National Institute of Diabetes and Digestive and Kidney Diseases
Juvenile Diabetes Research Foundation
Drug Screening for Hyperglycemic Cellular Injury Meeting

Bethesda Residence Inn
Bethesda, Maryland

February 78, 2005
Meeting Report

MEETING PARTICIPANTS

Presenters:  Jill Heemskerk (National Institute of Neurological Disorders and Stroke [NINDS], Bethesda, MD), Allan Tobin (High Q Foundation, New York, NY), Eva Feldman (University of Michigan, Ann Arbor, MI), Nigel Calcutt (University of California, San Diego), Michael Brownlee (Albert Einstein College of Medicine, Bronx, NY),  Thomas A. Hohman (Novartis, New York, NY), George King (Joslin Diabetes Center, Boston, MA), Kota Ramana (University of Texas, Galveston), Ann Marie Schmidt (Columbia University College of  Physicians and Surgeons, New York, NY), Teresa Jones (National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK], Bethesda, MD), Robert E. Pacifici (MRSSI/CHDI, Inc., Farmington, CT).

Attendees:  Kristin Abraham (NIDDK, Bethesda, MD), Michelle Cissell (Juvenile Diabetes Research Foundation [JDRF], New York, NY) Michael Clare-Salzler (University of Florida, Gainesville, FL), Peter Dudley (National Eye Institute, Bethesda, MD), Judith Fradkin (NIDDK, Bethesda, MD), Robert Goldstein, (JDRF, New York, NY), Anthony Horton (JDRF, New York, NY), James Inglese (National Human Genome Research Institute, Bethesda, MD), Richard Insel (JDRF, New York, NY), Aaron Kowalski (JDRF, New York, NY), Murray Loew (George Washington University, Washington, DC), John Porter (NINDS, Bethesda, MD), Cristina Rabadan-Diehl (National Heart, Lung, and Blood Institute, Bethesda, MD), Robert Stanton (Joslin Diabetes Center, Boston, MA), Myrlene Staten (NIDDK, Bethesda, MD), Grant Zimmerman (CombinatoRx, Boston, MA).

MEETING OVERVIEW

This meeting, cosponsored by NIDDK and the Juvenile Diabetes Research Foundation (JDRF), was initiated to bring together researchers on assay development and diabetes complications caused by hyperglycemic cellular injury (HCI).  NIDDK is interested in funding translational research in these areas and is seeking expert input on the state-of-the-science before making a commitment to support a consortium of public and private institutions.  Current NIDDK and National Institutes of Health (NIH) Roadmap Initiatives on assay development were presented as potential models for an HCI initiative.  Participants heard presentations on a NINDS initiative on drug-screening assays for neurodegeneration, which will be considered as a model for NIDDK in developing drug-screening assays for HCI.  Other invited speakers made short presentations on assays for HCI that provided background information and perspective on issues to be considered before NIDDK develops an initiative on drug screening for HCI.  The second day of the meeting was set aside for discussions of assays and libraries that might be useful for an NIDDK initiative.  Other issues also were discussed, and recommendations were made at the end of the meeting.

NINDS PROGRAM FOR DRUG SCREENING FOR NEURODEGENERATION

A potential model for NIDDK in developing assays for screening potential drugs for diabetes complications may be the NINDS drug-screening program for neurodegeneration.  Jill Heemskerk provided an overview of the NINDS programs, and Allan Tobin gave a presentation on “Screening Approved Drugs:  A Private Agency Perspective” that outlined his perspective on the role of collaborations and networking in this effort. The NINDS initiative was a collaboration among NINDS, the Huntington’s Disease Society of America, the Amyotrophic Lateral Sclerosis Association, and other interested research organizations.  Drugs tested included those approved by the U.S. Food and Drug Administration (FDA) and drugs that have been shown to be biologically active.  The goals of the NINDS program were drug discovery and understanding mechanisms-of-action of drugs that would allow further investigation of drug classes.  The NINDS program investigated 1,040 drugs (approximately 800 were FDA approved) using 29 assays; more than 1,000 drugs were screened in disease models using cell lines, worms, and fruit flies.  After 6 months of testing, 10 drugs were identified as showing promise in neurodegeneration; these included known drugs, natural products, and controlled substances.  

Although drugs were identified through positive results of assays, it is not possible to move these drugs directly from the laboratory to the clinic.  To be useful, the drugs identified through assays still most undergo toxicity and potency testing, development of analogues when feasible, chemical modification if necessary, and investigation in clinical trials using animal and then human subjects.  None of the drugs yet has been through the entire process, although Ceftriaxone, an antibiotic, soon may be investigated in a clinical trial.  Other screened drugs, such as Celastrol, had positive tests in assays and had positive clinical testing using animal models; chemical optimization studies on these drugs will begin shortly.  

NINDS drug screening procedures have been instructive in developing a process that can be adapted to the needs of NIDDK in screening drugs for diabetes complications.  An overarching principle of drug discovery is that it is unlikely that a single, effective drug will be discovered that meets all research objectives (i.e., a “home run” mentality).  Drug discovery requires the long-term funding and research commitment that will enable small gains that cumulatively result in reductions in complications from diabetes and mortality.  Another important lesson is that collaborations should to be formed and used to the fullest extent possible to advocate for the attention needed at the national and local levels to support this research.  
 
ASSAYS FOR HYPERGLYCEMIC CELLULAR INJURY

Assay development for HCI has been investigated for more than 25 years.  This research has increased the knowledge of the biologic pathways involved in injury, but only minimal success has been achieved in applying this knowledge to drug development for use in the clinic.  The Diabetes Control and Complications Trial (DCCT) showed a strong association between glycemia and HCI.  A series of presentations by George King, Ann Marie Schmidt, Thomas Hohman, Nigel Calcutt, Kota Ramana, and Michael Brownlee added perspective on past successes in understanding this relationship and approaches for future research to bring findings to the clinic.

Dr. King’s presentation, “Can cell-based assays be used to test drugs for treatments of diabetic complications?,”  pointed out that no clinical trials have replicated results from preclinical and animal studies of diabetes.  The effects of glucose on cells are well known, but there is little agreement on researchers’ definitions of hyperglycemia, the most appropriate cells lines for conducting research, or the breadth of inhibitory effects studied.  This makes identifying endpoints for such studies difficult.  The glucose pathways that result in HCI are complicated, and there is a need to develop a better understanding of the effects of altering specific steps in the pathway.  For example, hyperglycemia alters cell signaling through PKC, MAPK, JNK, or other genes.  Understanding these genetic factors and how they are altered to create vascular, endothelial, or monocyte dysfunctions is an area of research that may promote an understanding of how to stop or reverse HCI.  It may, however, be beneficial to ignore the middle steps in the pathways and to try to design investigations that address outcomes.  Key issues regarding cell-based assays for hyperglycemia include standardizing cell lines and concentrations, using realistic conditions for cell growth, and determining the correct concentration of glucose (e.g., less than 25 mM).

 Dr. Schmidt discussed “Cell Types and Relevance to Diabetes” in the context of diabetic complications, including nephropathy, neuropathy, retinopathy, wound healing, and inflammatory responses.  Different cell types are used to study each of the diabetic complications (e.g., Mueller cells for retinopathy), and it is accepted that studies in cell lines offer proof-of-concept for much of the research on diabetes complications.  The first glucose studies of cells were unsatisfactory because some of the cells that produce advanced glycation end products (AGEs) could not be grown in glucose.  AGEs are produced through the effect of reducing sugars on amino groups in proteins, lipids, and nucleic acids.  The failure to grow has been alleviated in part by developing cell lines, such as AGEs/N-carboxymethyl lysine (CML) cell lines, that have different ligands and can be studied with glucose exposure.  CML modifications of proteins activate key cell signaling pathways (e.g., NFB) to accelerate vascular and inflammatory complications associated with diabetes.  There is no standard cell line used to study HCI; this should be a priority for developing an initiative for assays.  In developing assays, promising areas to test include levels of reactive oxygen species and apoptosis versus proliferation.  Target validation appears to be important.  The use of zebra fish models with high-throughput assays appears promising, especially for increasing the understanding of small molecules.

Dr. Hohman discussed the “Regulation of Retinal Oxygenation Biomarkers for Diabetic Retinopathy.”  He is developing an alternative assay using an animal model to investigate the earliest changes in loss of retinal capillary pericytes.  The use of magnetic resonance imaging (MRI) with a carbogen inhalation challenge in animals can identify early changes that are predictive of future retinopathy.  Histopathology confirms these changes, and measurements relate well to oxygen saturation.  Diabetic animals show differences with exposure to oxygen and carbogen.  For assay purposes, oxygen saturation of the retinal-vitreous interface using MRI should be explored. 

Dr. Calcutt discussed “Human Diabetic Neuropathy” and focused on distal symmetrical polyneuropathy, including a discussion of potential models that consider the early pathology as a screen.  Within the time course of the pathology of diabetic neuropathy, epidermal fiber loss may be a potential screening target.  Epidermal fiber protein gene product (PGP) 9.5 is similar in mice and humans; the loss of PGP 9.5 has been studied for relevance in screening but has not yet been proven to be a biomarker for neuropathy.  Neurochemical markers, such as neuropeptides (e.g., sub P and CGRP), terminal vesicle docking proteins (Rab 3A-GTPase, SNAP-25B, synaptotagmin, and SNARE fusion complex), and terminal receptors/channels (VR1, p75, and trkA) are potential targets for screening.  A potential assay development plan for investigating epidermal fiber loss could include more than 400 compounds per year in 4,800 mice.  This would be realistic to accomplish and should be possible to complete using a pathological model in mice.  

Dr. Ramana made a presentation on “Screening of the Drugs That Inhibit Aldose Reductase (AR)-Catalyzed Reduction of Lipid Aldehyde-Glutathione Conjugates but not Lipid Aldehydes.”  AR reduces glucose and takes part in important cellular functions such as detoxification and cell signaling.  Because all known AR inhibitors block the carbonyl binding site responsible for the detoxification of toxic lipid aldehydes, it would be beneficial to have an inhibitor that can bind at the glutathione binding site and prevent signaling processes without affecting the detoxification of lipid aldehydes.  In screening for drugs that inhibit AR, attenuated high glucose, TNF-, and growth factors are potential markers, as is 4-Hydroxy-trans-2-nonenal  (HNE), the main aldehyde generated during lipid peroxidation.  High glucose-induced proliferation of vascular smooth muscle cells and apoptosis of vascular endothelial cells are prevented by TNF- antibodies or soluble-TNF receptors, suggesting that paracrine effects of TNF- are required for the propagation of hyperglycemic signals.  Based on studies of AR inhibition, screening should occur for drugs with AR activity toward lipid aldehydes and glutathione-lipid aldehyde conjugates.  Studies also should investigate the antiinflammatory effects of drugs that reduce lipid aldehydes but not glutathione conjugates.

Dr. Brownlee gave a short presentation on “Possible Assays for Drug Screening.”  The first step in the pathway for HCI assay development begins with cell based assays, necessary for screening membrane compounds.  After a compound has been modified to a drug, animal tests are performed, followed by testing in humans.  Each assay system has its own unique difficulties; additionally, data from these assays can be perplexing.  Dr. Brownlee identified four critical issues in HCI assay development:  (1) glucose metabolism is deviated in all transformed cell lines (i.e., the Warburg Effect); (2) phenotype may be profoundly influenced by genetic background; (3) epigenetic modification may be induced by prior environmental conditions; and (4) different targets may be dominant at different stages of diabetic complications.

Transformed cells’ glucose metabolism is altered such that they have a high rate of glycolysis and a lower rate of oxidative phosphorylation than nontransformed cells.  This may preclude the use of transformed cell model systems in studying hyperglycemia because usable information will not be produced.  To avoid metabolic problems in studying HCI, Dr. Brownlee suggested establishing conditionally immortalized cell lines.  An example of a conditionally immortalized cell line that could be used to study HCI is to grow a cell line under a certain set of growth conditions (i.e., higher or lower temperature, presence or absence of tetracycline).  For example, when one set of conditions exists (i.e., high temperatures), the cells may act as transformed cells; when another set of conditions exists (i.e., lower temperature), the cells redifferentiate and behave as primary cells do.  This addresses the first critical issue.

The second critical issue underscores the fact that cells from different animals have different phenotypes because they have different genetic backgrounds.  One way to reduce this problem is to use cell lines from and develop cell-based assays in several different inbred animal strains.  Ultimately, cell lines derived from different human donors should be used, as results in humans can vary greatly from results in cellular or animal models.  For example, a microarray study analyzing genes potentially involved in diabetes found that expression of CD36 was decreased in glomerular nephritis.  When human diabetic kidneys were stained for CD36, however, expression was increased dramatically; in a rodent model, expression was decreased.  This type of study underscores the importance of confirming results from cellular or animal model studies in humans.

The third critical issue involves the emerging knowledge being accumulated on the role of epigenetic changes caused by environmental events that alter, often permanently, patterns of gene expressioneven in animals and people with identical genomes.  Epigenetic events, such as modification of histones by the addition or removal of methyl or acetyl groups, can alter the transcription process.  For example, a recent study reported that 3T3-L1 adipocytes differentiated in 4 mM glucose are more insulin sensitive than those differentiated in 25 mM glucose and remain more sensitive long after differentiation has occurred.  

The fourth critical issue must be addressed by increasing the understanding of the sequela of diabetic complications.  There is no data confirming that the mechanisms active in later stage diabetes and the epigenetic changes that have occurred are the same as those in a naïve person.  A 3-year study of people with later-stage complications who are unhealthy may fail to demonstrate the efficacy of a drug that might be useful for diabetes diagnosed 2 rather than 25 years ago.  Although difficult, it is necessary to target the different stages of diabetic complications and to consider that improvement due to a given therapy may not be observed for many years.  Two possible ways to model this are to use cells derived from long-term rodent models at different stages or cells derived from human tissues at different stages.

CURRENT NIDDK AND NIH ROADMAP INITIATIVES ON ASSAY DEVELOPMENT

The NIH Roadmap (http://nihroadmap.nih.gov/overview.asp) is an integrated vision to deepen understanding of biology, stimulate interdisciplinary research teams, and reshape clinical research to accelerate medical discovery and improve people’s health.  This initiative, begun in 2002, identifies the most compelling opportunities in three main areas:  new pathways to discovery, research teams of the future, and reengineering the clinical research enterprise.  The goal of the NIH Roadmap that is relevant to assay development is the production of small-molecule probes for biomedical research.
  
Jill Heemskerk made a presentation on “NIH Assay Development,” and Theresa Jones made a presentation on “Development of Assays for High-Throughput Drug Screening.”  These presentations described the possibilities for using the integrated resources of the Roadmap for NIDDK’s initiative on developing screening assays.

Dr. Heemskerk described resources available through the Molecular Libraries Roadmap Initiative (MLRI).  These include more than 500,000 compounds a library, a network of high-throughput screening centers, and high-throughput assay development.  The NINDS initiative experience showed that identifying one compound for use in clinical trials required the screening of  approximately 100,000 compounds using high-throughput assays and 10 to 10,000 compounds using moderate-throughput assays; 10 compounds were then identified for further investigation in vivo.  As part of the MLRI, Requests for Applications (RFAs) are being issued for 1-year awards for late-stage assay validation for automated screening, new assay development, and moderate-throughput screens for assay validation.  For more information on the MLRI, visit http://nihroadmap.nih.gov/molecularlibraries/index.asp.

NINDS also has a Translational Research Program that issues RFAs for therapeutic development for neurological disorders.  Funding is provided for single and multicomponent therapeutics development programs, pilot projects and resource development (e.g., assay development), and projects are encouraged for high- and moderate-throughput assays and moderate-throughput screens.  In addition, NINDS accepts unsolicited grants under the R21 (i.e., high risk, innovative research) and R13 (i.e., product [assay] development) funding mechanisms.  Information on these funding opportunities may be found at http://www.ninds.nih.gov/funding/funding_announcements/allcurrent.htm.

Dr. Jones described a Program Announcement (PA04-068), “Development of Assays for High-Throughput Drug Screening” that requires a low-to-moderate throughput-screening assay with reproducibility and feasibility for adaptation to an automated high-throughput screening approach.  This initiative uses an R01 mechanism that allows for longer development time of novel approaches.  The goal is automation and use of the assays at one of the MLRI screening facilities.  These types of integrated research approaches may be the model that NIDDK will want to pursue for diabetic complication assay development.
  
DISCUSSION OF TOPICS

Summary of Discussion Topic #1:  Assays

Participants were asked to discuss specific questions regarding assays.  These are listed below.

	What are the range, quality, and quantity of high-throughput assays for hyperglycemic cellular injury?  Diabetic complications include a diverse set of conditions, which presupposes that there must be different types of assays for each condition.  It is important that assays are as specific as possible for specific conditions, and a key aspect of choosing an assay will be determined by defining study parameters.  A diversified portfolio is needed to cover each of the diabetic complications related to HCI.  One approach is to identify a target that can be validated and use systems biology to investigate pathways, as well as cell-based phenotypic research with very tight restrictions.  It would be useful for funding purposes if a set of guidelines and principles were developed to guide research groups in applying for research grants.
	What kinds of assays could be considered for inclusion in a screen for hyperglycemic injury (e.g., primary cell cultures versus cell lines, phenotypic versus protein target versus gene expression assays)?  A core principle of this research should be that a hierarchy of primary cellshuman, large animals, rodents, and cell linesis used as the focus of research and funding.  Before beginning an initiative on HCI assays, a decision must be made regarding whether the research should function to produce a drug to alleviate HCI diabetic complications or should focus on increasing knowledge of HCI.  This is a key question to be decided by NIDDK before committing funds in this area.
	Can assays developed for different pathologies be adapted for hyperglycemia?  This certainly should be considered because of the various pathologies created by hyperglycemia.  It seems logical to consider cardiovascular, peripheral vascular, and kidney pathologies to see if they have evidence of hyperglycemic injury, even if the studies are not examining this.

	What are the cell types affected by hyperglycemia that should be considered for the screen?  Primary cells or conditionally transformed cells are desired for HCI investigations.  Glucose levels should be relevant for the cell type, and levels of glucose should be pathophysiologically or physiologically relevant.  It is premature to recommend a cellular glucose level for use in these investigations, but a standard level should be determined for use before beginning this research.  Examples of target cells and conditions of interest include neurons (e.g., primary, dorsal root ganglia, and Schwann cells); kidney cells (e.g., podocytes and mesangial cells); vascular cells (e.g., human peripheral blood monocytes, vascular smooth muscle cells, vascular endothelial cells, lymphocytes, and endothelial cells); diabetic (diseased) tissue at different stages of pathology; and cells grown in different media under different conditions.
	Would a combination of high-throughput and intermediate-throughput assays be useful?  The endpoints of assay development must be determined.  Suggestions included endpoints of molecular targets or phenotypes, which will direct research along different paths depending on the desired endpoint.  It also is unclear whether a goal of assay development should be creating a process amenable for use in either high-throughput (i.e., one million tests per run) or moderate-throughput (i.e., ~1,000 tests per run) microarrays.  The tactical approach for the use of high-throughput microarrays will need to be made clear after discussions with investigators who have used the technology, such as chemists.

In summary, there is no recommendation for specific assays; a recommendation to develop guidelines and principles to direct research on HCI in diabetic complications is a more feasible goal.  Assays need specific criteria and thresholds of acceptability.  Endpoints must be determined and specific assays for specific diabetic complications must be developed.   

Summary of Discussion Topic #2:  Libraries

The importance of establishing a library of compounds for drug screening assays is undeniable.  Choosing the compounds to include is a more difficult question to address.  The NINDS library included compounds that were identified initially from pharmaceutical company libraries.  These included mostly compounds that were FDA-approved and others from international sources.  It is sensible to consider adding known drugs, dietary supplements, vitamins, herbals, narcotics, and agricultural compounds to a library to give as many options as possible to researchers.  Adding as many compounds as possible to the initial library will require filtering out many more at the end of the process; this may be impractical purely for the volume of compounds available.  One approach may be to choose one or two compounds from each class for initial screening, although this approach ignores the possibility that compounds in the same class may have different mechanisms-of-action that could be missed if only a few compounds from each class are chosen for screening.

The following questions were addressed and answered during the discussion period:
	Are there additional FDA-approved compounds that should be considered for the screen, beyond those that were chosen for the NINDS screen?  The screen should be as inclusive as possible.  An ideal situation would allow for the screening of 1,000 to 2,000 compounds in the first 6 months.  A secondary goal should be to involve new investigators in this effort so there is an opportunity to develop new assays from different fields.  It makes sense to involve investigators with established laboratories for funding purposes.
	Should the library include chemicals besides FDA-approved compounds?  Because FDA-approved drugs have been tested in humans and may have identified analogues, it is recommended that FDA-approved drugs be entered into the library.  This also will allow the inclusion of compounds being investigated by diabetes researchers that may not be approved but are in the pipeline for development (e.g., -lipoic acid and antioxidants).
	Could preselection of a target group of compounds for low-throughput screening be useful as an adjunct to the program?  At this time, it does not make sense to choose a target group of compounds.  This can be decided later. 

 Summary of Discussion Topic #3:  Other Issues

Participants identified various other issues to address regarding assay development and diabetic complications.  These are listed below.

	What are the next steps for positive hits in a screen?  Would it be useful to include assay development for this program?  It would be beneficial to create a flowchart to show when positive hits indicate it is appropriate to further test a compound or to consider the compound void of effects.  At this time, little is known about animal models and their ability to predict human disease, especially from HCI.  A first step to address this issue may be to talk with scientists in animal consortia to see what is available or known about the effects of certain compounds on diabetic complications (e.g., nephropathy and neuropathy).

	An important issue to address early in the process is whether the goal of assay development is prevention or intervention.  Once answered, this question will set the direction for identifying compounds for drug screening and for the type of animal model (e.g., swine, dog, or primate) to be used.

	Should the assays be performed in a central location, in individual laboratories, or a combination of both?  At the beginning of the initiative, it may be better to have little centralization.  As the project continues, however, centralization may be used.  Informatics can have a very large impact on data collection and analysis.
	What would be the optimal duration of the screening period?  An optimal duration for the screening period should be 6 months to a year, depending on the number of compounds screened.  This amount of time would foster enthusiasm for the project without having it viewed as static and immobile.
	How can the field of diabetes complications best utilize NIDDK and Roadmap Initiatives in this area?  The Roadmap Initiative should become a valuable resource for NIDDK in developing a drug screening initiative.  If a consortium is developed, it should take advantage of the resources available for assay development and informatics.  This would be an efficient and practical use of NIDDK funds.
	How would your institution deal with the intellectual property issues of the program?  It appears that most intellectual property (IP) issues will not apply to this initiative because screening is accomplished early in the process, before there is a product to market.  The consensus of the participants was that IP issues can be dealt with later in the process case-by-case for both compounds and for assays.  In addition, if work is done in academic institutions, patent rights do not become an issue.  This also is a perk of working with a consortium, because everyone shares resources and knowledge.
	The drugs in the proposed screens are already in use, and there may be a need to speak with patent-holders (e.g., pharmaceutical companies) to ensure that IP rights are not being infringed upon.  This is likely to be a bigger problem for new chemical entities.  NIDDK and JDRF will insist on full access to all compounds and assays for research purposes and on the “freedom to operate” for further development.  IP principles should include the following four tenets:  (1) results should be made available to other researchers as quickly as possible, subject to concern about peer-reviewed publication; (2) inventions should be patented only if such patents will enhance the timeliness and availability of diagnostic and therapeutic products; (3) such patents should not be asserted against anyone using the inventions for  research;  and (4) commercialization of patents should be done with the primary objective of making diagnostic and therapeutic products available to patients on a fair and timely basis.
 
	How could this program best work with industry?  It is important that NIDDK and JDRF include industry in planning and implementing this initiative. 
Dr. Jones raised an additional issue by asking if there are a minimum number of assays for NIDDK to consider funding.  Participants said that available resources rather than science should decide this.

Summary of Discussion Topic #4:  Recommendations

Participants reviewed what had been presented in this meeting on drug screening for HCI and assay development.  The following questions were discussed and recommendations were made:

	Given the state of the field, what should be the overall goal of such a program:  the identification of drugs for clinical testing, identification of new targets and mechanisms, or validation and development of cell assays?  The goal of an NIDDK initiative on drug screening for HCI could be anything from developing and validating assays to producing a clinical intervention for use in people.  Validation of targets and developing compounds that result in filing an Investigational New Drug (IND) application with the FDA both are reasonable goals.  There is, however, a broader range of goals that may become clearer as this research proceeds.  Investigators must have the ability to respond to new information in a way that often is impossible within NIH grant mechanisms.  

	A few of the potential interim results that could cause a change in research direction include

•	Differences uncovered during validation of assays between initiation and propagation of diabetic complications; 
•	Discovery of a novel assay, or a more appropriate assay, that closely recapitulates an intervention;
•	Unexpected hits could reveal new mechanisms and allow new drug discovery, resulting in a drug that could be taken directly to the clinic.

	Additionally, a mock flow scheme could be developed that allows researchers to know when an assay is producing viable results or when to stop testing a compound because of the lack of positive hits in the assays.  The best possible preclinical information is needed in a tiered mock flow system to provide a context for continuing to investigate a compound.  Much of the uncertainty in following a compound through the assay process can be alleviated by building a strong information technology component into the consortium and by creating systems that allow the sharing of information among consortium members.

	Should the NINDS program be modified to include a longer time for data collection, intermediate-throughput assays, or other features?  The danger in allowing the length of a research program to be extended is that investigators lose excitement and commitment to the program.  There also is the danger of losing enthusiasm unless positive results are achieved.  This often can be overcome in consortia, because sharing of information results in investigators seeing the overall program and what is being accomplished.

	Sharing data, even negative data, is key to the success of an initiative for assay development.  The NINDS initiative includes an information technology component; this may be an appropriate model for NIDDK.  Two types of data are needed.  The first is raw data that can be collected from early preclinical studies and submitted to a common database such as PubChem (http://pubchem.ncbi.nlm.nih.gov/).   PubChem, managed by the National Center for Biotechnology Information at the National Library of Medicine, is a repository of information on small organic molecules and their biological activity.  The second type of data is data for analysis, which could be used by consortium institutions to determine the benefits of assays.  Information collected in the NINDS initiative can be imported into PubChem, where it can become publicly available if the data can be standardized.

	Where are we on the continuum of developing a drug screening program for hyperglycemic cellular injury?  Are we ready to start a program similar to the NINDS program?  Do we need more assay development?  Do we need more research on mechanisms to develop more targets?  At the present time, there are 1,000 to 2,000 compounds identified and ready to begin preclinical assays.  Although there are no perfect assays, enough is known to begin looking at some of the compounds that have shown promise in past studies.  It will be important to manage expectations so researchers do not imagine they will produce a cure for diabetic complications in a few months.  The field of drug discovery needs attention and this type of project can answer many remaining questions, such as the type of cells, animals, or human risk factors to study.

To begin the process, participants encouraged NIDDK to begin work on the RFA for assay development.  JDRF is interested in working with NIDDK to support the initiative.  If possible, the RFA should be written to allow maximum flexibility and multiple pathways with various cell types and animal models.  The NINDS funding model is worth considering, but NIDDK should explore ways to begin this initiative in a timely fashion.