WO2015066285A2 - Compositions inhibant sgcα1 et méthodes de traitement de cancers à l'aide desdites compositions - Google Patents

Compositions inhibant sgcα1 et méthodes de traitement de cancers à l'aide desdites compositions Download PDF

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WO2015066285A2
WO2015066285A2 PCT/US2014/063089 US2014063089W WO2015066285A2 WO 2015066285 A2 WO2015066285 A2 WO 2015066285A2 US 2014063089 W US2014063089 W US 2014063089W WO 2015066285 A2 WO2015066285 A2 WO 2015066285A2
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seq
cancer
sgcal
peptide
cells
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PCT/US2014/063089
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WO2015066285A3 (fr
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Lirim Shemshedini
Shao-yong CHEN
Changmeng Cai
Chen-Lin Hsieh
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The University Of Toledo
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Publication of WO2015066285A3 publication Critical patent/WO2015066285A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • Androgen effects in prostate and other tissues are mediated by the androgen receptor (AR).
  • AR androgen receptor
  • This protein binds to and is activated by androgens.
  • the importance of the androgen receptor in the development of prostate cancer is demonstrated by the success of anti-androgen therapy at the early stages of prostate cancer.
  • Hormone ablation therapies continue to dominate the market, as they have proven to be the most effective at treating the early stage prostate cancer, which is hormone -dependent.
  • a representative method includes administering to a subject in need of treatment an effective amount of at least one effector agent.
  • the present invention is based, at least in part, on the discovery of a protein product which is able to down-regulate p53 activity in prostate cancer cells. Since disrupting mutations of p53 mainly occur in late-stage, hormone -refractory prostate tumors and are found in less than 50% of these tumors, this mechanism of p53 down-regulation is now believed to be important in those 50% of tumors that express wild-type p53 protein. It is also now believed that disrupting this mechanism of p53 down-regulation provides a new therapy against such tumors.
  • compositions are provided herein.
  • compositions of matter comprising a sGCal inhibitor. Specifically provided are those compositions wherein the inhibitor is capable of reducing sGCal -associated p53 inhibition. Also specifically provided are those compositions wherein the inhibitor is capable of increasing apoptosis of cancer cells capable of expressing sGCal and p53. Also specifically provided are those compositions wherein the inhibitor is capable of increasing apoptosis of sGCal-expressing cancer cells selected from the group consisting of: hormone refractory prostate cancer cells, metastatic prostate cancer cells, late stage prostate cancer cells, pancreatic cancer cells, and gastrointestinal cancer cells.
  • compositions wherein the inhibitor is selected from the group consisting of: a small molecule, a peptide, an sCG i mimic, an sCG i/sGCal dimerization inhibitor, a silencing RNA, and an antibody.
  • the inhibitor comprises a peptide. More specifically provided are those compositions wherein the peptide is selected from the group consisting of: A-8R [SEQ ID NO: 1], a functional variant of A-8R [SEQ ID NO: 1], a conserved variant of A-8R [SEQ ID NO: 1], B-8R [SEQ ID NO: 2], and a functional variant of B-8R [SEQ ID NO: 2].
  • the peptide comprises A-8R [SEQ ID NO: 1]. More specifically provided which comprises the peptide TFCKAFPFHII [SEQ ID NO: 5], or a conserved variants thereof, and means for translocating the peptide across a plasma membrane. Further provided are truncated A-8R [SEQ ID NO: 1] sequences and derivatives thereof.
  • compositions which are pharmaceutical formulations, and/or which further comprises a composition selected from the group consisting of: an adjuvant, a pharmaceutically-acceptable salt, a prodrug, a buffer, and a biomarker and/or which further comprises a chemotherapeutic agent, especially wherein the chemotherapeutic agent is etoposide.
  • compositions capable of increasing apoptosis of cancer cells comprising introducing at least one test composition to a plurality of sGCal-expressing cancer cells, and identifying whether the test composition increases apoptosis of the cells.
  • compositions capable of inhibiting cancer tumor growth comprising introducing at least one test composition to a plurality of sGCal -expressing cancer tumor cells, and identifying whether the test composition inhibits cancer tumor cell growth.
  • compositions capable of increasing cancer tumor regression comprising introducing at least one test composition to a plurality of sGCal- expressing cancer tumor cells, and identifying whether the test composition increases tumor cell regression.
  • compositions capable of inhibiting cancer cell proliferation comprising introducing at least one test composition to a plurality of sGCal- expressing cancer cells, and identifying whether the test composition inhibits cell proliferation.
  • compositions capable of treating cancer comprising introducing a test composition to a plurality of sGCal -expressing cancer cells, and identifying whether the test composition increases apoptosis of the cells.
  • the cancer is selected from the group comprising: neuroblastoma; lung cancer; bile duct cancer; non small cell lung carcinoma; hepatocellular carcinoma; lymphoma; nasopharyngeal carcinoma; ovarian cancer; head and neck squamous cell carcinoma; squamous cell cervical carcinoma; gastric cancer; colon cancer; uterine cervical carcinoma; gall bladder cancer; prostate cancer; breast cancer; testicular germ cell tumors; large cell lymphoma; follicular lymphoma; colorectal cancer; malignant pleural mesothelioma; glioma; thyroid cancer; basal cell carcinoma; T cell lymphoma; t(8;17)-prolyphocytic leukemia; mye
  • pancreatic cancer pancreatic cancer; t(5;14)(q35.1;q32.2) leukemia; malignant fibrous histiocytoma; gastrointestinal stromal tumor; and hepatoblastoma; colorectal; endometrial; ovarian; gastric; and urothelial.
  • compositions capable of treating hormone- refractory prostate cancer comprising introducing a test composition to a plurality of hormone- refractory prostate cancer cells, and identifying whether the test composition increases apoptosis of the cells.
  • compositions capable of treating metastatic prostate cancer comprising introducing a test composition to a plurality of metastatic prostate cancer cells, and identifying whether the test composition increases apoptosis of the cells.
  • compositions capable of treating late stage prostate cancer comprising introducing a test composition to a plurality of late stage prostate cancer cells, and identifying whether the test composition increases apoptosis of the cells.
  • compositions capable of treating pancreatic cancer comprising introducing a test composition to a plurality of sGCal -expressing cancer cells, and identifying whether the test composition increases apoptosis of the cells.
  • compositions capable of treating gastrointestinal cancer comprising introducing a test composition to a plurality of sGCal -expressing
  • test composition increases apoptosis of the cells.
  • compositions capable of treating breast cancer comprising introducing a test composition to a plurality of sGCal -expressing breast cancer cells, and identifying whether the test composition increases apoptosis of the cells.
  • [0025] Included are methods to affect apoptosis of sGCal -expressing cancer cells, comprising introducing an apoptosis-affecting amount of a composition capable of affecting sGCal activity to sGCal -expressing cancer cells.
  • Also provided are methods to increase apoptosis of sGCal -expressing cancer cells comprising introducing an apoptosis-increasing amount of a sGCal inhibitor to sGCal- expressing cancer cells.
  • Also provided are methods to inhibit tumor growth of sGCal -expressing cancer cells comprising introducing an tumor growth-inhibiting amount of a sGCal inhibitor to sGCal- expressing cancer cells.
  • Also provided are methods to increase regression of sGCal -expressing cancer cells comprising introducing an regression-increasing amount of a sGCal inhibitor to sGCal- expressing cancer cells.
  • Also provided are methods to inhibit proliferation of sGCal -expressing cancer cells comprising introducing an proliferation-inhibiting amount of a sGCal inhibitor to sGCal- expressing cancer cells.
  • Also provided are methods to affect p53 activity in sGCal -expressing cancer cells comprising introducing a p53 activity-affecting amount of a composition capable of affecting sGCal to sGCal -expressing cancer cells.
  • cells are selected from the group consisting of: LNCaP cells, C81 cells, Capan-2, and CWR22-Rvl cells.
  • cells are mouse cells.
  • cells are in a mammal selected from the group consisting of: mouse, rat, guinea pig, dog, cat, monkey and human.
  • cells are human cells.
  • a cancer patient is a candidate for treatment with an sGCal -inhibiting composition, comprising identifying a patient having cancer cells that express sGCal as a candidate for treatment with a sGCal -inhibiting composition.
  • Also provided are methods to predict the prognosis of a patient with cancer comprising identifying a patient having cancer cells that express sGCal as having a poor prognosis.
  • t(5;14)(q35.1;q32.2) leukemia malignant fibrous histiocytoma; gastrointestinal stromal tumor; and hepatoblastoma; colorectal; endometrial; ovarian; gastric; and urothelial.
  • methods of prophylactically treating cancer comprising administering to a subject in need of treatment an effective amount of at least one composition herein.
  • Kits for research and diagnostics are provided herein.
  • kits comprising: a volume a p53 regulator that disrupts sGCal-p53 interaction; and instructions for the use of the volume of p53 regulator in the treatment of a sGCal -expressing cancer in a subject.
  • the cancer is prostate cancer.
  • kits for conducting an assay to predict recurrence of prostate cancer in a biological sample comprising: materials for detecting sGCal.
  • kits comprising a peptide or nucleic acid herein as a biomarker.
  • identifying an anti-cancer agent comprising: culturing a plurality of cancer cells capable of expressing sGCal and p53 in the presence of a test compound, and measuring p53 expression levels, wherein an increase in the p53 expression levels relative to a control is indicative of the test compound being an anti-cancer agent.
  • Also provided are methods of identifying an anti-prostate cancer agent comprising: providing a test agent to a cell, and measuring the level of at least one p53 regulator associated with an altered expression levels in prostate cancer cells, wherein an altered level of a p53 regulator in the cell, relative to a control cell, is indicative of the test agent being an anti-prostate cancer agent.
  • compositions comprising at least one peptide which mimics sGC i heterodimerization domains which bind to and disrupts the pro-survival functions of sGCal, thereby leading to cell death.
  • compositions comprising Peptide A-8R— Ac-
  • TFCKAFPFHIIRRRRRRRR-OH [SEQ ID NO:l]
  • peptides comprising A-8R [SEQ ID NO: 1] which are useful to kill hormone-refractory prostate cancer cells, or an isolated variant or biologically-active fragment thereof.
  • Also provided are methods of determining the prognosis of a subject with prostate cancer comprising: measuring the sGCal expression level in a test sample from the subject, wherein the sGCal expression level relative to control is associated with an adverse prognosis in prostate cancer.
  • Also provided are methods of predicting recurrence of prostate cancer comprising the steps of: obtaining a sample from a patient; and measuring sGCal expression levels in the sample; wherein sGCal expression levels above or below pre -determined cut-off levels are indicative of predict recurrence of prostate cancer.
  • Also provided are methods of determining patient treatment protocol comprising the steps of: obtaining a sample from a patient; and measuring the sGCal expression levels; wherein sGCal expression levels above or below pre -determined cut-off levels are sufficiently indicative of risk of recurrence to enable a physician to determine the degree and type of therapy.
  • Also provided are methods of treating a patient comprising the steps of: obtaining a sample from a patient; measuring sGCal expression levels in the sample to determine risk; and treating the patient with adjuvant therapy if they are a high risk patient.
  • methods of treating a patient comprising the steps of: obtaining a sample from a patient; measuring sGCal expression levels in the sample to determine risk; and treating the patient with adjuvant therapy if they are a high risk patient.
  • a composition herein is used as a biomarker to determine sGCal expression levels.
  • cancer is prostate cancer.
  • nucleic acids, constructs, vectors, cells and transgenic animals are provided.
  • nucleic acids comprising a nucleic acid encoding an amino acid having the sequence TFCKAFPFHII [SEQ ID NO: 5].
  • isolated nucleic acids comprising a nucleic acid encoding an amino acid having the sequence of A-8R [SEQ ID NO: 1].
  • isolated nucleic acids comprising a nucleic acid encoding an amino acid having the sequence of B-8R [SEQ ID NO: 2]. Specifically provided are those nucleic acids herein, which further comprises a promoter operatively linked to the nucleic acid.
  • vectors comprising a nucleic acid herein.
  • cells comprising a vector herein.
  • transgenic mammals comprising a cell herein.
  • FIGS. 1A-1F Over-expression of sGCal inhibits p53 transcriptional activity. p53 activity was quantified by measuring luciferase activity. Bar graphs represent the average of three independent experiments plus standard deviation. All cells received the same amount of transfected expression plasmid and that pCHl 10 was used to control transfection efficiency.
  • FIG. 1A LNCaP cells were transfected with 0.1 ⁇ g p53-Luc reporter plasmid, with or without 0.5 ⁇ g p53, 0.5 ⁇ g pCHl 10, and 0.1 or 0.5 ⁇ g sGCal.
  • FIG. IB LNCaP were transfected with 50 nM sGCal siRNA, and 24 hrs later with 0.1 ⁇ g p53-Luc reporter plasmid and 0.5 ⁇ g pCHl 10.
  • FIG. 1C LNCaP cells were transfected with 50 nM control or sGCal siRNA and
  • FIG. ID LNCaP cells were transfected with 0.5 ⁇ g empty vector or sGCal and Western blotting was used to detect sGCa 1 and p53 expression, ⁇ -actin expression was used to standardize Western blot.
  • FIG. IE VCaP cells were transfected with 0.1 ⁇ g p53-Luc reporter plasmid, with or without 0.5 ⁇ g p53, 0.5 ⁇ g pCHHO, and 0.1 or 0.5 ⁇ g sGCal.
  • FIG. IF PC-3 Cells were transfected with 0.1 ⁇ g p53-Luc, 0.5 ⁇ g pCHl 10, 0.5 ⁇ g p53, and 0.1 or 0.5 ⁇ g sGCal. p53 transcriptional activity was measured by luciferase assay.
  • FIGS. 2A-2F The sGCal -mediated repression of p53 transcriptional activity is independent of mediators of NO signaling and guanylyl cyclase activity. Androgen-dependent
  • LNCaP cells were transfected with 0.5 ⁇ g pCHHO, 0.1 ⁇ g p53-Luc reporter plasmid, and subjected to different treatments:
  • FIG. 2A Cells were transfected with 0.5 ⁇ g pCHl 10, 0.1 ⁇ g p53-Luc reporter plasmid, and subjected to 5 or 50 mM ODQ.
  • FIG. 2B Cells were transfected with 0.5 ⁇ g p53 and 0.5 ⁇ g sGCal and/or sGC l.
  • FIG. 2C Cells were treated with different concentrations of C-PTIO.
  • FIG. 2D Cells were treated with different concentrations of SNP.
  • FIG. 2E Cells were treated with different concentrations of 8-Br-cGMP.
  • FIG. 2F Cells were transfected with 0.5 or 1 ⁇ g sGCal or sGCal(D531A). Bar graphs represent averages of three independent experiments plus standard deviations. All activities are relative to the first condition, and this activity was set to 1. All cells received the same amount of transfected expression plasmid and that pCHl 10 was used to control transfection efficiency.
  • FIGS. 3A-3E Endogenous sGCal associates with p53 in LNCaP cells: [0092] FIG. 3A-3B: Cytoplasmic extracts were prepared from LNCaP cells and subjected to immunoprecipitation using an anti-p53 (FIG. 3A) or anti-sGCal antibody (FIG. 3B). Western blotting was used to detect p53 and sGCal. The negative control IP was performed using an IgG antibody. "Input" represents extracts that were used in the IP experiments, while whole-cell extracts were positive controls for the Western blotting.
  • FIG. 3C LNCaP cytoplasmic extract was fractionated through a Sephacryl S-300 gel filtration column using HPLC. 2-ml fractions were collected and analyzed by Western blotting using either an anti-sGCal (Cayman Chemical) or anti-p53 (Santa Cruz Biotechnology) antibody.
  • FIG. 3D LNCaP cells were subjected to immunocytochemistry using anti-sGCal or anti-p53 antibody to measure subcellular localization of endogenous proteins. Images were viewed by confocal microscopy.
  • FIG. 3E Nuclear (N) and cytosolic (C) extracts were prepared from LNCaP cells and Western blotting was used to measure protein levels of p53, sGCal, the exclusively nuclear hRARa, and the cytosolic protein MLK3. ⁇ -actin was used as loading control.
  • FIGS. 4A-4C sGCal regulates the subcellular localization of p53:
  • FIG. 4A LNCaP cells were infected with a control empty adenovirus or sGCal- expressing virus (20 MOI of each) and nuclear and cytosolic extracts were prepared. These extracts were subjected to Western blotting to detect sGCal. Whole Cell Input represents total amount of cellular p53 before cell fractionation, ⁇ -actin was used as loading control.
  • FIG. 4B Nuclear (N) and cytosolic (C) extracts were prepared from LNCaP cells and Western blotting was used to measure protein levels of MDM2, p53, JAB 1 , and sGCal . ⁇ -actin was used as loading control.
  • FIG. 4C LNCaP cells were untransfected (upper panel) or transfected with sGCal siRNA (lower panel) and measured for subcellular localization of endogenous sGCal or p53 using antibodies against these two protein anti-p53 antibody. Images were viewed by confocal microscopy.
  • FIGS. 5A-5C E. coli-expressed sGCal and p53 interact in vitro.
  • Cell extracts were prepared from BL21 cells transformed with sGCal, p53, or sGC i. 500 ⁇ of each extract were mixed and subjected to immunoprecipitation using an anti-sGCal antibody (Cayman Chemical) or anti-p53 antibody (Santa Cruz Biotechnology), or anti- sGC i antibody (Cayman Chemical).
  • Western blotting was used to measure the levels of (FIG. 5A) p53, (FIG. 5B) sGCal, and (FIG. 5C) sGC i.
  • PBS was used to wash the Protein A-sepharose beads in the IP reactions. Extract represents bacterial extract before subjected to immunoprecipitation.
  • FIGS. 6A-6F sGCal affects the expression of p53-regulated genes in prostate cancer cells.
  • LNCaP cells were transfected with 50 nM control or sGCal siRNA and QRT-PCR was used to measure the expression of (FIG. 6A) p53AIPl, (FIG. 6B) PCBP4, (FIG. 6C) Survivin, (FIG. 6D) p21, and (FIG. 6E) sGCal, relative to GAPDH. Bar graphs represent averages of three independent experiments plus standard deviations. All activities are relative to control siRNA transfection, and this activity was set to 1. Student T test showed significant differences (P ⁇ 0.02), as indicated.
  • FIG. 6A p53AIPl
  • PCBP4 PCBP4
  • FIG. 6C Survivin
  • FIG. 6D p21
  • FIG. 6E sGCal affects the expression of p53-regulated genes in prostate cancer cells.
  • Bar graphs represent averages of three independent experiments plus standard
  • 6F - sGCal affects the expression of p53-regulated genes involved in apoptosis.
  • LNCaP cells were infected with empty adenovirus or adenovirus expressing sGCal and subjected to a p53 Signaling PCR array from Superarray. Shown are fold-changes in expression in p53-regulated genes p53AIPl, PCBP4, and BIRC5 in sGCal-over-expressing cells as compared to cells infected with empty virus.
  • FIG. 7A Viability of prostate cancer cells is affected by sGCal expression.
  • LNCaP cells were transfected with control siRNA or sGCal siRNA and grown for 0, 3, or 6 days in the absence of serum. Cell number was measured using the MTT assay. Each data point represents averages of three independent experiments plus standard deviations.
  • FIG. 7B Over-expression of sGCal protects prostate cancer cells from the lethal effects of etoposide.
  • LNCaP cells were infected with control empty adenovirus (-sGCal ; 20 MOI) or sGCal -expressing adenovirus (+ sGCal ; 2 or 20 MOI) and treated with 10 ⁇ etoposide. After two days of incubation, cell number was quantified by MTT assay. Bar graphs represent averages of three independent experiments plus standard deviations. All activities are relative to the first condition, and this activity was set to 100%. Student T test showed significant differences (P ⁇ 0.04), as indicated.
  • FIG. 8 sGCal is over-expressed in hormone refractory, metastatic prostate cancer.
  • RNA was prepared from 52 androgen-dependent (AD) and 11 androgen-independent prostate tumors and subjected to Affymetrix microarray analysis and expression was measured for sGCal, sGC i, and AR. Student T test was used to show significantly increased (P ⁇ 0.05) expression in AI tumors for sGCal and AR.
  • FIGS. 9A-C sGCal expression directly correlates with p53 expression in prostate tumors.
  • Total RNA was isolated from 9 metastatic prostate tumors and used to synthesize cDNA by reverse transcription. The cDNA was then used in a PCR reaction to measure gene expression
  • FIG. 9A RT-PCR was used to measure the expression of p53, sGCal , and GAPDH mRNAs. Note that GAPDH was used as an internal control.
  • FIG. 9B Three PCR products, ranging in size from 423 to 708 bp, were synthesized to cover the entire coding region of p53.
  • FIG. 9C Two PCR products, 360 and 460 bp, were synthesized to cover the central part of the p53 coding region.
  • FIG. 10A LNCaP cells were transfected with 0.1 ⁇ g p53-Luc reporter plasmid and 0.1 or 0.5 ⁇ g p53.
  • FIG. 10B LNCaP cells were transfected with 0.1 ⁇ g p53-Luc reporter plasmid and control or p53 siRNA.
  • FIG. IOC LNCaP cells were transfected with control or p53 siRNA and subjected to
  • FIG. 11A VCaP cells were treated with or without 1 nM R1881 and Western blotting was used to measure expression of sGCal. ⁇ -actin was used as a loading control.
  • FIG. 11B VCaP cells, treated with 1 nM R1881, were transfected with control or p53 siRNA and subjected to Western blotting to measure expression of sGCal. ⁇ -actin was used as a loading control.
  • FIG. 11 C VCaP cells were grown in the presence of 1 nM Rl 881 and transfected with control or sGCal siRNA. Cell density was measured at day 0, 3, and 6 using the MTT assay.
  • FIGS. 12A-B Peptides mimicking sGCpi dimerization domains with sGCal are toxic to cultured prostate cancer cells:
  • FIG. 12A The amino acid sequences of four synthetic peptides, Peptide A-D, fused to an 8-Arginine tag [SEQ ID NOS 1-4, respectively, in order of appearance] for membrane translocation, are shown. Note that these four peptide sequences mimic four known sGC i dimerization domains with sGCal.
  • FIG. 12B LNCaP cells were grown without androgen for 2 or 4 days in the presence of increasing concentration of Peptide A-8R [SEQ ID NO: 1] (upper left), Peptide B-8R [SEQ ID NO: 1] (upper left), Peptide B-8R [SEQ ID NO: 1] (upper left), Peptide B-8R [SEQ ID NO: 1] (upper left), Peptide B-8R [SEQ ID NO: 1] (upper left), Peptide B-8R [SEQ ID NO: 1] (upper left), Peptide B-8R [SEQ ID NO: 1] (upper left), Peptide B-8R [SEQ ID NO: 1] (upper left), Peptide B-8R [SEQ ID NO: 1] (upper left), Peptide B-8R [SEQ ID NO: 1] (upper left), Peptide B-8R [SEQ ID NO: 1] (upper left), Peptide B-8R [SEQ ID NO: 1] (upper left), Peptide B-8R [SEQ ID NO
  • FIGS. 13A-B A peptide mimicking a sGCal dimerization domain with sGCal is highly toxic to cultured prostate cancer cells:
  • FIGS. 13A LNCaP cells were grown with (Right) or without (Left) 1 nM androgen
  • FIG. 13B LNCaP cells were grown with (Right) or without (Left) 1 nM androgen
  • FIGS. 14A-B The cytotoxic activity of Peptide A-8R [SEQ ID NO: 1] requires a membrane translocation signal:
  • FIG. 14A LNCaP cells were grown with (Right) or without (Left) 1 nM R1881 for 3 or
  • FIG. 14B LNCaP cells were grown with (Right) or without (Left) 1 nM R1881 for 3 or 6 days in the presence of increasing concentration of Peptide A-8R [SEQ ID NO: 1] or vehicle and then cell density was measured.
  • FIGS. 15A-C Peptide A-8R [SEQ ID NO: 1] is toxic to hormone-refractory prostate cancer cells:
  • FIG. 15A LNCaP cells were grown for 1-8 hrs in the presence of 10 ⁇ Peptide A-8R
  • FIG. 15B C81 (androgen-refractory LNCaP) cells were grown for 1-8 hrs in the presence of 10 ⁇ Peptide A-8R [SEQ ID NO: 1] or vehicle and then cell density was measured.
  • FIG. 15C CWR22-Rvl (androgen-refractory) cells were grown for 1-8 hrs in the presence of 10 ⁇ Peptide A-8R [SEQ ID NO: 1] or vehicle and then cell density was measured.
  • FIGS. 16A-B sGCal is expressed in AR-positive prostate cancer cells, but not AR- negative prostate cancer or kidney cancer cells:
  • FIG. 16A LNCaP, C81, and CWR22-Rvl cells were grown in the absence or presence of 1 nM R1881 for two days and then measured for sGCal protein expression by Western blotting.
  • FIG. 16B LNCaP, PC-3, Cos, and ACHN cells were grown for two days and then measured for sGCal protein expression by Western blotting.
  • FIGS. 17A-C Peptide A-8R [SEQ ID NO: 1] is not toxic to cancer cells deficient in sGCal expression:
  • FIG. 17A PC-3 prostate cancer cells were grown for 3 or 6 days in the presence of increasing concentration of Peptide A-8R [SEQ ID NO: 1] or vehicle and then cell density was measured.
  • FIG. 17B Cos monkey kidney cancer cells were grown for 2 or 4 days in the presence of increasing concentration of Peptide A-8R [SEQ ID NO: 1] or vehicle and then cell density was measured.
  • FIG. 17C ACHN human kidney cancer cells were grown for 2 or 4 days in the presence of increasing concentration of Peptide A-8R [SEQ ID NO: 1] or vehicle and then cell density was measured.
  • FIGS. 18A-B Peptide A-8R [SEQ ID NO: 1] induces apoptosis of prostate cancer cells:
  • FIG. 18A LNCaP cells were grown for 1-24 hrs in the presence of vehicle, 10 mM
  • Peptide A-8R [SEQ ID NO: 1] or 20 mM Etoposide and then measured for Caspase 3/7 activity.
  • FIG. 18B LNCaP cells were grown for 4 hrs in the presence of increasing concentration of Peptide A-8R [SEQ ID NO: 1] with or without 40 mM Z-VAD-FMK and then cell density was measured.
  • FIGS. 19A-C Peptide A-8R [SEQ ID NO: 1] associates with sGCal in prostate cancer cells:
  • FIG. 19A LNCaP prostate cancer cells were grown in the presence of 25 mM Peptide A-8R-Biotin [SEQ ID NO: 1] and subjected to immunocytochemistry using an anti- sGCal antibody (green) or anti-Biotin antibody (red). DAPI was used to stain nuclei.
  • FIG. 19B LNCaP whole cell extract was incubated with 100 mg Peptide A-8R-Biotin [SEQ ID NO: 1] for4 hrs and then subjected to purification using streptavidin-agarose beads. As a negative control, the purification was repeated with extract alone (no Peptide A-8R-Biotin [SEQ ID NO: 1]).
  • FIG. 19C LNCaP cells were grown with for 12 or 48 hrs in the presence of increasing concentration of Peptide A-8R [SEQ ID NO: 1] (Left) or PeptideA-8R-Biotin [SEQ ID NO: 1] (Right) and then cell density was measured.
  • FIG. 20 Peptide A-8R [SEQ ID NO: 1] inhibits the growth of LNCaP prostate xenograft tumors.
  • LNCaP xenograft tumors were grown in nude mice to a size of 200-400 cubic mm and then treated with daily intratumoral injections of either Peptide A-8R [SEQ ID NO: 1] (80 mg/kg of animal) or Vehicle (DMSO) for seven days, after which the tumors were left untreated and allowed to grow for an additional 2-3 weeks. Each point represents the average tumor size of three animals plus/minus the standard deviations. Note that open squares or circles represent no treatment of either Peptide A-8R [SEQ ID NO: 1] or vehicle.
  • FIGS. 21A-B Peptide A-8R [SEQ ID NO: 1] is cytotoxic to pancreatic cancer cells:
  • FIG. 21A RT-PCR was used to measure the expression of sGCal, sGCpi, p53, and GAPDH mRNAs. Note that GAPDH was used as an internal control.
  • FIG. 21B Capan-2 pancreatic cancer cells were grown for 3 or 5 days in the presence of increasing concentration of Peptide A-8R [SEQ ID NO: 1] or vehicle and then cell density was measured.
  • FIG. 22 C81 cells were grown for 3 days in the presence of increasing concentration of the listed truncated peptides [SEQ ID NOS: 22, 24, 20, 18, 26, and 10, respectively, in order of appearance]. Cell growth was then measured.
  • FIG. 23 C81 cells were grown for 6 days in the presence of increasing concentration of the listed truncated peptides [SEQ ID NOS: 22, 24, 20, 18, 26, and 10, respectively, in order of appearance]. Cell growth was then measured.
  • FIG. 24 C81 cells were grown for 3 days in the presence of increasing concentration of the listed Peptide A derivatives [SEQ ID NOS: 32, 30, 28, 10, 16, 14, 12, and 1, respectively, in order of appearance]. Cell growth was then measured.
  • FIG. 25 C81 cells were grown for 6 days in the presence of increasing concentration of the listed Peptide A derivatives [SEQ ID NOS: 32, 30, 28, 10, 16, 14, 12, and 1, respectively, in order of appearance]. Cell growth was then measured.
  • FIG. 26 C81 cells were grown for 3 days in the presence of increasing concentration of the listed Peptide A derivatives [SEQ ID NOS: 20, 18, and 10, respectively, in order of appearance]. Cell growth was then measured.
  • FIG. 27 C81 cells were grown for 6 days in the presence of increasing concentration of the listed Peptide A derivatives [SEQ ID NOS 20, 18, and 10, respectively, in order of appearance]. Cell growth was then measured.
  • FIGS. 28A-C Peptide A-8R [SEQ ID NO: 1] inhibits the growth of C81 prostate xenograft tumors.
  • C81 (hormone -independent LNCaP) xenograft tumors were grown in nude mice to a size of about 200 cubic mm and then treated every other day with five intratumoral injections of either Peptide A-8R [SEQ ID NO: 1] (40 mm/kg of animal) or vehicle (DMSO) for seven days, after which the tumors were left untreated and allowed to grow for an additional 3 weeks before the tumors were excised. Each point represents the average tumor size of three animals plus or minus the standard deviations.
  • the arrow represents the last treatment of Peptide A-8R [SEQ ID NO: 1] or vehicle. There is a statistical significance (P ⁇ 0.0004) of Peptide A activity, relative to vehicle, for each treatment day.
  • Peptide A activity relative to vehicle, for each treatment day.
  • C Image showing the strong anti-tumor effect of Peptide A-8R [SEQ ID NO: 1], as compared to vehicle control, in one animal.
  • 8R refers to the sequence of 8 arginines, or RRRRRRRR [SEQ ID NO:
  • Androgen signaling is mediated by the androgen receptor (AR), whose transcriptional activity correlates with a higher risk and a higher grade of prostate cancer.
  • AR androgen receptor
  • This androgen dependency of early-stage prostate cancer is used to combat this disease with anti-androgen therapy.
  • the cancer is androgen-sensitive and thus responds to androgen-ablation therapy.
  • the cancer is androgen- insensitive such that androgen-ablation therapy fails to work.
  • functional AR is found in most prostate cancer cells, including late-stage cells that become androgen-independent.
  • sGCal soluble guanylyl cyclase alpha 1
  • sGCal is one subunit of sGC, a heterodimeric enzyme that catalyzes cGMP synthesis in response to nitric oxide. This signaling pathway is very important in mammalian physiology, particularly in the cardiovascular system. sGCal expression is androgen-regulated, is required for prostate cancer proliferation, and increases with increasing grade of prostate cancer.
  • sGCal can interact with cytoplasmic p53 and negatively regulate its transcriptional activity.
  • the p53 protein acts as a tumor suppressor, and is able to disrupt the growth process of cells by slowing the cell cycle or inducing apoptosis.
  • the p53 gene is the most commonly mutated locus in human cancers. While mutations at the p53 locus represent a common mechanism of p53 inactivation, there are at least half of human cancers that do not harbor such mutations. Indeed, in prostate cancer, p53 mutations are observed in late-stage disease and the mutation rate varies from 3% to 42%. Additional mechanisms are required to overcome or bypass the wild-type p53 protein in the more than half of tumors that express this protein.
  • the sGCal inhibition of p53 represents a mechanism for down-regulation in those 50% or so of prostate tumors that express wild-type p53 protein.
  • nucleic acids and amino acid sequences are disclosed. Those in the art are aware of the redundancy of the genetic code, and therefore, any nucleic acid which encodes for the amino acids herein are described as within the scope of the present invention. Moreover, conserved amino acid residue changes in the present amino acid compounds are also within the scope of the present invention, as are the corresponding nucleic acid changes and resulting nucleic acid sequences. These concepts are available in Alberts et al., Molecular Biology of the Cell, Fourth Edition (2002, Garland Science)
  • FIG. 1C is a graph showing the results where LNCaP cells were transfected with 50 nM control or sGCal siRNA and Western blotting was used to detect expression of sGCal and p53. ⁇ -actin expression was used to standardize Western blot.
  • FIG. ID is a graph showing the results where LNCaP cells were transfected with 0.5 ⁇ g empty vector or sGCal and Western blotting was used to detect sGCal and p53 expression, ⁇ - actin expression was used to standardize Western blot.
  • FIG. IF is a graph showing the results where PC-3 Cells were transfected with 0.1 ⁇ g p53-Luc, 0.5 ⁇ g pCH110, 0.5 ⁇ g p53, and 0.1 or 0.5 ⁇ g sGC ⁇ l.
  • NO interfering drugs were used. As shown in FIG. 2A, ODQ, which inhibits sGC enzyme activity, had no effect on sGCal-mediated repression of p53, even though these two concentrations strongly inhibited sGC -catalyzed cGMP synthesis (data not shown), showing that sGCal inhibits p53 activity independent of sGC enzyme activity.
  • FIG. 2B shows that transfected sGC i disrupts, rather than mediates, the negative activity of transfected sGCal on p53 (FIG. 2B), thus showing that the sGCal inhibitory activity is independent of sGC i.
  • C-PTIO is an NO scavenger and thus would be expected to enhance p53 activity if sGCal requires NO for its inhibitory activity; contrary to this, c-PTIO inhibit p53 activity of 100 nM of drug (FIG. 2C), suggesting that NO is not involved in sGCal inhibition.
  • mutant sGCal(D531A) which has greatly reduced cyclase activity (data not shown), is fully able to inhibit p53 transcriptional activity (FIG. 2F), demonstrating that sGCal activity on p53, as on proliferation (data not shown), does not depend on its guanylyl cyclase activity.
  • LNCaP cells were infected with an sGCal -expressing adenovirus or control empty virus and p53 subcellular localization was measured by Western blotting following cell fractionation.
  • adenovirus over-expression of sGCal led to higher cytosolic and lower nuclear levels of p53 protein, as compared to empty virus.
  • Western blotting showed no detectable expresssion for MDM2 and significant cytoplasmic expression of Jabl, two proteins known to be involved in p53 nuclear export (FIG. 4B).
  • Treatment of cells with the proteasome inhibitor MG132 significantly enhanced MDM2 cytoplasmic levels (FIG. 4B), suggesting that this protein is under active proteasome-dependent degradation.
  • FIG. 3 and FIG. 4 show that endogenous sGCal and p53 can co-associate in LNCaP cells.
  • the inventors have expressed the proteins in E. coli and carry out IP experiments.
  • an anti-p53 antibody can IP p53 from E. coli extract.
  • p53 and sGCal are mixed and an IP is performed using an anti-sGCal antibody, p53 is co-purified (FIG. 5A) showing that p53 and sGCal expressed in E. coli can associate with one another.
  • FIG. 6F shows that sGCal affects the expression of p53-regulated genes involved in apoptosis.
  • LNCaP cells were infected with empty adenovirus or adenovirus expressing sGCal and subjected to a p53 Signaling PCR array from Superarray. Shown are fold-changes in expression in p53-regulated genes p53AIPl, PCBP4, and BIRC5 in sGCal -over-expressing cells as compared to cells infected with empty virus.
  • FIG. 7A shows that the viability of prostate cancer cells is affected by sGCal expression.
  • LNCaP cells were transfected with control siRNA or sGCal siRNA and grown for 0, 3, or 6 days in the absence of serum. Cell number was measured using the MTT assay. Each data point represents averages of three independent experiments plus standard deviations.
  • LNCaP cells were infected with an sGCal -expressing adenovirus and treated with etoposide, an inducer of apoptosis.
  • Etoposide significantly reduced the viability of LNCaP cells (FIG. 7A), likely due to apoptosis.
  • over-expression of sGCal markedly enhanced cell viability, showing that sGCal desensitizes cells to apoptosis-inducing drugs (FIG. 7A).
  • This finding, together, with the PCR array data showing that sGCal can regulate Survivin expression, is consistent with results showing that Survivin desensitizes prostate cancer cells to etoposide.
  • Affymetrix microarray analysis was used to show that sGCal expression is significantly enhanced in hormone -refractory, metastatic prostate (AI) tumors as compared to hormone- dependent tumors (D) (FIG. 8).
  • AI metastatic prostate
  • D hormone- dependent tumors
  • sGC i levels are low in both types of tumor tissues (FIG. 8).
  • AR levels are dramatically higher in AI tumors than AD tumors (FIG. 8).
  • RT-PCR Reverse transcription-polymerase chain reaction
  • VCaP cells which express a wild-type AR and exhibit androgen-dependent proliferation, were also examined for sGCal expression.
  • FIG. 11A shows, R1881 significantly induced sGCal protein expression in VCaP cells, demonstrating that androgen-induced expression of sGCal is not unique to LNCaP cells or dependent on the mutant AR expressed in LNCaP cells.
  • Each peptide contained 8 arginines at the C-terminus, a sequence which is known to mediate plasma membrane translocation and cellular internalization. Treatment of LNCaP cells with different concentrations of peptides resulted in cell death induced by two of the four peptides.
  • Peptide B-8R [SEQ ID NO: 2] stopped the growth of LNCaP cells while Peptide A-8R [SEQ ID NO: 1] was highly cytotoxic, killing most of the cells. This cytotoxicity of Peptide A-8R [SEQ ID NO: 1] is clearly shown in FIG. 13A, in which 50 mM peptide treatment led to 100% cell death by day 6.
  • Peptide A-8R [SEQ ID NO: 1] induced LNCaP cell death in both the absence and presence of androgens (FIG. 13A).
  • an inactive peptide, Peptide C-8R [SEQ ID NO: 3] had no effect (FIG. 13B), demonstrating that the amino-acid sequence of Peptide A was required for the cytotoxic effect and excluding potential cytotoxicity induced by the 8-arginine sequence.
  • FIG. 14A When LNCaP cells were treated with Peptide A lacking 8 arginines, no effect was observed on cell growth (FIG. 14A), in contrast to the strong cytotoxic effect of Peptide A-8R [SEQ ID NO: 1] (FIG. 14B), strongly showing that cellular internalization is required for the peptide cytotoxic effect.
  • Endogenous sGCal is expressed in LNCaP, C81, and CWR22-Rvl cells (FIG. 16A), all of which are sensitive to the cytotoxic effect of Peptide A-8R [SEQ ID NO: 1] (see FIG. 15). These data show that the peptide effect requires endogenous sGCpi expression, an expected finding since sGCal is the designed target of Peptide A-8R [SEQ ID NO: 1]. To confirm, several different cancer cell lines were studied that do not express endogenous sGCal (FIG. 16B).
  • Peptide A-8R [SEQ ID NO: 1] had little to no effect on PC- 3 (prostate cancer), Cos (mouse kidney cancer), and ACHN cells (human kidney cancer), even at a high concentration of 100 ⁇ . These data strongly support the contention that the cytotoxic activity of Peptide A-8R [SEQ ID NO: 1] depends on endogenous sGCal protein.
  • Peptide A-8R [SEQ ID NO: 1] was able to induce Caspase 3/7 activity much faster than the well-studied apoptosis-inducing drug Etoposide.
  • the inventors used the pan-caspase inhibitor Z-VAD-FMK.
  • Pretreatment of LNCaP cells with Z-VAD-FMK resulted in complete relief of the Peptide A-8R [SEQ ID NO: 1] cytotoxic effect, even at a high peptide concentration of 50 ⁇ (FIG. 18B).
  • Z-VAD-FMK had no significant effect on Vehicle-treated cells (FIG. 18B).
  • Peptide A-8R [SEQ ID NO: 1 ] interacts with sGCal
  • Peptide A-8R [SEQ ID NO: 1] was synthesized with a Biotin tag at the C-terminus, giving Peptide A-8R-Biotin [SEQ ID NO: 1].
  • LNCaP cells were treated with Peptide A-8R-Biotin [SEQ ID NO: 1] and subjected to immunocytochemistry (FIG. 19A). Endogenous sGCal is found primarily or exclusively in the cytoplasm of LNCaP cells. Also, Peptide A-8R-Biotin [SEQ ID NO: 1] is also found in the cytoplasm and colocalizes with sGCal, as shown by the merged images.
  • FIG. 19C shows that addition of a Biotin tag to Peptide A-8R [SEQ ID NO: 1] does not affect the cytotoxic efficacy of this peptide.
  • Protein extracts were prepared by boiling the tumor tissue in 3 x SDS buffer, and evaluated by SDS-PAGE gel. The procedure was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Toledo. During the experiments, all animals were monitored daily for morbidity and all efforts were made to minimize suffering. At the end of the study, all animals were euthanized using inhalation of carbon dioxide, after which surgery was performed to excise tumors from all animals.
  • Peptide A-8R [SEQ ID NO: 1] stopped tumor growth and actually caused some tumor regression after five injections of peptide.
  • FIG. 28A and FIG. 28B. Remarkably, the tumors did not grow at all three weeks after peptide treatment was stopped, while the vehicle -treated tumors grew by more than three-fold.
  • FIG. 28A. To control for potential differences between animals, Peptide A-8R [SEQ ID NO: 1] and vehicle were tested in the same mouse having two tumors. The Peptide-treated tumor was markedly smaller than the vehicle-treated tumor.
  • FIG. 28C. In fact, the Peptide-treated tumor did not grow, as in the other animals, while the vehicle- treated tumor grew significantly.
  • Peptide A-8R [SEQ ID NO: 1] has potent anticancer activity, it can be further optimized for selective action on prostate cancer cells and in its overall 'drug-like' properties so that it is also suitable for clinical deployment.
  • Peptide A-8R [SEQ ID NO: 1] was used as a structural template to design additional peptides, peptidomimetics, and small molecule analogs. Initial analogs were screened by in vitro sGCal binding assays to generate structure-activity relationships (SAR) in order to better understand the target protein's interaction with A-8R [SEQ ID NO: 1] and the latter's inhibitory properties.
  • SAR structure-activity relationships
  • Such screening is useful to define the minimal size peptide required for activity while also establishing parameters that can be useful to transition compounds toward incorporated enhanced peptidomimetic character and drug-like properties.
  • probes displaying a range of activity can be used to validate the biochemical assay's link to computational chemistry strategies also seeking to further define the mechanism at the level of distinct molecular interactions and binding energies.
  • Active analog compounds can be additionally tested in a cell culture panel of normal mammalian tissues so as to establish structure -toxicity relationships (STR). Further design of compounds can then be able to move toward enhanced efficacy-related SAR and drug-like properties while moving away from STR.
  • truncated variants and derivatives of Peptide A Provided herein are truncated variants and derivatives of Peptide A.
  • the following sequences are truncated variants and derivatives of Peptide A: [00221] PI5 -- PFHII [SEQ ID NO: 8]
  • Peptide A-8R [SEQ ID NO: 1] can be truncated to a smaller peptide that retains the same type of biological activity profile.
  • truncating Peptide A-8R [SEQ ID NO: 1] results in greater potency upon conversion of some of the pharmacophore functionalities to peptidomimetic groups that can, in turn, enhance key interactions by lowering the net binding energy.
  • Peptide A-8R [SEQ ID NO: 1] was divided into two parts, using the central proline residue as the division point.
  • FF5 [SEQ ID NO: 9] (amide-capped peptide referred to in Table 2 as FF5-NH2);
  • FF5-F1A-8R [SEQ ID NO: 22] (amide-capped peptide referred to in Table 2 as AF5- 8R);
  • FF5-F5A-8R [SEQ ID NO: 24] (amide-capped peptide referred to in Table 2 as FA5- 8R);
  • FF5-K3A-8R [SEQ ID NO: 26] (amide-capped peptide referred to in Table 2 as FF5-
  • caps begin the transition toward peptidomimetic probes that can be continued by replacing some of the amino acid side chains within A-8R [SEQ ID NO: 1] by chemical surrogates that provide modified physicochemical properties.
  • the "F's" phenyl-groups can be replaced by naphthalene so as to be twice as hydrophobic, or by a t-butyl moiety so as to assess the importance of an aromatic or planar system
  • the "C's” sulfhydryl-group can be replaced by a hydroxyl- or thioether moiety to enhance selectivity by being less compatible with various other biological surfaces seeking to engage in hydrogen bonding as either donors or acceptors
  • "K's" primary amine can be replaced by a series having a range of basicity and steric features that can probe the sGCal pocket so as to potentially enhance selectivity upon optimization of these parameters for this very distinct locale.
  • potency While potency is diminished, potency is nonetheless comparable to Peptide A-8R [SEQ ID NO: 1] when less than half of the peptide sequence is retained (e.g., Peptides TF6-8R [SEQ ID NO: 14] and FF5-8R [SEQ ID NO: 10]). All active peptides induced ROS. Cells treated with these peptides are rescued by NAC, a ROS -sequestering agent, mimicking what was observed with Peptide A-8R [SEQ ID NO: 1].
  • Table 2 - Peptide A Derivatives [SEQ ID NOS: 5, 8, 7, 13, 15, 9, 9, 9, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35, respectively, in order of appearance] Cytotoxicity Compared to Peptide A-8R [SEQ ID NO: 1] (Table discloses sequence "Name(s)" as SEQ ID NOS: 1, 1, 11, 12, 14, 16, 10, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 36, respectively, in order of appearance.)
  • the following six groups each contain amino acids that are typical, but not necessarily exclusive, conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
  • Substantial changes in function or immunogenicity can be made by selecting substitutions that are less conservative than those listed in the table above, i.e. , by selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of substitution, (b) the charge or hydrophobicity of the polypeptide at the target site, or (c) the bulk of a side chain.
  • substitutions that can generally be expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, such as seryl or threonyl, is substituted by a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine or proline is substituted by any other residue; (c) a residue having an electropositive side chain, such as lysyl, arginyl, or histidyl, is substituted by an electronegative side chain, such as glutamyl or aspartyl; (d) a residue having a bulky side chain, such as phyenylalanyl, is substituted by a residue not having a side chain, such as glycyl; or (e) by increasing the number of sulfation or glycosylation.
  • a hydrophilic residue such as seryl or threonyl
  • LNCap protein extract containing partially purified sGCal can be incubated with a saturating concentration of FITC -labeled Peptide A-8R [SEQ ID NO: 1] together with increasing concentrations of competing unlabeled A-8R [SEQ ID NO: 1] (as a standard run) or the various probe compounds.
  • the amount of labeled material can be quantified for all runs and compared to the standard run so as to determine relative binding affinities.
  • One or more lead compounds can arise from screening this number of probe molecules according to the parallel testing approach.
  • the desired profile for classification as an initial lead compound is nM potency (biochemical assay) within the molecular context of a peptidomimetic structure having 'drug-like' properties, accompanied by a dose requirement of at least 100 ⁇ to produce significant effects on normal tissues (cell -based assay panel).
  • the compound library can be used to probe fundamental biology and establish SAR, the 'drug-like' specifications need not be overly stringent. Rather, they may serve as a guide according to the 'rule-of-five', avoidance of potential metabophore liabilities, and avoidance of blatant toxicophores.
  • the composite of SAR accumulating can also be used to guide the design of a probe molecule that can form a covalent bond within the A-8R [SEQ ID NO: 1] binding pocket on sGCal.
  • One approach is to incorporate a photoaffinity label that can be activated once the desired binding has occurred, rather than to use a chemically reactive functionality that could behave promiscuously in other bonding relationships. Both a benzophenone and an azide derivative can be utilized. Once a photoaffinity probe that retains a high level of binding activity is generated, the covalent interaction can be activated, and the so-bound probe-sGCal complex can be isolated and subjected to protein digestion protocols, followed by characterization of the resulting fragments by mass spectrometry analyses.
  • An x-ray of the H-NOXA domain is available from the species Nostoc punctiforme that has 35% sequence identity to the human form.
  • An x-ray of the CC domain is available from the rat sGCpi monomer.
  • VCaP expresses wild type AR, PSA, and TMPRSS2-ERG fusion gene (found in -50% of Pea patients) and VCaP CRPC xenograft model has been successfully established.
  • most of androgen synthetic enzymes are expressed in VCaP cells and xenografts, and more significantly, the expression of AKR1C3 and HSD17B6 are upregulated in the relapsed tumor, suggesting that the intratumoral androgen synthesis is elevated in relapsed VCaP xenograft.
  • the targets of this suppression functions include AR itself, androgen synthetic genes (AKR1C3 and HSD16B6), and a group of cell cycle genes (MCM7, FANC1, cycline El, etc.). Androgen deprivation treatments relieve the suppression on these genes and therefore increase the expressions in CRPC.
  • VCaP xenografts can be used to assess peptide effects on androgen-dependent tumors
  • relapsed VCaP xenografts can be used to assess effects on CRPC tumors.
  • alternative models such as LNCaP, CWR22, and LAPC4 can be used to validate and extend key findings in the VCaP xenografts.
  • the VCaP xenografts can be established through injecting 2 million cells (mixed with Matrigel) in the flank of male SCID mice (4-8 weeks). Tumor biopsies can be taken before the treatment.
  • Peptide A and truncated variants or derivatives thereof can be injected directly into tumors daily to each each group (6 per group) for up to 2 weeks, and then the mice can be sacrificed.
  • the peptide doses will begin with 40 mg peptide/kg of animal, which was been shown previously (in Example I, above) to be highly effective against xenograft tumors from C81 cells.
  • BrdU can be injected 2 hours before sacrifice to assess S-phase fraction. Paraffin sections of tumor tissue can be analyzed by immunohistochemistry for Ki67 and BrdU to assess effects on growth, and for cleaved caspase 3 and TUNEL staining for apoptosis.
  • Protein extracted from frozen sections can be analyzed by Western blotting for sGCal, sGCpi, p53, Survivin, and other apotosis pathway proteins, and for activation status of pathways including PI3K/AKT and NFkB. Without wishing to be bound by theory, it is believed that AKT protein levels will drop and the other proteins will remain unchanged. The expression of any proteins identified as involved in peptide -induced ROS generation can also be measured. ROS generation in the tumors can be measured to assess Peptide A activity, which has been shown to kill PCa cells by enhancing ROS levels. cGMP levels can also be monitored to confirm that peptide treatment does not affect cGMP levels or NO signaling in general.
  • RNA extracted from frozen sections can be assessed by RNA-seq (or alternatively, expression microarray) in the pre-treatment biopsy samples versus the post-treatment tumors.
  • the expression of genes downstream of AKT signaling is anticipated to change in response to peptide treatment.
  • the effects on proliferation and apoptosis can also be studied, but it is to be understood that these effect are likely to be dependent on the spectrum of genes that are activated or repressed, and the magnitude of these effects.
  • PK/PD testing of these drugs can be performed in the plasma/tissues from the mice. These blood samples can be collected pre- and post-treatments and sent for pharmacokinetic studies. PK tests can be performed in male mice with established xenograft tumors. Agents can be evaluated after intratumor and intravenous delivery at a single efficacious dose. Oral and/or intraperitoneal delivery can be included for certain agents with improved properties for absorption. One or more final lead agents and back-up agents can be tested at three different doses per route. Blood samples can be collected via terminal cardiac puncture under anesthesia, with tumor tissues collected immediately after euthanasia.
  • Blood samples can be centrifuged and serum stored at -80 °C, while tumor tissue can be flash-frozen in liquid nitrogen and stored at -80 °C until analysis.
  • Tumor tissues can be homogenized with a pestle in lysis buffer.
  • Serum and tumor homogenate samples can be analyzed by LC -MS/MS assay on a system consisting of a Waters 2795 Alliance HT separations module, a Quattro Micromass triple quadruple mass spectrometer, and MassLynx software for instrument control, data acquisition, and analysis. Samples can be collected from five animals at each time point for each delivery route. A total of 5-8 time points after dosing can be evaluated to estimate elimination half-life, tumor half-life, serum and tumor AUC, and PO/IP bioavailability.
  • sGCal The expression of sGCal is increased in CRPC cell lines and human tissue samples versus primary PCa, while expression of sGC i remains low. Therefore, the pro-proliferative, pro-survival functions of sGCal may be a mechanism for castration resistance and targeting sGCal with Peptide A, or a truncated variant or derivative thereof, may be a treatment of human CRPC.
  • Peptide A is able to stop xenograft tumor growth from C81 cells, which is a model for CRPC, as shown in Example I above.
  • the well-established CRPC xenograft models including VCaP and LAPC4 can be used.
  • Subcutaneous xenografts can be established in the flanks of male SCID mice, and the mice can then be castrated. When the tumors relapse (usually in 6-8 weeks), biopsies can be taken to assess baseline gene expression. Groups of mice bearing these castration-resistant tumors (6 per group) can be treated with selected Peptide A analogs for about 2 weeks, and can then be sacrificed. While the previous xenograft evaluation with Peptide A have utilized direct intratumoral injections, other delivery methods are possible, including intraperitoneal, intravenous, or oral delivery. This model can develop drug-like compounds with increased stability and selectivity for prostate tumors. The IV delivery method is best for PK/PD studies.
  • Biological triplicates for each model at an effective treatment can be further analyzed on RNA-seq or Affymetrix microarrays (in comparison with the matched pre -treatment biopsy) to globally assess effects on gene expression.
  • VCaP xenograft can develop resistance to long- term exposure of arbiraterone, which is a FDA-approved CYP17A1 inhibitor to treat human CRPC
  • the effect of Peptide A truncated variants and derivatives can be assessed in a combination with abiraterone treatment (for example, three weeks of abiraterone and two weeks of co- treatment) on abiraterone-resistant VCaP CRPC xenograft (abiraterone pre-treated for 4 weeks).
  • cancers or neoplastic diseases and related disorders such as, but not limited to prostate cancer
  • other disorders or conditions such as, but not limited to prostate cancer
  • the methods and compositions can be used in the treatment of human cancers.
  • compositions can be employed as part of a treatment of prostate cancer by administering a therapeutically effective amount of at least one of the compounds of the present invention as a single agent or in combination with another anti-cancer agent.
  • embodiments include methods of regulating androgen receptors levels in human prostate cancer cells by administering an effective amount of at least one effector agent to a subject in need of treatment.
  • a method for regulating expression of the tumor suppressor gene p53 in a subject in need thereof comprising administering an effective amount of a composition comprising a soluble guanylyl cyclase alpha 1 (sGCal)-type compound.
  • the subject is suffering from prostate cancer.
  • a composition for regulating wild-type p53 protein in a subject in need thereof comprising a soluble guanylyl cyclase alpha 1 (sGCal)- type compound.
  • a method for mediating the proliferation of androgen-dependent and androgen-independent prostate cancer cells comprising administering an effective amount of a composition comprising soluble guanylyl cyclase alpha 1 (sGCal)-type compound.
  • sGCal soluble guanylyl cyclase alpha 1
  • a method for disrupting soluble guanylyl cyclase alpha 1 (sGCal) interaction with p53 comprising administering an effective amount of a composition comprising a soluble guanylyl cyclase alpha 1 (sGCal)-type compound.
  • composition comprising a soluble guanylyl cyclase alpha 1 (sGCal)-type compound.
  • sGCal soluble guanylyl cyclase alpha 1
  • a method of reactiving p53 in cancer cells comprising administering an effective amount of a composition comprising a soluble guanylyl cyclase alpha 1 (sGCal)-type compound.
  • a method of treating cancer comprising administering to a subject in need of treatment an effective amount of at least one soluble guanylyl cyclase alpha 1 (sGCal)-type compound.
  • sGCal soluble guanylyl cyclase alpha 1
  • the cancer is chosen from breast cancer and genital cancer.
  • the cancer is prostate cancer.
  • the prostate cancer is advanced prostate cancer characterized by androgen-independence.
  • the prostate cancer cells express wild-type p53.
  • soluble guanylyl cyclase alpha 1 (sGCal)-type compound induces apoptosis in the prostate cancer cell by p53 accumulation.
  • a method for inducing p53 accumulation in a subject having prostate cancer comprising the step of administering a soluble guanylyl cyclase alpha 1 regulator to a subject in need thereof, whereby the induction of p53 accumulation aids the treatment of prostate cancer characterized by androgen-insensitivity.
  • the method can further include treating the subject with at least one conventional anticancer treatment chosen from radiation and chemotherapy.
  • the method can further include treating the subject with at least one conventional anticancer agent.
  • Suitable anticancer agents include, but are not limited to, chemotherapeutic agents; cytotoxins; antimetabolites; alkylating agents; protein kinase inhibitors; anthracyclines; antibiotics; antimitotic agents (e.g.
  • antiiubuJin agents corticosteroids; radiopharmaceuticals; proteins such as cytokines, enzymes, or interferons; biological response modifiers such as krestin, lentinan, sizofiran, picibanil, ubenimex; anti- angiogenic compounds such as aciireiin, fenretinide, thalidomide, zoiedronic acid, angiostatin, apiidine, ciiengtide, combretastatin A-4, endostatin, halofuginone, rebimastat, removab, Revlimid, squalamine, ukrain, or Vitaxin; platinum- coordinated compounds such as cisplatin, carboplatin, nedaplatin, or oxaliplatin; camptothecin derivatives such as camptothecin, 10-hydroxycamptothecin, 9-aniinocaniptothecin, irinotecan, SN-38,
  • interferons include, but are not limited to interferon alpha, interferon alpha-2a, interferon, alpha-2b, interferon beta, interferon gamma- la, interferon gamma-lb (Actimmune), interferon gamma-nl, or combinations thereof.
  • the anticancer agent is one or more of filgrastim, lentinan, sizofiian, TheraCys, ubenimex, WF-10, aldesleukin, aiemtuzumab, BAM-002, dacarbazine, daclizumab, denileukin, gemtuzumab ozogamicin, ibritumomab, imiquimod, lenograsiim, lentinan, Corixa, molgramostim, OncoVAX-CL, sargramostim, tasonermin, tecleukin, thymaiasin, tositumomab, Virulizin, Z-100, epratuzumab, mirumomab, oregovomab, pemtumomab (Y- muHMFGI), Provenge (Dendreon), alitretinoin, ampiigen,
  • Bosentan calcitnol, exisulmd, finasteride.fotemustine, ibandronic acid, miltefosine, mitoxantrone, 1-asparaginase, procarbazine, dacarbazine, hydroxycarbamide, pegaspargase, pentostatin, tazarotne, Telcyta (TLK-286, Telik Inc.), Velcade (bortemazib, Millenium), tretinoinor, or combinations thereof.
  • a chemopreventative method of prophylactically treating cancer comprising administering to a subject in need of treatment an effective amount of at least one soluble guanylyl cyclase alpha 1 (sGCal)-type compound.
  • sGCal soluble guanylyl cyclase alpha 1
  • sGCal soluble guanylyl cyclase alpha 1
  • a pharmaceutically acceptable carrier wherein the at least one soluble guanylyl cyclase alpha 1 (sGCal)-type compound is present in a dosage level effective to treat cancer.
  • a method of decreasing androgen-associated cancer by regulating androgen receptors present in host cells comprising exposing the host cells to an effective amount of at least one composition comprising a soluble guanylyl cyclase alpha 1 (sGCal)-type compound.
  • sGCal soluble guanylyl cyclase alpha 1
  • a method of regulating androgen receptors in a subject in need thereof comprising administering an effective amount of at least one composition to the subject, wherein the composition includes at least one soluble guanylyl cyclase alpha 1 (sGCal)-type compound.
  • sGCal soluble guanylyl cyclase alpha 1
  • a method of down regulating p53 levels in human prostate cancer cells comprising administering an effective amount of at least composition to a subject in need of treatment, wherein the composition includes at least one soluble guanylyl cyclase alpha 1 (sGCal)-type compound.
  • a pharmaceutical composition comprising at least one composition in combination with a pharmaceutically acceptable carrier, wherein at least one composition is present in a dosage level effective to down regulate androgen receptors present in host cells, wherein the composition includes at least one soluble guanylyl cyclase alpha 1 (sGCal)-type compound.
  • a novel p53 regulator comprising a composition that disrupts sGCal-p53 interaction in host cells.
  • a method for treating a disease condition in a subject comprising: providing a p53 regulator that disrupts sGCal-p53 interaction; and administering the p53 regulator to the subject in an amount sufficient to treat the disease condition.
  • kits comprising: a volume a p53 regulator that disrupts sGCal-p53 interaction 1; and instructions for the use of the volume of p53 regulator in the treatment of a disease condition in a subject.
  • the volume of p53 regulator is included in a composition that further comprises an additional component selected from the group consisting of a vehicle, an additive, a pharmaceutical adjunct, a therapeutic compound, a carrier, agents useful in the treatment of disease conditions, and combinations thereof.
  • a method of identifying an anti-prostate cancer agent comprising providing a test agent to a cell and measuring the level of a p53 regulator that disrupts sGCal-p53 interaction associated with altered expression levels in prostate cancer cells, wherein an increase or a decrease in the level of the p53 regulator in the cell, relative to a control cell, is indicative of the test agent being an anti-prostate cancer agent.
  • a method of determining the prognosis of a subject with prostate cancer comprising measuring the level of at least one p53 regulator in a test sample from the subject, wherein: the p53 regulator is associated with an adverse prognosis in prostate cancer; and an alteration in the level of the at least one p53 regulator in the prostate test sample, relative to the level of a corresponding p53 regulator in a control sample, is indicative of an adverse prognosis.
  • a method of treating prostate cancer in a subject who has a prostate cancer in which at least one p53 regulator is down-regulated or up- regulated in the cancer cells of the subject relative to control cells comprising:(l) when the at least p53 regulator is down-regulated in the cancer cells, administering to the subject an effective amount of at least one p53 regulator, or an isolated variant or biologically-active fragment thereof, such that proliferation of cancer cells in the subject is inhibited; or,(2) when the at least p53 regulator is up-regulated in the cancer cells, administering to the subject an effective amount of at least one compound for inhibiting expression of the at least p53 regulator, such that proliferation of cancer cells in the subject is inhibited.
  • a method of identifying an anti-prostate cancer agent comprising providing a test agent to a cell and measuring the level of at least p53 regulator associated with an altered expression levels in prostate cancer cells, wherein an altered level of the p53 regulator in the cell, relative to a control cell, is indicative of the test agent being an anti-prostate cancer agent.
  • the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more of the effector agents described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8) pulmonarily, or (9) nasally.
  • oral administration for example, drenches (aqueous or non-a
  • embodiments of the present compounds may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with
  • salts refers to the relatively nontoxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like.
  • the pharmaceutically acceptable salts of the compounds of the present invention include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non- toxic organic or inorganic acids.
  • such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulionic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.
  • the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases.
  • pharmaceutically-acceptable salts in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a
  • Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
  • Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.
  • 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: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated
  • hydroxyanisole BHA
  • BHT butylated hydroxytoluene
  • lecithin propyl gallate
  • alpha- tocopherol alpha- tocopherol
  • metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier 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 carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • compositions suitable for parenteral administration comprise one or more compounds of the invention 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 sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples 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.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the compounds of the present invention may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • absorption of the drug from subcutaneous or intramuscular injection This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.
  • the rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the compounds of the present invention 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 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 suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • a compound may also be administered as a bolus, electuary or paste.
  • the compounds When the compounds are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier. Regardless of the route of administration selected, the compounds, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
  • compositions of the present 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 employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, 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 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. Generally, oral, intravenous, intracerebroventricular and
  • subcutaneous doses of the compounds 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.
  • 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. Preferred dosing is one administration per day. While it is possible for a compound to be administered alone, it is preferable to administer the compound as a
  • composition composition
  • compositions can, where appropriate, be conveniently presented in discrete unit dosage forms and/or kits and can be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combination thereof, and then, if necessary, shaping the product into the desired delivery system.
  • the compound can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.
  • a person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation.
  • a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.
  • the dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • Effector agents may be suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal, or parenteral (including subcutaneous, intramuscular, subcutaneous, intravenous, intradermal, intraocular, intratracheal, intracistemal, intraperitoneal, and epidural) administration.
  • Effector agents may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association one or more effector agents of the present invention and one or more pharmaceutical carriers or excipients.
  • the total daily usage of the effector agents of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors, including for example, the disorder being treated and the severity of the disorder; activity of the specific effector agents employed; the specific effector agents employed, the age, body weight, general health, sex and diet of the patient; the time of administration; route of administration; rate of excretion of the specific effector agents employed; the duration of the treatment; drugs used in combination or coincidental with the specific composition employed; and like factors well known in the medical arts.
  • Effector agents of the present inventions are preferably formulated in dosage unit form for ease of administration and uniformity of dosage.
  • dosage unit form refers to a physically discrete unit of the effector agents appropriate for the subject to be treated.
  • Each dosage should contain the quantity of effector agents calculated to produce the desired therapeutic affect either as such, or in association with the selected pharmaceutical carrier medium.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub- dose, or an appropriate fraction thereof, of the administered effector agent. In this regard, studies were performed to assess the dosage regimen.
  • Beneficial combinations may be suggested by studying the activity of the test compounds with agents known or suspected of being valuable in the treatment of a particular disorder. This procedure can also be used to determine the order of administration of the agents, i.e. before, simultaneously, or after delivery.
  • the present invention relates to a method of treating or
  • preventing prostate cancer in a subject comprising administering to a subject in need thereof a therapeutically effective amount of any one or more of the aforementioned compounds.
  • one or more compounds of the present invention are used to treat or prevent cancer or neoplastic disease in combination with one or more anti-cancer,
  • one or more compounds of the present invention can be used to treat or prevent cancer or neoplastic disease in combination with one or more chemotherapeutic or other anti-cancer agents including, but not limited to radiation.
  • the chemotherapeutic agent and/or radiation therapy can be administered according to therapeutic protocols well known in the art. It will be apparent to those skilled in the art that the administration of the chemotherapeutic agent and/or radiation therapy can be varied depending on the disease being treated and the known effects of the chemotherapeutic agent and/or radiation therapy on that disease. Also, in accordance with the knowledge of the skilled clinician, the therapeutic protocols (e.g., dosage amounts and times of administration) can be varied in view of the observed effects of the administered therapeutic agents (i.e., antineoplastic agent or radiation) on the patient, and in view of the observed responses of the disease to the administered therapeutic agents, and observed adverse affects.
  • the administered therapeutic agents i.e., antineoplastic agent or radiation
  • compounds of the present invention and the chemotherapeutic agent do not have to be administered in the same pharmaceutical composition, and may, because of different physical and chemical characteristics, have to be administered by different routes.
  • compounds of the present invention may be administered intravenously to generate and maintain good blood levels, while the chemotherapeutic agent may be administered orally.
  • the determination of the mode of administration and the advisability of administration, where possible, in the same pharmaceutical composition, is well within the knowledge of the skilled clinician.
  • the initial administration can be made according to established protocols known in the art, and then, based upon the observed effects, the dosage, modes of administration and times of administration can be modified by the skilled clinician.
  • chemotherapeutic agent or radiation will depend upon the diagnosis of the physicians and their judgment of the condition of the patient and the appropriate treatment protocol.
  • a compound of the present invention, and chemotherapeutic agent and/or radiation may be administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially, depending upon the nature of the proliferative disease, the condition of the patient, and the actual choice of chemotherapeutic agent and/or radiation to be administered in conjunction (i.e., within a single treatment protocol) with a compound of the present invention.
  • a compound of the present invention and the chemotherapeutic agent and/or radiation is not administered simultaneously or essentially simultaneously, then the optimum order of administration of the compound of the present invention, and the chemotherapeutic agent and/or radiation, may be different for different tumors. Thus, in certain situations the compound of the present invention may be administered first followed by the administration of the
  • chemotherapeutic agent and/or radiation may be administered first followed by the administration of a compound of the present invention.
  • This alternate administration may be repeated during a single treatment protocol.
  • the determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during a treatment protocol, is well within the knowledge of the skilled physician after evaluation of the disease being treated and the condition of the patient.
  • the chemotherapeutic agent and/or radiation may be administered first, especially if it is a cytotoxic agent, and then the treatment continued with the administration of a compound of the present invention followed, where determined advantageous, by the
  • the practicing physician can modify each protocol for the administration of a component (therapeutic agent, i.e., compound of the present invention, chemotherapeutic agent or radiation) of the treatment according to the individual patient's needs, as the treatment proceeds.
  • a component therapeutic agent, i.e., compound of the present invention, chemotherapeutic agent or radiation
  • the term "admixing” generally refers to the mixing the two components, and any additional optional components, together. Depending upon the properties of the components to be admixed, there may or may not be a significant chemical or physical interaction between two or more components when they are mixed. For example, if one component is an acid, and the other component is a base, upon Admixing, the two components may, depending on the strength of the acids and bases, react to form a salt comprising the anion corresponding to the acid and the protonated cation corresponding to the base, or an equilibrium mixture of the original acids and bases, and their salts.
  • compositions may be claimed in terms of the components known to be present after the admixing process, or alternatively may be claimed in terms of the components admixed in a product-by-process claim format, especially if the exact nature of the product resulting from the process of admixing the components is unknown or only poorly known or understood.
  • administration and variants thereof (e.g., “administering” a compound) in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of the animal in need of treatment.
  • a compound of the invention or prodrug thereof is provided in combination with one or more other active agents (e.g., a cytotoxic agent, etc.)
  • “administration” and its variants are each understood to include concurrent and sequential introduction of the compound or prodrug thereof and other agents.
  • estrogen receptor generally refers to a protein whose function is to
  • mutant androgen receptors include androgen receptors with amino acid additions, insertions, truncations and deletions, as long as the function is sufficiently preserved.
  • cancer and “cancerous” generally refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers. By “early stage cancer” or “early stage tumor” is meant a cancer that is not invasive or metastatic or is classified as a Stage 0, 1, or II cancer.
  • pre -cancerous refers to a condition or a growth that typically precedes or develops into a cancer.
  • a "pre -cancerous” growth will have cells that are characterized by abnormal cell cycle regulation, proliferation, or differentiation, which can be determined by markers of cell cycle regulation, cellular proliferation, or differentiation.
  • dysplasia is meant any abnormal growth or development of tissue, organ, or cells. Preferably, the dysplasia is high grade or precancerous.
  • metalastasis is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the
  • non-metastatic is meant a cancer that is benign or that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site.
  • a non-metastatic cancer is any cancer that is a Stage 0, 1, or II cancer, and occasionally a Stage III cancer.
  • primary tumor or “primary cancer” is meant the original cancer and not a metastatic lesion located in another tissue, organ, or location in the subject's body.
  • tumor or “benign cancer” is meant a tumor that remains localized at the site of origin and does not have the capacity to infiltrate, invade, or metastasize to a distant site.
  • tumor burden is meant the number of cancer cells, the size of a tumor, or the amount of cancer in the body. Tumor burden is also referred to as tumor load.
  • tumor number is meant the number of tumors.
  • chemotherapeutic agent generally refers to a chemical compound useful in the treatment of cancer.
  • Non-limiting examples of chemotherapeutic agents include one or more chemical compounds useful in the treatment of cancer.
  • compositions as used herein also includes
  • composition is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
  • the various compounds, compositions and/or effector agents disclosed herein can comprise a "carrier” molecule and/or the corresponding "carrier” functional group or residues that are either directly or indirectly bonded to another functional group or residue comprising one or more protease inhibitors.
  • Such compounds, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions.
  • These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein.
  • each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • any subset or combination of these is also specifically contemplated and disclosed.
  • the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • This concept applies to all aspects of this disclosure including, but not limited to, steps in methods of making and using the disclosed compositions.
  • steps in methods of making and using the disclosed compositions are if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
  • subgenera, and species of compounds described herein, including their prodrugs and/or pharmaceutically acceptable salts, and their various pharmaceutical compositions and kits prepared thereof, can be used to treat or prevent prostate cancer.
  • carrier molecule as defined herein is any compound or functional group or residue thereof that can facilitate the delivery of the protease inhibitor into a muscle tissue.
  • the carrier molecule can be any endogenous molecule.
  • the carrier molecule can be a derivative of an endogenous compound.
  • any of the carrier molecules or residues, linkers, and/or protease inhibitors described herein, and the compounds derived therefrom, can be employed in the form of a pharmaceutical composition, or used to prepare or manufacture pharmaceutical compositions or medicaments.
  • cytotoxic agent generally refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g., I 131 , 1 125 , Y 90 and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
  • the term "effective amount" of a subject compound generally refers to an amount of the antagonist in a preparation which, when applied as part of a desired dosage regimen brings about, e.g., a change in the rate of cell proliferation and/or rate of survival of a cell according to clinically acceptable standards for the disorder to be treated.
  • effector agent generally refers to as any small molecule that interacts with a receptor, either directly or indirectly, in a manner that alters its ability to bind a ligand. A positive effector enhances binding activity while a negative effector reduces it.
  • Effector agents and compositions containing the same can be used to treat conditions such as, but not limited to, cancer and cancer-related diseases.
  • effector agents can be used prophylactically as chemopreventative compositions that can be used to inhibit the development and/or slow the development of the cancer and cancer-related conditions and/or advanced stages of cancer and cancer-related conditions.
  • the "effector agents” can be used to treat these cancers and other cancers at any stage from the discovery of the cancer to advanced stages.
  • effector agents can be used in the treatment of the primary cancer and metastases thereof.
  • Effector agents may be used as the active ingredient in combination with one or more pharmaceutically acceptable carrier mediums and/or excipients. Except insofar as any conventional carrier medium is incompatible with the effector agents used in practicing embodiments of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with one or more of the effector agents of the pharmaceutical composition, its use is contemplated to be within the scope of the embodiments of this invention.
  • host cells include non-cancerous and cancerous cells.
  • cancer cells include, but are not limited to, cancer cells, neoplastic cells, neoplasia, tumors, and tumor cells, which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype, characterized by a significant loss of control of cell proliferation.
  • salt form generally refers to those salt forms that retain the biological effectiveness and properties of the effector agent.
  • Non-limiting examples of such salts include: (1) acid addition salt which is obtained by reaction of the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, 5 phosphoric acid, sulfuric acid, and perchloric acid and the like, or with organic acids such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or malonic acid and the like, preferably hydrochloric acid or (L)-malic acid such as the L-malate salt of sunitinib; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion
  • Exemplary ions include aluminum, calcium, lithium, magnesium, potassium, sodium and zinc in their usual valences.
  • Preferred organic base include protonated tertiary 15 amines and quaternary ammonium cations, including in part, trimethylamine, diethylamine, ⁇ , ⁇ '- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.
  • salts are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio and effective for their intended use.
  • the salts can be prepared in situ during the final isolation and purification of one or more effector agents, or separately by reacting the free base function with a suitable organic acid.
  • esters refers to those esters of one or more effector agents which are suitable, within the scope of sound medical judgment, for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use
  • prodrugs refers to those prodrugs of one or more effector agents which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use.
  • Pharmaceutically acceptable prodrugs also include zwitterionic forms, where possible, of one or more compounds of the composition.
  • prodrug refers to compounds that are rapidly transformed in vivo to yield the parent compound, for example by hydrolysis in blood.
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form.
  • radiation therapy can include the use of directed gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether. It will be appreciated that there will be many ways known in the art to determine the dosage and duration of treatment. Typical treatments are given as a one time administration and typical dosages range from 10 to 200 units (Grays) per day.
  • Reduce or inhibit is meant the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. Reduce or inhibit can also refer to the symptoms of the disorder being treated, the presence or size of metastases, the size of the primary tumor, or the size or number of the metastatic tumor.
  • a "safe and effective amount” refers to the quantity of a component that is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.
  • subject generally refers to an animal, typically a mammal or a human, that has been the object of treatment, observation, and/or experiment.
  • subject is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
  • the subject is a human.
  • the term is used in conjunction with administration of a compound or drug, then the subject has been the object of treatment, observation, and/or administration of the compound or drug.
  • compositions and effector agents of the present invention can be used prophylactically as chemopreventative agents for these conditions
  • terapéuticaally effective amount means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.
  • efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and/or quality of life.
  • an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation.
  • an effective amount is an amount sufficient to delay development.
  • an effective amount is an amount sufficient to prevent or delay occurrence and/or recurrence.
  • An effective amount can be administered in one or more doses.
  • the effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of disease, preventing spread (i.e., metastasis) of disease, delaying or slowing of disease progression, amelioration or palliation of the disease state, and remission (partial or total) whether detectable or undetectable.
  • stabilization i.e., not worsening
  • preventing spread i.e., metastasis
  • delaying or slowing of disease progression i.e., metastasis
  • amelioration or palliation of the disease state e.e., remission
  • remission partial or total
  • treatment can also refer to both therapeutic treatment and prophylactic or preventative measures.
  • Those in need of treatment include those already having a benign, precancerous, or non-metastatic tumor as well as those in which the occurrence or recurrence of cancer is to be prevented.
  • treating cancer refers to administration to a mammal afflicted with a cancerous condition and refers to an effect that alleviates the cancerous condition by killing the cancerous cells, but also to an effect that results in the inhibition of growth and/or metastasis of the cancer.
  • Up and/or down regulation or “up or down regulating” can be defined as an increase or decrease in the number of ligand receptors or other cellular proteins within or on the surface of a host cell. Such up- or down-regulation occurs after host cells have been exposed to an effector agent, either as a result of a direct interaction of the effector agent with the receptor or other protein, or through indirect interactions.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des substances et des méthodes utiles pour traiter divers cancers exprimant sGCα1. Lesdites substances comprennent des peptides interférant avec les fonctions favorables à la survie de sGCα1, ce qui engendre l'apoptose des cellules exprimant sGCα1. La présente invention concerne en outre des dosages utilisables à des fins de criblage, des dosages utilisables à des fins de diagnostic, des méthodes de pronostic, des méthodes de traitement et des kits associés.
PCT/US2014/063089 2013-11-01 2014-10-30 Compositions inhibant sgcα1 et méthodes de traitement de cancers à l'aide desdites compositions WO2015066285A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US14/069,790 2013-11-01
US14/069,790 US9301991B2 (en) 2009-05-30 2013-11-01 sGCalphal inhibiting compositions

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WO2015066285A2 true WO2015066285A2 (fr) 2015-05-07
WO2015066285A3 WO2015066285A3 (fr) 2015-06-25

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WO2009149278A1 (fr) * 2008-06-04 2009-12-10 Synergy Pharmaceuticals Inc. Agonistes de guanylate cyclase utiles dans le traitement de troubles gastro-intestinaux, d'une inflammation, d'un cancer et d'autres troubles
WO2010141349A1 (fr) * 2009-05-30 2010-12-09 University Of Toledo Peptides a1 et a-8r de type guanylyl cylase soluble utilisables en tant que marqueurs diagnostics et cibles thérapeutiques dans le cas d'un cancer de la prostate

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