The Assessment of the Effect of Metformin and Its Serum Concentration on the Concentration of Substances Associated With the Production of Nitric Oxide in Patients With Impaired Carbohydrate Metabolism
Phase: PHASE4
Conditions: PreDiabetes, Impaired Fasting Glucose (IFG), Impaired Glucose Tolerance (IGT)
Sponsor: Wroclaw Medical University

STUDY PROTOCOL 
Title: Metformin and Its Impact on the Substances Associated With NO Production in Prediabetes Patients. 
NCT03398356 
Main Researcher: Dr Sutkowska Edyta   
Sponsor: Wroclaw Medical University 
Document Date: Jun 13,2017 
Aim:  
The study evaluated the effect of different doses of metformin on the intermediates of NO (nitric oxide) 
biosynthesis in people with pre-DM (pre-diabetes). 
Material and Methods:  
Population and groups 
The treatment-naïve patients (regarding to hypoglycemic drugs) with new-diagnosed pre-DM were recruited 
from Diabetes Centre between October 2017 and December 2018.  
Inclusion criteria for the patients:  
• Pre-diabetes based on FG (fasting glucose) and / or OGTT (oral glucose tolerance test) according to 
the local (similar to EASD- European Association for the Study of Diabetes) criteria  
• Age 40-65 yo  
•  Any gender, profession, place of residence  
•  Negative history for diabetes 
•  No hypoglycemic drugs in the past 
•  Negative history for cardio-vascular diseases 
•  Written consent to participate in the study 
Criteria for exclusion for the patients: 
• 
Age <40 or >65 years 
• 
History of diabetes –from patients, patients family and/or medical records if available 
• 
FG>125 mg% (6.9mmol/L) and or glucose after 2 hours in OGTT >199mg% (11mmol/L) 
• 
History of treatment with any hypoglycemic drug  
• 
History of cardio-vascular disease  
• 
Contraindications to the use metformin according to the Product Characteristics 
• 
Known gastro-intestinal disease which could be potentially responsible for metformin intolerance (eg 
IBD- irritable bowel disease, GERD- gastro-esophageal reflux  disease) 

• 
Known potential difficulties in collaboration with patients (dementia, mental disorders, distance from 
patients residence if considered as potentially problematic) 
• 
No consent to participate in the study 
• 
Patient participation in another study 
Inclusion criteria for * healthy volunteers: 
• 
Age 40-65 yo  
• 
Any gender, profession, place of residence  
• 
Negative history for diabetes, pre-diabetes and FG<100 mg% (5.6 mmol/L) 
• 
No hypoglycemic drugs in the past 
• 
Negative history for cardio-vascular diseases 
• 
Written consent to participate in the study 
 
Methods: definitions, information collected, lab-tests performed, treatment scheme, lab-tests technique, 
statistical analysis   
1. Pre-diabetes diagnosis definitions: 
 IFG (impaired fasting glycaemia): FG (fasting glucose): 100-125 mg% (5.6- 6.9 mmol/L);    
 IGT (impaired glucose tolerance): glucose 140-199 mg% (7.8- 11.0 mmol/L) after 2 hours of 75 glucose     
solution taken  
2. Negative history for cardio-vascular diseases definitions:  
Patients without ischemic heart disease and/or stroke and/or PAOD (peripheral arterial occlusive disease) in 
a history 
3. Demographic information collected: age, sex  
4. Baseline parameters collected: weight, height, BMI (body mass index), information about fatty liver 
(based on the available ultrasonography organs’ description), family history for DM (YES/NO), 
hypertension (YES/NO), active nicotinism (YES/NO); and biochemical: FPG, lipids profile, creatinine, 
AlaT (alanine transaminase), creatinine, HbA1c (glycated hemoglobin) if available. 
5. Before the first dose of the metformin and after: 6, 12, 15 weeks of the treatment, the blood samples were 
collected for the assessment of the:  
a. concentration of the metformin  
b. concentration of the compounds related to the production of  the NO: L-arginine, L-citrulline, ADMA – 
asymmetric dimethylarginine, DMA – dimethylamine, SDMA – symmetric dimethylarginine, ornithine 
* For healthy volunteers only once concentration for the compounds related to the production of  the NO 
was assessed and no blood samples for metformin concentration were taken.  

The admissible delay in taking blood for lab-testing was set at 4 days. The blood samples were taking 
randomly (no hours or gaps between metformin taking were defined). 
6. Patients with pre-DM were treated 3 weeks with an increasing dose of the immediate-release (regular 
form) metformin to 3 x 500 mg, and then randomly assigned to: 
group A – continuation of a dose of 3 x 500 mg or  
group B – increase dose to: 3 x 1000 mg (also by titration); after 12 weeks  this group returned to the dose 3 
x 500 mg for the next 3 weeks  
7. If potential side effects appeared in group B, the patients could take a lower dose (3 x 500 mg if well-
tolerated) and thus change to group A. 
8. If the smallest dosage (3 x 500mg) was not tolerated the patient was excluded from the study. 
9. During titration period (by phone) and blood collection visits (personally), patients were asked about the 
compliance and drug’s tolerance. The patients were also encouraged to contact in any case of the problem 
with metformin tolerance.  
10. The technique used to assess concentration of the metformin in serum and the wider panel of L-
arginine/NO pathway metabolites: LC-MS/MS (liquid chromatography-mass spectrometry). 
11. Safety outcomes: Adverse events were reported at each clinic visit and revised via telephone contact 
during treatment period. 
12. SAP- Statistical Analysis Plan 
- to prove the homogeneity of the compared groups, analysis of the baseline parameters was done 
- to show the potential differences between people with pre-DM and healthy people the comparison of the 
baseline parameters was done 
- to follow the metformin intake by the patients, the metformin concentration with its change during the 
therapy was done  
- for the analysis of the impact of the metformin dosage on the concentration of the compounds related to the 
production of the NO the difference of these compounds between two groups was done   
- as an extra analysis, the correlations of the serum drug concentration with BMI and body mass, after 6 and 
15 weeks of the treatment was done, while research was ongoing 
The Statistica 13 program was used with cut-off point for statistical significance (P) at 0.05.  
The normality of the distribution of the examined quantitative variables was checked using the Shapiro-Wilk 
test. In order to determine the significance, statistical tests were used in accordance with the distribution of 
variables and the nature of the data (Student t test, Welch test, Mann-Whitney test, chi – square test, the 
Wilcoxon signed-rank test, Kruskal – Wallis test,  Friedman test, F test). To determine the correlation the 
Spearman's rank-order correlation and the Pearson correlation was used. 
 
 
 

The flow chart of the management of the patients 
              Met “Inclusion criteria”: YES;   Written consent: YES 
 
            Initial visit: blood samples for: arginine, citrulline, ADMA, DMA, SDMA, ornithine 
                                                                Metformin start    (1 x 500 mg) 
                                                                                                 3 weeks titration to the dose: 1500mg/day 
                                                                                                               
                                                                 Good dose tolerance: YES 
                                                                                                  3 weeks continuation of the dose 1500mg/day 
 
Control visit: blood samples for: arginine, citrulline, ADMA, DMA, SDMA, ornithine 
                                          and metformin 
                                                                 Randomization 
  Group B                                                                                                                Group A 
  3 weeks titration to the dose:                                                                           6 weeks continuation of the dose 1500/day 
     3000mg/day 
                                                                                                                                   
                                              Good dose tolerance: YES        NO            
    3 weeks continuation of the dose                                                                                   
   3000mg/day 
                       Control visit: blood samples for: arginine, citrulline, ADMA, DMA, SDMA, ornithine 
                                          and metformin 
                                                                 
                                                                                                 3 weeks continuation of the dose 1500mg/day     
                                                                               
                        Final visit: blood samples for: arginine, citrulline, ADMA, DMA, SDMA, ornithine 
                                                and metformin  
 
 
 

Title: Metformin and Its Impact on the Substances Associated With NO Production in 
Prediabetes Patients.  
NCT03398356 
Main Researcher: Dr Sutkowska Edyta     
Sponsor: Wroclaw Medical University 
Document Date: Jun 13,2017 
OŚWIADCZENIE BADANEGO (see below for English version) 
– zgoda na udział w badaniach 
 
Nazwisko i imię osoby badanej........................................................................... 
.............................................................................................................Lat........... 
 
Adres:................................................................................................................. 
............................................................................................................................ 
 
Temat badań: Ocena wpływu metforminy i jej stężenia w surowicy na stężenie substancji 
związanych z produkcją tlenku azotu u pacjentów z zaburzeniami gospodarki cukrowej. 
Niniejszym oświadczam, że zostałem/am szczegółowo poinformowany/na       o sposobie 
przeprowadzenia badań i moim w nich udziale (Informacja dla Pacjenta). Rozumiem, na czym 
polegają badania i do czego potrzebna jest moja zgoda. Zostałem poinformowany/na, że mogę 
zadawać pytania dotyczące projektu w trakcie jego trwania oraz odmówić uczestnictwa w 
badaniach na każdym ich etapie bez podawania powodu. 
Wszystkie informacje jakie zostaną przekazane na cel badania będą poufne (nie będzie 
możliwości identyfikacji mojej osoby).  
 
Wyrażam świadomą i dobrowolną zgodę na uczestnictwo w powyższym badaniu. 
 
 
.........................................                                  ....................................... 
podpis badacza               
 
    podpis badanego 
 
 
 
 
 
miejscowość...................                                data  ........................................ 
 
 
 
 
 
 
 

Title: Metformin and Its Impact on the Substances Associated With NO Production in 
Prediabetes Patients.  
NCT03398356 
Main Researcher: Dr Sutkowska Edyta     
Sponsor: Wroclaw Medical University 
Document Date: Jun 13,2017 
 
STATEMENT OF THE PARTICIPANT 
- consent to participate in the research 
 
 
Surname and first name of the participant ............................................. 
................................................. .................................................. Years .........  
 
Address:................................................ .................................................. 
................................................ .................................................. ........................ 
 
Research topic: Metformin and its impact on the substances associated with nitric oxide (NO) 
production in pre-diabetes patients. 
 
I hereby declare that I have been informed in detail about the way of conducting the study. I 
understand what the research is for and what my consent is needed for. The purpose and nature 
of the study have been explained to me in writing and I have been able to ask questions about 
the study. I understand that even if I agree to participate now, I can withdraw at any time or 
refuse to answer any question without any consequences of any kind. 
I understand that all information I provide for this study will be treated confidentiall (no 
identification of my personal data).  
 
I express my informed and voluntary consent to participate in the above research. 
 
 
 
 
.........................................                                       ......... ..............................  
signature of researcher                                            signature of participant 
 
 
 
 
 
 
 
place……………………………,                               date  ............................ ..... 
 
 
 
 
 
 
 
 

Title: Metformin and Its Impact on the Substances Associated With NO Production in 
Prediabetes Patients.  
NCT03398356 
Main Researcher: Dr Sutkowska Edyta     
Sponsor: Wroclaw Medical University 
Document Date: Jun 13,2017 
 
 
 
OŚWIADCZENIE BADANEGO (see below for English version) 
- zgoda na przetwarzanie danych 
 
 
Nazwisko i imię osoby badanej........................................................................... 
.............................................................................................................Lat........... 
 
Adres:................................................................................................................. 
............................................................................................................................ 
 
Temat badań: Ocena wpływu metforminy i jej stężenia w surowicy na stężenie substancji 
związanych z produkcją tlenku azotu (NO) u pacjentów z zaburzeniami gospodarki cukrowej (stan 
przedcukrzycowy) 
 
Niniejszym oświadczam, że wyrażam zgodę na przetwarzanie moich danych osobowych w 
zakresie niezbędnym do realizacji projektu badawczego,          w którym biorę udział.  
 
 
 
 
.........................................                                  ....................................... 
podpis badacza                                      podpis badanego 
 
 
 
 
 
miejscowość....................                                data  ........................................ 
 
 
 
 
 
 

Title: Metformin and Its Impact on the Substances Associated With NO Production in 
Prediabetes Patients.  
NCT03398356 
Main Researcher: Dr Sutkowska Edyta     
Sponsor: Wroclaw Medical University 
Document Date: Jun 13,2017 
 
 
STATEMENT OF THE PARTICIPANT 
- data processing agreement 
 
 
Surname and first name of the participant ............................................. 
................................................................................ ............. Years ......... ........... 
 
Address:................................................ .................................................. 
............................................ .................................................. ........................ 
 
Research topic: Metformin and its impact on the substances associated with nitric oxide (NO) 
production in pre-diabetes patients. 
 
I hereby declare that I agree to the processing of my personal data to the extent necessary for 
the implementation of the research project in which I participate. 
 
 
 
 
......................................... ...                              ...... .............................. 
signature of researcher                                     signature of participant 
 
 
 
 
 
 
 
place…………………….                                         date  ...........................