Immunizations: Regressive subset, Friendly, thirsty, flat footed,


3. Autism has Diverse Presentation
- Variable symptoms, IQ
- Multiple brain regions identified
- Seizures in 30%
- Blood serotonin elevated in 35%
- GI symptoms in 40-50%
Diverse Biology = Multiple Approaches
Notes: Talked that there were several theories on the cause of Autism, the treatment of autism, but the "autistic" population is so diverse, that multiple approaches may be used and are helpful.


4. Reasonable Expectations [of any treatment/drug]
- Meaningful improvement in at least one of the major symptoms of autism
- Works in at least 10% of patients: 10% ASD = more patients than CF or leukemia
- Toxicity has been evaluated in ASD patients and pediatric animals
No drug meets "reasonable expectations" yet

5. Phase 2 Clinical Trial of Secretin
[text slide]

6. Secretin
- Natural hormone: stimulates the pancreas
- Porcine secretin approved as a diagnostic
- Activity in autism detected in 1996 following a GI exam of an autistic child
--diarrhea resolved
--imitation of simple words
--improved task focus

7. Secretin – anecdotal Observations
Secretin used "off label" in > 2,000 autistic children. Some parents & doctors reported:
- eye contact, social interaction
- some language gains
- physiological changes: diet, GI, sleep
- effect lasts 3-5 weeks
- few side-effects: hyperactivity?

Notes: off-label means it is being used for some other purpose than what is stated on the label. Hyperactivity was seen the most, but whether this was interpreted to be positive or negative was debatable.

8. FDA Clinical Trial Process
- Phase 1 – Establish safety: 10-30 patients, single dose
- Phase 2 – Establish dosing and endpoints: 50-200 patients, multiple doses, dose levels
- Phase 3 – Statistical proof of efficacy: 100-2,000 patients, full dosing regimen
Only one Phase 2 completed in autism
Notes: These are the phases of the FDA clinical trial procedure. Only one Phase 2 has been done in autism.

9. Phase 2 Clinical Trial Questions - 1
Is there a subset of patients who can be defined by symptoms or biochemical markers?
[text slide only]

10. Secretin Phase 2 Clinical Trial Inclusion Criteria
- age of 3.0-6.9 years
- gastrointestinal symptoms
- moderate to severe autims
- no functional language
Notes: He did not specify the same GI symptoms for all the children in the trial, just that they had some known GI problem

11. Phase 2 Clinical Trial Questions - 2
What is the best way to measure changes in social interactions, speech and behavior?
[text slide]

12. Showed a slide with a before and after picture of a boy. The first shot was the boy looking "blankly" out into space, the second was the same boy later actively looking and smiling at the camera. How do we measure "connectedness"? He talked about this aspect being hard to quantify and measure.

13. Measurement of Symptom Changes
- global assessment vs. specific symptoms
- parent vs. clinician as an information source
- social vs. behavior vs. communication
- how to avoid a large placebo effect?
Notes: He talked about how to set up criteria for evaluating and rating changes in symptoms. Was the parent more accurate than the clinician? Should we go for social or behavioral or communication more? How are these things to be measured and compared?

14. Comparison of Assessment Tools
- Childhood Autism Rating Scale (CARS)
- Gilliam Autism Rating Scale (GARS)
- Autism Diagnostic Observation Schedule (ADOS)
- Clinical Global Impression of Change: Parent (CGI-P) versus Rater (CGI-R)
- MacArthur Communication Development Inventory
Notes: These are some of the assessment tools already used. They were trying to determine which if not all would be best in determining results.

15. Secretin Phase 2 Clinical Trial
- Double-blind, placebo-controlled
- 126 patients evaluated at 5 US centers
- 3 doses of secretin or placebo at 3 week intervals
- 66 patients received secretin, 60 patients received placebo

16. Autism Diagnostic Tools
- Childhood Autism Rating Scale (CARS): psychologist rates 14 symptoms on severity compared to a NT child; there is low test retest stability = random results.
- Gilliam Autism Rating Scale (GARS): Parent checklist rates severity of 56 symptoms; two administrations prior to dosing = highly variable results particularly in social domain

17. Statistics
- "Statistically significant" means there is a 95% probability that the difference between drug (or treatment) and placebo is due to activity of the drug and not random variation in symptoms
p = .10 means 90% confidence the difference is due to the treatment, called a trend
p = .05 means 95% confidence the difference is due to the treatment, called significant
p = .01 means 99% confidence the difference is due to the treatment

- Statistical confidence is proportional to size of treatment effect AND number of patients (that is, the larger the sample size the more accurate this "confidence" will be)

18. Evaluation of Symptoms – 1
Clinical Global Impression of Change
- Independent evaluations by Rater & Parent (they used this rating scale)
1 = very much improved
2 = much improved
3 = minimal improvement
4 = no change
5 = minimal worsening
6 = much worse
7 = very much worse

- FDA required assessment for depression, Alzheimer's, schizophrenia, OCD (this assessment scale is common and was used for the conditions listed)

19-23. Clinical Global Impression All 126 Patients
Notes: He showed a series of slides with bar charts showing that the parents rating was much more accurate that the expert raters (professionals representing doctors in this area). He said often it is said that parents are hopeful for their children and this skews their observations and reports, but that parents would be hopeful whether their child received the secretin or the placebo. They found that the observed changes in the children by the parents were far more accurate than the observed changes in the children than the raters. Most probably because the parents know their children's behavior and are around them all day whereas the professional would only be seeing the child for minutes at a time. From this they
learned that the parents were a very reliable source of information and would provide the most accurate ratings to be used in official clinical trials. He made a HUGE point about this.
P = 0.22 for the raters
P = 0.02 for the parents

24. Evaluation of Symptoms – 2
Autism Diagnostic Observation Schedule
- this is the "gold standard" diagnosis of autism
- evaluates social and communicative skills in a series of structured activities
- there are four modules which are adjusted for child's skill level

25. ADOS-Social Changes
[showed a chart with little dots on it I didn't understand. Sorry.  The point was to show that some of the patients responded positively to secretin with a confidence rate of p = 0.12 for these results]

26. Language Assessment
slide showing a book saying User's Guide and Technical Manual on the cover. This is used to evaluate language.

27. Evaluation of Symptoms – 3
The MacArthur Communicative Development Inventory measures
- words & gestures module: designed to assess emergent language, typical of 8-16 months
- vocabulary inventory of 396 words
- determines words understood and words used

28. MacArthur Language Inventory
Notes: showed bar graph. Using this measure, the number of new words used by the children in the trial with secretin increased: expressive language with a confidence of p = 0.16 and receptive language with a confidence of p = 0.15

29. Evaluation of Symptoms – 4
GI Symptom Score
Notes: graph showing some decrease in GI symptoms for both the placebo and secretin, with no numeric values or labels. Not sure what it represented except they did make an effort to quantify and measure all these things.

30. Secretin Phase 2 Clinical Trial – Subset analysis
- improvement not correlated with GI symptom severity at baseline (shown in previous slide)
- improvements not confined to patients with diarrhea
- improvements not correlated with age
Notes: they tried to break out subsets of children based on particular criteria to see if there was a pattern, and did not find any on the GI symptoms

31. Secretin Phase 2 Clinical Trial – Biological analysis
- chymotrypsis in stool: pancreatic enzyme released by secretin; this is a marker of "pancreatic insufficiency"
- Calprotectin in stool: neutrophil anti-microbial protein; this is a marker of intestinal inflammation, "colitis"

32. Secretin Phase 2 Clinical Trial – Biological analysis
- Chymotrypsis: 29% of patients had low levels
- Calprotectin: 26% of patients had elevated levels
- 64 patients with normal levels of both proteins
Patients with abnormal levels had highly variable symptoms Notes: They tried to find particular markers to better determine who would be a better candidate for success with secretin – looking for patterns – but did not find goods ones here

33-35. showed 3 slides with charts on Clinical Global Impressions among those patients with Normal Calpro and Chymo.

36. Symptom Assessment Summary of All patients vs the Normal Calpro/chymo Patients
This showed when all the patients were rated with statistical confidence,: the parents came in at 98%,
the raters were 78%,
the ADOS Social test was 88%
MCDI was 85%
When the Normal Calprotectin, Chymotrypsis patients were evaluated:
The parents came in at 99.9%
the raters were 98%,
the ADOS Social test was 99%
MCDI was 98%
This showed that the parents were consistently accurate, but the
other methods was only accurate with a certain subgroup of the patients.

37. Responder analysis: Normal Chymo/Calpro slide showing data, don't remember what it was supposed to show

38. Secretin Safety.

39. Secretin Safety Phase 2 Extension Study
- All 124 patients have the option to receive 6 additional doses of secretin at 3 week intervals
- Continued surveillance for adverse events, blood chemistries, antibody formation
- retest patients with ADOS

40. Secretin Phase 2 Summary
(these are the things they accomplished with this first clinical study)
- Identified biomarkers for patient selection
- Identified behavioral assessment tools which capture secretin effect
- established statistically significant drug effect on social and communicative symptoms
- established safety profile consistent with chronic dosing


41. Phase 1 Trials of Secretin
(these are the things they needed to look at with the next clinical study)
- small number of subjects
- single dose protocol
- diverse patient population: level of function, IQ, age, diagnosis, other symptoms (GI, seizures)
- Inadequate assessment tools: designed for diagnosis, not stable for test-retest, insensitive to change
- calculation and design efforts, bias (need to account for these)

42. Phase 1: Thoms Rehabilitation Hospital
(this is the second study)
28 secretin and 29 placebo patients; 33% PDD
- age 3-14 years
- all function levels; ?% with GI symptoms
- one dose: evaluations at days 0, 1, 7, 14, 28
-autism behavior checklist
All patients substantially improved on day 1. There was no difference between secretin and placebo.

43. NIH Autism Coordinating Committee
Psychopharmacology Working Group
- The appropriate length of a trial will vary depending on the target and goals of the treatment, but generally should be longer than the few weeks or months usually reported in the literature.
- Studies should use narrower age ranges or stratify on age to prevent the clouding of results by developmental differences.
National Institute of Mental Health Website

44. Language Assessment
showed covers of two manuals for evaluating language.
The MCDI is for 8-16 months,
The PLS is for 0-7 years

45. Meta-analysis of Phase 1 trials
text slide: "There are significantly more "responders" in the secretin groups than in the placebo groups.

46. Next Step: Phase 3 Clinical Trials
(this is what is being planned)
- up to 200 patients at 10-12 sites
- GI positive and GI negative patients
- longer dosing period
- additional bioprofiling
- 10-12 clinical sites

47. Neurobiology of Secretin
[text slide]

48. Effect of Secretin on Body Temperature
Two diagrams showing that body temperature increased after a first and second dose of secretin to just under 99 degrees and stayed elevated over time.

49. Brain Activation in Rats
showed a slide of a rats brain were the site in the brain activated by secretin is located. There is a definite region to look at.

50. Secretin Stimulates the Vagus Nerve
When secretin is added, it stimulates this nerve which sends a signal to the brain, which activates this particular region in the brain which sends response signals to the gut. (I think I got that right.)

51. Brain Anatomy
showed slide labeling the parts of the brain

52. Secretin Activates the Amygdala in Rates
showed slide with 8 photos or rat brain where the region was activated and where it wasn't

53. Function of the Amygdala region in the brain
- face recognition
- emotion
- fear, anxiety, stress response
- control of autonomic nervous system: body temperature, digestion and GI function, sensory input (taste, pressure), cardiac and pulmonary function

54. Does Secretin Act Directly on the Brain?
- secretin receptors in the brain
- secretin crosses the BBB in mice
- secretin activates the brain in rats

55. GI Symptoms in Autism:
Gut-Brain or Brain –Gut?

Genetics/Environmental Insult leads to neural dysfunction which leads to either autism or GI symptoms

Notes: He said the question is does the gut activity affect the brain, or does the brain activity affect the gut (primarily)

56. diagnostic Secretin Products
- porcine secretin approved for in vivo diagnosis of pancreatic disease, Ferring withdrew product in 1998
- Repligen licensed synthetic products: Porcine – approvable letter November 2000, Human – NDA submitted Q2 2001
- FDA requested more QC data (quality control data)

57. BioProfiling in Autism
[text slide]

58. BioProfiling: Systematic assessment of biomarkers
(they can look at these potential markers)
- genetics: HOX A1, reelin
- Proteins: immune – interleukins, TNF, ACTH; endocrine – oxytocin, GH
- Metabolites: > 200 amino acid, metals, purines, neuroltransmitters, etc.

59. Why Do BioProfiling?
- It is a research tool to identify subsets of patients with similar disease characteristics
- gives laboratory confirmation of clinical observations
- may suggest new interventions
- may provide evidence of treatment effects

60. Secretin Phase 2 Clinical Trial: 1,250 Biological Samples Collected from Highly Characterized Patients
Notes: They are going to collect blood, urine, and stool samples are regular intervals during the patients' treatment with secreting to assist in bioprofiling.

61. BioProfiling in Autism: Initial Results from the Phase 2 Patients
Urine: Opioid Peptides, Uric acid, 7-methyl Xanthine
Blood: Cytokines, Zinc
Stool: Calprotectin, chymotrypsin

62. Opioid Peptides in Urine
- these are small peptides derived from casein (dairy) or gluten (wheat) having opioid activity
- Hypothesis: incomplete digestion leads to opioids in gut; peptides cross gut into blood and urine; peptides cross the blood-brain-barrier

63. Opioid Peptides in Urine
- analyzed for three casomorphins and gliadomorphin by HPLC-MS
- No opioid peptides detected (<10ng/ml) in pre-treatment samples from 120 patients
- previously used HPLC-UV methods may identify "false positives"
- food intolerances may have immune or metabolic basis

64. Uric Acid (Urate)
- urate is the end product of purine metabolism which is excreted into the urine
- urine levels elevated in diseases of purine metabolism (Lesch-Nyhan)
- urate was reported to be elevated in 20% of ASD urine by Mary Coleman in 1976
- several patients treated with uridine

65. Uric Acid in Phase 2 Samples
showed a bar graph - not sure what the point was

66. "Purine Autism"
text slide saying: Does elevated urate define a set of patients with a common biological defect? If so, can uridine therapy improve symptoms?

67-68. [This next series of slides was presented as one text slide and one bar chart showing the text]
Immune Cytokines in Serum
- IFN-gamma, TNF-a, IL-2, IL-4, IL-5, IL-9, IL-10
- measured before and 4 weeks after dosing
17 out of 126 patients with elevated IFN-g
5 out of 126 patients with less than 3 elevated cytokines
no relationship to symptoms, GI, age
no effect of secretin on cytokine levels

69-70. Zinc
- early patients had low serum zinc levels
- zinc deficiency can lead to diarrhea
- reasonable to consider a role of the pancreas in zinc absorption form food
- many biological processes are zinc dependent

71-73. Calprotectin
- anti-microbial protein release by neutrophils (a type of white blood cell)
- not degraded in the gut; detected in stool
- used to monitor gut inflammation in Crohn's disease, ulcerative colitis, etc.
- first used in ASD by Wakefield, et al
- 26% of Phase 2 patients showed abnormally high calprotectin at baseline: > 75 ug/gm

74-75. Chymotrypsin
- enzymes released by pancreas in response to secretin/CCK as food enters the intestine
- measured in stool as a marker for "pancreatic insufficiency"
- 29% of patients showed low chymotrypsis at baseline: <8 units/gm
BUT
- All 36 patients with low chymotrypsin had normal levels of SIRT (serum immunoreactive trypsinogen)
- 20 patients with low chymotrypsin has normal levels of elastase
Low chymotrypsin may be a result of degradation due to increased
transit time or altered gut flora

80. BioProfiling Summary
- opioid peptides – none detected
- uric acid – 25% have elevated uric acid
7-methyl Xanthine – 40% have no 7-MX in urine
Cytokines – 5% have elevated cytokines
Zinc – no abnormal values
Calprotectin – 26% have elevated values
Chymotrypsin – 29% have depressed levels
Calpro & Chymo unrelated to GI severity or diet

81. Acknowledgements
[lots of references]

82. Repligen

Submitted by Karen from the autismandenzymes  Yahoo Group.

2: November 24, 2001