies specific for phosphorylated Pinl" is intended to include antibodies that preferentially bind to an antigen of Pinl that contains a phosphorylated residue.
  • Antibodies that are specific for phosphorylated Pinl bind to Pinl with at least twice the affinity that they bind to a nonspecific antigen (e.g., BSA or casein).
  • an antibody that is specific for phosphorylated Pinl has more affinity for phosphorylated Pinl than it does unphosphorylated Pinl .
  • the antibody specific for phosphorylated Pinl binds with at least 2, 3, 4, 5, 10, 20, 50, 100, 500, or 1000 times the affinity to phosphorylated Pinl as it does unphosphorylated Pinl.
  • the antibody specific for phosphorylated Pinl recognizes a Pinl molecule that is phosphorylated on serine 16 of SEQ ID NO:l.
  • the phrase "antibodies specific for unphosphorylated Pinl" is intended to include antibodies that preferentially bind a Pinl polypeptide that is not phosphorylated.
  • Antibodies that are specific for unphosphorylated Pinl bind to Pinl with at least twice the affinity that they bind to a nonspecific antigen (e.g., BSA or casein). Further, an antibody that is specific for unphosphorylated Pinl has more affinity for unphosphorylated Pinl than it does phosphorylated Pinl. In certain embodiments, the antibody specific for unphosphorylated Pinl binds with at least 2, 3, 4, 5, 10, 20, 50, 100, 500, or 1000 times the affinity to unphosphorylated Pinl as it does phosphorylated Pinl .
  • the antibody specific for unphosphorylated Pinl recognizes an epitope of Pinl that comprising a residue that is capable of being phosphorylated, e.g., serine 16 of SEQ ID NO:l.
  • Polyclonal antibodies are produced by immunizing animals, usually a mammal, by multiple subcutaneous or intraperitoneal injections of an immunogen (antigen) and an adjuvant as appropriate.
  • animals are typically immunized against a protein, peptide or derivative by combining about 1 ⁇ g to 1 mg of protein capable of eliciting an immune response, along with an enhancing canier preparation, such as Freund's complete adjuvant, or an aggregating agent such as alum, and injecting the composition intradermally at multiple sites.
  • Animals are later boosted with at least one subsequent administration of a lower amount, as 1/5 to 1/10 the original amount of immunogen in Freund's complete adjuvant (or other suitable adjuvant) by subcutaneous injection at multiple sites.
  • Such populations of antibody molecules are refened to as "polyclonal" because the population comprises a large set of antibodies each of which is specific for one of the many differing epitopes found in the immunogen, and each of which is characterized by a specific affinity for that epitope.
  • An epitope is the smallest determinant of antigenicity, which for a protein, comprises a peptide of six to eight residues in length (Berzofsky, J. and I. Berkower, (1993) in Paul, W., Ed.. Fundamental Immunology.
  • the polyclonal antibody fraction collected from mammalian serum is isolated by well known techniques, e.g. by chromatography with an affinity matrix that selectively binds immunoglobulin molecules such as protein A, to obtain the IgG fraction.
  • the specific antibodies may be further purified by immunoaffinity chromatography using solid phase-affixed immunogen.
  • the antibody is contacted with the solid phase-affixed immunogen for a period of time sufficient for the immunogen to immunoreact with the antibody molecules to form a solid phase-affixed immunocomplex.
  • Bound antibodies are eluted from the solid phase by standard techniques, such as by use of buffers of decreasing pH or increasing ionic strength, the eluted fractions are assayed, and those containing the specific antibodies are combined.
  • "Monoclonal antibody” or “monoclonal antibody composition” as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • Monoclonal antibodies can be prepared using a technique which provides for the production of antibody molecules by continuous growth of cells in culture. These include but are not limited to the hybridoma technique originally described by ohler and Milstein (1975, Nature 256:495-497; see also Brown et al. 1981 J.
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.
  • a monoclonal antibody can be produced by the following steps. In all procedures, an animal is immunized with an antigen such as a protein (or peptide thereof) as described above for preparation of a polyclonal antibody. The immunization is typically accomplished by administering the immunogen to an immunologically competent mammal in an immunologically effective amount, i.e., an amount sufficient to produce an immune response.
  • the mammal is a rodent such as a rabbit, rat or mouse.
  • the mammal is then maintained on a booster schedule for a time period sufficient for the mammal to generate high affinity antibody molecules as described.
  • a suspension of antibody-producing cells is removed from each immunized mammal secreting the desired antibody.
  • the animal e.g., mouse
  • antibody-producing lymphocytes are obtained from one or more of the lymph nodes, spleens and peripheral blood.
  • Spleen cells are prefened, and can be mechanically separated into individual cells in a physiological medium using methods well known to one of skill in the art.
  • the antibody-producing cells are immortalized by fusion to cells of a mouse myeloma line.
  • Mouse lymphocytes give a high percentage of stable fusions with mouse homologous myelomas, however rat, rabbit and frog somatic cells can also be used.
  • Spleen cells of the desired antibody-producing animals are immortalized by fusing with myeloma cells, generally in the presence of a fusing agent such as polyethylene glycol.
  • a fusing agent such as polyethylene glycol.
  • Any of a number of myeloma cell lines suitable as a fusion partner are used with to standard techniques, for example, the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines, available from the American Type Culture Collection (ATCC), Rockville, MD.
  • ATCC American Type Culture Collection
  • the fusion-product cells which include the desired hybridomas, are cultured in selective medium such as HAT medium, designed to eliminate unfused parental myeloma or lymphocyte or spleen cells.
  • selective medium such as HAT medium
  • Hybridoma cells are selected and are grown under limiting dilution conditions to obtain isolated clones.
  • the supernatants of each clonal hybridoma is screened for production of antibody of desired specificity and affinity, e.g., by immunoassay techniques to determine the desired antigen such as that used for immunization.
  • Monoclonal antibody is isolated from cultures of producing cells by conventional methods, such as ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography (Zola et al, Monoclonal Hybridoma Antibodies: Techniques And Applications, Hurell (ed.), pp. 51-52, CRC Press, 1982).
  • Hybridomas produced according to these methods can be propagated in culture in vitro or in vivo (in ascites fluid) using techniques well known to those with skill in the art.
  • a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening an antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No.
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Patent No.
  • Labeled antibody includes antibodies that are labeled by a detectable means and includes enzymatically, radioactively, fluorescently, chemiluminescently, and/or bioluminescently labeled antibodies.
  • an antibody can be detectably labeled is by linking the same to an enzyme.
  • This enzyme when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means.
  • Enzymes which can be used to detectably label the Pinl -specific or a cancer associated polypeptide-specific antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-NI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • Detection may be accomplished using any of a variety of immunoassays. For example, by radioactively labeling an antibody, it is possible to detect the antibody through the use of radioimmune assays.
  • a description of a radioimmune assay (RIA) may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T. S., et al., North Holland Publishing Company, NY (1978), with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T.
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by audioradiography.
  • Isotopes which are particularly useful for the purpose of the present invention are: 3 H, 131 1, 35 S, 14 C, and preferably 125 I. It is also possible to label an antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. An antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series.
  • metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
  • DTP A diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • An antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label an antibody of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • the invention provides methods for evaluating subjects for the level and phosphorylation state of Pinl . These results can be used to preselect a subject for treatment with a Pinl modulator.
  • the amount of phosphorylated and unphosphorylated Pinl in a biological sample may be determined by a radioimmunoassay, an immunoradiometric assay, enzyme immunoassay, and /or by immunohistochemistry using antibodies specific for phosphorylated Pinl and unphosphorylated Pinl, respectively.
  • Radioimmunoassay is a technique for detecting and measuring the concentration of an antigen using a labeled (i.e. radioactively labeled) form of the antigen.
  • radioactive labels for antigens examples include 3 H, 14 C, and 125 I.
  • concentration of phosphorylated and unphosphorylated Pinl in a sample is measured by having the antigen in the sample compete with a labeled (i.e. radioactively) antigen for binding to an antibody to the antigen.
  • a labeled antigen i.e. radioactively
  • the labeled antigen is present in a concentration sufficient to saturate the binding sites of the antibody. The higher the concentration of antigen in the sample, the lower the concentration of labeled antigen that will bind to the antibody.
  • the antigen-antibody complex In a radioimmunoassay, to determine the concentration of labeled antigen bound to antibody, the antigen-antibody complex must be separated from the free antigen.
  • One method for separating the antigen-antibody complex from the free antigen is by precipitating the antigen-antibody complex with an anti-isotype antiserum.
  • Another method for separating the antigen-antibody complex from the free antigen is by precipitating the antigen-antibody complex with formalin-killed S. aureus.
  • Yet another method for separating the antigen-antibody complex from the free antigen is by performing a "solid-phase radioimmunoassay" where the antibody is linked (i.e.
  • IRMA immunoradiometric assay
  • the multivalent antigen conjugate must have at least 2 antigen residues per molecule and the antigen residues must be of sufficient distance apart to allow binding by at least two antibodies to the antigen.
  • the multivalent antigen conjugate can be attached to a solid surface such as a plastic sphere.
  • Unlabeled "sample” antigen and antibody to antigen which is radioactively labeled are added to a test tube containing the multivalent antigen conjugate coated sphere.
  • the antigen in the sample competes with the multivalent antigen conjugate for antigen antibody binding sites. After an appropriate incubation period, the unbound reactants are removed by washing and the amount of radioactivity on the solid phase is determined.
  • the amount of bound radioactive antibody is inversely proportional to the concentration of antigen in the sample.
  • the most common enzyme immunoassay is the "Enzyme-Linked Immunosorbent Assay (ELISA).”
  • the "Enzyme-Linked Immunosorbent Assay (ELIS A)” is a technique for detecting and measuring the concentration of an antigen using a labeled (i.e. enzyme linked) form of the antibody.
  • an antibody i.e. to phosphorylated and unphosphorylated Pinl
  • a solid phase i.e. a microtiter plate
  • a labeled i.e. enzyme linked
  • a labeled is then bound to the bound-antigen (if present) forming an antibody-antigen-antibody sandwich.
  • enzymes that can be linked to the antibody are alkaline phosphatase, horseradish peroxidase, luciferase, urease, and ⁇ -galactosidase.
  • the enzyme linked antibody reacts with a substrate to generate a colored reaction product that can be assayed for.
  • a “competitive ELISA” antibody is incubated with a sample containing phosphorylated and unphosphorylated Pinl .
  • the antigen-antibody mixture is then contacted with an antigen-coated solid phase (i.e.
  • a labeled (i.e. enzyme linked) secondary antibody is then added to the solid phase to determine the amount of primary antibody bound to the solid phase.
  • an "immunohistochemistry assay" a section of tissue for is tested for specific proteins by exposing the tissue to antibodies that are specific for the type of Pinl protein that is being assayed (e.g., phosphorylated and unphosphorylated Pinl . The antibodies are then visualized by any of a number of methods to determine the presence and amount of the protein present.
  • Examples of methods used to visualize antibodies are, for example, through enzymes linked to the antibodies (e.g., luciferase, alkaline phosphatase, horseradish peroxidase, or ⁇ -galactosidase), or chemical methods (e.g., DAB/Substrate chromagen).
  • enzymes linked to the antibodies e.g., luciferase, alkaline phosphatase, horseradish peroxidase, or ⁇ -galactosidase
  • chemical methods e.g., DAB/Substrate chromagen.
  • immunohistochemsitry assays are provided in the Examples. Once the levels of phosphorylated and unphosphorylated Pinl in a biological sample are determined the subject can be classified based on the level and/or ratio of phosphorylated and/or unphosphorylated Pinl .
  • Exemplary classifications provided in the Examples are nPinlStr-cPinlneg, nPinlStr-cPinlpos, nPinl Weak-cPinlneg, nPinl Weak-cPinlpos, nPinlNeg-cPinlneg, and nPinlNeg-cPinlpos.
  • nPinl is used to indicate nuclear Pinl
  • cPinl is used to indicate cytoplasmic Pinl.
  • the data presented in the Examples indicates that the majority of phosphorylated Pinl localizes to the nucleus while the majority of unphosphorylated Pinl localizes to the cytoplasm.
  • subjects with high levels of unphosphorylated Pinl localized in the cytoplasm are preselected for treatment with a Pinl modulator.
  • the instant invention provides method of determining the prognosis of a subject with a Pinl associated disorder, e.g., a cell proliferative disorder.
  • the Pinl associated disorder is a type of cancer, e.g., breast cancer.
  • the instant invention provides for the determination of the prognosis of a subject by evaluating the levels of pPinl in the subject at one or more points in time.
  • the level of pPinl in a subject can be compared to the statistical mean level in a population of subjects with similar diseases and the prognosis of the subject can be determined based on the level of pPinl relative to the statistical mean.
  • the prognosis of the individual is considered poor, e.g., the subject will likely not survive for as long as the mean length of survival of the population. If the level of pPinl in a subject is higher than the mean , the prognosis of the individual is considered good, e.g., the subject will survive for as long as the mean length of survival of the population, or longer.
  • the term "statistical mean” is used herein in a manner consistent with the well- understood definitions in the art of statistics. The statistical mean can be determined by quantitating the level of pPinl in a statistically significant number of subjects and determining the mean value of that population.
  • the prognosis of an individual can be determined by evaluating the level of pPinl in biological samples isolated at different time points. If the levels of pPinl decrease from a first sample to a second sample, the prognosis of said subject is poor. If the levels of pPinl in a sample stay the same, or increase, the prognosis is good.
  • the levels of pPinl can be determined and compared with a survival curve generated with data from a statistically significant number of subjects having a similar disease. The comparison of the pPinl levels in a subject to the survival curve will determine the prognosis of a subject, i.e., the chance the subject has to survive for a given amount of time.
  • the levels of pPinl in a biological sample can be determined and used in combination with the levels of other known prognostic markers to determine the prognosis of a subject.
  • the levels of pPinl and one or more known cancer markers e.g., Her2, ER, PR, EGFR, or CCND1
  • Exemplary cancer markers used to predict the prognosis of a subject in combination with the levels of pPinl are provided in the Examples. Survival curves are provided for subjects based on the determination of pPinl levels and Her2, ER, PR, EGFR, or CCND1 levels.
  • subject is intended to include living organisms, e.g., prokaryotes and eukaryotes. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. Most preferably the subject is a human.
  • subject that would benefit from treatment with a Pinl modulator is intended to include subjects having a Pinl associated disorder wherein treatment of said subject with a Pinl modulator would alleviate, reduce or eliminate one or more symptoms of the Pinl disorder.
  • effective amount of the compound is that amount necessary or sufficient to treat or prevent a Pinl associated state, e.g.
  • an effective amount of a Pinl modulator of the invention is the amount sufficient to inhibit undesirable cell growth in a subject.
  • an effective amount of the Pinl modulator compound is the amount sufficient to reduce the size of a pre-existing benign cell mass or malignant tumor in a subject.
  • the effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular Pinl binding compound. For example, the choice of the Pinl modulator compound can affect what constitutes an "effective amount".
  • One of ordinary skill in the art would be able to study the aforementioned factors and make the determination regarding the effective amount of the Pinl modulating compound without undue experimentation.
  • a Pinl modulator compound can be administered to the subject either prior to or after the onset of a Pinl associated state. Further, several divided dosages, as well as staggered dosages, can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. Further, the dosages of the Pinl modulator can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • the term "treated,” “treating” or “treatment” includes the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated. For example, treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder.
  • composition includes preparations suitable for administration to mammals, e.g., humans.
  • modulators are administered as pharmaceuticals to mammals, e.g., humans, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable canier.
  • pharmaceutically acceptable canier is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals.
  • the earners include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each canier must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulf ⁇ te and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, ⁇ -tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulf ⁇ te and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
  • Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a canier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 per cent to about 30 per cent.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the canier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid earners, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • the active ingredient is mixed with one or more pharmaceutically acceptable earners, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pynolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol monostearate
  • the pharmaceutical compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by 7 incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric su-bstances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and e
  • the oral compositions can also incLude adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may coaitain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.
  • Formulations of pharmaceutical compositions for use in the methods of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable noninitating excipients or earners comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will rrxelt in the rectum or vaginal cavity and release the active compound.
  • Dosage forms for the topical or transdermal administration of a compound include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable canier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin.
  • the rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.
  • Pharmaceutical compositions suitable for parenteral administration comprise one or more compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous caniers which may be employed in the pharmaceutical compositions for use in the methods of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled.
  • biodegradable polymers examples include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • formulations may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration is prefened.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracistemally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • intravenous and subcutaneous doses of the compounds of this invention for a patient when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day, and still more preferably from about 1.0 to about 100 mg per kg per day.
  • An effective amount is that amount treats an Pinl associated state.
  • the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • Immunohistochemistry assay was used to detect expression of unphosphorylated and phosphorylated PINl in tissue microanays (TMAs). Freshly cut tissue microanay sections were used to avoid the possibility of reduced assay sensitivity due to age-dependent oxidation of stored slides.
  • Protocol for unphosphorylated Pinl detection The following steps were taken to analyze the level of unphosphorylated Pinl in breast tissue samples.
  • Protocol for phosphorylated Pinl detection The following steps were taken to analyze the level of phosphorylated Pinl in breast tissue samples.
  • a breast cancer tissue microanay containing 2517 premalignant and malignant breast tissue samples was analyzed. The breakdown of histopathological features is presented in the results section.
  • a Pinl antibody that preferentially recognizes unphosphorylated Pinl was used for IHC analysis to determine the level of unphosphorylated Pinl.
  • a Pinl antibody that is specific for phosphorylated Pinl was used for IHC analysis to determine the level of phosphorylated Pinl.
  • weak and moderate groups were combined and are denoted as "weak" Pinl .
  • Table 1 pPINl expression and tumor phenotype in breast tissues. pPINl nuclear staining
  • uPinl unphosphorylated Pinl
  • pPinl phosphorylated Pinl
  • Example 2 Survival of Cancer Subjects Based on Nuclear Pinl Levels. Nuclear Pinl levels were determined as described in Example 1. Subjects survival was monitored as a function of nuclear Pinl levels as measured by immunohistochemistry ( Figure 5). The data indicate that the higher the levels of nuclear Pinl in a subject the better the prognosis, and, further, the presence of nuclear Pinl at any level is indicative of a better prognosis than the absence of nuclear Pinl.
  • Example 3 Survival of Cancer Subjects Based on pPinl Levels Nuclear pPinl level were determined as described in Example 1. Subjects survival was monitored as a function of nuclear pPinl ( Figure 6). The data indicate that generally, the higher the levels of nuclear pPinl in a subject the better the prognosis, and further, the presence of nuclear Pinl at any level is indicative of a better prognosis than the absence of nuclear Pinl. The levels of pPinl and nuclear Pinl were also conelated ( Figure 7). The data indicate that the level of nuclear Pinl increases as a function of pPinl levels.
  • Example 4 Survival of Cancer Subjects Based on Nuclear Pinl Levels and Her2 Levels Nuclear Pinl level were determined as described in Example 1. Her2 levels were determined by immunohistochemistry ( Figure 8) and fluorescence in situ hybridization ( Figure 10). For the fluorescence in situ hybridization experiments, commercially available DNA probes (Vysis, Inc. Downers Grove, IL) directed against the Her2 were used. Tissue microanay slides were pretreated according to Vysis' s recommended protocol. Probes were hybridized to the microanay sections overnight at 37°C in a wet chamber. Differentially labeled probes specific for the centromeric regions of chromosomes 11 and 17 were included as internal controls. Her2 levels were determined using a bright-field immunohistochemistry assay.
  • Freshly cut tissue microanay sections were used to decrease the possibility of reduced assay sensitivity due to age-dependant oxidation of stored slides.
  • the sections were de- paraff ⁇ nized, rehydrated and subjected to high-temperature antigen retrieval procedures prior to incubating with the primary antibody overnight.
  • Sections were washed and incubated with a secondary biotinylated antibody, followed by avidin-peroxidase- conjugate (Vector ABC Elite system).
  • the bound antibody was visualized by incubating the slides with osmium enhanced diaminobenzidine solution resulting in dark brown color indicating Her2 positivity.
  • the slides were analyzed by a pathologist and staining was given a score of 0, 1, 2, or 3.
  • the data indicate that the number of Her2 positive samples decreases as the levels of nuclear Pinl increase. Subjects survival was monitored as a function of nuclear Pinl and Her2 ( Figure 9 and 11). The data indicate that, generally, subjects that are Her2 negative have a better survival prognosis than those that are Her2 positive. The data further indicate that within each Her2 classification, subjects with high levels of Pinl have a better prognosis than those who are negative for nuclear Pinl .
  • Example 5 Survival of Cancer Subjects Based on Nuclear Pinl Levels and ER Levels Nuclear Pinl level were determined as described in Example 1.
  • Estrogen receptor (ER) levels were determined by immunohistochemistry. ER levels were determined using a bright-field immunohistochemistry assay. Freshly cut tissue microanay sections were used to decrease the possibility of reduced assay sensitivity due to age-dependant oxidation of stored slides. The sections were de- paraffinized, rehydrated and subjected to high-temperature antigen retrieval procedures prior to incubating with the primary antibody overnight. Sections were washed and incubated with a secondary biotinylated antibody, followed by avidin-peroxidase- conjugate (Vector ABC Elite system).
  • the bound antibody was visualized by incubating the slides with osmium enhanced diaminobenzidine solution resulting in dark brown color indicating ER positivity.
  • the slides were analyzed by a pathologist and staining was given a score of 0, 1, 2, or 3.
  • Tissue samples were considered positive for ER if at least 10% of the cells showed at least level 1 staining.
  • the data indicate that the number of ER positive samples increases as the levels of nuclear Pinl increase ( Figure 12).
  • Subjects survival was monitored as a function of nuclear Pinl and ER ( Figure 13). The data indicate that subjects that are ER positive have a better survival prognosis than those that are ER negative.
  • the data further indicate that within the ER positive subjects, those with high levels of nuclear Pinl have a better prognosis than those who are negative for nuclear Pinl .
  • Example 6 Survival of Cancer Subjects Based on Nuclear Pinl Levels and PR Levels Nuclear Pinl level were determined as described in Example 1.
  • Progesterone receptor (PR) levels were determined by immunohistochemistry. PR levels were determined using a bright-field immunohistochemistry assay. Freshly cut tissue microanay sections were used to decrease the possibility of reduced assay sensitivity due to age-dependant oxidation of stored slides. The sections were de- paraffinized, rehydrated and subjected to high-temperature antigen retrieval procedures prior to incubating with the primary antibody overnight. Sections were washed and incubated with a secondary biotinylated antibody, followed by avidin-peroxidase- conjugate (Vector ABC Elite system).
  • the bound antibody was visualized by incubating the slides with osmium enhanced diaminobenzidine solution resulting in dark brown color indicating ER positivity.
  • the slides were analyzed by a pathologist and staining was given a score of 0, 1, 2, or 3. Tissue samples were considered positive for ER if at least 10% of the cells showed at least level 1 staining.
  • the data indicate that the number of PR positive samples increases as the levels of nuclear Pinl increase (Figure 14). Subjects survival was monitored as a function of nuclear Pinl and PR ( Figure 15). The data indicate that subjects that are PR positive have a better survival prognosis than those that are PR negative. The data further indicate that within each classification of PR subjects, those with high levels of nuclear Pinl, generally, have a better prognosis than those who are negative for nuclear Pinl .
  • Example 7 Survival of Cancer Subjects Based on Nuclear Pinl Levels and EGFR Levels Nuclear Pinl levels were determined as described in Example 1.
  • Epidermal growth factor receptor (EGFR) levels were determined by immunohistochemistry. EGFR levels were determined using a bright-field immunohistochemistry assay. Freshly cut tissue microanay sections were used to decrease the possibility of reduced assay sensitivity due to age-dependant oxidation of stored slides. The sections were de- paraffinized, rehydrated and subjected to high-temperature antigen retrieval procedures prior to incubating with the primary antibody overnight. Sections were washed and incubated with a secondary biotinylated antibody, followed by avidin-peroxidase- conjugate (Vector ABC Elite system).
  • the bound antibody was visualized by incubating the slides with osmium enhanced diaminobenzidine solution resulting in dark brown color indicating ER positivity.
  • the slides were analyzed by a pathologist and staining was given a score of 0, 1, 2, or 3.
  • Tissue samples were considered positive for EGFR if at least 10% of the cells showed at least level 1 staining.
  • the data indicate that the number of EGFR positive samples decreases as the levels of nuclear Pinl increase ( Figure 16).
  • Subjects survival was monitored as a function of nuclear Pinl and EGFR ( Figure 17). The data indicate that, generally, subjects that are EGRF negative have a better survival prognosis than those that are EGFR positive.
  • Example 8 Survival of Cancer Subjects Based on Nuclear Pinl Levels and CCNDl Levels Nuclear Pinl levels were determined as described in Example 1 cyclin Dl (CCNDl) levels were determined by fluorescence in situ hybridization. Commercially available DNA probes (Vysis, Inc. Downers Grove, IL) directed against the CCNDl were used. Tissue microanay slides were pretreated according to Vysis's recommended protocol. Probes were hybridized to the microanay sections overnight at 37°C in a wet chamber.
  • Differentially labeled probes specific for the centromeric regions of chromosomes 11 and 17 were included as internal controls.
  • the data indicate that the number of CCNDl positive samples decreases as the levels of nuclear Pinl increase (Figure 18).
  • Subjects survival was monitored as a function of nuclear Pinl and CCNDl ( Figure 19).
  • the data indicate that, generally, subjects that are Pinl positive have a better survival prognosis than those that are Pinl negative.
  • the data further indicate that within the Pinl negative classification subjects that have high levels of CCNDl have a worse survival prognosis than those that have normal levels of CCNDl .
  • Within the group of subjects that are Pinl positive CCNDl levels do not appear to confer a beneficial or detrimental effect on survival.
  • Example 9 Levels of pPinl and Pinl in Ductal Carcinoma
  • the levels of pPinl and Pinl were determined as described above.
  • Figure 20 depicts the levels of pPinl and Pinl as a function of normal, grade 1, grade 2, grade 3 and grade 4 ductal carcinoma tissues. The data indicate that the 61% of normal subjects have strong levels of pPinl and that the percentage of subjects with strong levels of Pinl decreases as a function of increasing tumor grade. Only 13% of subjects with grade 4 ductal carcinoma have high levels of pPinl.
  • Figure 20 further indicates that percentage of subjects expressing strong levels of Pinl remains fairly constant as a function of tumor grade.

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Abstract

L'invention concerne des procédés de sélection de sujets qui pourront bénéficier d'un traitement avec un modulateur Pin 1 par détermination du niveau et de l'état de phosphorylation de Pin 1 présent dans un échantillon biologique prélevé sur le sujet. Cette invention porte aussi sur des procédés de détermination du pronostic d'un sujet par détermination du niveau de pPin 1 dans un échantillon biologique. Elle se rapporte aussi à des procédés de traitement de sujets atteints d'un trouble associé à Pin 1, et à des procédés de contrôle de l'efficacité du traitement.
PCT/US2005/003703 2004-02-05 2005-02-04 Procedes de determination du pronostic et du traitement de sujets atteints de troubles lies a pin 1 Ceased WO2005076958A2 (fr)

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US54262904P 2004-02-05 2004-02-05
US60/542,629 2004-02-05
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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WULF G.M. ET AL: 'Pin1 is overexpressed in breast cancer and cooperates with Ras signaling in increasing the transcriptional activity of c-Jun towards cyclin D1' THE EMBO JOURNAL vol. 20, no. 13, 02 July 2001, pages 3459 - 3472, XP002276442 *

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