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Autism Research and On-Going Clinical Trials in Arkansas

Autism Research and On-Going Clinical Trials in Arkansas. S. Jill James, PhD Professor, Department of Pediatrics Director, Autism Metabolic Genomics Laboratory Arkansas Children’s Hospital Research Institute University of Arkansas for Medical Sciences Little Rock, AR. OVERVIEW.

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Autism Research and On-Going Clinical Trials in Arkansas

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  1. Autism Research and On-Going Clinical Trials in Arkansas S. Jill James, PhD Professor,Department of Pediatrics Director, Autism Metabolic Genomics Laboratory Arkansas Children’s Hospital Research Institute University of Arkansas for Medical Sciences Little Rock, AR

  2. OVERVIEW A little basic biochemistry: folate/methionine/glutathione Abnormal metabolic profile in children with autism Efficacy of methylB12 and folinic acid treatment Parent Metabolic profiles Specific Aims of our 5 year NIH-funded study Placebo-controlled double-blind cross-over study of broad spectrum nutritional supplementation Autism Treatment Network, Department of Defense Grant Autism: Beyond the Brain

  3. Methionine Transsulfuration to Cysteine and Glutathione Methionine Methylation Potential (SAM/SAH) THF SAM MTase 2 1 5,10-CH2THF Cell Methylation MS SAH B12 5-CH3THF SAHH Adenosine Homocysteine 1 Folate Cycle Methionine Cycle Transsulfuration Pathway CBS B6 B6 Cystathionine 2 Antioxidant Redox Potential (GSH/GSSG) B6 3 Cysteine 3 GSH GSSG

  4. Vital Importance of these Interdependent Metabolic Pathways Methionine METHYLATION THF SAM MTase Cellular Methylation Reactions 2 1 5,10-CH2THF MS SAH B12 5-CH3THF SAHH Purines and Thymidylate Adenosine Homocysteine B6 Cystathionine DNA SYNTHESIS REDOX HOMEOSTASIS 3 Cysteine PROLIFERATION GSH GSSG

  5. Evidence for increased oxidative stress and impaired methylation capacity in children with autism Results of intervention trial with methylB12 and folinic acid treatment American Journal of Clinical Nutrition 2004 American Journal of Medical Genetics 2006 American Journal of Clinical Nutrition 2008

  6. Fasting Plasma Transmethylation Metabolites Autistic Control p value n=80 n=75 Methionine (µM/L) 20.6 ± 5.2 28.0 ± 6.5 <0.0001 SAM (nM/L) 84.3 ± 11 93.8 ± 18 <0.0001 SAH (nM/L) 23.3 ± 7.9 18.8 ± 4.5 <0.0001 SAM/SAH Ratio 4.0 ± 1.75.5 ± 2.8 <0.0001 Homocysteine 5.7 ± 1.26.0 ± 1.3 0.03 Means ±SD

  7. INTERPRETATION • The significant decrease in methionine and SAM • and increase in SAH levels in autistic children provides metabolic evidence that methylation capacity may be reduced in autistic children.

  8. Fasting Plasma Transsulfuration Metabolites Autistic Control p value n=80 n=75 Cysteine (µM/L) 165 ± 14 207 ± 22 <0.0001 Total GSH (µM/L) 5.1± 1.2 7.5 ± 1.7 <0.0001 Free GSH (µM/L) 1.4 ± 0.5 2.2 ± 0.9 <0.0001 GSSG (µM/L) 0.4 ± 0.2 0.24 ± 0.1 <0.0001 Free GSH/GSSG 4.9 ± 2.2 7.9 ± 2.5 <0.0001 Means ±SD

  9. INTERPRETATION • The significant decreases in homocysteine, cysteine and glutathione suggest that the transsulfuration pathway is insufficient for adequate glutathione synthesis. • 2. The increase in GSSG (oxidized inactive glutathione) and decrease in GSH (active antioxidant glutathione) is strong evidence that oxidative stress is increased in autistic children.

  10. AN OPEN LABEL TRIAL OF METHYLCOBALAMIN AND FOLINIC ACID IN AUTISTIC CHILDREN

  11. Can supplementation with methyl-B12 and folinic Acid improve glutathione levels and core behaviors in autistic children? Intervention: MethylB12 (75µg/Kg every 3 days) (3 months) Folinic Acid (400 µg bid) Inclusion Criteria: Autistic Disorder (DSM-IV; CARS) Age 3-7 No previous supplements GSH < 6.0 Endpoints: Methylation and glutathione metabolites Vineland Adaptive Behavioral Scales

  12. Methyl B12 Methionine Folinic Acid THF SAM MTase Cellular Methylation Reactions 2 1 5,10-CH2THF MS SAH B12 5-CH3THF SAHH Purines and Thymidylate Adenosine Homocysteine Folinic Acid B6 Cystathionine DNA SYNTHESIS 3 Cysteine GSH GSSG

  13. Intervention Trial with MethylB12 and Folinic Acid

  14. Cysteine Micromol/L

  15. GSSG Micromol/L

  16. GSH/GSSG REDOX RATIO Micromol/L

  17. SUMMARY OF METABOLIC RESULTS • Baseline metabolites were significantly different • from age-matched controls • 2. The treatment did not significantly improve • levels of methionine, SAM or SAM/SAH • The treatmentdid significantly improve cysteine, • glutathione, and GSH/GSSG • Although significantly improved, glutathione and • GSH/GSSG did not reach levels in control children

  18. Behavioral Evaluation The Vineland Adaptive Behavior Scales (VABS) provides a numerical score for adaptive functioning in the areas of communication, socialization, daily living skills, motor skills, and an adaptive behavior composite (ABC) score. The data are presented as the mean score for each category before and after intervention.

  19. BEHAVIOR SCORES

  20. SUMMARY OF BEHAVIOR RESULTS Although treatment with methylB12 and folinic acid significantly improved core behaviors, they did not reach standard scores for unaffected children (100 ± 15) CONCLUSIONS Improvement in measures of both metabolic and behavioral endpoints converge to suggest that some children may benefit from targeted nutritional intervention. This open label trial provides strong preliminary evidence for a double-blind placebo-controlled clinical trial.

  21. What about the parents? Journal Of Autism and Developmental Disabilities 2008

  22. Maternal Methionine Cycle Metabolites: Autism Moms Control Moms (n = 46) (n= 200) Methionine (µM/L) 24 ± 5 26 ± 6 SAM (nM/L) 80 ± 19 83 ± 13 SAH (nM/L) 33 ± 14* 23 ± 8.4 SAM/SAH Ratio 3.1 ± 1.7* 4.0 ± 1.4 Homocysteine(µM/L) 11 ± 3.9* 7.6 ± 1.6 *statistically significant

  23. Maternal Transsulfuration Metabolites Autism Moms Control Moms Cysteine (µM/L) 232 ± 40 231 ± 20 Total GSH (µM/L) 5.1 ± 1.7* 7.3 ± 1.5 Free GSH (µM/L) 1.5 ± 0.5* 2.6 ± 0.6 GSSG (µM/L) 0.30 ± 0.08* 0.24 ± 0.04 Total GSH/GSSG 17 ± 8 31 ± 10* *statistically significant

  24. Metabolite imbalance and the risk of being a mother of a child with autism

  25. IMPORTANT CAVEAT It is not possible to determine from this data whether the abnormal metabolic profile in parents is genetically determined or whether it simply reflects the stress of living with an autistic child

  26. METABOLIC BIOMARKERS OF AUTISM: PREDICTIVE POTENTIAL AND GENETIC SUSCEPTIBILITY A 5 YEAR NIH-FUNDED STUDY (2006-2011)

  27. SPECIFIC AIM 1:METABOLITES AND BEHAVIOR Specific Aim 1: To determine whether the observed metabolite imbalance is associated with quantitative measures of autistic behavior An expanded database of metabolic profiles will allow us to determine whether the metabolite imbalance is associated with abnormal behaviors.

  28. SPECIFIC AIM 2:PROSPECTIVE STUDY Specific Aim 2:To investigate whether the abnormal metabolic profile is present before the diagnosis of autism among toddlers 18-30 months of age who are identified in developmental delay clinics to be at increased risk of developing autism.

  29. SPECIFIC AIM 2:PROSPECTIVE STUDY An autism screening test (MCHAT) and plasma metabolic biomarkers will be measured at Visit 1 and children will be followed for subsequent diagnosis of autism (case) or developmental delay (control). Metabolic data will be analyzed statistically to determine whether metabolic abnormalities precede the behavioral diagnosis of autism and could serve as predictive biomarkers for risk of autism.

  30. AUTISM PROSPECTIVE STUDY DESIGN Visit 1: M-CHAT (18-30 months) FAIL = High Risk PASS = Developmental Delay and Normal CONTROLS Metabolic Profile Metabolic Profile (1-6 months) (6 months) Visit 2:M-CHAT Repeat M-CHAT Repeat FAIL PASS Not Autism Visit 3:DSM-IV; CARS; ADOS Autism Diagnosis Control

  31. IMPLICATIONS OF AIM 2 AUTISMPROSPECTIVE STUDY If the metabolic profile is found to precede the behavioral diagnosis of autism, subsequent studies would determine whether early intervention to normalize the metabolic profile can reduce or prevent the development of autism.

  32. SPECIFIC AIM 3: CELLULAR CONSEQUENCES Specific Aim 3: To establish whether cells from children with autism exhibit evidence of increased oxidative stress and oxidative damage. This mechanistic aim will determine whether lymphocytes from autistic children are inherently more vulnerable to oxidative stress than control cells

  33. EXPERIMENTAL PROCEDURES Lymphoblastoid cell lines from autistic individuals with at least one affected sibling were compared with lymphoblastoid cell lines from unaffected controls* Pairs of autistic and control cells lines were cultured under identical conditions. Rate of free radical generation, GSH/GSSG were measured at baseline and after exposure to thimerosal as oxidative stress.

  34. Baseline intracellular glutathione status in autistic and control lymphoblastoid cell lines * * * * p < 0.05

  35. Cells from autistic children generate more free radicals than control cells

  36. Cells from autistic children have lower GSH/GSSG ratio than control cells

  37. MITOCHONDRIAL REDOX IMBALANCE IN LYMPHOBLASTOID CELL LINES Autistic GSH/GSSG RATIO Control (X 10)

  38. PERCENT DECREASE IN MITOCHONDRIAL MEMBRANE POTENTIAL WITH NITRIC OXIDE EXPOSURE

  39. CONCLUSION Since both cell lines were cultured at the same time under identical conditions with identical media, the differences at baseline and after exposure to oxidant stress must reflect inherent genetic or epigenetic differences. These results provide experimental evidence that cells from autistic children may be more vulnerable to pro-oxidant environmental exposures.

  40. SPECIFIC AIM 4: METABOLIC GENETICS Specific Aim 4: Using a case-control design, we will determine whether the frequency of relevant genetic polymorphisms is increased among autistic children and whether specific genotypes are associated with the abnormal metabolic phenotype. We have access to 500 trios (child, mother, father) from NIH genetic repository to look at relevant SNP frequencies and transmission

  41. A Targeted Approach to Autism Genetics: Using the Metabolic Endophenotype as a Guide to Candidate Genes Methionine THF SAM Methyl Acceptor DMG Methyltransferase B12 COMT 5,10-CH2-THF TC II Methylated Product MTHFR 5-CH3-THF SAH RFC Adenosine Homocysteine Cystathionine CBS Cysteine GCL GST Glutathione

  42. Treating Oxidative Stress and the Metabolic Pathology of Autism A RANDOMIZED DOUBLE-BLIND PLACEBO- CONTROLLED CROSS-OVER STUDY

  43. HYPOTHESIS A significant proportion of autistic children have impaired methylation and antioxidant/detoxification capacity that results in chronic oxidative stress. Targeted nutritional intervention that is designed to correct the metabolic imbalance will significantly improve their metabolic profile and improve measures of autistic behavior.

  44. SPECIFIC AIMS Specific Aim 1. We will screen children with a diagnosis of autism for evidence of impaired methylation (↓SAM/SAH) and impaired antioxidant capacity (↓GSH/GSSG) Specific Aim 2. Children who exhibit evidence of impaired methylation and antioxidant capacity will be randomized into a double blind placebo-controlled cross-over trial of targeted nutritional intervention designed to correct metabolic deficiencies and to improve scores on standardized behavioral evaluation tests.

  45. RANDOMIZED DOUBLE-BLIND PLACEBO- CONTROLLED CROSS-OVER DESIGN A is supplement first, placebo second B is placebo first, supplement second WASHOUT A B B A Thiols, Complete Lab, Thiols, Complete Lab, Thiols, Complete Lab, Behavioral Testing Behavioral Testing Behavioral Testing Children are randomly assigned to either the placebo first or the treatment first for 3 months before 1 month wash out period and cross-over

  46. The supplements have been selected to impact three core cellular functions that are altered with chronic oxidative stress (www.clinicaltrials.gov) • 1) Decreased SAM/SAH ratio and cellular • methylation capacity • 2) Antioxidant and detoxification support • (mitochondrial and cytosolic) • 3) Cell membrane integrity

  47. OUTCOME MEASURES • Behavioral testing: ADOS; Vineland; PLS-2; SRS • Behavioral testing will be videotaped and administered by PhD psychologists • 2. Metabolic evaluation: • Plasma: Thiol metabolic profile; CBC; amino acid profile, P5P, B12; sulfate; nitrotyrosine; vitamin D; • uric acid • Urine: Sulfate, organic acids; creatinine; FIGlu, MMA • Cellular: RBC membrane phospholipids; GSH/GSSG • 3. Immunologic evaluation: • Flow cytometry for intracellular cytokine expression

  48. AUTISM TREATMENTNETWORK (ATN) IN ARKANSAS

  49. The ATN The ATN is a consortium of 15 national sites composed of experts in developmental pediatrics, neurology, genetics, metabolism, sleep, and gastroenterology who are dedicated to improving the standard of care of children with autism. The ATN believes that treatment of medical issues can improve core behaviors and improve quality of life for children and adults with autism and their parents.

  50. DEPARTMENT OF DEFENSE AUTISM IDEA DEVELOPMENT AWARD 2008-2011

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