Golimumab Effect in the Modulation of Gut Microbiota in Ulcerative Colitis: Pilot Study
Conditions: Ulcerative Colitis
Sponsor: Institut d'Investigació Biomèdica de Girona Dr. Josep Trueta

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Golimumab effect in the modulation of gut microbiota in 
Ulcerative Colitis. Pilot Study. 
 
Protocol Code: GOLI2015 v1    10/11/2015 
Promotor: Merck & Co., Inc. 
Financing: This project will be funded by Merck & Co., Inc 
Investigador principal: Dr. Xavier Aldeguer Manté, Head of Gastroenterology Service, 
Hospital Universitari de Girona, Dr. Josep Trueta. 
Centers where the study will take place: 
Hospital Universitari de Girona, Dr. Josep Trueta 
Av de França S/N, 17007, Girona, Spain 
 
Hospital de Santa Caterina 
Carrer del Dr. Castany, s/n, 17190 Salt, Girona, Spain 
 
Collaborating centers: Centers of Catalonia, that will wish to participate after the 
approval of this project 
 
Duration: 26 months, Starts in May 2016 
 
 
1. 
BACKGROUND  
 
Recently, a consistent body of evidence indicates that the gut microbiota ability to modulate and 
direct the host immune response is considered one of the pivotal factors that modulate the 
delicate balance between health and disease in digestive diseases (1). In the case of 
inflammatory bowel disease (IBD), the gut microbiota has a central role in maintaining the 
microbiological homeostasis of the patient (2, 3). Recent studies describe the existence of 
different patterns of intestinal microbiota from patients with IBD and healthy individuals (4, 5). 
The alteration of the microbiological pattern not associated with disease is known as dysbiosis 
and is directly related to the degree of inflammation of the intestinal mucosa, and, thus, the 
clinic status: a greater dysbiosis, greater clinical correlation with disease state, so that the gut 
microbiota is a direct marker of the state of colonic inflammation (6). 
The state of dysbiosis is measured through different microbiological indices of the total 
population of bacteria in the colon. Changes in rates of these indices are the parameters that 
are measured to characterize the gut microbiota of the patients. In the case of ulcerative colitis 
(UC) studies have shown that intestinal microbiota is a central factor in maintaining the balance 
between deep remission and the presence of a flare-up (7, 8). Despite advances, in-depth 
knowledge of how the gut microbiota interacts with the intestinal mucosa and alters the 
intestinal barrier and the molecular/genetic mechanism is still unknown. Some genes and 
associated cofactors have been identified but the exact mechanism of interaction has not yet 
been described. The data indicate that, presumably, the mechanism of regulation is a 
multifactorial process (9, 10). 
In this way, a deep understanding of gut microbiota and its interactions with the host and the 
colon will be of great interest, in order to shape the future of IBD treatment, especially of UC. 
Thus, IBD patients’ intestinal microbiota could be either a therapeutic target itself (fecal 
transplantation) or be consider as an adjustable adjuvant with immunomodulators (11, 12). 
The group of anti-TNFα drugs are the most recently incorporated in the therapeutic arsenal of 
UC, in some cases changing the natural curse of the disease. However, it is still unknown how 
these drugs modulate the intestinal microbiota and how they interfere with it, although, probably, 

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they develop a role. Recent results from studies by our group, indicate that on entering clinical 
remission, gut microbiota is modified to patterns less related with dysbiosis (13, 14,15, 16). 
Given the increasing importance of the use of anti-TNFα drugs, it is of great interest to 
discriminate between the patterns associated with dysbiosis and those related with healthy 
mucosa, and how they are modified as a result of the use of anti-TNFα drugs. In this way, 
previous results of our group analyzing the changes of intestinal microbiota associated with 
Adalimumab anti-TNFα drug treatment have shown that during the progression of the patient 
into remission, the mucosal dysbiosis pattern changes (17). On the other hand, our group has 
also observed that after drug treatment failure, the gut microbiota returns to a pattern closer to 
dysbiosis. For that reason, gut microbiota could be considered as an excellent indicator of the 
real drug effectiveness in the patient. 
Regarding Golimumab, a recently introduced anti-TNFα drug therapy in UC, it is still unknown 
how it is able to modulate the intestinal microbiota to remission-related patterns, since  to date 
there are not available studies about the relationship between Golimumab and this 
phenomenon.  
The use of prebiotics and probiotics has shown some effectiveness as adjuvants in the 
treatment of UC (18). For that reason, further characterization of the gut microbiota patterns is 
very important to develop new strategies for adjuvant ability to modulate it, especially in patients 
receiving anti-TNFα drugs and do not achieve complete remission. Similarly to recent studies, 
we suggest that the modulation of gut microbiota could optimize the response outcomes in 
patients treated with Golimumab.  
In conclusion, based on current trends in the literature, we suggest that modulation of the 
intestinal microbiota and the characterization of remission-related patterns, will have a huge 
impact on the management of patients with UC. Moreover, the modulation of gut microbiota 
together with the anti-TNFα drug effectiveness could be the most promising field in the 
management of inflammatory bowel disease. 
 
2. HYPOTHESIS AND OBJECTIVES 
 
2.1 Hypothesis 
 
1. Intestinal microbiota profile change according UC activity. 
 
2. The Intestinal microbiota profile correlated to clinical remission is represented by 
stable intestinal microbiota biodiversity. 
 
3. Determination of Intestinal Microbiota is a useful tool to measure Golimimab 
efectiveness in patients naïve to anti-TNF treatment and patients recurrent to anti-TNF 
treatment. 
 
2.2 Objectives 
 
1. To correlate clinical remission under Golimumab treatment with stable intestinal 
microbiota profile biodiversity 
 
2. To characterize the pattern of intestinal microbiota associated with the use of 
Golimumab and temporal dynamics of microbial change. 
 
3. Assess the effect of golimumab on the degree of colic dysbiosis in the treatment of 
CU naive to Golimumab.  
 
 
 
 
 
 
 
 
 
 

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3. MATERIAL AND METHODS 
 
3.1 Type of study/design 
 
Multicentrer transversal pilot Study 
 
3.2 Study population 
The proposed study will include 15 UC anti-TNFα naïve patients from Hospital 
Universitari Dr. Josep Trueta, Hospital Santa Caterina and Hospitals collaborators. 
We will consider remission when patients have an endoscopic Mayo score ≤1, and 
activity index score, Mayo= 0 points. 
 
3.2.1 
Inclusion criteria 
- 
> 18 years 
- 
Signed the informed consent 
- 
Anti-TNFα naïve  
- 
Screening for opportunistic infections 
 
 
3.2.2 
Exclusion criteria 
- 
Active tuberculosis or another chronic infections 
- 
Antibiotic treatment prior 1 month 
- 
Probiotics & Prebiotics 
- 
Gestation and lactation 
- 
Heart disfunction 
- 
Colectomy 
 
3.3 Interventions 
 
3.3.1 
Treatments 
GOLIMUMAB induction with 200mg at week 0, and 100mg at week 2. Under 70kg, 
the follow up treatment will be 50mg/month, and 100mg/month in patients over 
70kg, as clinical practice. 
 
 
3.4 Variables 
 
3.4.1 
Demographic variables 
 
- 
Age expressed in years (y) 
- 
Sex Male (M)/ Female (F)  
- 
Weight expressed in IMC 
- 
Ethnic: Caucasian/ African/ Asiatic/ American 
- 
Tobacco: YES / NO / EX-Smoker 
- 
Age of UC diagnosis expressed in years (y) 
- 
Familiar Antecedents (YES/NO) which kind of disease? 
- 
Localization Left (L)/ Righ (R) / extended (E) 
 
 
 
 
 
 
 
 
 
 
 
 
 

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3.4.2 
Clinical variables 
 
- Mayo clinic score (Clinical colitis activity index) (Schroeder KW et al. NEngl 
J Med 1987; 317: 1625–9). Score range: 0-9 points 
- CRP (C-reactive protein): blood C-reactive protein concentration (mg/L) 
- Faecal calprotectin: stool sample expressed in concentration of calprotectin 
in ug for g of feces (ug/g) 
-Standard Analysis: 
Hemoglobin: blood hemoglobin concentration (g / dl) 
   Platelets: blood platelets concentration (x103/ ul) 
   Leucocytes: blood leucocytes concentration (x103/ ul) 
     Albumin: blood albumin concentration (g / dl) 
  Creatinine levels: blood creatinine concentration (mg/ dl) 
   
 
3.4.3 
Endoscopic variables 
 
- Mayo (Endoscopic score of Ulcerative Colitis) (Schroeder KW et al. 1987). 
Mayo Endoscopic Score is based only on Endoscopic Findings. Mayo 
Score range from 0 to 3. 
 
Note: We will send the image in a centralization digital platform, where we will 
reevaluate the Mayo score with 2 independent professionals, in order to have 
an objective score. 
 
 
 
3.4.4 
Microbiological variables 
 
- Operational Taxonomic Units (OTUs) 
- Abundance and bacterial load. 
 
 
3.5 Methods 
 
The proposed study will include 15 UC patients over 18 years, that have signed the informed 
consent, subjected to treatment with anti-TNFα by clinical practice and who have made 
screening for opportunistic infections. Patients will be anti-TNFα naïve patients. 
 
Stool samples will be collected before starting Anti-TNF treatment (M0), and then every 3 
months (M1, M2, M3 and M4) to complete the study. The monitoring period shall be one year. 
We will be also collected at M0, M1, M2, M3 & M4 demographic variables (age, sex, tobacco, 
age of diagnosis, localization,…), clinical data (Mayo clinical score, CRP, albumin, hemoglobin, 
creatinine, 
leucocytes, 
platelets…), 
microbiological 
variables, 
also 
a 
4 
calprotectin 
determinations (at baseline, at week12, at 6 months and at the end of the study as a clinical 
practice) and Mayo endoscopic index (at baseline and at the end of the monitoring period as a 
clinical practice). Follow-up visits will also take place within routine clinical practice. For better 
follow-up the evolution of the patient we propose an additional test as a clinical practice, but 
optional, a rectosigmoidoscopy at week 12 after starting Anti-TNF treatment. 
 
We will consider remission when patients have an endoscopic Mayo score ≤1, and activity index 
score, Mayo clinical score =0 points. 
 
Moreover, depending on the evolution of the patient, additional tests shall also be performed as 
routine clinical protocol during the monitoring period. 
 
 

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3.5.1 
Sample processing 
 
DNA Extraction: 
Before microbiological analyses, genomic DNA of 16s RNA gene will be extracted using 
NucleoSpin® Soil Kit (Machery-Nagel GmbH & Co., Germany). DNA concentration will be 
determined with Qubit® BR (Invitrogen) Kit. 
 
Bacterial 16S rRNA Gene Amplification by Pyrosequencing  
 
For pyrosequencing purposes, the 16S rRNA gene was partially amplified from extracted 
genomic 
DNA 
using 
the 
universal 
bacterial 
primers 
GC-357F 
5’- 
CGCCCG 
CCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCC- CCT ACG GGA GGC AGC AG-3’ 
(341Y357) and 907R 5’-CCG TCA ATT CCT TTG AGT TT-3’ (907Y926). PCR was performed 
with a GeneAmp PCR System ® 2700 (AppliedBiosystems)). Then we purify the PCR product 
with kit Ampure (Agencourt AMPure, Beckman Coulter Inc), and quantify the PCR products with 
Qubit® BR (Invitrogen) Kit. The pyrosequencing will be performed with a 454 Life Genome 
Sciences Sequencer FLX. 
 
Sequence Editing and Analysis 
 
High-quality consensus sequences were obtained and manually refined with the Bioedit 
software package. Alignments were carried out with ClustalW24 software. Consensus 
sequences were compared with those in GenBank and the Ribosomal Database Project by 
using BLASTN 2.2.10. Sequences were grouped by number of operational taxonomic units or 
phylotypes with the DOTUR program26 using the farthest neighbor method at a precision level 
of 0.01, i.e., 99% minimum similarity for any pair of sequences to belong to the same phylotype, 
on a distance matrix with the Jukes-Cantor correction calculated with the DNADIST program of 
the Phylip software package. 
 
 
3.6 Statistical analysis 
 
Statistical analysis was performed with the SPSSx version 11.0. Significance of distances 
between groups was checked using an analysis of variance. Pearson_s x2 test was used to 
compare the prevalence of genus and species. 
 
Clinical and laboratory data will be correlated with the values of quantitative microbial indices 
using Receiver Operating Characteristic (ROC) curves. 
 
 
4. BIBLIOGRAPHY  
 
 
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Characterization of bacterial community shift in human Ulcerative Colitis patients 
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non-inflamed regions of the intestine in inflammatory bowel disease. BMC Microbiol. 
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Vermeiren J, van de Wiele T, Licht TR. Microbiotas from UC patients display altered 
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Louvriex R, Miquel-Cusachs JO, Garcia-Gil JL, Aldeguer X. Anti-tumour Necrosis 
Factor Treatment with Adalimumab Induces Changes in the Microbiota of Crohn's 
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BUDGET 
 
 
DNA extraction
750 €
PCR reactives 
2.100 €
Pyrosequencing analysis
9.000 €
Fungible
950 €
Sample transport
339 €
Technical
4.800 €
Overhead IDIBGI (30%)
5096,7
Total
23.036 €
MATERIAL
SERVICES
OTHERS