Bettie M. Steinberg, PhD Chief Scientific Officer The Feinstein Institute for Medical Research

1. Bettie M. Steinberg, PhD Chief Scientific Officer The Feinstein Institute for Medical Research Dean, Elmezzi Graduate School of Molecular Medicine Professor and Chair, Department of Molecular Medicine Hofstra North Shore-LIJ School of Medicine “Designing and Preparing a Grant Application for Clinical Studies; Initial Results of a Funded Study"

2. Disclosure Statement The Presenter, Dr. Bettie Steinberg, has the following conflict: Receives a grant from Pfizer, Inc. for celecoxib and matched placebo for clinical trial

3. Learning Objectives Hypothesis Definition Types Using the medical literature identifying a clear, structured, searchable clinical question to be addressed Applying research to clinical care/grantsmanship Planning and presenting assessment of study design, performance & analysis

4. Choosing Your Project:Long-term and Short-term views • Long term • Pick area where you wish to become the expert • Based on clinical interest or research you have already done • Chose topic with high impact • Select area with gaps no one is filling • Create 10 year plan for research goals • Divide into 3-4 sections • Short term • Actual study will address first section of plan • Identify immediate pressing problem – important and unique • Write one sentence on project, impact and hypothesis

5. Hypothesis Two types of studies: Hypothesis Testing: study tests idea to see if probably correct Hypothesis Generating: study collects data, look for patterns that might generate a hypothesis Hypothesis: Explanation or “educated guess” that is proposed in order to explain certain events or observations, to provide guidance for further investigation A hypothesis must be capable of being tested Two types Null hypothesis: treatment will not differ from control Alternative hypothesis: treatment will be effective

6. Generating a Hypothesis Can be based on your own observations Can be based on reading literature Or (usually is) combination of both Your expertise – pediatric nephrology Epidemiology studies show that high sodium diets correlate with elevated blood pressure in adults, although the efficacy of sodium restriction for hypertension is not clear cut. Can you create a hypothesis to test in a study of kids?

7. When You Have Your Hypothesis Define exactly what you propose to do Think of all problems/complications you can There will be plenty you did not think of Get statistical help early – especially when a clinical study Identify funds to do study Best source outside grant Write grant application, get funds Conduct study Analyze data Write paper(s) Write next grant

8. Writing A Good Grant Is Not Magic

9. The Grant – Based on A Good Idea The IDEA must be: Novel/Original/Creative/Significant Want a leap of concept, not just obvious next step Reviewer should think “I wish I had thought of that!” Potential results should move field forward

10. What You Need to Write a Successful Grant Communication skills Must write clearly, simply, organized Tell a story “Good writing cannot make a good grant out of a bad idea, but bad writing will make a bad grant out of a good idea” – no spelling or grammar errors! Get help if writing is not your strength Persistence, persistence, and more persistence

11. Four Basic Elements To Writing The Grant What you want to study? What is the question or problem to be studied? What need do you plan to fill? Why does it matter? Why you should get the money Not somebody else How you’re going to do it Approach, study design

12. “Over-ambitious” is Common Major Error Don’t propose more than is reasonable to do in time allotted Very rare for grant to be criticized because it does not propose enough – common to be criticized as over-ambitious or unfocused, even if it includes exciting ideas

13. You As the Investigator Can study be done? by you? here? Have you or someone else published methods or preliminary data? Do you have the facilities, patients, etc.? Include collaborators for strength don’t try to be an expert at everything

14. Scientific Parts Of Most Grants Description (abstract) – must fit in allotted space on form Specific Aims Research Strategy Significance, Innovation, and Approach Resources/facilities – no limit, but normally 1-2 pages Descriptive Appropriate safeguards human subjects, animal welfare Consortium/contractual arrangements, letters of support, etc. References Appendix (if permitted) questionnaires, informed consent forms, etc.

15. NIH Review Criteria -Write All Grants to Address These Points Significance: Important problem? How will scientific knowledge or clinical practice be improved? Innovation: Novel concepts, approaches, methods? Challenge or shift research or practice paradigms? Approach: Strategy, methodology and analyses appropriate for study? Potential problems presented and managed? Protection of human subjects, inclusion of children and minority plans OK? Investigators: Well suited? Sufficient experience and training? Collaborators? Environment: Will it enhance likelihood of success?

16. Ways to Kill A Grant Major ethical or design problem Central scientific question not important Ideas not original, important or exciting PI and/or research team lacks the experience or ability to carry out proposed work Preliminary data weak or absent – no justification for the hypothesis Proverbial house of cards Scope too ambitious, or with multiple hypotheses Don’t follow instructions Write over weekend

17. Description (Abstract) Summary of the whole grant Only part that is public knowledge on NIH Grants Critical First Page First thing reviewer reads I leave to the end, after the whole grant is finished If do first, and then change grant, must rewrite

18. Structure of Specific Aims Page Narrative Paragraph Define problem and significance State long and short term goals, how address problem Summarize expected outcome(s) State hypothesis (can put in bold or italic to emphasize) Specific Aims Each should test part of hypothesis – otherwise why do Each should be linked but independent of other aims – don’t set up house of cards Aims should address mechanism, not correlations Keep grant narrow, keep it focused and keep the number of aims small (3-4 aims for 4-5 years work)

19. Research StrategySignificance and Innovation This is where you set up the rationale and novelty for your proposal Explain importance of problem or critical barrier to progress that you will address Describe how concepts, methods, technologies or treatments in field will be changed if proposed aims are achieved Need a conceptual leap, not just a “better mousetrap” Explain how project will improve scientific knowledge, technical capability, and/or clinical practice Review of current literature is part of this –All this in about 1-2 pages for most grants

20. Preliminary Data Do you need? Very helpful, even for exploratory and small grants Supports your idea Proves you (your mentor/collaborator) can do what you propose Amount of preliminary results depends on grant type Preliminary data must be very high (publication) quality - don’t include poor quality data “There are no preliminary results, just unpublished ones” Organize so reviewer can see your points

21. Sample Preliminary Data B. 6 6 6 * * 2 levels Normal Normal 5 5 5 - Papilloma Papilloma 4 4 4 3 3 3 2 2 2 Relative COX 1 1 1 Normal A Papilloma 0 0 0 COX-2 Negative control “We first asked whether COX-2 was expressed in papilloma tissues” (gives flow and thought process) A: Paraffin embedded tissues were incubated with anti-COX-2 (Santa Cruz) or PBS (control), briefly counterstained with hematoxylin and detected using DAB stain. B: Extracts from normal and papilloma tissues were analyzed by western blot. Membranes were reprobed for β-actin to confirm equal loading and transfer, and intensity determined by densitometry. Graph shows data from 6 papillomas and 7 normal tissues. * p=<0.01 by Student t-test. COX-1 levels were extremely low, and comparable in both types of tissue (not shown).

22. Research Strategy Describe overall strategy, methodology, and analyses to accomplish specific aims Discuss potential problems, alternative strategies, and benchmarks for success If in the early stages of development, describe any strategy to establish feasibility Address management of any high risk aspects Point out anything that may be hazardous and precautions to be taken

23. Overall Strategy, Methodology, Analyses Clinical Study Several essential sections – I put in a small table of contents for this section Research question pg __ Study Design/Approach pg __ Patient Sample pg __ Attrition pg __ Detailed Treatment Plan (for efficacy studies) pg __ Data Management pg __ Time Periods for Study pg __ Biostatistical Considerations pg __ Sample Size Determination pg __ Include Case Report Forms (CRFs), Consent form, Data Safety Monitoring Plan (DSMP), etc. in appendix

24. Research Question – one short paragraph, clearly stated Study Design – use a diagram! If you cannot draw one, you have a problem Overall Strategy Months on Study Drug -6 -3 0 3 6 9 12 15 18 21 24 Arm 1 Celebrex Placebo Pre Treat Celebrex Placebo Arm 2 (Endoscopy, biopsies, blood sample collection at each 3-month time point)

25. Overall Strategy, con. Patient sample inclusion, exclusion criteria; how recruit; enrollment procedure accrual plan – what if problem? Attrition -must address, predict rate Detailed treatment plan narrative - who will do what, when maybe a table with all lab tests, evaluations, etc Time periods for study figure showing first enrollment and last, interim data analysis, etc Biostatistical analyses – work with an expert Sample size calculations – critical for feasibility

26. Clinical Protocol

27. Finally, The Abstract Page Take key sentence(s) on significance of problem from Significance section Summarize in 1-2 sentences the essential background State or paraphrase the hypothesis or goal State the Specific Aims and the general approach you plan to use Must fit in space defined

28. Respiratory Papillomas –Caused by HPVs 6 and 11 Tracheal disease Moderate disease Severe disease Mild disease Pulmonary disease

29. Respiratory Papillomas Express COX-2 B. 6 6 6 * * 2 levels Normal Normal 5 5 5 - Papilloma Papilloma 4 4 4 3 3 3 2 2 2 Relative COX 1 1 1 Normal A Papilloma 0 0 0 COX-2 Negative control

30. Cyclooxygenase -2 (COX-2) Arachidonic Acid COX-2 PGG2 COX-2 PGH2 Inducible synthases PGD2 PGE2 PGF2a PGI2 TXA2 • Elevated in many premalignant and malignant tumors • If express in transgenic mouse skin, get tumors • Stimulates EGF Receptor • Stimulates tumor angiogenesis – induces VEGF • Inhibits apoptosis • Suppresses TH1 immune response

31. Inhibiting COX-2 Reduces Papilloma Cell Proliferation and Increases Apoptosis 1.2 * * * * Normal 6 1.0 * * * * 5 Papilloma 0.8 * * 4 * 0.6 Relative Proliferation Relative Apoptosis 3 0.4 2 0.2 1 0.0 0 0 2.5 5.0 7.5 0 2.5 5 7.5 Celecoxib ( µ M) Celecoxib ( M) µ * p < 0.05 ** p < 0.01

32. COX-2 and PGE2 Regulate Expression of HPV In Vitro * * control PGE2 celecoxib Relative E6/E7 mRNA * * HPV16 HPV11

33. Subject 3 Subject 1 Growth Rate Growth Rate Months after randomization Months after randomization Subject 2 Growth Rate- 12 months 18 months Trachea, Subject 3 Months after randomization Clinical Celecoxib Study Design Months on Study Drug -6-30369 1215182124 Scheduled surg. Celebrex Placebo Arm 1 Pre Treat Placebo Celebrex Arm 2

34. Preliminary Results Laryngeal Response – 28 patients Tracheal Response

35. Preliminary Results Summary Enrolled to date: 50 subjects Patients with >6 months follow-up after randomization: 29 13 with clinical response – reduction in severity >50% 9 free of disease for > 6 months 5 free of disease for a year or more 4 free of disease for > 3 months, but not disease-free at two successive surgeries marked reduction in disease severity 16 with no significant remission of laryngeal disease One patient with persistent laryngeal disease now free of tracheal disease

36. Current Questions – In Progress Is celecoxib an effective adjunct treatment for RRP? Is improvement maintained when celecoxib therapy stops? Do clinical characteristics (juvenile vs. adult onset, gender or HPV type) correlate with response? Are molecular markers consistent with a COX-2 dependent mechanism of response? Does celecoxib treatment reduce the prevalence of latent HPV DNA? How does COX-2/PGE2 impact on local immune response to HPV? Does COX-2/PGE2 also play a role in HPV+ tonsil cancers?

37. Thank You Clinicians Allan Abramson Mark Shikowitz Ginny Mullooly Physicians at participating sites Laboratory Studies Alexandra Lucs Rong Wu Ray Pica Ali Afzal All Participating RRP Patients