The Clinician Researcher: How to Fund your Habit

The Clinician Researcher: How to Fund your Habit Neville D Yeomans AM, MD, DSc (hon.), FRACP Director of Research, Austin Health / Austin LifeSciences Emeritus Professor, University of Western Sydney Professorial Fellow, University of Melbourne

What I will cover • Strategies at different career stages: • While still a research novice • Post research doctorate (i.e. ‘trained’) • Just back from overseas, with international mentors • Writing grant applications

While still a research novice • Approach someone who is well established and already has funding • Perhaps join a clinical trial team(pro bono). • Good research practice (GRP) • How to get Ethics approval • Statistics • Apply for job or scholarship on established program

Post research doctorate (i.e. ‘trained’) • Local collaborators still very important ... even though must establish your own reputation • Negotiate with hospital / head of service etc. for some protected research time and PROTECT it! • Make sure all your work thus far is published • Look for every conceivable source of funds • Hospital; University; Professional Society; Philanthropic; Industry (clin. trials)

Just back from overseas • See whether you can get your overseas mentor(s) to join with you in at least one joint collaboration • Helps show your ‘international’ status to local funding bodies • International collaborative research with strong ‘named’ researchers attractive to local funding bodies • May be able to get international funds (e.g. NIH)

Writing a Grant Application

Factors you (and whoever are assessing it) need to consider: • Novelty and Importance (“Excitement of project”) • Appropriateness of research strategy (to answer the questions) • Feasibility – i.e. likelihood that YOU can deliver on it • Track record: (a) overall, (b) in area of project • Skills and cohesiveness of the team • Availability of equipment, antibodies etc. • Budget (value for money)

BACKGROUND • Informative about basics of the area (for the potentially many non-experts who may have to read it) • Convey the logic and the rationale for WHY this study really needs to be done ….. and by YOU • Generate some excitement in the reader about your field and how you could contribute to it (obviously important in Experimental Plan section as well) • All is lost if it is not easily READABLE

BACKGROUND (Note: references prefixed A to D are in CIA to CID’s lists of recent publications) The gastric mucosa is continually threatened by damaging factors such as endogenous acid and bacteria (e.g. Helicobacter pylori), as well as ingested irritants and drugs. Fortunately, the stomach has sophisticated protective and reparative capabilities, although many of the details of process and control are still unclear. When cells at the mucosal surface are destroyed, their surviving neighbours rapidly dedifferentiate and migrate to cover the defect [Yeomans, 1973 #9]. This process, termed restitution, normally prevents deeper ulcers developing, and is also an important step in the initial repair of an established ulcer. Our group and others have shown that a number of growth factors and peptides, whose expression is induced when the stomach is injured, play a part in stimulating the generation of new cells to replace those lost, and in the remodeling and re-differentiation that reconstitutes the gastric architecture [refs – include trefoil paper of Greg’s]. We have also been intrigued by the phenomenon of adaptation [St John, 1973 #8; Skeljo, 1992 #5; Skeljo, 1996 #4289 (but cite as Ax from Greg’s list of CIA publications], where the amount of new injury markedly reduces after the stomach has been repeatedly damaged for several days. We have characterized the conditions under which adaptation occurs, and elucidated some of the likely mechanisms [Alderman, 2000 #1 (cite as Bx); Alderman, 2000 #2(cite as Bx)]. This proposal explores these mechanisms further, and looks for links with the other central theme of the present proposal – the role of cyclooxygenase-2 in gastric defence. Cyclooxygenase (COX) Inhibitors The clinical problem. Nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin, are valuable analgesic, anti-inflammatory and anti-thrombotic agents, used very widely by the world’s ageing population. In Australia, at any time more than 20% of individuals aged 65 years and over are taking NSAIDs by prescription [Day, 1992 #10]. A major drawback to their use is the high frequency of gastroduodenal damage (erosions and ulcers), which leads to significant morbidity, occasional mortality, and substantial health costs. Conservative calculations give an estimate of around 5,000-10,000 patients hospitalised for NSAID-related gastro-intestinal complications in Australia annually [Sung, 2000 #427-but cite as Dx]. NSAID gastric damage is via COX inhibition. In 1971, Vane reported that the principal mechanism of action of aspirin and other NSAIDs was through inhibition of COX, the enzyme responsible for prostaglandin (PG) production [Vane, 1971 #11]. He postulated that this was the basis of both the anti-inflammatory effects and the gastric mucosal damage, and suggested that PGs played an important part in gastric defences. The molecular characterization of two COX isoforms in 1991 [Kujubu, 1991 #12] [Xie, 1991 #13], later led to a central tenet that PGs important in gastrointestinal and renal function (‘good COX’) are produced solely via COX-1, while PGs that mediate inflammation, fever and pain (‘bad COX’) are produced solely via COX-2. Selective COX-2 inhibitors. This hypothesis led to an intensive effort to develop selective inhibitors of COX-2. It was postulated that they would have anti-inflammatory and analgesic properties similar to those of conventional NSAIDs (which nonselectively block both isoforms). However, by sparing COX-1 activity, the selective COX-2 inhibitors were predicted to have greatly reduced toxicity, particularly in the gastrointestinal tract [Greg, please check this ref.;can’t find it in Medline]6. This hope has been substantially realised. Two highly selective COX-2 inhibitors, celecoxib and rofecoxib, are now in clinical use. They have been shown to relieve pain and inflammation in arthritis patients with efficacy similar to nonselective NSAIDs, while producing substantially fewer gastroduodenal ulcers and complications [refs]. However, the clinical trials have excluded patients with current or recent ulcers, and preliminary data from our group and others (see below) challenges the view that COX-2 inhibition is harmless to the stomach [Yeomans, 1998 #16as Dx]. A further complexity has arisen as a result of one publication adducing evidence for a 3rd isoform of COX [ref]; its function and whether it is blocked by current inhibitors are presently unknown. At 3:00 in the morning, would you like to read this?

BACKGROUND(Note: references prefixed A to D are in CIA to CID’s lists of recent publications) The gastric mucosa is continually threatened by damaging factors such as endogenous acid and bacteria (e.g. Helicobacter pylori), as well as ingested irritants and drugs. Fortunately, the stomach has sophisticated protective and reparative capabilities, although many of the details of process and control are still unclear. When cells at the mucosal surface are destroyed, their surviving neighbours rapidly dedifferentiate and migrate to cover the defect [Yeomans, 1973 #9]. This process, termed restitution, normally prevents deeper ulcers developing, and is also an important step in the initial repair of an established ulcer. Our group and others have shown that……………………. This proposal explores these mechanisms further, and looks for links with the other central theme of the present proposal – the role of cyclooxygenase-2 in gastric defence. Cyclooxygenase (COX) Inhibitors The clinical problem. Nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin, are valuable analgesic, anti-inflammatory and anti-thrombotic agents, used very widely by the world’s ageing population. In Australia, at any time more than 20% of individuals aged 65 years and over are taking NSAIDs by prescription [Day, 1992 #10]. A major drawback to their use is the high frequency of gastroduodenal damage (erosions and ulcers), which leads to significant morbidity, occasional mortality, and substantial health costs. Conservative calculations give an estimate of around 5,000-10,000 patients hospitalised for NSAID-related gastro-intestinal complications in Australia annually [Sung, 2000 #427]. NSAID gastric damage is via COX inhibition. In 1971, Vane reported that the principal mechanism of action of aspirin and other NSAIDs was through inhibition of COX, the enzyme responsible for prostaglandin (PG) production [Vane, 1971 #11]. He postulated ……. The molecular characterization of two COX isoforms in 1991 [Kujubu, 1991 #12] [Xie, 1991 #13], later led to a central tenet that PGs important in gastrointestinal and renal function (‘good COX’) are produced solely via COX-1, while PGs that mediate inflammation, fever and pain (‘bad COX’) are produced solely via COX-2. Selective COX-2 inhibitors. This hypothesis led to an intensive effort to develop selective inhibitors of COX-2. It was postulated that they would have anti-inflammatory and analgesic properties similar to those of …………………... A further complexity has arisen as a result of one publication adducing evidence for a 3rd isoform of COX [ref]; its function and whether it is blocked by current inhibitors are presently unknown. • Use headings and subheadings well • Add a little space between sections, e.g. use the Word paragraph command to add 6 pts after last para in a section

Summarize, and signpost where you are heading The gastric mucosa is continually threatened by damaging factors such as endogenous acid and bacteria (e.g. Helicobacter pylori), as well as ingested irritants and drugs. Fortunately, the stomach has sophisticated protective and reparative capabilities, although many of the details of process and control are still unclear. When cells at the mucosal surface are destroyed, their surviving neighbours rapidly dedifferentiate and migrate to cover the defect [Yeomans, 1973 #9]. This process, termed restitution, normally prevents deeper ulcers developing, and is also an important step in the initial repair of an established ulcer. Our group and others have shown that a number of growth factors and peptides, whose expression is induced when the stomach is injured, play a part in stimulating the generation of new cells to replace those lost, and in the remodeling and re-differentiation that reconstitutes the gastric architecture [refs]. We have also been intrigued by the phenomenon of adaptation [St John, 1973 #8; Skeljo, 1992 #5; Skeljo, 1996 #4289], where the amount of new injury markedly reduces after the stomach has been repeatedly damaged for several days. We have characterized the conditions under which adaptation occurs, and elucidated some of the likely mechanisms [Alderman, 2000 #1; Alderman, 2000 #2]. This proposal explores these mechanisms further, and looks for links with the other central theme of the present proposal – the role of cyclooxygenase-2 in gastric defence.

Feel free to use ‘Scientific American’ language where it will help the (non-expert) assessors NSAID gastric damage is via COX inhibition. In 1971, Vane reported that the principal mechanism of action of aspirin and other NSAIDs was through inhibition of COX, the enzyme responsible for prostaglandin (PG) production [Vane, 1971 #11]. He postulated ……. The molecular characterization of two COX isoforms in 1991 [Kujubu, 1991 #12] [Xie, 1991 #13], later led to a central tenet that PGs important in gastrointestinal and renal function (‘good COX’) are produced solely via COX-1, while PGs that mediate inflammation, fever and pain (‘bad COX’)are produced solely via COX-2. Selective COX-2 inhibitors. This hypothesis led to an intensive effort to develop selective inhibitors of COX-2. It was postulated that they would have anti-inflammatory and analgesic properties similar to those of …………………... A further complexity has arisen as a result of one publication adducing evidence for a 3rdisoform of COX [ref]; its function and whether it is blocked by current inhibitors are presently unknown.

PRELIMINARY RESULTS In this section we summarize some recent experiments that form the immediate basis for several of the experiments in the Research Plan. COX-2’s ROLE IN GASTRIC MUCOSAL REPAIR Celecoxib, a selective COX-2 inhibitor, delays gastric ulcer healing Animal models of gastric ulceration allow the study of mechanisms involved in the repair of chronic mucosal injury. Serosal application of glacial acetic acid (GAA) is a widely used model for the study of gastric ulcer formation and healing21,22 and has been validated in our laboratory23,24. Preliminary data from our laboratory has shown that celecoxib (CEL), a selective COX-2 inhibitor, delivered daily for 7 days post maximal ulceration, delays the progressive healing of GAA induced gastric ulcers (fig.1). Whether the extent of healing retardation is significantly different to non-selective NSAIDs needs to be clarified, but this striking delay in healing suggests that the resolution of existing gastric ulcers may be compromised by selective COX-2 inhibitor use. Importance of Preliminary Results to show you have some methods working and are in hot pursuit

Importance of Preliminary Results to show you have some methods working and are in hot pursuit PRELIMINARY RESULTS In this section we summarize some recent experiments that form the immediate basis for several of the experiments in the Research Plan. COX-2’s ROLE IN GASTRIC MUCOSAL REPAIR Celecoxib, a selective COX-2 inhibitor, delays gastric ulcer healing Relax, we’re using a robust, well-tried approach Animal models of gastric ulceration allow the study of mechanisms involved in the repair of chronic mucosal injury. Serosal application of glacial acetic acid (GAA) is a widely used model for the study of gastric ulcer formation and healing21,22 and has been validated in our laboratory23,24. Preliminary data from our laboratory has shown that celecoxib (CEL), a selective COX-2 inhibitor, delivered daily for 7 days post maximal ulceration, delays the progressive healing of GAA induced gastric ulcers (fig.1). Whether the extent of healing retardation is significantly different to non-selective NSAIDs needs to be clarified, but this striking delay in healing suggests that the resolution of existing gastric ulcers may be compromised by selective COX-2 inhibitor use.

Importance of Preliminary Results to show you have some methods working and are in hot pursuit PRELIMINARY RESULTS In this section we summarize some recent experiments that form the immediate basis for several of the experiments in the Research Plan. COX-2’s ROLE IN GASTRIC MUCOSAL REPAIR Celecoxib, a selective COX-2 inhibitor, delays gastric ulcer healing Animal models of gastric ulceration allow the study of mechanisms involved in the rpair of chronic mucosal injury. Serosal application of glacial acetic acid (GAA) is a widely used model for the study of gastric ulcer formation and healing21,22and has been validated in our laboratory23,24. Preliminary data from our laboratory has shown that celecoxib (CEL), a selective COX-2 inhibitor, delivered daily for 7 days post maximal ulceration, delays the progressive healing of GAA induced gastric ulcers (fig.1). Whether the extent of healing retardation is significantly different to non-selective NSAIDs needs to be clarified, but this striking delay in healing suggests that the resolution of existing gastric ulcers may be compromised by selective COX-2 inhibitor use. It works in our lab too … and furthermore we’ve published with it

Importance of Preliminary Results to show you have some methods working and are in hot pursuit PRELIMINARY RESULTS In this section we summarize some recent experiments that form the immediate basis for several of the experiments in the Research Plan. COX-2’s ROLE IN GASTRIC MUCOSAL REPAIR Celecoxib, a selective COX-2 inhibitor, delays gastric ulcer healing Animal models of gastric ulceration allow the study of mechanisms involved in the rpair of chronic mucosal injury. Serosal application of glacial acetic acid (GAA) is a widely used model for the study of gastric ulcer formation and healing21,22 and has been validated in our laboratory23,24. Preliminary data from our laboratory has shown that celecoxib (CEL), a selective COX-2 inhibitor, delivered daily for 7 days post maximal ulceration, delays the progressive healing of GAA induced gastric ulcers (fig.1). Whether the extent of healing retardation is significantly different to non-selective NSAIDs needs to be clarified, but this striking delay in healing suggests that the resolution of existing gastric ulcers may be compromised by selective COX-2 inhibitor use. But there are important questions to be answered and we’re keen to do it

Importance of Preliminary Results to show you have some methods working and are in hot pursuit PRELIMINARY RESULTS In this section we summarize some recent experiments that form the immediate basis for several of the experiments in the Research Plan. COX-2’s ROLE IN GASTRIC MUCOSAL REPAIR Celecoxib, a selective COX-2 inhibitor, delays gastric ulcer healing A graph or a table has high impact and is rapidly taken in Animal models of gastric ulceration allow the study of mechanisms involved in the repair of chronic mucosal injury. Serosal application of glacial acetic acid (GAA) is a widely used model for the study of gastric ulcer formation and healing21,22 and has been validated in our laboratory23,24. Preliminary data from our laboratory has shown that celecoxib (CEL), a selective COX-2 inhibitor, delivered daily for 7 days post maximal ulceration, delays the progressive healing of GAA induced gastric ulcers (fig.1). Whether the extent of healing retardation is significantly different to non-selective NSAIDs needs to be clarified, but this striking delay in healing suggests that the resolution of existing gastric ulcers may be compromised by selective COX-2 inhibitor use. But do proof read!

Project Grant Synopsis – and the Aims section of main application • Think hard about setting these out – they are probably sections that assessors will come back to several or many times to keep them on track • Clarity of thought and conciseness of prose are invaluable • Keep looking, yourself, at what you wrote here all the time you are writing your application

RESEARCH PLAN Aim 1: To examine the effect of specific and non-specific COX inhibition on gastric ulcer healing Experiment 1 – effect of COX inhibition on exacerbation of ulcers Four groups of 7 rats will be dosed orally with either: (i) aspirin (ASA - 120mg/kg), (ii) indomethacin (INDO - 10mg/kg), (iii) celecoxib (CEL - 10mg/kg), or (iv) methyl cellulose (MC – 1%), at 24 and 48 hrs post GAA ulcer induction. Rats will be sacrificed 4 hours post the last dose. Ulcer size will be measured macroscopically using digital planimetry, and histologically using a colourQuantimet image analysis systemGC8. Mucosa at the site of ulceration will be rapidly removed and stored at -70ºC for subsequent measurement of protein and mRNA for COX-1 and COX-2. Protein will be quantitated by Western blot analysis (WBA)GC5,12 and immunohistochemistry (IHC)GC5,14 using specific COX antibodies. Message will be quantitated by reverse transcription polymerase chain reaction (RT-PCR)GC12, ribonuclease protection assay (RPA)GC12, and in situ hybridisationGC10,14, using COX-1 and COX-2 primers and antisense probes already established in the laboratory. GAPDH and ribosomal L32 levels will serve as internal controls. COX-1 activity will be assessed measuring thromboxane B2 production from whole blood platelets. This will be undertaken in collaboration with Prof. C. Hawkey (University Hospital, Nottingham, UK). COX-2 activity will be assessed by measuring mucosal generation of PGE2 using a commercial radioimmunoassay kit (New England Nuclear). Experiment 2 – effect of COX inhibition on delayed ulcer healing Eight groups of 7 rats will be dosed orally with: (i) ASA (120mg/kg), (ii) INDO (10mg/kg), (iii) CEL (10mg/kg), or (iv) MC (1%), for 7 or 21 days beginning at day 2 post GAA ulceration (time of maximal ulceration. Rats will be sacrificed 4 hours post the last dose. Ulcer size, and COX-1 and COX-2 protein, mRNA and activity levels will be assessed as in Experiment 1. Aim 2: To examine the mechanism of action of COX-2 in ulcer healing The repair of deep mucosal injury is a complex process that involves cellular processes such as restitution, proliferation, apoptosis and differentiation. While COX-2’s role in mediating mucosal repair processes is not known, the time course of delayed ulcer healing exhibited by celecoxib in our pilot studies suggests that COX-2 may be important in mediating early reparative processes such as restitution, proliferation and apoptosis. Experiment 1– effect of selective COX-2 inhibition on restitution in vitro HGF is known to facilitate restitution of gastric mucosal cells in vitro10,12. We will use this growth factor to examine the role of COX-2 in gastric epithelial restitution. Confluent gastric epithelial cells (RGM1 and IMGE-5) will be wounded linearly with a pipette tip then treated with: (i) media alone for 1, 4, 8, 12 or 24 hrs post wounding (in the presence of 10µg/ml mitomycin C to block DNA synthesis), ii) media alone, 30 minutes prior to HGF I’ve used deliberately small font size for body text here … just to show the heading structure better It is very useful to keep tying the structure of Research Plan back to your original Aims set out in the Synopsis and at start of the detailed application Experiment 2– effect of selective COX-2 inhibition on restitution in vivoRestitution of the gastric mucosa will be assessed by in vivo measurement of gastric mucosal potential difference - a validated marker of gastric mucosal integrityGC4,6,8. Three groups of 7 rats will be dosed intragastrically with: (i) 0.5ml 1% MC 30 minutes prior to 1ml ethanol (70%) alone, (ii) 0.5ml CEL

RESEARCH PLAN Aim 1: To examine the effect of specific and non-specific COX inhibition on gastric ulcer healing Experiment 1 – effect of COX inhibition on exacerbation of ulcers Four groups of 7 rats will be dosed orally with either: (i) aspirin (ASA - 120mg/kg), (ii) indomethacin (INDO - 10mg/kg), (iii) celecoxib (CEL - 10mg/kg), or (iv) methyl cellulose (MC – 1%), at 24 and 48 hrs post GAA ulcer induction. Rats will be sacrificed 4 hours post the last dose. Ulcer size will be measured macroscopically using digital planimetry, and histologically using a colourQuantimet image ………………………………………….. ……………………………………….. Experiment 2 – effect of COX inhibition on delayed ulcer healing Eight groups of 7 rats will be dosed orally with: (i) ASA (120mg/kg), (ii) INDO (10mg/kg), (iii) CEL (10mg/kg), or (iv) MC (1%), for 7 or 21 days beginning at day 2 post GAA ulceration (time of maximal ulceration. Rats will be sacrificed 4 hours post the last dose.Ulcer size, and COX-1 and COX-2 protein, mRNA and activity levels will be assessed as in Experiment 1. Aim 2: To examine the mechanism of action of COX-2 in ulcer healing The repair of deep mucosal injury is a complex process that involves cellular processes such as restitution, proliferation, apoptosis and differentiation. While COX-2’s role in mediating mucosal repair processes is not known, the time course of delayed ulcer healing exhibited by celecoxib in our pilot studies suggests that COX-2 may be important in mediating early reparative processes such as restitution, proliferation and apoptosis. Experiment 1– effect of selective COX-2 inhibition on restitution in vitro HGF is known to facilitate restitution of gastric mucosal cells in vitro10,12. We will use this growth factor to examine the role of COX-2 in gastric epithelial restitution. Confluent gastric epithelial cells (RGM1 and IMGE-5) will be wounded linearly with a pipette tip then treated with: (i) media alone for 1, 4, 8, 12 or 24 hrs post wounding (in the presence of 10µg/ml mitomycin C to block DNA synthesis), ii) media alone, 30 minutes prior to HGF • Often very useful to make the rationale of the experiment • clear before you get into its detail • Keep explaining and summarizing throughout

Choose your collaborators well

Factors you (and the assessor and GRP) need to consider: • Novelty and Importance (“Excitement of project”) • Appropriateness of research strategy (to answer the questions) • Feasibility – i.e. likelihood that YOU can deliver on it • Track record: (a) overall, (b) in area of project • Skills and cohesiveness of the team • Availability of equipment, antibodies etc. • Budget (value for money)

Get someone (or many) to read your application, ….while you’ve still got plenty of time to take their comments on board. Could they understand it easily? Were they excited by it?

The Clinician Researcher: How to Fund your Habit