**Drug Screening for Diabetic Complications**

**Co-Sponsored by National Institute of Digestive and Diabetes and
Kidney Disorders (NIDDK) and Juvenile Diabetes Research Foundation
(JDRF)**

**October 5-6, 2006**

**Sheraton Suites**

**Alexandria, VA**

***Summary Report***

**[October 5, 2006]{.underline}**

**Welcoming Remarks**

*Judith Fradkin, M.D., Director, Division of Diabetes, Endocrinology,
and Metabolic Diseases, National Institute of Diabetes and Digestive and
Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda,
MD,* and *Helen Nickerson, Ph.D., Juvenile Diabetes Research Foundation
(JDRF), New York, NY*

**Introduction and Summary of Results**

*Teresa L. Z. Jones, M.D., Diabetes Complications Program Director,
NIDDK, NIH*

Dr. Jones provided an overview of the agenda, including presentations on
results from PubChem, assay descriptions, and future plans regarding
animal models. She acknowledged individuals and organizations who worked
to plan the meeting and who have been instrumental in implementing the
drug screening initiative.

Acknowledgements include Jill Heemskerk, National Institute of
Neurological Disorders and Stroke (NINDS), for their drug screening
program on neurodegeneration; the Juvenile Diabetes Research Fund (JDRF)
and Antony Horton, Alzheimer's Drug Discovery Foundation, for advocating
for the program; Dr. Judith Fradkin, NIDDK, for the Type 1 Diabetes
funding program that supports the initiative; Steve Bryant and Svetlana
Dracheva at Pubchem for their help; and the investigators who are
participating in this program.

Dr. Jones informed participants of the confidentiality agreements
required for participation in the program. Each investigator must sign
the confidentiality agreement, which covers assays and data generated by
laboratories in this program until October 5, 2007. The goal for the
program is to develop a manuscript on the program and release all drug
screening assay data at that time. Individual investigators or groups
can publish the data generated in their own laboratory.

PubChem will be the eventual repository for all data generated in the
program. A balance will need to be maintained between protecting
confidentiality and facilitating the utility and comprehensiveness of
the website. Specific formats will be required for submission to
PubChem. For PubChem assay descriptions, protocols will not be placed on
the website. Submission of Gene ID and Protein ID numbers will allow
cross-referencing, and assay descriptions can be edited through the
PubChem Deposition Gateway website using an assigned user name and
password. All data files will be required, including activity
determination scores (i.e., 1=inactive, 2=active and 3=inconclusive),
with activity scores of 100 having the greatest activity and 0 the
least.

Dr. Jones described the types and number of assays being conducted in
the 16 participating laboratories. These include:

-   Four investigating growth and development;

-   Two investigating apoptosis/cell death;

-   Three investigating enzyme activity;

-   Four investigating gene and protein expression; and

-   Three investigating phosphorylation and binding.

As for the types of biological material being investigated, one
laboratory is investigating assays of the whole animal, four
laboratories are investigating tissue/multiple cells, 10 laboratories
are investigating single cells, and one laboratory is investigating
proteins. Among assays for cell and tissue types, five are investigating
endothelial cells, three are investigating neurons, and one laboratory
each is investigating vascular smooth muscle cells, pericytes,
macrophages, fetal islets, mesangial cells, embryonic fibroblasts, and
*Drosophila melanogaster* larvae.

**Assay Presentations**

**[Growth and Development Assays]{.underline}**

> *Tom Baranski, M.D., Ph.D., and Ross Cagan, Ph.D., Washington
> University School of Medicine, St. Louis, MO*
>
> *Paul Fernyhough, M.D., University of Manitoba, Canada*
>
> *Royce Mohan, Ph.D., University of Kentucky, Lexington, KY*
>
> **[Apoptosis Assays]{.underline}**
>
> *Charles Mobbs, Ph.D., Mount Sinai School of Medicine, New York, NY*
>
> *Rakesh Chibber, Ph.D., King's College, London, UK*
>
> **[Introduction to PubChem]{.underline}**
>
> *Steve Bryant, National Library of Medicine, NIH, Bethesda, MD*
>
> **[Enzyme Activity Assays]{.underline}**
>
> *Rosario Scalia, M.D., Ph.D., Thomas Jefferson University,
> Philadelphia, PA*
>
> *David Clemmons, MD. University of North Carolina Chapel Hill, NC*
>
> *Andrea Vincent, M.D., Ph.D., and Eva Feldman, M.D., Ph.D., University
> of Michigan, Ann Arbor, MI*

**[October 6, 2006]{.underline}**

**Assay Presentations (continued)**

> **[Gene and Protein Expression Assays]{.underline}**
>
> *Michael Brownlee, M.D., Albert Einstein College of Medicine, Bronx,
> NY*
>
> *Fred Levine, M.D., University of California San Diego, CA*
>
> *Yuqing Eugene Chen, M.D., Ph.D., University of Michigan Medical
> School, Ann Arbor, MI*
>
> *George King, M.D., Joslin Diabetes Center, Boston, MA*
>
> *Jeffrey Kopp, NIDDK, NIH, Bethesda, MD*
>
> **[Phosphorylation and Binding Assays]{.underline}**
>
> *Fred Schroeder, Ph.D., Texas A & M University, College Station, TX*
>
> *Elia Duh, M.D. The Johns Hopkins University. Baltimore, MD*
>
> *Shulamit Lerner, M.D., and Ann-Marie Schmidt, M.D., Columbia
> University, New York, NY*

**Opportunities to Pursue Promising Compounds and Assays: NIH Roadmap**

*Dr. Jones*

Dr. Jones informed participants of available funding and NIH resources
that may be used for the drug screening program. A recent funding
opportunity under the NIH Roadmap Opportunities in Molecular Libraries
initiative has been announced. This is PAR-06-545: Solicitation of
Assays for High Throughput Screening (HTS) in the Molecular Libraries
Screening Centers Network (R03-NIH Small Research Grant). Receipt dates
for funding are October 26, 2006; February 26, 2007; June 26, 2007; and
October 26, 2007. The funding will total \$25,000 for assay
implementation. The Molecular Libraries Screening Centers Network will
screen the repository compounds for biological activity, and the Small
Molecule Repository has a collection of about 100,000 compounds that
will continue to grow to about 500,000 compounds.

Dr. Myrlene Staten, NIDDK, presented information on the NIH Rapid Access
to Interventional Development (NIH-RAID) program. She stressed that
funding is available for researchers who have good animal data. This
program will only provide resources rather than money to conduct
research. Applications are available online at
http://nihroadmap.nih.gov/raid/. She indicated that these types of
funding mechanisms show that NIH is interested in pursuing relationships
with the academic community that may not have been part of NIH research
in the past.

**Animal Models of Diabetic Complications**

*Nigel Calcutt, Ph.D., Associate Professor, Molecular Pathology Program,
School of Medicine,*

*University of California at San Diego, California*

Dr. Calcutt provided an overview of his presentation, including a
description of his research facility at the University of California at
San Diego (UCSD), which is contracted with the T1D RAID program to
assess the efficacy of test compounds in rodent models of diabetic
neuropathy, nephropathy, and retinopathy. He described animal
maintenance, treatment, testing, tissue collection, and assays performed
at UCSD, and their ability to subcontract to specialists in nephropathy
and retinopathy as necessary.

Animal studies are designed with a review of existing data,
identification of subcontractors if required, development and
finalization of a study protocol, and approval by T1D RAID program
staff. At each step in the design process, UCSD faculty work with the
applicant, NIH program staff, and subcontractors. After approval, the
study is initiated.

Dr. Calcutt discussed the issues related to determining the correct
study to use. Practical issues include drug availability, composition,
and delivery route, and the known *in vivo* effects of the test
compound. For example, streptozotocin (STZ) and alloxan are
diabetogenic, and colchicine, paclitaxel, and hexachlorophene are
neurotoxins; these drug characteristics must be considered in designing
a study. The focus of the therapy also is an important issue. It should
be determined beforehand if the focus of the therapy is on a target of
the *in vitro* screening assay is complication-specific or
pathology-specific, and whether the literature contains information that
is important in consideration of an agent, for example, if the study
endpoints vary between animal species.

For monitoring diabetes, the following assessments are recommended:

-   Body weight

-   Blood/plasma glucose

-   HbA1c

-   Plasma insulin

-   Urine volume and glucose

Dr. Calcutt reviewed models for retinopathy and neuropathy that have had
some success in previous studies. For retinopathy, suggested models
include the adult male Sprague-Dawley rats with STZ, and adult C57 Bl/6J
mice and STZ. *In vivo* assays include ERG, which is in development.
Histology in these models should include whole mount retinal preparation
or whole eye preparation, immersion or perfusion fixed, which are
shipped to a subcontractor for assessment of retinal thickness,
capillary cell apoptosis, capillary drop out, neovascularization, and
neurodegeneration.

For nephropathy, suggested models include adult male Sprague-Dawley rats
and STZ, adult C57 Bl/6J mice and STZ, or adult C57 Bl/Ks db/db mice.
*In vivo* assays in these models include glomerular filtration rate
(GFR) by insulin clearance, urine albumin, creatinine, glucose, and
volume. Histology in these models should include perfusion or immersion
fixed for paraffin, which are shipped to the subcontractor for
assessment by periodic acid-Sciff (PAS) for mesangial expansion,
arteriolar hyalinosis, tubular interstitial damage, and glomerular
basement membrane thickening. Nephropathy endpoints include degenerative
neuropathy (i.e., nerve conduction velocity \[large fiber\], nerve
morphometry \[large fiber\], nerve blood flow, nerve blood vessel
density, sensory loss/hypoalgesia \[small fiber\], and epidermal fiber
density \[small fiber\]) and painful neuropathy (i.e., tactile allodynia
\[large fiber\], thermal hyperalgesia \[small fiber\], and formalin
hyperalgesia \[spinal cord involvement\]).

Dr. Calcutt reviewed established research assays, which included the
following:

-   Gas chromatography (sugars)

-   Spectrophotometry (enzyme activity)

-   Enzyme immunoassays (EIAs) (neuropeptides, neurotrophins)

-   Western blots (proteins)

-   Immmunocytochemistry

-   Confocal and electron microscopy

-   Axon and blood vessel regeneration after crush injury

Research assays in development include those for lipid, protein, and DNA
oxidative damage; endoneurial blood flow; skin blood flow; and epidermal
nerve fibers by EIA.

Dr. Calcutt discussed the advisability of using rats or mice for
diabetes research. The advantages of using rats include the existence of
the early pain phenotype, which includes thermal hyperalgesia at 2 to 6
weeks, tactile allodynia at after 1 week, and formalin hyperalgesia
after 1 week. In addition, rats may have a later degenerative phenotype
that includes NCV slowing at approximately 4 weeks, thermal hypoalgesia
after 8 weeks, epidermal fiber loss after 8 weeks, and reduced axonal
caliber after 8 weeks. The type of rat used for research purposes will
influence what endpoints are included. For example, STZ causes phenotype
progression and presentation consistent between sex and strain (e.g.,
Sprague-Dawley and Wistar) for most end points, and STZ + insulin can be
used to prolong thermal hyperalgesia. There is a need to be cautious
about the age of diabetes induction. Among insulin-treated BB Wistar/ZDF
rats, the literature suggests that pain phenotype persists and there is
no epidermal fiber loss.

Advantages of using mice for research include the presence of a rapid
onset degenerative phenotype, which includes thermal hypoalgesia and
tactile hypoalgesia after 1 to 2 weeks; formalin hypoalgesia after 4
weeks; NCV slowing after 1 to 2 weeks; and epidermal fiber loss at 4
weeks. Among the types of mouse used for research is the STZ mouse and
the db/db mouse, which is similar to the STZ mouse on a C57 Bl/6J
background. Dr. Calcutt presented slides of results of investigations
using these strains of mice.

Dr. Calcutt concluded by suggesting that recent experiments in cats may
offer another animal model for research on diabetes. There is an
epidemic of diabetes in pet cats with frequent complications; neuropathy
presents as NCV slowing and degenerative pathology indistinguishable
from humans; cat owners are compliant; and there is the potential for
use as preclinical trial subjects prior to clinical trials of drugs that
target degenerative neuropathy.

**Animal Models of Diabetic Complications Consortium (AMDCC)**

*Kristin Abraham, Ph.D., Director, Cell Signaling and Diabetes Center
Programs, NIDDK, NIH, Bethesda, Maryland*

Dr. Abraham presented a brief description of the AMDCC. It is an
NIDDK-supported program that focuses on pre-clinical animal model
development. The AMDCC is now in its second phase, for use in drug
development. For example, a mouse model for diabetic neuropathy could be
used to test for compounds in the second phase of the AMDCC program.
This could be based on the Akita and db/db models. The AMDCC is
currently working on 15 different models; the models are characterized
by different pathways for investigation.

It is hoped that a collaborative effort can be developed between the
AMDCC and the Mouse Phenotyping Centers to investigate different
phenotypes. Dr. Abraham asked participants to provide input to her on
what they think would be a useful avenue of research, including the
types of animals and compounds to investigate. NIDDK will listen to
suggestions and act on those that seem promising.

**Approaches to Pursue Consensus Hits and Charge to the Break-out
Sessions**

*Dr. Jones and Dr. Antony Horton, Ph.D., Alzheimer's Drug Discovery
Foundation*

Dr. Jones described the breakout session. There will be three breakout
sessions, one each on vascular, neuronal, and basic mechanisms. These
will be moderated by Drs. Helen Nickerson, Antony Horton, and Kristin
Abraham, respectively. Each breakout session will prioritize compounds
for future study, recommend animal models for testing, and provide other
recommendations for future studies within their topic area.

Dr. Horton provided a template for reporting from the break-out groups
that included requests to create priority lists for compounds,
protocols, animal, and data to be recommended for drug screening. He
also reported that the JDRF will be developing a Request for Application
(RFA) on drug testing in animal models. Other sources of funding in this
area are available through the NIH T1D-RAID program, as well as other
sources.

**Reports from the Break-out Sessions and Discussion of Future Plans**

**[Breakout Session: Vascular]{.underline}**

*George King, M.D., Joslin Diabetes Center*

Dr. King reported that prioritization is of utmost importance regarding
assays. The ranking of compounds was discussed in the breakout session,
and it was determined that there is not enough information known at this
time to rank the compounds. There are, however, certain criteria that
can be used to develop a consistent ranking system. These include:

-   Developing assay-specific prioritization.

-   Determining the maximum effect at the lowest concentration versus
    the EC50 of each compound.

-   Ranking compounds according to their positive and/or negative
    effects, such as their ability to stimulate protease.

-   Making a determination between active compounds versus inactive
    compounds in relation to specific tests. It should be possible to
    generate two assays; one for positive effects (e.g., increased
    phosphorylation) and one for negative effects (e.g., decreased
    phosphorylation).

-   Including an additional category for the opposite effect.

-   Defining the criteria and prioritizing hits for each assay, as each
    assay is distinct.

In the assay process, it is important to look for lines of logic for
individual hits in the assay. For example, in a specific assay, the
following should be considered:

-   The positives meet statistical significance.

-   Is the compound ranked against a known inhibitor?

-   Determine if it is possible to rank each compound according to low
    versus high glucose activity.

-   Did the compounds all use high glucose?

-   Any number higher than 1 percent may be important.

-   For example, in Big Pharma, it may take 10,000 assays to reduce
    those with potential to approximately 50 considering the structure
    activity relationship (SAR).

-   There need to be different categories for active positive versus
    detrimental effects.

Next steps include taking the main leads regarding dose response, or
other criterion, and reconfirming the results in another assay (e.g.,
cell based). Potential criteria include the following:

-   Use another assay in a screen with more comparable doses.

-   Use alternative endpoints within the same lab/pathway.

-   Set endpoints in alternative cell types, and define specificity.

-   Make sure the biology is consistent (e.g., the compounds are in a
    similar biological family member, or the compound is a family
    member).

-   Investigate the reversibility of inhibition.

-   Study kinetics.

-   The second steps need to funded, with a sound statistical rationale
    for the secondary assays.

Dr. King commented that when the group reconvenes, there will be a need
to review confirmed compounds. Areas to review include SAR; toxicity in
the short- and long-term; chemistry analysis, which could be conducted
through the NIH Roadmap; collaboration with experts; and animal testing
if it is appropriate once the whole analysis is complete. The end goal
should be a set of compounds that have been identified to send forward
for further derivatives. There is a question whether animal model
testing should be conducted before medicinal chemistry; low IC50s may
preclude this.

Recommendation for assays included the following:

-   Increased number of assays validated for complications.

-   The use of primary cells are often needed, which limits the
    possibility for high throughput screening.

-   Diverse endpoints: therefore the strength of individual assays
    should be pursued.

-   Examine leads for other pathway events with alternative assays
    (e.g., specific gene regulation pathways).

-   Initially target pre-treatment, then look at secondary intervention.

-   Kinetic assays will be important.

-   More information is need about the stability of compounds. Are
    compounds still good after freeze thaw? Companies should be able to
    answer these types of questions.

On the topic of a common publication, the breakout group suggested that
individuals include in the common publication the assay hits of interest
and further works. Included in the publication should be the list of
compounds, a description of the study, and statistical information. The
group should try to develop a publication within 1 year from this
meeting. There is a need for more information before a publication of
interest could be completed, but there is enough time to complete it in
a year. Dr. King emphasized that the breakout group would need to
request assistance in statistical support to develop a publication,
especially for the second layer of assay analysis.

**[Breakout Session: Neuropathy]{.underline}**

*Antony Horton, Ph.D., Alzheimer's Drug Discovery Foundation*

Dr. Horton provided feedback from the breakout session on drug screening
for neuropathy. The following are recommendations for prioritizing
compounds:

-   Develop ranges of thresholds for drug screening.

-   Conduct dose response experiments on hits from initial screening.

-   Investigate structural homology (e.g., SMA Project), and develop
    structural motifs to investigate up-regulation of specific
    homologous gene sequences (e.g., reading frames).

-   Choose drug classes (e.g., anti-inflammatory class) where there is
    known pharmacologic and toxicologic effects from the literature.

-   Target specific molecules that have known pharmacologic and
    toxicologic effects from the literature.

-   Investigate mechanisms in parallel with animal model testing (e.g.,
    rodent poking).

-   Follow up on compounds that have multiple hits. Diversity of the
    NIDDK/JDRF screen assays needs to be addressed, and look for overlap
    in assays (i.e., conduct sub-group assays by mechanism or by
    complication).

-   Develop a list of further assays for consideration.

-   Develop group listings (i.e., categories versus the top 100).

For data analysis, it is recommended that a follow-up workshop best
scheduled in approximately 6 months. The purpose would be to discuss
data integration and to brainstorm on next steps based on results of
completed assays. Communications among workshop participants would be
facilitated by using a website and by scheduled conference calls.

To optimize data from drug screening, there should be an identification
of thresholds for determining cutoffs for positives and
antagonistic/toxic effects. There is a great need for developing
biostatistics models for use in drug screening similar to those used by
NINDS. Quality control, including positive controls, appear to be
lacking in some screening investigations, which should be corrected. In
addition, issues of drug stability affected by the collection and
storage of sample aliquots (i.e., the freeze/thaw issue) must be
addressed.

Dr. Horton addressed the need for the development and use of animal
models. Recommendations include the following:

-   Prioritize the "hit" list first by searching the literature to avoid
    duplication.

-   In pre-clinical work, use defined endpoints, such as those used by
    the AMDCC and through existing NIH resources (e.g., Mouse Model
    Phenotyping Consortium).

-   Create an external advisory board to conduct integrative review, and
    to develop a priority list for quality control and protocol
    development.

For lead optimization, it is suggested that the following be considered
by workshop participants for meeting future research needs.

-   Conduct a PubChem search to determine tests for similar compounds.
    There may need to be seed funding to acquire and test compounds
    without known results.

-   Consider inviting medicinal chemists to the next meeting. Collect
    information regarding ongoing medicinal chemistry initiatives, such
    as those at RAND.

-   Training in informatics is needed for collection and analyses of
    data.

-   Consider researching what is known about the absorption,
    distribution, metabolism, and excretion of test compounds (i.e.,
    disposition of a compound within an organism, and how that
    influences pharmacological activity).

-   Consider how the drug screening can collaborate with existing NIH
    and non-NIH entities, such as T1D-RAID.

Dr. Horton reiterated that there may be funding opportunities for drug
screening, such as the JDRF RFA on drug testing in animal models, the
NIH T1D-RAID program, and other sources.

**[Breakout Session: Basic Mechanisms]{.underline}**

*Dr. Abraham*

Dr. Abraham reported on the breakout session on basic mechanisms. She
commented that the recommendations and suggestions reported by the
previous two groups are many of the same things that this group
discussed. The group agreed overall that it is not possible to develop a
prioritization for basic mechanisms at this point in time.

The following are recommendations that the group did think could be
offered at this point.

-   Each group of researchers should develop a priority list of
    approximately 10 to 30 hits to focus on, especially those hits that
    are giving EC50s of 10 mol or so in the primary screen.

-   There is an apparent need for secondary assays based on the primary
    assays. This will validate the primary assays.

-   Secondary assays may be useful on *Drosophila melanogaster*,
    combinatorial assays, and identified classes of compounds.

-   In a discussion of possible compounds that could be ready for animal
    testing, two of the compounds may be ready for animal studies.

Dr. Abraham agreed that it may be good to meet in approximately 6 months
to review data and spend time prioritizing from these results. At that
time, enough should be known to have a discussion of the comparative
utility and specificity. An external advisory board would then take a
look at the results and advise the group on next steps. After being
advised, there would be at least three different paths for further
research. They are:

-   A possibility may exist to propose mechanistic experiments and to
    obtain some hypothesis-driven research funding.

-   The identified compounds could be useful for manipulation with
    medicinal chemistry.

-   Another track would be to take the compounds directly to animal
    studies if the results are scientifically sound.

**Final Discussion and Adjournment**

A brief discussion ensued where participants reiterated the need to look
at similar programs around the country, both at NIH (i.e., National
Institute on Aging and NINDS) and in academic and research centers
(e.g., University of Wisconsin project with aging rats). Other points
that were re-emphasized included the need for biostatistician
involvement, the use of PubChem to search for structural similarities
among compounds, and the need for an external advisory board to support
and review the work of the drug screening group. Collaborations, in
general, were encouraged, as were the need for participating in existing
NIH funding sources, such as the T1D NIH-RAID program.

Dr. Jones thanked participants for the many good ideas put forth during
the workshop. She advised the group that she would be meeting with
colleagues in the next weeks to develop these ideas, and that she would
be in contact with workshop participants regarding next steps.