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Type 1 Diabetes Pre-Clinical Research

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BRM is a successful biopharmaceutical company formed in 1996 by Dennis Guberski and Dr. Arthur Like of the University of Massachusetts Medical School (UMass). Over the course of 20 years the founders developed proprietary diabetes research models under the sponsorship of the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK). BRM licensed this intellectual property portfolio from UMass in 1998 and since that time has used these proprietary tools to become one of the leading sources of customized preclinical contract research specializing in type 1 and type 2 diabetes.

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Type 1 Diabetes Pre-Clinical Research

  1. 1. Biomedical Research Models, Inc. Founded 1996 www.brmcro.com 67 Millbrook St., Suite 422 Worcester, MA 01606
  2. 2. Diabetes Mellitus is Not a Single Disease ABSOLUTE INSULIN DEFICIENCY TYPE 2 (NIDDM) MODY (Maturity Onset Diabetes of the Young) TYPE 1b (IDDM) TYPE 1a (IDDM) LADA (Latent Autoimmune Diabetes in Adults) RELATIVE INSULIN DEFICIENCY DIABETES SPECTRUM HYPERGLYCEMIA
  3. 3. Type 1 Diabetes
  4. 4. Genetics, Environment and T1A Diabetes • The environment seems to be important in human T1Diabetes • Monozygotic twin with the disorder: ~50% concordance • The incidence of type 1 diabetes is increasing • Finland, Sardinia, the Baltic States, Poland • Is this genetics, environment, or both?
  5. 5. Candidate Environmental Agents • Toxins • e.g. nitrosoureas • Diet • e.g. cow milk protein • Toll-like receptor ligands (e.g. LPS) • Infection • e.g. mumps, rubella, measles • “Hygiene”
  6. 6. Epidemiology of Type 1A Diabetes • Seasonal Variation • Lower incidence in Finnish boys in June with a spike in November-December • Correlations of Type 1A diabetes onset with: • Coxsackie B • Mumps • Rubella • CMV,EBV, Retrovirus are less clearly associated
  7. 7. Viruses and Human T1D • Epidemiological studies suggest association • “Causality” is unproven • No evidence of direct viral infection of islets • Other putative mechanisms include: • Bystander T-cell activation • Molecular mimicry • T cell activation by viral superantigen
  8. 8. Changing Incidence of AutoimmunityIncidenceofInfectionsDiseases(%) 1950 1990198019701960 2000 Rheumatic fever Mumps Measles Tuberculosis Hepatitis A 0 100 50 IncidenceofImmuneDiseases(%) 1950 1990198019701960 2000 Multiple sclerosis Crohn’s Disease Asthma Type 1 diabetes 100 200 300 400
  9. 9. “Spontaneous” Type 1 Diabetes Human NOD BBDP Rat MHC Multiple I-Ag7 RT1B/Du Other Loci ~18 ≥27 >5 Gender M=F F>M M=F Insulitis + + + DKA + +/- + AutoAbs + + +
  10. 10. Rat Models of Autoimmune Diabetes: • Spontaneous • BBDP/Wor • BBZDP/Wor • Long Evans Tokushima Lean (LETL) • Komeda Diabetes Prone (KDP) • LEW.1AR1/ Ztm-iddm • Induced • YOS • PVG • PVG.RT1u • PVG.R8 • WAG • BBDR/Wor • WF.iddm4 congenic • Both • MAD (LEW.1WR1) • Transgenic • None Strains in blue are maintained by BRM
  11. 11. Insulitis in Human, Rat, and Mouse BBDP/Wor Human Pancreas NOD Mouse
  12. 12. The Diabetes Prone BB Rat • Oldest Model of Spontaneous Type 1 Diabetes • Evidence for Autoimmunity • Pancreatic insulitis • Class II MHC associated (RT1u) • T cell dependent • Preventable by immunosuppression • Transferable to adoptive recipients • Autoantibodies • Lymphocytic thyroiditis
  13. 13. Tolerance in the Presence of Genetic Susceptibility A AA R β Autoimmunity CD4+ART2+ Treg Transfusion A A R R RA β Tolerance BBDP/Wor Rat
  14. 14. Environmental Considerations • Viruses have been shown to alter the tempo of diabetes among BBDP/Wor rats • Some viruses delay the onset of diabetes • (SDAV,LCMV and Sendai) • Others accelerate (RCMV) • Incidence of diabetes in virus free colony ~85% • In BBDR rats virus can induce resistant rats to develop autoimmune disease • Hashimoto’s Thyroiditis • Type 1 Diabetes • No spontaneous diabetes among virus free rats
  15. 15. • Parvovirus in the BBDR Rat • Kilham’s rat virus (KRV) • Single stranded DNA virus • 3 overlapping structural proteins, VP1, VP2 and VP3 • 2 overlapping nonstructural proteins, NS1 and NS2 • Parvovirus B19 associated with autoimmune rheumatoid arthritis in humans (Simpson et al 1984 Science 223:1425) • BBDR rats are an animal model for Rheumatoid Arthritis (Watson et al. 1990 J.Exp.Med 172:1331)
  16. 16. Susceptibility and Mechanism of Parvovirus Induced Diabetes • Protocol • 21-25 day old male and female animals • 1 x 107 PFU KRV i.p. on day 0 • Monitor plasma glucose and body weight • Experimental data demonstrates that the virus • working through TLR-9 • creates and immune imbalance
  17. 17. Diabetes in Parvovirus (KRV) infected BBDR Rats Days After Infection 0 10 20 30 40 CumulativeDiabetes-Free Survival(%) 0 20 40 60 80 100 KRV Alone (N=24)
  18. 18. Immunological Balance Tolerance and Autoimmunity in BB rats A AA R β Autoimmunity CD4+ART2+ Transfusion “Treg” Cells A A R R RA β Tolerance ART2+ Treg Depletion Virus Infection BBDP BBDR TLR Ligation
  19. 19. Diabetes in Parvovirus vs. H-1 Infected BBDR Rats Days After Infection 0 10 20 30 40 CumulativeDiabetes-Free Survival(%) 0 20 40 60 80 100 KRV Alone (N=24) H-1 Alone (N=6)
  20. 20. Parvovirus Reduces Splenic Treg Cells BBDR WF CD4+CD25+ TregSpleenCells(%) 0% 1% 2% No Virus KRV H-1 N.D.
  21. 21. BBDR Summary 1 • BBDR rats show virus-specific susceptibility to the triggering of T1D • Virus-induced Treg modulation may be one mechanism of viral induction of diabetes
  22. 22. Innate Immune Responses Trigger T1D in Rats • Adaptive Immunity • Pathogen-specific defense • Involves the MHC and T Cell Receptor • Long lasting immunity • Innate Immunity • First line of defense • Activation of immune responses through toll-like receptors (TLRs) that recognize pathogen associated molecular patterns (PAMPs)
  23. 23. Hypothesis and Method • Innate immune responses produced by TLR ligation will induce T1D in resistant rats • Polyinosinic:polycytidylic acid (poly I:C) – A synthetic double-stranded polyribonucleotide – Ligand of toll-like receptor 3 (TLR3) – Strong inducer of cytokines – A simple tool for testing multiple strains
  24. 24. TLR3 Ligation Can Induce Diabetes Strain RT1 B/D Diabetes MAD (LEW.1WR1) u 22/22 BBDR u 20%-100% PVG.RT1u u 26/30 LEW.1AR1 u 4/20 PVG.R8 u 6/9 LEW.1AR1-iddm u 0/10 WF u 1/22 WAG u 1/9 Treatment with poly I:C alone for 2-3 weeks; insulitis data concordant Data from Ellerman and Like, Tirabassi et al., and Hedrich et al.
  25. 25. Synergy: Diabetes After Virus Infection and Activation of Innate Immunity Days After Infection 0 10 20 30 40 CumulativeDiabetes-Free Survival(%) 0 20 40 60 80 100 KRV Alone (N=24) KRV after Poly I:C (N=6) H-1 Alone (N=6) H-1 after Poly I:C (N=10)
  26. 26. Parvovirus (KRV) Itself May Act via TLR9 Stimulus CpG KRV Poly I:C IL-12p40(%ofControl) 0 20 40 60 80 100 0.1 µg/ml 1 µg/ml 10 µg/ml The ability of spleen cells to respond to KRV by producing IL12-p40 is inhibited by iCpG, an inhibitor of TLR9 (Dr. D Zipris)
  27. 27. BBDR Summary 2 • Innate immune activation can trigger T1D in many but not all rat strains with a high risk MHC haplotype • Innate immune activation can synergize with viral infection to increase the penetrance of T1D • One diabetogenic virus, KRV, may act in part via activation of TLR9
  28. 28. New Model of Type1 Diabetes The MAD Rat (LEW.1WR1) • Recombinant inbred congenic strain • Same MHC class II RT1B/Du haplotype required for autoimmune diabetes in BB, Komeda, and LEW.1AR1/Ztm-iddm rats • In the colony of rats maintained at BRM, Inc.: • Spontaneous diabetes absent from acquisition in 1989 until 1999 • Now occurs at low frequency (0.5-3%) • Develop autoimmune insulitis and diabetes when treated with poly I:C, a ligand of toll-like receptor 3 (TLR3)
  29. 29. Extending the Scope of Viral Triggers: The MAD Rat • The BBDR + KRV combination is not unique • LEW.1WR1 Rats – MHC-Congenic LEW rats – High risk MHC class II RT1B/Du haplotype – Normal immunological phenotype – Diabetes occurs at consistently but at low frequency (~2.5%)
  30. 30. Hypothesis • Given its genetic predisposition to spontaneous T1D: • Viral infection will trigger T1D in MAD rats
  31. 31. Rat Cytomegalovirus (RCMV) • Beta-herpesvirus homologous to human cytomegalovirus • Persistent and latent infections • No reported associated diseases • Stocks are prepared from salivary glands
  32. 32. Relevance to Human Disease • Human Cytomegalovirus (HCMV) • Associated with autoimmune diseases • Ubiquitous pathogen causing unapparent infections in immunocompetent individuals • Peptide from HCMV protein stimulate CD4+ T cells that recognize GAD • Organ transplantation • Currently no effective vaccine
  33. 33. MAD Rats are Sensitive to Virus-Induced Diabetes Treatment N % Diabetic Grade None 6 0 n.a. Kilham Rat Virus 8 38 3.3 H-1 Virus 20 0 n.a. Rat Cytomegalovirus 16 44 4.0 Vaccinia virus 10 0 n.a. Coxsackie B4 10 0 n.a. Tirabassi, et. al. 2004. Diabetes. 53 Supplement 2:A301
  34. 34. Diabetes in BBDR and MAD Rats Summary of Induction Studies Method BBDR/Wor MAD Spontaneous - + Anti-Art2a - + TLR3 ligands - + Parvovirus 21days + + RCMV -/+ + + TLR 3 + Art2a + + Parvovirus 45days - +
  35. 35. Diabetogenicity of five TLR agonists in MAD rats Group Test Article TLR Induction of Diabetes Induction Of Arthritis Gender Bias 1 Poly I:C (primarily HMW) TLR3 ++++ No ♀ = ♂ 2 LMW poly I:C (purified) TLR3 ++++ 3 HMW poly I:C (purified) TLR3 ++++ 4 Zymosan TLR2 +++ 5 R848 TLR7 ++ 6 CpG oligodeoxynucleotide TLR9 - The dosing regimen was 3 times weekly by intraperitoneal injection beginning at 21-24 days of age. Compounds were administered to 6 female and 6 male rats at each dose of compound. Animals were treated over a 30 day period and were monitored for diabetes from 7 to 40 days after the initiation of treatments
  36. 36. Serial Viral Infection Alters Diabetes Incidence Virus % Diabetic BBDR MAD Parvovirus 41% 38% RCMV 6.3% 44% Parvovirus→ RCMV 0% 40% RCMV→KRV 75% 90%
  37. 37. RCMV accelerates diabetes onset in Diabetes Prone BB rats 0 5 0 20 40 60 80 100 30 40 50 60 70 80 90 100 110 120 RCMV/mock infection + RCMV - RCMV RCMV INFECTED (n = 47) CONTROLS (n = 29) Age (days) %DiabeticBB-DPrats p = .0043 (Kaplan-Meier Log Rank) van der Werf et al 2003: Clin Dev Immunol. 10:153 Hillebrands et al 2003: Clin Dev Immunol 10:133
  38. 38. Virus N Diabetes Virus N Diabetes None 6 0 (0%) H-1 20 0 (0%) Parvo 32 11 (34%) Coxsackie B4 18 0 (0%) RCMV 38 14 (37%) Vaccinia 10 0 (0%) Triggering of T1D in MAD Rats is Virus-Specific Latency to onset 16-30 Days Dose of virus varied from 104 to 107 PFU
  39. 39. RCMV Infected Cells in Salivary Glands But Not in Pancreatic Islets Immunohistochemistry for RCMV early antigen (mAb 8) Salivary Gland (mAb8) Islet (H&E) Islet mAb8
  40. 40. Conclusions Virus Triggering of T1D in Resistant Rats • T1D can be triggered in the rat by: – Specific viral infections – Two infections that are synergistic – TLR ligation – Certain viruses only in an immune system “pre- activated” by TLR ligation
  41. 41. Topic 2 Prevention by Immunization • If viral infections do promote diabetes onset in susceptible rats with normal immune systems… • Then immunization may prevent the disease
  42. 42. “Immunized Pups” X Immunized Female Naïve Male Virus Non-Diabetic Virus Diabetes? A “Maternal Immunization” Protocol MAD rat Females
  43. 43. Maternal Immunization Protects Weanling MAD Rats from Virus-Induced Diabetes 0 CumulativeDiabetes-Free Survival(%) 0 20 40 60 80 100 Days After Infection 10 20 30 Pups born to RCMV+KRV Immunized Dam (N=10) KRV+RCMV co-infection Ordinary pups (N=10) Pups born to RCMV Immunized Dam (N=26) Ordinary pups (N=13) RCMV
  44. 44. Conclusion • Maternal immunization can provide effective and specific protection from virus-induced diabetes in weanling rats • Diabetes may be preventable by vaccines that target candidate pathogens • Rats like the MAD can be used to test diabetes vaccination strategies
  45. 45. T-1D RAID Programs • Rapid Access to Interventional Development for Type 1 diabetes • BRM responded to two USPHS RFP’s in June of 2005. • Our requested budgets totaled > $11.3 MM • BRM was notified in December 2005 that it was technically competent and within the Competitive Range for Both RFP’s – Only company competing for both contracts
  46. 46. T-1D RAID Programs • Rapid Access to Interventional Development for Type 1 diabetes • Preclinical Studies of Efficacy in Animal Models of Type 1 Diabetes (Pathogenesis) N01-DK-6-2909 – BRM Awarded $4.9Million Contract 2006-2012 – Selected over a Top 25 Research Institute
  47. 47. T1D RAID Process Potential therapeutics submitted by US scientists 5 selected yearly by T1D RAID board Successful compounds screened and identified by BRM Entry into TrialNet New successful drug identified
  48. 48. Trials to Prevent or Treat Type 1 Diabetes: Clinical Considerations  Tight glycemic control is correlated with C-peptide preservation in type 1 diabetes clinical trials.  Interventions are more likely to succeed if done early, while some beta cell function is preserved.
  49. 49. NOD Model Problem 1: Treatment of diabetes in new onset NOD mice  Diabetic NODs are treated with poorly- characterized insulin pellets or by insulin injection resulting in variable or inadequate glycemic control.  Poor control may contribute to rapid beta cell demise or loss of functionality, and therefore a poorer response to immune interventions designed to reverse disease may be observed.
  50. 50. NOD Model Problem 1: Solutions  Contract is developing better ways to control glycemia in NOD diabetic mice  Contract is developing ways to predict imminent diabetes in order to intervene prior to beta cell loss.
  51. 51. Interventions to Maximize Success • Tight glycemic control of diabetic animals for periods of 3-6 weeks – Provides immune modulatory therapies maximum opportunity for success • Contract is developing ways to predict imminent diabetes to intervene prior to complete beta cell loss
  52. 52. Model Standardization • Use optimized rodent models for standardized preclinical testing of agents to prevent or reverse diabetes – Testing in multiple models (mice and rats) – Development of standardized testing protocols – Development of insulin treatment protocols for reversal studies
  53. 53. Example Testing Schemes Treat 21-110 days Treat 21-160 days Follow 50 days Preventative Protocol Read-outs Preventative/Reversal Protocol Treat when turns diabetic Treat 2 nondiabetic littermates Read-outs Pulse Protocol Read-outs
  54. 54. Year BRM Jackson 2006 68% 65% 2007 (at 27 weeks) 86% 95% 2008 (at 23 weeks) 75% 75% Incidence of Diabetes Among NOD Mice
  55. 55. Therapeutic Evaluations in the NOD Mouse Study Designs Reversal Initiate treatment after frank diabetes onset. Insulin therapy to maintain euglycemia. Late Prevention Initiate treatment to animals with impaired GTT prior to frank diabetes. Insulin therapy not used. Early Prevention Initiate treatment prior to disease onset. Insulin therapy not used.
  56. 56. Control and Prediction • Contract is developing better ways to control glycemia in NOD diabetic mice (and BB rat) • Contract is developing ways to predict imminent diabetes to intervene prior to complete beta cell loss
  57. 57. Using the GTT test to predict diabetes onset (Protocol 2) • Impaired glucose tolerance (IGT) expected to be early manifestation of impending diabetes – Perform GTT (ip injected glucose) on fasted nondiabetic female mice at 12, 14, or 16 weeks of age, then follow for 30 days for diabetes onset – Analyze AUC of animals that became diabetic or remained non-diabetic – Determine the best parameters for prediction
  58. 58. *** P<0.001 * P<0.05 *** * NOD Mice with Impaired Glucose Tolerance Develop Diabetes
  59. 59. NOD Mice with Impaired Glucose Tolerance Develop Diabetes GTT performed on animals at 14 weeks of age. IGTT= Impaired Glucose Tolerance, NGTT= Normal Glucose Tolerance. 9/19 animals with IGTT turned diabetic within 30 days after test. 4/31 animals with NGTT turned diabetic within 30 days after test.
  60. 60. GTT Conclusions • GTT has high predictive value for diabetes when performed at 14 weeks of age Sensitivity Specificity Pos. Pred. Value 14 Weeks 0.69 0.73 0.47
  61. 61. Study Design Late Intervention Perform GTT at 14 weeks Select mice with impaired IGTT Randomize into groups: Rx, vehicle or no rRX
  62. 62. Development of Protocols to Optimize Treatment of Spontaneous Type 1 Diabetes in NOD Mice
  63. 63. Metabolic Control in NOD Mice • Factors to consider – Total daily dose (2- 4 Units) 25 g mouse – Type of Insulin – Frequency of dosing QD, BID, TID – % of daily dose administered each injection – Injection time relative to “fed state” • Many pilots done to test insulin formulation (Humulin 50/50, Humulin 70/30, PZI), dose, use of diluted insulin and BID dosing
  64. 64. Continuous Insulin Release to Control Blood Glucose in Diabetic NOD Mice • Continuous release of insulin using Alzet osmotic pumps – Implant subcutaneously – Use Humulin R at 0.2, 0.3, 0.4U/day – Monitor BG daily, with an intensive (every 3 hours) 24 h monitoring on Days 3, 7 and 14 • Continuous release insulin controls blood glucose rapidly with few glycemic excursions
  65. 65. Daily Blood Glucose Averages 0 100 200 300 400 500 600 0 5 10 15 20 BloodGlucose(mg/dL) Day Post Pump Insertion 0.2U 0.3U 0.4U Mice implanted subcutaneously with pumps releasing 0.2-0.4U Humulin R/day. Daily blood glucose measured approx. 8-9 hours after lights on. N=6 for 0.2 and 0.3U groups; N=12-15 for 0.4U group.
  66. 66. Intensive BG Monitoring – 0.4U Mice implanted subcutaneously with pumps releasing 0.4U Humulin R/day. BG measured every 3 hours for 24 hours on Days 3, 7 and 14 post pump insertion. N=15 for Days 3 and 7; N=12 for Day 14. 0 100 200 300 400 500 600 0 2 4 6 8 10 12 14 16 18 20 22 24 BloodGlucose(mg/dL) Hour in Study 3 Day 7 Day 14 Day
  67. 67. Intensive BG Monitoring – 0.3U 0 100 200 300 400 500 600 0 2 4 6 8 10 12 14 16 18 20 22 24 BloodGlucose(mg/dL) Hour in Study 3 Day 7 Day 14 Day Mice implanted subcutaneously with pumps releasing 0.3U Humulin R/day. BG measured every 3 hours for 24 hours on Days 3, 7 and 14 post pump insertion. N=15 for Days 3 and 7; N=12 for Day 14
  68. 68. Daily Blood Glucose Mean +/- SEM 0 100 200 300 400 500 600 -1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 BloodGlucose(mg/dL) Days Post Pump Insertion .2U .25U .3U Diluent Mice implanted subcutaneously with 1002 Alset pumps releasing 0.2-0.3U Humulin R/day. Daily blood glucose measured approx. 5-7 hours after lights on. N=12 for 0.2U and 0.25U groups; N=14 for 0.3U group; N=7 for Diluent group.
  69. 69. Day 3 Intensive BG (Mean +/- SEM) 0.0 100.0 200.0 300.0 400.0 500.0 600.0 3 3.5 4 BloodGlucose(mg/dl) Days Post Pump Insertion .2U .25U .3U Diluent Mice implanted subcutaneously with 1002 Alset pumps releasing 0.2-0.3U Humulin R/day. Daily blood glucose measured 3 days post insertion over 24 hours. N=12 for 0.2U and 0.25U groups; N=14 for 0.3U group; N=7 for Diluent group.
  70. 70. Day 7 Intensive BG (Mean +/- SEM) 0.0 100.0 200.0 300.0 400.0 500.0 600.0 7 7.5 8 BloodGlucose(mg/dl) Days Post Pump Insertion .2U .25U .3U Diluent Mice implanted subcutaneously with 1002 Alset pumps releasing 0.2-0.3U Humulin R/day. Daily blood glucose measured 7 days post insertion over 24 hours. N=12 for 0.2U and 0.25U groups; N=14 for 0.3U group; N=7 for Diluent group.
  71. 71. Day 14 Intensive BG (Mean +/- SEM) 0.0 100.0 200.0 300.0 400.0 500.0 600.0 14 14.5 15 BloodGlucose(mg/dl) Days Post Pump Insertion .2U .25U .3U Diluent Mice implanted subcutaneously with 1002 Alset pumps releasing 0.2-0.3U Humulin R/day. Daily blood glucose measured 14 days post insertion over 24 hours. N=12 for 0.2U and 0.25U groups; N=14 for 0.3U group; N=7 for Diluent group.
  72. 72. Digitized images of islets following 3 weeks of insulin Rx by Alzet pumps
  73. 73. Impact of insulin pumps on β-cell mass Diluent 0.2U 0.3U 0.4U 0.00 0.03 0.06 0.09 Treatment Group FractionalInsulinArea(%)

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