Trial Outcomes & Findings for Desensitising Celiac Disease Patients With the Human Hookworm (NCT NCT01661933)

Results Overview

Biopsies were fixed in neutral buffered formalin, processed and carefully orientated and embedded in paraffin wax. Sections (3 µm) were stained with H\&E. Slides from both time-points were de-identified, shuffled and graded by Dr John Croese after which results from poorly orientated slides were verified by Dr Andrew Clouston. The Vh:Cd ratios were measured on 5 randomly selected well-orientated sites. The null hypothesis is that hookworm infection will not protect against mucosal damage following 12-week exposure to gluten in celiac disease.

Recruitment status

COMPLETED

Study phase

PHASE1/PHASE2

Target enrollment

12 participants

Primary outcome timeframe

Week -24 to -36

Results posted on

2014-10-20

Participant Flow

Of 20 potential candidates, 2 could not be contacted (address changed), 1 was not interested, and 5 were unavailable (travel-2, pregnancy-2 or study-1 commitments). Twelve enrolled in September 2012. 8 committed to 3 endoscopic procedures and 2 committed to two endoscopies.

Participant milestones

Participant milestones
Measure	Necator Americanus, Gluten Challenge Single arm, vertical. Necator americanus: Subjects were inoculated with 20 3rd stage Na larvae (10 + 10 over 4-8 weeks). After hookworm colonization, a micro-dose gluten challenge of 10 mg daily for 6 weeks, followed by 50 mg daily for 6 weeks was completed. After this, a detailed assessment including histology was performed to establish it safe for the participant to proceed to a low-dose gluten challenge of 25 mg daily and 1 G (15-20 G of pasta) twice weekly for 12 weeks. After low-dose challenge, a further evaluation was undertaken before inviting participants to undertake a gluten challenge of 3 G daily over for 2 weeks (preceded by a micro-dose 2 week lead-in).
Gluten Micro-challenge STARTED	12
Gluten Micro-challenge COMPLETED	12
Gluten Micro-challenge NOT COMPLETED	0
Low-dose Gluten Challenge STARTED	12
Low-dose Gluten Challenge COMPLETED	10
Low-dose Gluten Challenge NOT COMPLETED	2
High-dose Gluten Challenge STARTED	10
High-dose Gluten Challenge COMPLETED	8
High-dose Gluten Challenge NOT COMPLETED	2

Reasons for withdrawal

Reasons for withdrawal
Measure	Necator Americanus, Gluten Challenge Single arm, vertical. Necator americanus: Subjects were inoculated with 20 3rd stage Na larvae (10 + 10 over 4-8 weeks). After hookworm colonization, a micro-dose gluten challenge of 10 mg daily for 6 weeks, followed by 50 mg daily for 6 weeks was completed. After this, a detailed assessment including histology was performed to establish it safe for the participant to proceed to a low-dose gluten challenge of 25 mg daily and 1 G (15-20 G of pasta) twice weekly for 12 weeks. After low-dose challenge, a further evaluation was undertaken before inviting participants to undertake a gluten challenge of 3 G daily over for 2 weeks (preceded by a micro-dose 2 week lead-in).
Low-dose Gluten Challenge Physician Decision	2
High-dose Gluten Challenge Withdrawal by Subject	2

Baseline Characteristics

Desensitising Celiac Disease Patients With the Human Hookworm

Baseline characteristics by cohort

Baseline characteristics by cohort
Measure	Gluten Micro-challenge n=12 Participants Single arm, vertical. All twelve healthy adults enrolled subjects were successfully inoculated with hookworm and there was no serious adverse response. Individual hemoglobin levels were all normal and the group mean had significantly increased unexpectedly at completion of the study. Histology was not graded until after low-dose challenge but retrospectively confirmed enrollment Marsh scores of M0-8, M1-1, M2-2 and M3a-1. All participants were complying with a gluten-free diet, were symptomatically well and had a normal anti-tTG.
Age, Categorical <=18 years	0 Participants n=5 Participants
Age, Categorical Between 18 and 65 years	11 Participants n=5 Participants
Age, Categorical >=65 years	1 Participants n=5 Participants
Age, Continuous	53 years n=5 Participants
Sex: Female, Male Female	9 Participants n=5 Participants
Sex: Female, Male Male	3 Participants n=5 Participants

PRIMARY outcome

Timeframe: Week -24 to -36

Population: All 10 of 12 participants who completed micro-challenge successfully progressed and completed low-dose challenge.

Biopsies were fixed in neutral buffered formalin, processed and carefully orientated and embedded in paraffin wax. Sections (3 µm) were stained with H\&E. Slides from both time-points were de-identified, shuffled and graded by Dr John Croese after which results from poorly orientated slides were verified by Dr Andrew Clouston. The Vh:Cd ratios were measured on 5 randomly selected well-orientated sites. The null hypothesis is that hookworm infection will not protect against mucosal damage following 12-week exposure to gluten in celiac disease.

Outcome measures

Outcome measures
Measure	Necator Americanus, Gluten Challenge n=10 Participants Single arm, vertical. Necator americanus: Subjects were inoculated with 20 3rd stage Na larvae (10 + 10 over 4-8 weeks). After hookworm colonization, a micro-dose gluten challenge of 10 mg daily for 6 weeks, followed by 50 mg daily for 6 weeks was completed. After this, assessments including histology were performed to establish it safe for the participant to proceed to a low-dose gluten challenge of 25 mg daily and 1 G (15-20 G of pasta) twice weekly for 12 weeks. After low-dose challenge, a further evaluation was undertaken before inviting participants to undertake a gluten challenge of 3 G daily over for 2 weeks (preceded by a micro-dose 2 week lead-in).
Duodenal Villus Height:Crypt Depth Low-dose gluten	2.73 Ratio Interval 2.31 to 3.15
Duodenal Villus Height:Crypt Depth Baseline	2.73 Ratio Interval 2.31 to 3.15

SECONDARY outcome

Timeframe: Week-24 and -36

Biopsies were fixed in neutral buffered formalin, processed and carefully orientated and embedded in paraffin wax. Sections (3 µm) were stained with anti-CD3. All slides were de-identified and graded by Dr John Croese. The IEL percentages were measured on 2 or more randomly selected well-orientated villi. The null hypothesis is that hookworm infection will not protect against mucosal IEL influx following 12-week exposure to gluten in celiac disease.

Outcome measures

Outcome measures
Measure	Necator Americanus, Gluten Challenge n=10 Participants Single arm, vertical. Necator americanus: Subjects were inoculated with 20 3rd stage Na larvae (10 + 10 over 4-8 weeks). After hookworm colonization, a micro-dose gluten challenge of 10 mg daily for 6 weeks, followed by 50 mg daily for 6 weeks was completed. After this, assessments including histology were performed to establish it safe for the participant to proceed to a low-dose gluten challenge of 25 mg daily and 1 G (15-20 G of pasta) twice weekly for 12 weeks. After low-dose challenge, a further evaluation was undertaken before inviting participants to undertake a gluten challenge of 3 G daily over for 2 weeks (preceded by a micro-dose 2 week lead-in).
Intraepithelial Lymphocyte Count Baseline	33.40 Percentage of epithelial cells Interval 27.12 to 39.68
Intraepithelial Lymphocyte Count Low-dose gluten	35.00 Percentage of epithelial cells Interval 28.72 to 41.28

SECONDARY outcome

Timeframe: Longitudinal change between week-24 and week-36

Population: The outcome score was the number with a 2-point increase in Marsh 3 score post GC-1g.

The Marsh score is a defined but qualitative assessment assigned a value to allow for comparison. The scores were evaluated by consensus between the primary (chief) investigator and the study pathologist. The Marsh score was graded 0, 1, 2, 3A (assigned-4), 3B (-5) and 3C (-6); rage 1-6 with normal=0 and severe inflammation=6. Because the scoring is vulnerable to artefact, only a 2-point shift was regarded as a significant intra-individual change. The scores were graded after week-36 on biopsies de-identified shuffled. An upward shift was interpreted to reflect a significant worsening of gluten-associated inflammation. The comparison reported evaluated changes from baseline (week-24) to post-low-dose gluten challenge (week-24; GC-1g). The objective for using the Marsh score was to identify individuals who might have experienced a severe worsening in pathology due to GC-1g that might not be reflected in the Vh:Cd group analysis.

Outcome measures

Outcome measures
Measure	Necator Americanus, Gluten Challenge n=10 Participants Single arm, vertical. Necator americanus: Subjects were inoculated with 20 3rd stage Na larvae (10 + 10 over 4-8 weeks). After hookworm colonization, a micro-dose gluten challenge of 10 mg daily for 6 weeks, followed by 50 mg daily for 6 weeks was completed. After this, assessments including histology were performed to establish it safe for the participant to proceed to a low-dose gluten challenge of 25 mg daily and 1 G (15-20 G of pasta) twice weekly for 12 weeks. After low-dose challenge, a further evaluation was undertaken before inviting participants to undertake a gluten challenge of 3 G daily over for 2 weeks (preceded by a micro-dose 2 week lead-in).
Number of Participants With 2 Points Increase in Marsh Score Post GC-1g	1 participants

SECONDARY outcome

Timeframe: Anti-tTG IU/mL levels pre-trial, mid-trial and after 3 gram/day gluten challenge

Population: 2 of 12 participants did not progress past baseline micro-challenge, 2 of 10 participants did not progress past low-dose challenge.

The trial was extended with pre-trial and mid-trial anti-tTG antibody levels used to compare with the post-trial levels. Anti-tTG is a serological measure of tissue transglutaminase-2 antibodies. In active celiac disease, levels are increased. In treated disease, levels are low (normal cut-off was \<15 IU/mL). A significant increase compared to baseline in tTG can be expected 2 weeks after consuming 3g of gluten daily for 2 weeks in people with celiac disease who have been maintaining a gluten-free diet, but who are not taking other treatment.

Outcome measures

Outcome measures
Measure	Necator Americanus, Gluten Challenge n=8 Participants Single arm, vertical. Necator americanus: Subjects were inoculated with 20 3rd stage Na larvae (10 + 10 over 4-8 weeks). After hookworm colonization, a micro-dose gluten challenge of 10 mg daily for 6 weeks, followed by 50 mg daily for 6 weeks was completed. After this, assessments including histology were performed to establish it safe for the participant to proceed to a low-dose gluten challenge of 25 mg daily and 1 G (15-20 G of pasta) twice weekly for 12 weeks. After low-dose challenge, a further evaluation was undertaken before inviting participants to undertake a gluten challenge of 3 G daily over for 2 weeks (preceded by a micro-dose 2 week lead-in).
Serum Anti-tissue Transglutaminase Antibodies Measured as International Units/mL (IU/mL) Baseline	4.12 International Units/mL Interval 3.12 to 5.1
Serum Anti-tissue Transglutaminase Antibodies Measured as International Units/mL (IU/mL) Low-dose gluten	2.99 International Units/mL Interval 2.0 to 3.94
Serum Anti-tissue Transglutaminase Antibodies Measured as International Units/mL (IU/mL) After 3 G gluten daily	2.11 International Units/mL Interval 1.16 to 3.07

Adverse Events

Gluten Micro-challenge

Serious events: 0 serious events

Other events: 2 other events

Deaths: 0 deaths

Serious adverse events

Adverse event data not reported

Other adverse events

Other adverse events
Measure	Gluten Micro-challenge n=12 participants at risk Single arm, longitudinal study of responses to escalating gluten doses in people with celiac disease after infection with Necator americanus, a human hookworm.
Gastrointestinal disorders Symptomatic gluten intolerance	8.3% 1/12 • Number of events 1
Gastrointestinal disorders Active CeD	8.3% 1/12 • Number of events 1

Additional Information

Dr John Croese. The Chief Investigator

The Prince Charles Hospital

Phone: +61733716164

Email: [email protected]

Results disclosure agreements

Principal investigator is a sponsor employee
Publication restrictions are in place