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Autism Research in Arkansas: On-going clinical trials and the Arkansas Autism Alliance

Autism Research in Arkansas: On-going clinical trials and the Arkansas Autism Alliance. 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

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Autism Research in Arkansas: On-going clinical trials and the Arkansas Autism Alliance

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  1. Autism Research in Arkansas: On-going clinical trials and the Arkansas Autism Alliance 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 Review of metabolic pathways: folate/methionine/glutathione Efficacy of methylB12 and folinic acid treatment on glutathione redox status and core behaviors in autism Parent Metabolic Profiles Specific Aims of our 5 year NIH-funded study Placebo-controlled double-blind cross-over study of broad spectrum nutritional supplementation AAA and ATN in Arkansas

  3. Methionine Transsulfuration to Cysteine and Glutathione Methionine THF 1 5,10-CH2THF MS B12 MTHFR 5-CH3THF Homocysteine B6 THF: tetrahydrofolate Enzymes

  4. Methionine Transsulfuration to Cysteine and Glutathione Methionine Methylation Potential (SAM/SAH) THF SAM MTase 2 1 5,10-CH2THF Cell Methylation MS SAH B12 MTHFR 5-CH3THF SAHH Adenosine Homocysteine B6 THF: tetrahydrofolate Enzymes

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

  6. Methionine Transsulfuration to Cysteine and Glutathione Methionine Methylation Potential (SAM/SAH) THF SAM MTase 2 1 5,10-CH2THF Cell Methylation MS SAH B12 MTHFR 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

  7. 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

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

  9. 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

  10. STUDY DESIGN Each child served as their own control in the open label trial in which both parents and investigators were aware that the child was receiving supplements ofmethyl-B12 and folinic acid for a period of three months. Plasma metabolites in the transmethylation and transsulfuration pathways were measured at baseline and again after the 3 month intervention period. The study nurse administered and scored the Vineland Adaptive Behavior Scales parent questionnaire before and after the 3 month intervention.

  11. 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

  12. METABOLIC DATA

  13. x

  14. SUMMARY OF METABOLIC RESULTS • All baseline metabolites were significantly different • from age-matched controls (except for SAH) • 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

  15. 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.

  16. BEHAVIOR SCORES

  17. 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

  18. What about the parents?

  19. 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 It would be a very good idea to ask your physician to check your “total” homocysteine

  20. 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

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

  22. 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

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

  24. 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 severity and specificity of the metabolite imbalance is associated with the severity and specificity of behavioral abnormalities.

  25. SPECIFIC AIM 2:PROSPECTIVE STUDY Specific Aim 2: To investigate whether the abnormal metabolic profile precedes 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.

  26. SPECIFIC AIM 2:PROSPECTIVE STUDY M-CHAT autism screening test 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.

  27. AUTISM PROSPECTIVE STUDY DESIGN Visit 1: M-CHAT (18-30 months) FAIL = High Risk PASS = Developmental Delay and Normal CONTROLS Metabolic Profile Metabolic Profile

  28. 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

  29. 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

  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 FAIL = High risk Regression PASS Not Autism Visit 3:DSM-IV; CARS; ADOS Autism Diagnosis Control

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

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

  33. 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

  34. EXPERIMENTAL PROCEDURES Lymphoblastoid cell lines from autistic children with at least one affected sibling were compared with unaffected control lymphoblastoid cell lines* 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. *Preliminary data supported by SafeMinds

  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. 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 sensitive to pro-oxidant environmental exposures.

  39. 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

  40. 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

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

  42. 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.

  43. 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 blindplacebo-controlled cross-over trial of targeted nutritional intervention designed to correct metabolic deficiencies and to improve scores on standardized behavioral evaluation tests.

  44. 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

  45. 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

  46. OUTCOME MEASURES • Behavioral testing: ADOS; Vineland; PLS-2; SRS • Behavioral testing will be videotaped and administered by PhD psychologists • Metabolic evaluation: • Plasma: Thiol profile; CBC; amino acid profile, P5P, HoloTCII; • sulfate; nitrotyrosine; lactate/pyruvate; 25-hydroxy vitamin D; uric acid; • Urine: Sulfate, organic acids; creatinine; FIGlu, MMA • Cellular: RBC membrane phospholipids; leukocyte GSH/GSSG. • Immunologic evaluation: • Flow cytometry for CRP, cytokine mRNA expression and protein levels for TNFα; g-IFN, IL-1; IL-4, IL-6; IL-10; IL-13; T-regs

  47. AUTISM TREATMENTNETWORK (ATN) IN ARKANSAS

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