Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • G—PHYSICS
  • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
  • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
  • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• This invention is related to evaluation of molecular pharmacodynamics markets in response to carboxyamidotriazofe orotate (CTO) in ex vivo human anagen hairs -obtained fr m healthy subjects or patients with different diseases.
• CTO is an orally active agent with antineoplastic activity, that inhibits non-voltage operated Ca2+ channels, blocking both Ca2 ⁇ - iniiux into cells and Ca2+ release from intracellular stores and resulting in the disruption of calcium-mediated signal transduction and inhibition of vascular endothelial growth factor
• VEGF vascular endothelial growth factor
• .multiple tyrosine kinase signaling including A.KT, MEK-ERK, or Ber-
• the present invention relates to the evaluation of molecular phannacodynamics bio.mark.ers of overall signaling output by transcriptomic assessment of response to CTO given to patients or when added to ex vivo cultured human anagen hairs in vi ro.
• HDAC deacetylases
• the P53 transcription factor is a major tumor suppressor protein that serves as a gatekeeper of cellular fate in multicellular organisms. P53 is activated in response to a variety of stress signals and initiates cell cycle arrest, senescence or apopiosis via pathways involving tnmsactivation of P53 target genes. Stamho.lse V et at, Mot Cell: 317- 325 (2001 ). This universal protection of genetic integrity is however impaired in many huma cancers. The new paradigm is to develop agents that target the precise molecular signaling that maintains normal cell cycle, growth and proliferation.
• chemothempendc agents based on knowledge of the molecular abnormalities in particular cancers, together with the understanding of the feedback loops that apply upon blockade of a given pathway, as well as enhancing the tumor suppressor signaling pathways of p53 transcription factor and PTEN in normal and cancer ceils. .
• Carboxyaniidotriazole orotate (CTG), an orotate salt of carboxysmidotriazole CA.I) is an inhibitor of receptor-operated calcium channel-mediated calcium influx, and is shown to have anti-proliferative and anti-invasive functions in several human cancer cell lines, including human glioblastoma cells. Ge S et al. 5 Clin Cancer Res 6: 1248-1254 (2000). By interrupting calcium mobilization as a second messenger.
• CAI can inhibit calcium-sensitive signal transduction pathways, including the release of araehidonic acid and its metabolites; nitric oxide release; the generation of inositol phosphates; and tyrosine phosphorylation Kohn E C et al,Cancer Res 52:3208-3212 ( 1992); Kohn E C et al., Froe Na l Acad Sci 92: 1307- 131 1 (1995); Feider CF et al, J Pharmacol Exp Therap 257: 967-971 (l990);Hupe DJ ei al, J Biol Chero 266: 10136- 10142 ( 1991); Mignen O ei al., J Cell Sc 1 18: 5615-5623 (2005); and Enfissi E et al., Cell Calcium 36; 459-467 (2004), CAi inhibits phosphorylation of cellular proteins STATS and CrfcL, and induces apoptosis in imatinib mesylate-resist
• i is therefore important to develop the complete molecular signatures of CTO in view of its effec t on signaling of multiple kinases, tyrosine kinases and calcium signal transduction pathways.
• the invention is re lated to evaluation, of the response of molecular
• anagen hairs are obtained before dosing the patient with CTO, and at. different time points after the daily dosing of a therapeutic amount of CTO is given, The patient's clinical status and blood levels of CAI are monitored during this period.
• the aaagen hairs are obtained from an untreated subject and the hairs are treated in ex vivo cultures with different doses of CTO which represent the range of doses required for therapeutic efficacy.
• R A is extracted from the bulbs at the end of anagen hairs
• cD A is then prepared from the RNA and global transcriptional or gene expression levels are determined by microarray analysis or by quantitative PCR (qPCR).
• B formatic analysis is then conducted to identify CTO induced gene expression changes in anagen hairs.
• a protocol can also be applied to issues other than anagen hair obtained from healthy subjects or patients,
• the present invention describes in greater detail, uses of the plucked hair biornurker assay to study effects of CTO on mRNA and protein expression levels in vitro.
• Plucked scalp hair is an ideal surrogate for measuring direct, response to treatment with CTO.
• Highly vascularized, hair follicle can respond within hours of exposure.
• Gi ven this vascularization, their epithelial, nature and rapid rate of proliferation, the cells in the hair bulb at the base of die plucked hair and the outer root sheath are highly relevant surrogate marker tissue tor solid tumors.
• Highly vascularized, the hair follicle can respond to drug treatment within hours of exposure. Bioinformaiie analysis was conducted to identify drug-induced changes in hairs.
• the present invention relates to the evaluation of molecular pharmacodynamic biomarkers of multiple signaling pathways in response to CTO given in vivo or in vitro, in ex vivo cultured human anagen hairs.
• Using a commercially available plucked hair molecular platform assay (Episiem Ltd, Manchester, UK.) direct response to treatment with different doses of CTO equivalent to levels of carboxyamidotriazole (CAl) that are therapeutically achieved in patients, was evaluated to test targeting intracellular signaling pathways in oncology and other therapeutic areas.
• the present invention used plucked scalp hair from subjects, extracted the RNA from the bulbs at the end of the anagen hairs, prepared the c-DNA from the RNA, determined the gene expression levels by microarray analysis or quantitative PCR iqPCR) and conducted biointormatics analysis to identify drag induced gene expression changes for CTO and other drugs, for example, Tarceva® (EGFR umibitor ⁇ or BEZ23S (a FBK inhibitor).
• the invention relates to development of .molecular pharmacodynamics bioroarkers of signaling output by transcriptomic assessment of response to CTO in ex vivo cultured human anagen hairs from human subjects with or without cancer or other diseases.
• the molecular phannacodynamics biomarkers of CTO expose include RAS, GFS (PI3K.PBK TOR), EK, HDAC, NOTCH, WNT- ⁇ catenin, HSP90, EGFR, F5.1 CAIIPA.
• CAI ex vivo Calcium Signaling Non-voltage dependent, Calcium signaling all genes. Calcium signaling ex vivo, Canonical Calcium Signaling, Canonical Calcium ex vivo. Calcium all genes non-voltage dependent, EG I , PTEN, TGFp, CEACAML or Dystonin.
• the invention provided a method of inhibiting RAS pathway signatures in response to treatment with CTO.
• 1x3 a further aspect the invention provided a method of inhibiting GFS pathway signatures in response to treatment with CTO.
• the invention provided a method of inhibiting MEK pathway signatures in response to treatment with CTO,
• the invention provided a method of inhibiting HDAC pathway signature i response to treatment with CTO. In a further aspect the invention provided a method of inhibiting NOTCH pathway signatures i response to treatment with CTO.
• the invention provided a method of inhibiting WNT- ⁇ eaten in pathway signatures in response to treatment with CTO.
• the invention provided a method of inhibiting HSP90 signatures in response to treatment with CTO.
• the invention provided a method of inhibiting EGF.R pathway signatures in response to treatment with CTO.
• the invention provided a method of inducing P53 pathway signatures in response to treatment with CTO.
• the invention provided a method of inhibiting genes associated with non-voltage dependent calcium signaling in response to treatment with CTO.
• the invention provides Signature Scores for the CAI Ingenuity Pathway Analysis, the CAI ex vivo pathway, the calcium signaling pathway and the canonical calcium signaling pathways as pharmacodynamics markers of response to CTO.
• the invention provided a method of up regulating EGRl pathway signatures in response to treatment with CTO.
• the invention provided a method of up regulating PTEN pathway signatures in response to treatment with CTO,
• the invention provided a method of inducing TGF- ⁇ pathway signatures in response to treatment with CTO.
• the invention provided a method of down regulating CEACAM! pathway in response to treatment with CTO.
• the invention pro vided a method of down regulating dystonm pathway in response to treatment with CTO.
• the invention also relates to pharmaceutical compositions including CTO and another agent, combined to improve sensitivity and efficacy and reduce toxicity while regulating one or more gene signatures including EGFR, MEK, VEGF, HDAC, HSP90.
• E K, BCK-AB p53, ERG 1 , CEACAM I , dystonia or genes associates with .oon ⁇ voltage dependent, calcium signaling, by monitoring the molecular pharmacodynamics biomarkers of signaling outpu t in response to CTO in ex vivo cultured anagen hairs from a treated mammal.
• the invention provides a method of treating or preventing a condition in a mammal in which the regulation of one or more gene signatures including EGFR, MEK, VEGF, HDAC, HSP9 , ERK, BCR-A8L, p53, ERG 1 , CEACAM .1 , dyston , or genes associates with non-voltage dependent calcium signaling , prevents, inhibits or ameliorates a pathology or a symptomology of the condition, the method including administration of a therapeutically effective amount of CTO as monotherapy or as combinatorial therapy, and monitoring the molecular pharmacodynamics biomarkers of signaling output in response to CTO in ex vivo cultured anagen hairs irons a treated mammal, in another aspect the present invention pro vides a method of preventing or treating a proliferative condition in a subject, the method including administration of a therapeutically effective amount of CTO alone or in combination with another agent, and monitoring the molecular pharmacodynamic
• Gene expression patterns can be established in other tissues as well to distinguish between tissues in different disease states or to predict prognosis of a disease such as cancer in response to one or more therapies.
• Another paradigm is to develop a platform of pharmacodynamics markers to design specific and customized formulation base on the gene expression pattern.
• the invention provides a paradigm for rational selection and development of combination treatments based on knowledge of the molecular abnormalities in. particular diseases or cancer, together with the understanding that CTO may be combined with other targeted agents that are. constructed to the molecular makeup of the disease or cancer, together with the understanding that of the feedback loops that apply upon blockade of a given pathway blocked by the targeted drug and how these feedback loops may be prevented, to maintain sustained inhibition of the molecular targets that drive the disease as wet! as in some instances to induce suppressor genes io optimize the treatment outcome.
• the invention provides a shift in the method of developing combinatorial drug regimens inhibiting several oncogenic targets with CTO in order to overcome cancers that are driven by several abnormalities, as well as to prevent or neutralize the development of drug resistance by monitoring the pharmacodynamics biomarkers of signaling output in response to CTO and carefully picked combination drugs, by transeriptomie assessment in ex vivo cultured anageo hairs.
• the current poly- pi m ecology approach using several targeted drugs cocktails has not been successful, and has posed new problems due to cumulative toxicities of the drugs in the cocktail.
• An important embodiment of the invention is the development of the panel of 15-20 gene mRNA expression signatures from hair samples and deploy this on the cDNA samples generated from patients by AiYymetrix array data to identify genes differentially expressed in scalp harr samples from untreated and treated patients.
• FIG. I a illustrates the different stages of the plucked hair hiomarker platform
• FIG. lb describes A.NOV A analysis of results of transcription response for all contrasts in response lo different doses o CTO at 8 hx and 24 hr and for 1 dose of 8EZ235 (P13 inhibitor) and Tsrceva® (EGFR inhibitor) for 24 hr only.
• FIG. 2a illustrates the results of different doses of CTO on multivariate signatures at 8hr and 24hr periods for 1 1 different. signatures starting with the strongest to lowest inhibition- with EGF i, MEKi, HSP 0i, non-voltage dependent CAl related calcium signaling.
• HDACL GF and .AS, WNT/fT-eatenin was inhibitory at 24hrand no activity was noted for P13K1 P!3K/mTOF, OTCH/GSI.
• P53 was induced and F53 stabilization w s noted especially at 24hr.
• FIG. 2b lists 13 different signatures studied.
• FI 3a illustrates the Signature Score for the RAS pathway m response to different doses of CTO at 8hr and 24hr compared with BEZ235 and Tareeva#
• FIG 3b illustrates the Signature Score for the Growth Factor Signature (GFS) pathway in response to different doses of CTO at.8hr and 24hr compared with ⁇ 2235 and Tareeva® for 24hr.
• GFS Growth Factor Signature
• FIG 3c illustrates the Signature Score for the P13 pathwa in response to different closes of CTO at 8hr and 24hr compared with BEZ235 and TareevaS? for 24hr,
• FIG 3d illustrates the Signature Score for the PBK/mTOR pathway in response to different doses of CTO at 8hr and 24hr compared with BEZ235 and Tarceva® for 24hr,
• FIG 3e illustrates the Signature Score for the MEK. pathway in response to different doses of CTO at 8hr and 241ir compared with BEZ235 and Tarcevg® for 24hr.
• FIG 4a illustrates the Signature Score for the HDAC pathway in response to different doses, of CTO at 8hr and 24hr compared with 8EZ235 and Tarceva® for 24hr.
• FIG 4b illustrates the Signature Score tor the NOTCH pathway in response to different doses of CTO at 8hr and 24.hr compared with BEZ235 and Tareeva'D for 24hr.
• FIG 4c illustrates the Signature Score for the W ' T ⁇ -catenin pathway in response to different doses of CTO at 8hr and 24hr compared with BEZ235 and Tarceva® for 24hr.
• FIG 4d illustrates the Signature Score for the HSP9G pathway in response to different doses of CTO at 8hr and 24hr compared with BEZ235 and Tarceva ⁇ for 24hr.
• FIG 4e illustrates the Signature Score for the EGFR pathway in response to different doses of CTO at 8hr and 24hr compared with BEZ235 and Tarceva® for 24hr.
• FIG 4f illustrates the Signature Score for the P53 pathway in response to different doses of CTO at Sfsr and 24hr compared with BE2235 and Tarceva® for 24hr.
• FIG 5a illustrates the Signature Score for the CAI IP A pathway in response to different doses of CTO at 8hr and 24hr compared with BEZ235 and Tarceva® for 24hr, Using the Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, Redwood City, CA) lor genes influenced by CAI, some suppression of CAI signature in response to CTO treatment was observed, except at 10 ⁇ .
• IPA Ingenuity Pathways Analysis
• FIG 5b illustrates the Signature Score for the CAI.
• Ex vivo pathway in response to different doses of CTO at 8hr and 24hr compared wit BE2235 and Tarceva® for 24hr. With the tissue specific direction of change information a strong dose dependent suppression of the IPA CAI list was observed.
• FIG 5c illustrates the Signature Score for the Calcium signaling pathway in response to different doses of CTO at 8hr and 24hr compared with BEZ235 and Tarceva'tD for 24hr.
• Non-voltage dependent calcium signaling genes were identified from data sets in literature to
• FIG 5d illustrates the Signature Score for the Calcium signaling for ail gene pathways in response to different doses of CTO at 8hr and 24hr compared with BEZ235 and Tareeva® for 24.hr. The estimated regulation direction for ail genes was identified.
• FIG 5e illustrates the Signature Score for the Calcium Signaling pathway in response to different doses of CTO at 8hr and 24hr compared with BEZ235 and Tareeva® for 24hr.
• a NOV A was used to filter informative genes ⁇ FDR ⁇ 0,05 and .5 FC).
• FIG 6a illustrates the Signature Score for the Canonical Calcium signaling using the KEOG Calcium Signaling and I.PA to predict regulation of calcium signaling in response to different doses of CTO at Shr and 24h.r compared with BEZ235 and Tareeva® for 24hr.
• FIG 6b illustrates the Signature Score for the Canonical Calcium signaling ex vivo using the KBGG Calcium Signaling and IPA to predict regulation of in response to different doses of CTO at Shr and 24hr compared with BF.Z235 and Tareeva® for 24hr. Suppression noted for 6/78 canonical pathway genes only.
• FIG. ?a illustrates the Signature Score for all Signaling genes after merging of the ANOVA filtered gene sets for both the CA.I signature ( FIG. 5b) and the non-voltage dependent (NVD) gene sets (FIG, 5c).
• FIG 7b illustrates results in a panel of 3.1 CAl/Cafcium related genes capable of separating the different CTO doses, both by signature view and by PC A,
• FIG.8 illustrates the EGR1 Signaling Pathway.
• This invention is related to carboxyamidotriazo!e orotate (CTO).
• CTO carboxyamidotriazo!e orotate
• an orally active agent with antineoplastic activity that inhibits non-voltage operated Ca2-t" channels, blocking both Ca2+ influx into cells and Ca2-f release from intracellular stores and resulting in the disruption of calcium-mediated signal transduction and inhibition of vascular endothelial growth factor (VEGF) signaling, multiple tyrosine kinase signaling, including ART, ME - ERK, or BCR-ABL.
• VEGF vascular endothelial growth factor
• the present invention relates to the evaluation of molecular pharmacodynamics biomarkers of signaling output by transcriptomie assessment of response to CTO in vivo and in vitro cultures in ex vivo cultured human anagen hairs.
• Anagen hairs are obtained at different time points from subjects given varying doses of CTO treatment. This is the approach used in. clinical trials of CTO in patients. Alternately, anagen hairs are obtained from untreated subjects and the hairs are treated with varying doses of CTO in vitro-this approach is not subject to physiological conditions of absorption and delivery and was chosen.
• signaling through the phosphatidylinositol 3-kinase (POK) pathway is activated by multiple growth factors through receptor tyrosine kinases and has effects on multiple processes, including cell growth and survival metastatic potential, and drug resistance through gene amplification and/or by pass or new mutations.
• Many pharmaceutical, companies arc developing specific inhibitors of one or more signaling pathways and proposing combinations of several specific inhibitors to inhibit multiple oncogene pathways.
• a major limitation to the overall benefit from targeted therap is the development of drag resistance. Resistance can occur because of imitations that render the drug target insensitive to the inhibitor or when cancer cells change then dependency on the pathway that is targeted.
• resistance can be overcome by developing new drugs that effectively inhibit resistance-associated mutants, as in the example of dasatinib and niiotinib which are effective on SC -ABL mutants that confer resistance to imatinib.
• a second approach is to target multiple signaling pathways simultaneously, and thus prevent the cancer from changing its dependency to another significant pathway, for example combining inhibitors of mitqgen-activated protein ( APl-sxtraceMu!ar signal regulated kinase (ERK) kinase with inhibitors of P.O kinase.
• the third approach is to enhance efficacy of the targeted therapy to simultaneously target downstream proteins that protect tumor cells from apoptosis.
• gene expression signatures for pathway activation and/or inactivation may be used as i) pharmacodynamics biomarkers to monitor the drug induced pathway inhibition, in tumors or surrogate markers such, as anagen hair, ii) as prediction biomarkers to identify tumors with high level of a particular pathway; and early efficacy biomarkers to get an early readout of efficacy or prevention.
• Carboxyamidotriaxoie orotate (CTG).
• an oroiate salt of earboxyam dotriazole (CAl) is an inhibitor of receptor-operated calcium channel-mediated calcium influx, and is shown to have anti-proiifer tive and anti-invasive functions in several human cancer cell lines, including human glioblastoma cells (Fiorio Fla et al, 2008; Ge et al 2000).
• CAl By interrupting calcium mobilization a a second messenger, CAl can inhibit ca!einm-sensittve signal transduction pathways, including the release of arachidonic acid and its metabolites; nitric oxide release; the generation of inositol phosphates; and tyrosine phosphorylation (Ge et al, 20Q0; Kohn et al, 1 92).
• CAl inhibits phosphorylation of cellular proteins STATS and CrkL, and induces apoptosis in iraatinib niesylate-rcsistant chronic myeloid leukemia cells by down-regulating bcr ⁇ abl (Aiessandro et al, 2008).
• pharmacodynamics biomarkers of signaling outputs of CTO to design combinatorial regimens against molecular targets in different types of cancers.
• Current ' methods for assessing pathway activation in tumors involve the measurement of the drug targets, known oncogenes or known tumor suppressors. However, one pathway can he activated at multiple points so it is not feasible to assess pathway activation by evaluating just known cancer associated genes.
• the plucked hair biomarker assay is used to study effects of CTO on mRNA and protein expression levels in vitro. Plucked scalp hair is an ideal surrogate for measuring direct response to treatment with CTO. Highly vascularized, hair follicle can respond within hours of exposure.
• the cells in the hair bulb at the base of the plucked hair and the outer root sheath are highly relevant surrogate marker tissue for solid tumors. Bioinforrnaiic analysis is conducted to identify drag- induced changes in hairs.
• the invention relates to development of molecular pharmacodynamics iamarkers of signaling output by transcriptomie assessment of response to CTO in ex vivo cultured human anagen. hairs from human subjects with or without cancer or other diseases.
• This invention provides methods, pharmacodynamics biomarker signatures for multiple signaling pathways in a cell sample such as anagen hair, in response to
• CTO car oxyamidotriazole orotate
• pharmacodynamics biomarkers of multiple signature pathways comprising: a) Obtaining a cell sample obtained from a subject and exposing the cell sample to varying doses of CIO alone, to CTO in combination with another agent or to other agents for different time periods;
• RAS gene products are involved in kinase signaling pathways that control the transcription of genes, which then regulate cell growth and differentiation.
• the conversion of RAS from a proto-oncogene usually occurs through a point mutation in the gene, and the altered function can affect the cell in different ways because RAS is involved in many signaling pathways that control cell division and cell death.
• Mutant ras has been identified in cancers of man -origins, including pancreas, colon, hmg, thyroid, bladder ovarian, breast, skin, liver kidney and some leukemias. Song, S et a!.. PLOS ONE 7: 1 1 (2012).
• GFS is responsive to phosphatidyiinosito.1 3 -kinase ( ⁇ !3 ⁇ pathway perturbation and related to phosphatase and tensiri homolog (PTEN) degradation.
• PTEN phosphatase and tensiri homolog
• the invention provided a method of inhibiting EGFR pathway signatures in response to treatment with C ' FO.
• Dntgs targeting the EGF receptor (EGFR)- antibodies binding the extracellular domain and small-molecule tyrosine kinase inhibitors have expanded treatment options for several solid tumors.
• the EFGR gene is frequently up regulated in carcinomas of the breast, kidney, ovary, cervix, and in squamous cell carcinomas. ' The up regulation is typically due to gene amplification or overexpressiou.
• EGFR up regulation m gliomas is most often associated with the rearrangement of the EGFR gene resulting in alterations of its transcript so that such gliomas express both wild type endogenous EGFR as well as episomal mutant form.
• the EGFR gene is amplified in >50% of glioblastomas.
• E FR-targeted monoclonal antibodies Cetuximab® and P&mtumumab® have been extensively studied in metastatic colorectal cancers.
• the clinical efficacy of EGFR-targeted antibodies is limited by the development of acquired secondary resistance which typically occurs within 3 to 1.2 months of starting therapy.
• Multiple mechanisms of secondary resistance to ami-EGFR antibodies have been reported such as expression of EGFR ligands, HER2 amplification, and deregulation of the EGFR recycling process, RAS mutations arise and are responsible for acquired resistance in half the patients who initially respond to cet ximah or panituroumab.
• the invention provided a method of inhi it ng ME pathway signatures in response to treatment with CTO.
• the MAP pathway is commonly activated in human cancers and then activates R.AF -MEK-ER kinase cascade which leads to activation downstream of substrates involved in cell proliferation, survival, transformation, translations! control and cytoskeletal rearrangements.
• Small molecule inhibitors targeted this pathway, such as sllosteric inhibitors of MEK exhibit anticancer efficacy in vitro and in vivo.
• the invention provided a method of inhibiting HDAC pathway signatures in response to treatment with CTO.
• Histone acetylation is a reversible
• HDACs histone deaeetylases
• HDACs histone deaeetylases
• HDACs are represented by 18 genes in humans -and are divided into four distinct classes.
• Several classes of HDAC inhibitors are being evaluated in clinical investigations and indicate that certain HDAC family members are aberrantly expressed in several tumors, Unselective HDAC inhibitors show promising result ' s in leukemia* and solid tumors, for example Vormostat# approved for cutaneous T cell Lymphoma. Witt O et al.. Cancer Letter 277: 8-2 1 (2008).
• pan-HDAC inhibitors may cause numerous side effects thus requiring selective targeting of HDACs with oncogenic function in cancer cells.
• the invention provided a method of inhibiting WNT pathway signatures in response to treatment with CTO.
• the WNT family of signaling molecules regulates numerous processes in animal development, and WNT malfunction is implicated in various forms of disease including cancer and degenerative diseases.
• the canonical WNT signaling pathway is regulated at many levels and ⁇ here is increasing evidence .from other systems for crosstalk between WNT signaling and other pathways important in
• B-eatenin is a multifunctional protein with distinct molecular roles in cell adhesion at the plasma membrane and in transcription within, the nucleus.
• An increasing number of studies suggest that elevated WNT signaling in glioblastoma ⁇ GBM ⁇ is initiated by several alternative mechanisms that are involved in different steps of the disease, De Robertis A et al., Mo! Cancer Ther 12: 1180-1 189 (2013); and Nusse R, Cell Res 15: 28-32 ⁇ 2005). Therefore, inhibition of WNT signaling may represent a therapeutically relevant approach tor GBM treatment.
• the invention provided a method of inhibiting HSP90 pathway signatures in .response to treatment with CTO, Heat shock protens serve as molecular chaperones required for stability, post translation modification, and function of multiple client proteins. Expression of MSP is increased at times of physiologic stress, and these effects are believed to support ceil survival. HSP90 is over expressed in many tumor types indicating that it may play a role in the survival of cancer cells and thus making it an attractive target for an anticancer agent. Increased HSP90 has been linked to worse prognosis in patients with non-small cell lung cancer.
• NSCLC arises as a result of several driver mutations, for example EOF receptor mutations are seen in about 10% ofNSCLC.
• HSPs play an important role in
• the invention provided a method of inhibiting genes associated with non-voltage dependent calcium signaling in response to treatment with CTO.
• Kohn E C et aLCaneer Res 52:3208-3212 (1.992); ohn E C e ah, Proc Natl Acad Sci 92: 1307-1311 ( 1995); Felder CF et al, J Phasrmacol Exp Therap 257: 967-971 (J 0Hupe DJ ei ah, J Biol Chem 266: 10136-10142 ( 1991 ); Mignen O et ai.
• the invention provided a method of inhibiting genes associated with TGF- ⁇ signaling In response to treatment with CTO.
• TGF- ⁇ is part of a large fomsly of structurally related cytokines that include hone raorphogenic proteins, growth and differentiation factors, activins and inhibits.
• TGF- ⁇ functions as a tumor suppressor, where it inhibits proliferation, induces apoptosis and mediates
• TGF- ⁇ promotes turn or progression through increasing tumor cell invasion and metastasis.
• ccaarreeiinnooeemmbbrryyiioommcc a annttiiggeenn ( (CCEEAA)) g geennee f faammiillyy ooff !gg--iiiikkee cceellll--cceellll, aaddhheessiioonn m moolleeccuulleess.
• PPTTEENN i iss a a ttuummoorr ssuupppprreessssoorr aanndd iiss aa nneeggaattiivvee rreegguullaattoorr ooff P P..O13K-/''PP .8//AAKTT---ddeeppeennddeenivit cceelllluullaarr ssuurrvviivvaall..
• the early growth response 1 (EGRI ) gene product is a transcription factor with roles in differentiation and growth.
• the transcription factor EGR 1 is a direct regulator of multiple tumor suppressor including TGFpl , PTEN.and fibronectm. Baron V et aL Cancer gene Therapy 13: 1 15- 124 ⁇ 2006 ⁇ .
• EGR I exhibits prominent tumor suppressor function and many human tumor cel.! lines express little or no EGR 1 in contrast to their normal counterparts.
• EGR is decreased ot undetectable in non small cell lung cancers, breast tumors and human gliomas.
• Reexpression of ERG S in human tumor ceil lines inhibits transformation. The mechanism of suppression involves the direct induction of TGF- ⁇ leading to increased fibronectm- and plasminogen, activator inhibitor. Liu. C et ai Proc Natl Acad Sci 93; 1 1831 -1 1836 ' (1996).
• EGR! is implicated in the regulation of PS3 in melanoma cells leading to apoptosis and the proapoptotic suppressor gene PTEN is also directl regulated by EGRI .
• the invention also relates to pharmaceutical compositions including CTO and another agent combined to improve sensitivity and efficacy and reduce toxicity while regulating one or more gene signatures including E FR, MEK, VEGF, HDAC, HSP9Q, ERK, BCR-ABL, p53, ERGI , CEACA l , dystonia, or genes associates with non-voltage dependent calcium signaling, by monitoring the molecular pharmacodynamic biomarkers of signaling output in response to CTO in ex. vivo cultured anangen hairs from a treated mammal
• the invention provides a method of treating or preventing a condition in & mammal in which the regulation of one or more gene signatures including EGFR, MEK, VEGF, HDAC, HSP90, ERR, BCR-ABL, p53, ERG I , CEACAM l , dystonia, or genes associates with non-voltage dependent calcium signaling , prevents, inhibits or ameliorates a pathology or a symptomoiogy of the condition, the -method including administration of a therapeutically effective amount of CTO as monotherapy or as combinatorial therapy, and monitoring the molecular pharmacodynamics bio arkers of signaling output in response to CTO in ex vivo cultured anangen hairs from a treated mammal.
• the regulation of one or more gene signatures including EGFR, MEK, VEGF, HDAC, HSP90, ERR, BCR-ABL, p53, ERG I , CEACAM l , dystonia, or genes associates with
• the present invention provides a method of preventing or treating a proliferative condition in a subject, the method including administration of a therapeutically effective amount of CTO alone or in combination with another agent, and .monitoring the molecular pharmacodynamics biomarkers of signaling output in response to CTO in ex vivo cultured anagen hairs from a treated mammal.
• Gene expression patterns can be established in other tissues as well to distinguish between tissues in different disease states or to predict prognosis of a disease such as cancer in response to one or more therapies.
• Another paradigm is to develop a platform of pharmacodynamics markers to design specific and customized formulation base on the gene expression pattern.
• the invention provides a paradigm for rational selection and development of combination treatments based on knowledge of the molecular abnormalities in particular diseases or cancer, together with the understanding that CTO may be combined with other targeted agents that are constructed to the .molecular makeu of the disease or cancer, together with the understanding that of the feedback loops that apply upon blockade of a given pathway blocked by the targeted drag and .how these feedback loops may be prevented, lo maintain sustained inhibition of the molecular targets that drive the disease as well as m some instances to induce suppressor genes to optimize the treatment outcome.
• the invention provides a shift in the method of developing combinatorial drug regimens inhibiting several oncogenic targets with CTO in order to overcome cancers that are driven by several abnormalities, as well as to prevent or neutralize the development of drug resistance by monitoring the pharmacodynamics biomarkers of signaling output in response to CTO and carefully picked .combination drugs, by transcriptomic assessment in ex vivo cultured snagen hairs.
• the current poiy- harmacology approach using several targeted drugs cocktails has not been successful and has posed new problems due to cumulative toxicities of the drugs in the cocktail
• the invention provides a rescue solution for targeted and non- targeted drug combinations that fail due to drug resistance mechanisms by combining them with CTO from the start or even after drug resistance is noted, to maintain sineitivit and effectiveness.
• An important embodiment of the invention is the development of the pane! of up to 15-20 gene mR A expression signatures from hair samples and deploy this on the cDNA samples generated from patients by AfTymetrix array data to identify genes differentially expressed in scalp hair samples from untreated and treated patients.
• CTO carfooxyatnidotriazole orotate
• CTO is an orally active agent with antineoplastic activity, that inhibits non-voltage operated a channels, blocking both Ca2+ influx into cells and Ca2+ release from intracellular stores and resulting in the disruption of calcium-mediated signal transduction and inhibition of vascular endothelial growth factor (VEGF) signaling, multiple tyrosine kinase signaling, including A T, MEK- ERK, o BCR-ABL.
• VEGF vascular endothelial growth factor
• the present invention relates to the evaluation of molecular pharmacodynamics biomarkers of signaling output by transcriptomic assessment of response to CTO in ex vivo cultured human anagen hairs.
• Figure la describes the overall process steps.
• Plucked scalp hair is an ideal surrogate for measuring direct response to CTO treatment, It is a non-invasive and can also be used using samples from patients who have been treated with CTO, in which case the hairs do .not need to be treated ex vivo. Hairs growing on the scalp in growth stage (anagen) and which have highly vascularized follicles are suitable.
• Donor hairs were plucked from 5 male donor volunteers, immediately transferred to maintenance medium cultures and exposed to varying doses of CTO equivalent to 2 uM, 5 ⁇ , and 10 ⁇ carboxyaimdotriazole (CAl). The cultures were either maintained for Shrs or 24hrs. Controls were used, for example, Tareeva® (JSGFR inhibitor) at I ⁇ . ⁇ and BEZ235 iPB inhibitor) at I uM for 24 hrs only. Once in culture hairs were collected at the specified periods for n .NA isolation or protein analysis to ensure quality control. Small amounts of RNA (about up to SOOng) are extracted irons the bulbs at the end of the anagen hairs. Representative cD A was prepared from the RNA and gene expression levels determined by microarray analysis. Biotin labeling, fragmentation and hybridization to 048 Asymetrix 11133 plus 2.0 array was done. Bkunformatic analysis was done to identify C O induced gene expression changes. The whole procedure is given in FIG. .1 .
• Results obtained show strong transcriptional response for all contrasts. High level of differential expression of transcripts was observed. Tarcev&# showed less differential probes than CTO or BE2235 at this threshold. For CTO differential probes increased in a dose related maimer. CTO showed the greatest effect transcriptionally in anagen hair at all doses compared with B.EZ235 or Tarceva®. Results are presented in detail in Figure lb. Biologically relevant alteration of the hair bulb transcriptome ranging from - lOOfo!d to - : -25 fold differential expression was observed at clinically relevant levels of CTO.
• the signature scores were assessed as described in detail by Loboda A., et a! (Clinical Pharmacology & Therapeutics 86:92-96 (2009) for identification of growth factor gene signatures, and by Lobo a A et al, BMC Medical Genomics 3: 1 - 1 !( 2010) for identification of signatures of RAS pathway and PO .
• These references are incorporated herein. They describe the Ingenuity Pathway Analysis (ingenuity Systems, Redwood City, CA); htip : / www. ingennity.com software tool to identify signaling pathways that are statistically enriched among growth factor signature genes.
• the RAS pathway was analyzed using publicly available and literature datasets, e.g., .https.7/amiy.nei, nih.gov/can'ay project woost- 00041.
• FIG 3a describes results of multivariate signatures for RAS and Growth Factor Signatures. Briefly, modest down regulation in RAS and Growth Factor signatures (GPS . ) were observed, the inhibition being most obvious at S rs, Table 2 gives List of RAS and GFS signatures.
• FIG. 3c describes results; on P13K in response t CTO. Strong suppression oi ' PDK signature was observed w ith control BEZ23S treatment but not with CTO.
• FiG, 3d describes results on PI3K/mTOR signature. Strong suppression of PBK/mTO signature was observed with control BEZ235 treatment but not with CTO.
• FIG. 3e describes strong inhibition of MEKi signature m all CTO treatments at 8hrs and :24.hrs.
• FiG. 4a gives results showing weak suppression of HDAC signature m response to CTO treatment.
• the Signature Score for the HDAC pathway in response to different doses of CTO at 8hr and 24hr is compared with BEZ235 and Tarceva® for 24hr.
• FIG. 4b describes no suppression of Notch signature in response to CTO treatment.
• the Signature Score for the NOTCH pathway in response to different doses of CTO at 8hr and 24hr is compared with BEZ2.35 and Tarceva® for 24hr.
• FiG, 4c describes results showing WNT ⁇ -caienin signature shows modes suppression in 24hrs exposure to CTO.
• the Signature Score for the WNT ⁇ -ea ersin pathway in response to different doses of CTO at 8hr and 24hr is compared with BEZ233 and Tarceva® for 24hr.
• FIG. 4d describes strong suppression of HSP90 signature with CTO treatmen which is dose dependent.
• the Signature Score for the HSP90 pathway in response to different doses of CTO at 8hr and 24hr is compared with BEZ235 and Tarceva® for 24hr.
• FIG. 4e describes very strong suppression of EGF.R signature with CTO ' treatment and importantly this is stronger than that in Tarceva® at all doses of CTO and time points.
• the Signature Score for the EGFR pathway in response to different doses of CTO at 8h and 24hr is compared with BEZ235 and Tarceva® for 24hr
• FK1 4f describes in u tion of P53 signature with CTO treatment at all doses which is higher at the 24hr time point.
• the Signature Score for the P53 pathway in response to different dos.es of CTO at 8hr and 24hr is compared wit BE2235 and Tarceva® for 24hr,
• FIG 5a illustrates the Signature Score for the CAI ⁇ . ⁇ pathway in response to different doses of CTO at Shr and 24hr compared with BEZ235 and Tareeva® for 24hr.
• ingenuity Pathways Analysis if A) (Ingenuity Systems, Redwood City, CA> for genes influenced by CAI, some suppression of CAI signature in response to CTO treatment was observed, except at 10 p.M.
• 29 genes were .filtered from ⁇ for FDA ⁇ 0.0.5 and 1..5FC in CTO data set, to select informative genes and determine the direction of change, a 14 gene set resulted.
• FIG 5b illustrates the Signature Score for the CAI Ex vivo pathway in response to different doses of CTO at Shr and 24hr compared with BEZ235 and Taxceva® for 24hr, With the tissue specific direction of change information a strong dose dependent suppression of the IPA CAI list was observed.
• Table 4 lists the 14 gene set.
• FIG 5e illustrates the Signature Score for the Calcium signaling pathway in response to different doses of CTO at Shr and 24hr compared with B.EZ235 and Tarceva® for 24hr.
• Non- voltage dependent calcium signaling genes were identified from datasets in literature to inform on regulation. The list is presented in Table 5.
• FIG 3d illustrates the Signature Score for the Calcium signaling for all gene pathway in response to different doses of CTO at 8hr and 24m- compared with BEZ235 and Tarceva® for 24hr.
• the estimated regulation direction for all genes was identified. The list is provided in Table 6.
• FIG 5e illustrates the Signature Score for the Calcium Signaling pathway in response to different doses of CTO at 8kr and 24hr compared with BEZ235 and Tarceva® for 24hr.
• AN VA was used to filter informative genes (FDR ⁇ 0,05 and 1.5 FC),
• FIG 6a illustrates the Signature Score for the Canonical Calcium signaling using the KEGG Calcium Signaling and IPA to predict regulation of calcium sigaa!ittg in response to differentdoses of CTO at 8hr and 24hr compared with BEZ235 and. Taroeva® for 24hr. Table 8,
• FIG 6b illustrates the Signature Score for the Canonical. Calcium signaling ex vivo using the KEGG Calcium Signaling and IPA to predict regulation of n response to di fferent doses of CTO at Shr and 24hr compared with BEZ235 and Tareeva® for 24hr. Suppression noted for 6/78 canonical pathway genes only. Table 9
• FIG. 7a illustrates the Signature Score for ail Signaling genes after merging of the A OV'A filtered gene sets for both the CA1 signature (FIG. 5b) and the non-voltage dependent (NVD) gene sets (FIG. 5c).
• F G 7b illustrates results in a panel of 31 CAI/Cakium related genes capable of separating the different C'TO doses, both by signature view and by PCA.
• Table 10 provides the list of 31 genes
• FIG. 8 illustrates the EGRl Signaling Pathway
• CEACA i carcinoerrsbryoxiie antigen-related cell adhesion, molecule
• Dystonia was down regulated by 60 fold, in response to CTO treatment
• TGF- ⁇ signaling was inhibited in response to treatment with CTO.

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Analytical Chemistry (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Physics & Mathematics (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Biophysics (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Microbiology (AREA)
• Oncology (AREA)
• Hospice & Palliative Care (AREA)
• Evolutionary Biology (AREA)
• Bioinformatics & Computational Biology (AREA)
• Spectroscopy & Molecular Physics (AREA)
• Medical Informatics (AREA)
• Theoretical Computer Science (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Enzymes And Modification Thereof (AREA)

Priority Applications (8)

Applications Claiming Priority (2)

Publications (1)

Family

ID=52428197

Family Applications (1)

Country Status (9)

Cited By (1)

Families Citing this family (2)

Citations (4)

Family Cites Families (6)

• 2013
  • 2013-08-02 US US13/957,720 patent/US10378059B2/en active Active
• 2014
  • 2014-07-30 CN CN201480050189.3A patent/CN105531380B/zh active Active
  • 2014-07-30 CA CA2919690A patent/CA2919690C/en active Active
  • 2014-07-30 AU AU2014296212A patent/AU2014296212B2/en active Active
  • 2014-07-30 KR KR1020167002926A patent/KR102389934B1/ko active Active
  • 2014-07-30 WO PCT/US2014/048905 patent/WO2015017545A1/en not_active Ceased
  • 2014-07-30 EP EP14832909.7A patent/EP3027776B1/en active Active
  • 2014-07-30 JP JP2016531859A patent/JP6913459B2/ja active Active
• 2016
  • 2016-02-01 IL IL243894A patent/IL243894B/en active IP Right Grant

Patent Citations (4)

Non-Patent Citations (27)

Also Published As

Similar Documents

Legal Events