Human Pilot Study - HA35 (Hyaluronan Molecular Weight 35) Dietary Supplement for Promoting Intestinal Health
Phase: NA
Conditions: Gastrointestinal Microbiome
Sponsor: The Cleveland Clinic

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“Human pilot study - HA35 dietary supplement for promoting intestinal 
health” 
 
Detailed Protocol   
 
Twenty healthy adult volunteers, ten with a lean BMI between 19-25 and ten with 
an obese BMI between 30-35, will complete this study. There will be no restriction 
to gender or race. Age limits will be 18-45 years for this pilot study, since ageing 
may affect intestinal immunity, microbiome composition, prevalence of pre-clinical 
disease confounders1,2. The screening visit will include medical history, physical 
examination, diet history (including probiotics and food allergies) using the Diet 
History Questionnaire II 3, and laboratory testing (liver function testing, basic 
metabolic panel, complete blood count, indirect calorimetry). Once entered to the 
study, subjects will be asked to refrain from chronic NSAIDs (i.e. more frequent 
dosing than twice per week) and acid suppressants, and notify the study team if 
they are prescribed new systemic (oral, intravenous) or local enteric (laxatives, 
enemas) medications. Starting at D1, subjects will ingest 140 mg of HA35 
dissolved in purified sterile water that is provided in individual pre-made doses 
(supplied by the CRU team) once daily, 30 min. before breakfast. The initial dosage 
was chosen from the functional dose we determined in mice and using intestinal 
surface area ratio calculations4 scaled to an average 70k human, and multiplied 
by 2 for the maximal potential weight of a study participant. The dosage derived by 
this method is slightly higher, but still in general agreement with the calculated 
dose recommended by body surface area conversion methods5. The seven HA35 
doses will be made in the kitchen of the Clinical Research Center just prior to the 
start of each volunteer’s treatment course. 
 
Study subjects will be asked to keep a daily log of gastrointestinal symptoms, a 
food diary, and a stool frequency chart6. At days D-0, D-7 and D-28 subjects will 
provide a stool sample from the entire day. If subjects do not have daily bowel 
movements they can provide a stool sample on D-1 or D0, D7 or D8, and D27 or 
D28.  Stool samples will be refrigerated immediately after collecting and stored 
long-term at -80° C until analyzed (samples will be homogenized before sampling) 
for microbiome and antimicrobial peptide determination. Additionally subjects will 
provide blood samples at the same time points for analysis as outlined in primary 
and secondary outcomes (see below). Subjects will be seen at the Clinical 
research Unit at the Cleveland Clinic at D0, D8 and D28 of the study and donate 
samples. 
 
Inclusion criteria: 
• BMI 19-25 (lean), and BMI 30-35 (obese) 
• Age 18-45 years old 
• Willingness to take oral supplement and adhere to study requirements 
 
Exclusion criteria: 
• Diabetes  

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• Oral antibiotics within 4 weeks of study initiation 
• History of cardiac disease, and medications for cardiac disease 
• Use statins and antihypertensive drugs 
• Inflammatory bowel disease including irritable bowel syndrome 
• History of intestinal surgery, excluding hernia repair and appendectomy 
• Active cancer diagnosis (except skin cancer) 
• Chronic acid suppression treatment (proton pump inhibitors, histamine H2 
receptor antagonists) 
• Immune 
modulatory 
treatments 
(e.g. 
chronic 
immunosuppressive 
medications, chronic NSAIDs) 
• Vegetarian or vegan diet7 
• Abnormal liver or kidney function as measured by routine serum chemistry 
testing 
• Severe anemia or significant white blood cell or platelet abnormalities 
• No additional blood or blood product donations during the study 
 
Objectives 
• To evaluate whether oral HA35 supplementation modifies the human gut 
microbiome 
• To confirm the safety profile of oral HA35 treatment  
• To evaluate whether oral HA35 increases intestinal defensin levels, and 
decreases intestinal inflammation and permeability 
• To assess the health benefits that may result from oral HA supplementation  
(improved BMI, serum panels) 
 
Study Endpoints 
Study outcomes include: 1) stool microbiome diversity and phylogenetic 
distribution (primary); 2) assessment of comprehensive metabolic panel (CMP), 
complete blood count (CBC); 3) indirect calorimetry measurements using the 
respiratory quotient (RQ); 4) indices of fecal intestinal antimicrobial peptide 
secretion (calprotectin, human beta-defensin; 5) serum indicators of intestinal 
permeability (serum HA, serum LPS, serum I-FABP); 6) serum indicators of 
inflammation and injury (high-sensitivity C-Reactive Protein, IL6 and TNFa). Oral 
HA35 supplementation may prove to have a prebiotic effect and modulate 
intestinal as well as systemic inflammation through a modulation of the intestinal 
microbiome and epithelial defense system. 
 
Methods 
Composition of stool microbiome as measured by 16S pyrosequencing will 
beconducted by Second Genome on a fee for sample basis. The CMP and CBC 
will be performed by the Cleveland Clinic Department of Laboratory Medicine (fee/ 
sample). Indirect calorimetry measurement to determine changes in resting energy 
expenditure will be conducted in the CRU using Vmax Encore sampler and Care 
Fusion conversion software8. Stool calprotectin levels, stool HBD2 levels, serum 
HA, serum I-FABP, serum high-sensitivity CRP and serum LPS (ELISA) will be 
determined using commercially available ELISA kits and performed in the de la 
Motte laboratory.  
 
 

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Statistical analysis 
We have calculated that we need at least 20 subjects, and up to 30 subjects, 
providing several samples to be able to detect an effect of our intervention on the 
microbiome primary endpoint, i.e. microbial diversity and phylogenetic distribution. 
This is in agreement with interventional studies investigating the modification of 
intestinal microbiome in obese individuals that have analyzed between 9 and 28 
subjects7,9,10. We propose to recruit 20 subjects in order to ensure sufficient power.  
 
 
 
 
Safety 
Serpil Erzurum, MD, director of the Cleveland Clinic Clinical Research Unit, is the 
safety and emergency contact physician for this study. Dr. Erzurum and can be 
reached at  
 (216) 445-5764. 
 
 
 
Section 6 references: 
1. Salazar N, Arboleya S, Valdés L, Stanton C, Ross P, Ruiz L, Gueimonde 
M, de Los Reyes-Gavilán CG. The human intestinal microbiome at extreme 
ages of life. Dietary intervention as a way to counteract alterations. Front 
Genet. 2014; 5:406. 
2. Man AL, Gicheva N, Nicoletti C. The impact of ageing on the intestinal 
epithelial barrier and immune system. Cellular immunology 2014;289:112-
118. 
3. Diet history questionnaire, version 2.0. National Institutes of Health, Applied 
Research Program, National Cancer Institute. 2010. 
4. Casteleyn C, Rekecki A, Van der Aa A, Simoens P, Van den Broeck W. 
Surface area assessment of the murine intestinal tract as a prerequisite for 
oral dose translation from mouse to man. Lab Anim. 2010; 44:176-83. 
5. Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human 
studies revisited. FASEB J. 2008; 22:659-61. 
6. Lewis SJ, Heaton KW. Stool form scale as a useful guide to intestinal transit 
time. Scandinavian Journal of Gastroenterology 1997; 32:920-924. 
7. David LA, Maurice CF, Carmody RN, Gootenberg DB, Button JE, Wolfe BE, 
Ling AV, Devlin AS, Varma Y, Fischbach MA, Biddinger SB, Dutton RJ, 
Turnbaugh PJ. Diet rapidly and reproducibly alters the human gut 
microbiome. Nature 2014; 505:559-563. 
8. Boullata J, Williams J, Cottrell F, Hudson L, Compher C. Accurate 
determination of energy needs in hospitalized patients. J Am Diet Assoc. 
2007; 107:393-401. 
9. Leber B, Tripolt NJ, Blattl D, Eder M, Wascher TC, Pieber TR, Stauber R, 
Sourij H, Oettl K, Stadlbauer V. The influence of probiotic supplementation 

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on gut permeability in patients with metabolic syndrome: An open label, 
randomized pilot study. European Journal of Clinical Nutrition 2012; 
66:1110-1115. 
10. Vrieze A, Van Nood E, Holleman F, Salojarvi J, Kootte RS, Bartelsman JF, 
Dallinga-Thie GM, Ackermans MT, Serlie MJ, Oozeer R, Derrien M, 
Druesne A, Van Hylckama Vlieg JE, Bloks VW, Groen AK, Heilig HG, 
Zoetendal EG, Stroes ES, de Vos WM, Hoekstra JB, Nieuwdorp M. Transfer 
of intestinal microbiota from lean donors increases insulin sensitivity in 
individuals with metabolic syndrome. Gastroenterology 2012; 143:913-916.