PARP-1 Inhibitors In Oncology
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PARP-1 Inhibitors In Oncology

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Discovery and development of CEP-9722, currently Phase 2 for adjuvant cancer therapy.

Discovery and development of CEP-9722, currently Phase 2 for adjuvant cancer therapy.

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  • A small-molecule prodrug of CEP-8983, a novel 4-methoxy-carbazole inhibitor of the nuclear enzymes poly(ADP-ribose) polymerase (PARP) 1 and 2, with potential antineoplastic activity. Upon administration and conversion from CEP-9722, CEP-8983 selectively binds to PARP 1 and 2, preventing repair of damaged DNA via base excision repair (BER). This agent enhances the accumulation of DNA strand breaks and promotes genomic instability and apoptosis. CEP-8983 may potentiate the cytotoxicity of DNA-damaging agents and reverse tumor cell chemo- and radioresistance. PARP catalyzes post-translational ADP-ribosylation of nuclear proteins that signal and recruit other proteins to repair damaged DNA and can be activated by single strand breaks in DNA.
  • Inhibitors of PARP-1 have potential therapeutic utility in oncology through potentiation of the anti-tumor activity of radiation or chemotherapeutic DNA damaging agents. Until more recently, the potentiation observed in-vitro and in-vivo could not be unequivocally linked to inhibition of PARP due to limitations with respect to potency, selectivity, toxicity, and deliverability of compounds.
  • Poly (ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that catalyzes the synthesis of poly-ADP-ribose chains from NAD+ as part of the DNA repair process.
  • Poly (ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that catalyzes the synthesis of poly-ADP-ribose chains from NAD+ as part of the DNA repair process.
  • Poly (ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that catalyzes the synthesis of poly-ADP-ribose chains from NAD+ as part of the DNA repair process.
  • Poly (ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that catalyzes the synthesis of poly-ADP-ribose chains from NAD+ as part of the DNA repair process.
  • Poly (ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that catalyzes the synthesis of poly-ADP-ribose chains from NAD+ as part of the DNA repair process.
  • A model of PARP overactivation-mediated cytotoxicity. Reactive oxygen species (ROS) such as nitric oxide (NO z ), superoxide anion (O2--), peroxynitrite (ONOO2), or hydroxyl radical (OH z ) are generated during inflammation or ischemia–reperfusion. These ROS damage cellular molecules like proteins, lipids, and DNA. DNA damage activates PARP , synthesizing poly(ADP-ribose) polymer from its substrate b-nicotinamide adenine dinucleotide (NAD1). Limited amounts of DNA damage can be repaired by cellular DNA repair enzymes, and PARP, in this case, acts as a sensor for DNA damage, modulating the repair process. In apoptotic cell death, activated caspases cleave PARP protein to inhibit a futile DNA repair process and preserve cellular ATP, which is essential for the apoptotic process. Extensive DNA damage can lead to overactivation of PARP . With excessive activation of PARP, NAD1 is depleted, and in efforts to resynthesize NAD1, ATP is also depleted. Hence, necrotic cell death involves energy loss. Depletion of ATP can transform an ongoing apoptotic process into necrosis with intracellular ATP levels regulating the mode of cell death.
  • - Colorectal Carcinoma with Irinotecan #2 choice HNSCC with radiotherapy #3 choice
  • More recent competition?
  • Screening of Cephalon’s internal library identified pyrrolocarbazole CEP-3498 as a potent inhibitor of PARP-1 with an IC50 value of 35 nM, although with poor cellular permeability and solubility. The chemistry objective was to improve the enzyme and cell potency, and the physical-chemical properties to allow either oral or i.v. delivery in the clinic.
  • Key H-bond interactions between imide C=O and N-H groups with backbone N-H and C=O of Gly863 ; H-bond between indole-NH and C=O of Glu988 side-chain; aromatic pi-stacking of aryl rings of Tyr896 and Tyr907 with B and D rings of the carbazole, resp.; cyclopentyl ring fits closely into fold formed by Lys861 side-chain, Ala898, Trp861, and Asn987; open pocket around 3- and 4-positions of the inhibitor provided opportunity for SAR development.
  • Key H-bond interactions between lactam C=O and N-H groups with backbone N-H and C=O of Gly863 ; H-bond between indole-NH and C=O of Glu988 side-chain; aromatic pi-stacking of aryl rings of Tyr896 and Tyr907 with B and D rings of the carbazole, resp.; cyclopentyl ring fits closely into fold formed by Lys861 side-chain, Ala898, Trp861, and Asn987; open pocket around 3- and 4-positions of the inhibitor provided opportunity for SAR development.
  • But with limited opportunity elsewhere on the core template, the aryl group was retained for further SAR development.
  • 1-Aza analog CEP-9397 modeled with PARP-1 active site, postulated as more potent due to an additional H-bond between the 1-aza group and the amine side chain of Met890.
  • Need reference!

PARP-1 Inhibitors In Oncology Presentation Transcript

  • 1. PARP-1 Inhibitors in Oncology The Discovery and Development of CEP-9722, an Orally Active Prodrug for the Treatment of Cancer
  • 2. Rationale for PARP-1 Inhibitors in Oncology
    • PARP comprises a family of at least 14 related enzymes some
    • of which play a pivotal role in DNA repair.
    • The primary member, Poly (ADP-ribose) polymerase 1 (PARP-1),
    • is a nuclear enzyme that catalyzes the synthesis of poly-ADP
    • ribose chains from NAD+ as part of the DNA repair process.
    • Signals and recruits other proteins to repair damaged DNA and can
    • be activated by single strand breaks in DNA.
    • Inhibitors of PARP-1 have shown promise in oncology through
    • potentiation of the anti-tumor activity of radiation or
    • chemotherapeutic DNA damaging agents.
  • 3. PARP-1 - Background
    • Inverse correlation of PARP-1 activity with the degree of cell
    • differentiation. Tumors have increased PARP activity as compared
    • to the corresponding normal tissue.
    • PARP inhibitors as chemopotentiators: Benzamides, Isoquinolones,
    • Nicotinamide derivatives in vitro and in vivo.
      • Potentiates the activity of TMZ, bleomycin, cisplatin, and
      • radiation in human and murine tumor models
  • 4. Role of PARP-1 in DNA Repair N C N C A. de Murcia & M. de Murcia (1994) TIBS 19 , 172 DNA Damage ADP-ribose PARP Activation NAD + ATP
  • 5. Role of PARP-1 in DNA Repair N C N C A. de Murcia & M. de Murcia (1994) TIBS 19 , 172 DNA Damage ADP-ribose PARP Activation NAD + ATP DNA damage repaired
  • 6. Role of PARP-1 in DNA Repair N C N C A. de Murcia & M. de Murcia (1994) TIBS 19 , 172 DNA Damage ADP-ribose PARP Activation NAD + ATP DNA damage repaired Healthy cell
  • 7. Role of PARP-1 in DNA Repair N C N C A. de Murcia & M. de Murcia (1994) TIBS 19 , 172 PARP Inhibitor DNA damage persists DNA Damage ADP-ribose PARP Activation NAD + ATP Damage repaired, Healthy cell
  • 8. Role of PARP-1 in DNA Repair N C N C A. de Murcia & M. de Murcia (1994) TIBS 19 , 172 PARP Inhibitor DNA damage persists DNA Damage ADP-ribose PARP Activation NAD + ATP Damage repaired, Healthy cell Apoptosis, Cell death
  • 9. PARP activity, NAD + , and ATP levels are interdependent Ha, H. C.; Neurobiology of Disease 7, 225–239 (2000)
  • 10. Clinical Path Forward
    • Glioblastoma w/TMZ - # 1 Choice
      • o Poor single agent response rate (~5%) allows for clear
      • improvement using combination therapy with p.o. TMZ
      • o Reasonable size and duration of clinical trials with TMZ
      • o Unmet therapeutic need for GBM; other therapeutic indications for
      • TMZ- sarcoma, melanoma, colon carcinoma and CNS lymphoma
      • o Substantial supportive pre-clinical data with TMZ
    • Therapeutic Endpoints: Improved Response Rate and “Time to
    • Event” outcome
  • 11.
    • Known PARP inhibitors mimic
    • nicotinamide binding at the NAD+
    • site and are planar aromatic ring
    • systems containing a bidentate
    • H-bonding group
    PARP-1 inhibitors based on NAD + substrate 4-ANI 3-AB Ki = 4  M Ki ~ 150nM
  • 12. Earlier Competitor PARP-1 Scaffolds
  • 13. o inc. radiation sensitivity of Chinese hamster V79 cells o Radiat Res; 126(3), 367 (1991) o potentiates TMZ and TP growth inhib. in human tumor cell lines o Clin Cancer Res; 6, 2860 (2000 ) Proof of Concept o Clinical trials candidate
  • 14. o Potentiates TMZ, Cisplatin, radiation in syngeneic and xenograft tumor models o Clin Cancer Res; 13, 2728 (2007) o Completing Phase 2 trials w/TMZ o Irreversible inhibitor o Excellent Phase 2 results o First PARP inhibitor in Phase 3 trials o Failed primary endpoint o Cancelled Phase 3 trials for breast cancer o Commencing Phase 3 for ovarian cancer o Phase 1 trial for various cancers o Well tolerated o Commencing Phase 2 for Mantle Cell Lymphoma In the Clinic
  • 15. Early CEP Library Screening Hit - Pyrrolocarbazole Imide
    • Screening of Cephalon’s internal library identified a pyrrolocarbazole
    • as a potent inhibitor of PARP-1.
    • Improvements Needed:  Potency
    •  Cellular Permeability/Activity
    •  Solubility
  • 16. 4-ANI PARP-1 Modeling
    • Apply structure-based design to optimize potency and cell activity
    • Obtained 2PAX from PDB (Protein Data Bank) - Catalytic Fragment Of
    • Poly(ADP-Ribose) Polymerase complexed with 4-amino-1,8-naphthalimide
    • Chicken PARP (PARP-CF), 87% homologous with the human form
  • 17.  
  • 18. Modeling of CEP-3499 with PARP-CF Wells, Bihovsky; BMCL, 16, 1151 (2006)
  • 19. PARP Inhibitor Discovery Flow In Vitro Cytotoxicity Assays (PARP inhibitors + Chemotherx.) In Vivo Chemo-Potentiation Studies GBMs /TMZ, HT-29/Irinotecan Significant shift in tumor versus normal cell kill versus chemotherx. alone In Vivo PAR Accumulation Assay No enhanced human myelotoxicity in vitro Biochemical efficacy in vivo Cmpd Scale-Up Significant potentiation of anti-tumor efficacy versus chemotherx. alone; acceptable systemic tolerability. Go/No Go Decision PK and Tolerability in Rodents rh PARP Inhibition Assay PC12 cells/H 2 O 2 insult Assay for Inhibition of NAD+ Depletion In vitro and in vivo evaluation on normal tissues; clinical chemistry and histopathology IC 50 < 50 nM 50% recovery @ < 1 uM > 90% max. recovery Criteria
  • 20. General Route to Pyrrolocarbazoles
  • 21. PARP-1 Activity of Pyrrolocarbazole Lactam Isomers
    • PARP activity resides in the 7-oxo isomer, the 5-oxo is inactive for PARP
    • Imide CEP-3498 is 3-fold more potent than the 7-oxo lactam CEP-3499
    • (enhanced H-bonding?)
    35 Imide (CEP-3498) ~10, 000 5-oxo (CEP-3500) 90 7-oxo (CEP-3499) PARP IC 50 (nM) Structure CEP
  • 22. Pyrrolocarbazole Right-Hand Modifications
    • The cyclopentyl ring is critical for potency
    ~ 10,000 5848 > 10,000 2520 ~10,000 5558 35 3498 PARP IC 50 (nM) R1, R2 CEP
  • 23. PARP-1 Inhibition of Pyrrolocarbazoles
    • Ring-opened analogs showed decreased potency
    Tao, Wells; BMCL ; 16, 938 (2006) 35 -CH 2 CH 2 CH 2 - 3498 ~2000 Methyl H 5729 > 10,000 Propyl Ethyl 5706 5000 H Methyl 5674 700 Methyl Methyl 5653 ~ 10, 000 H H 1526 PARP IC 50 (nM) R2 R1 CEP
  • 24. PARP-1 Inhibition of Benzofuran/Benzothiophene Analogs
    • Indole N-H an essential binding function for potency
    • Corresponding N-Me also inactive
    35 NH 3498 O S X > 10, 000 6373 > 10, 000 6297 PARP IC 50 (nM) CEP
  • 25. PARP-1 Inhibition of Truncated Analogs
    • Des-Aryl CEP-6012 was equipotent with carbazole CEP-3498
    • SAR supports the model and the importance of the N-H interaction
    750 2220 40 PARP IC 50 (nM) 5775 6011 6012 Structure CEP
  • 26. Indole-Cyclopentyl Series Tao, Wells; BMCL; 16, 938 (2006)
    • Smaller MW - potentially improved physical properties
    • Single digit nM leads
  • 27. SAR of Methoxy Analogs
    • Data confirms model for 3- and 4-substituents for optimal
    • activity
    • CEP-8983 is a potent, high permeability compound
    • (PAMPA = 7.3 x 10 -6 cm/sec)
  • 28. 3-Aminoalkyl-1-Carba-Series
    • Generally good solubility, potency and cell activity
    • CEP-6800-HCl demonstrated >10 mg/mL aq. solubility
    • CEP-6800 showed toxicity, low brain levels, poor PK
  • 29. 3-Alkoxy-1-Carba-Series
    • Evaluated amino and ether alkyl spaced groups
    • Morpholino-CEP-8969 showed good enzyme activity
    • and solubility, but low cellular activity
  • 30. 3-Carbamate and Amide Derivatives Wells, Bihovsky; BMCL, 16, 1151 (2006)
    • Identified potent, cell permeable inhibitors with good water solubility
    • Low brain levels observed with carbamates
  • 31. General Synthesis of Alkoxy Analogs
  • 32. Summary SAR
  • 33. PARP-1 model for 1-Aza analog of CEP-8983
    • Favorable H-bond postulated between 1-aza group and amide of Met890
  • 34. Synthetic Approach to Aza-analog CEP-8315 CEP-8315 (IC 50 = 3 nM)
    • Further confirms model, binding pose
    • Most potent analog prepared
  • 35. Proposed 1-Aza-4-Alkoxy Series
    • Good cellular activity
    • Better potency
    • Best of both worlds?
  • 36. 1-Aza-4-Alkoxy Synthetic Challenges o Expen$ive o Limited supply
    • Chloride displacement tricky, requires sealed (“bomb”) reactor
    • Ethers thermally sensitive, give variable yields and purity profile
  • 37. 1-Aza-4-Alkoxy Series Synthetic Challenges
    • Series requires alternate approach
  • 38. 1-Aza-4-Alkoxy Series Synthetic Challenges
  • 39. Diene Problem “Solved”
    • Suitable method for small (mg-gm) quantities
  • 40. SAR of 1-Aza analogs
    • 1-Aza group confers order-of-magnitude greater potency
    • 3- and 4-substituents tolerated – opportunity for solubility, improved PK
  • 41. SAR of 1-Aza-4-Subst’d- analogs
    • Non-basic 4-substituents well-tolerated
    • Aza-series ultimately discontinued due to synthetic challenges,
    • non-scalability, expense, and poor solubility
  • 42. Synthetic Process for Drug Candidate CEP-8983
  • 43. Synthetic Process for CEP-8983 (cont.)
  • 44. Synthetic of Prodrug CEP-9722
    • Other related prodrug analogs (amide, sulfonamide, carbamate, urea,
    • N,O-aminal) were more or less stable and/or soluble
  • 45. Solubility and Aqueous Stability of N-Methylpiperazinyl Aminal Prodrug Analogs
    • CEP-16345 and CEP-9722 met solubility and stability criteria
    • at a pH sufficient for I.V. delivery in the clinic
  • 46. Stable, Soluble Mannich Base Gluconates
    • Gluconic acid salt gave optimal aqueous solubility and stability
    • Decomposes in-vivo to CEP-8983/9397, formaldehyde, and
    • methylpiperazine
  • 47. Single Crystal Structure of CEP-9722
    • Confirms bonding of prodrug moiety at imide, not indole
  • 48. hERG Structure-Activity Relationships: 4-Alkoxy SAR
    • Diether substitution at the 4-position reduced
    • hERG channel activity in patch clamp assay
  • 49. Dose Escalation Study with CEP-9722/8983 in Rats
    • i.v. administration of CEP-9722 (3, 10, 30 mg/kg dose equivalents) to
    • rats showed dose related increases in plasma level exposure of CEP-8983
  • 50. The Effects of CEP-8983 and CEP-9397 on Temozolomide Mediated Toxicity in U251MG Cells
    • CEP-8983 and CEP-9397 potentiated the growth inhibitory
    • effects of TMZ in U251MG cells
  • 51. * p≤0.5, ** p≤0.01-TMZ alone as compared to TMZ + 0.3 µM CEP-8983; * p≤0.5 ** p≤0.01, **** p≤0.0001- p≤0.01-TMZ alone as compared to TMZ + 1.0 µM CEP-8983; *** p≤0.001 **** p≤0.0001- p≤0.01-TMZ alone as compared to TMZ + 3.0 µM CEP-8983; ** p≤0.01 **** p≤0.0001-TMZ alone as compared to TMZ + 10.0 µM CEP-8983 by Mann-Whitney Rank Sum Test or t-test where appropriate. *p≤0.5, ** p≤0.01-TMZ alone as compared to TMZ + 0.1 µM CEP-9397 * p≤0.5, ** p≤0.01-TMZ alone as compared to TMZ + 0.3 µM CEP-9397; * p≤0.5 ** p≤0.01, *** p≤0.001- p≤0.01-TMZ alone as compared to TMZ + 1.0 µM CEP-9397; ** p≤0.01 *** p≤0.001 **** p≤0.0001- p≤0.01-TMZ alone as compared to TMZ + 3.0 µM CEP-9397; ** p≤0.01, *** p≤0.001, **** p≤0.0001-TMZ alone as compared to TMZ + 10.0 µM CEP-9397 by Mann-Whitney Rank Sum Test or t-test where appropriate. The Effects of CEP-8983 and CEP-9397 on Temozolomide Mediated Toxicity in NB1691 Cells
    • CEP-8983 and CEP-9397 potentiated the growth inhibitory
    • effects of TMZ in TMZ-resistant tumor cell lines
  • 52.
      • Phase 1 commenced June 2009
        • Open-label study to evaluate the safety, pharmacokinetics, and
        • pharmacodynamics as single-agent oral therapy and as combination
        • therapy with temozolomide in patients with advanced or metastatic
        • solid tumors.
        • Expected completion May-June 2011
    • Phase 2 IND filed Jan 2011
      • Evaluate safety and tolerability of maximum tolerated
      • dose (MTD) found in Phase 1, and investigate CEP-9722
      • oral efficacy as a single agent.
      • Additional combination studies with Gemcitabin/Cisplatin planned
      • Expected completion July 2013
    CEP-9722 Advanced to Clinical Trials
  • 53. Oncology Candace Burns Jennifer Grobelny Kathryn Hunter Sonya Pritchard Hugh Zhao Susan Jones-Bolin Bruce Ruggeri Acknowledgements Chung Ho Park Dandu Reddy Sankar Chatterjee Ron Bihovsky Gregory Wells Chemistry Mary Birchler Laura Gwinn Jean Husten Bruce Jones Biochemistry Seetha Murthy Damaris Rolon-Steele Kelli Zeigler Lisa Aimone Mark Ator Jim Diebold Ming Tao Derek Dunn Allison Zulli Bob Hudkins Fox Chase Cancer Center Andres Klein-Szanto
  • 54. Extra slides
  • 55. TMZ – Hydrolysis gives active form Temozolomide is not directly active but undergoes rapid nonenzymatic conversion at physiologic pH to the reactive compound 5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide (MTIC). The cytotoxicity of MTIC is thought to be primarily due to alkylation of DNA. Alkylation (methylation) occurs mainly at the O 6 and N 7 positions of guanine.
  • 56. 29th Annual J.P. Morgan Healthcare Conference January 10-12, 2011 Cephalon Oncology Pipeline
  • 57.  
  • 58. Wang; Am J Cancer Res; 1(3):301-327 (2011)
  • 59. Wang; Am J Cancer Res; 1(3):301-327 (2011)