Effect of Linagliptin + Metformin vs Metformin Alone on the Role of Pancreatic Islet Function, Insulin Resistance and Markers of Cardiovascular Risk in Patients With Prediabetes: Randomized Clinical Trial
Phase: PHASE4
Conditions: Prediabetic State, Insulin Resistance
Sponsor: Universidad de Guanajuato

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Ccombination of Linagliptin, metformin and lifestyle to prevent type 2 diabetes 
in high risk patients with prediabetes  
 
PRELLIM Project (Diabetes Prevention with Lifestyle, Linagliptin and Metformin) 
 
 
 
 
 Research Protocol 
 
 
Rodolfo Guardado-Mendoza MD PhD 
 Principal Investigator 
University of Guanajuato 
Hospital Regional de Alta Especialidad del Bajío, León, Guanajuato, México 
 
 
 
 
 
Collaborators 
Alberto Aguilar-García MD 
Edgar Durán-Pérez MD 
Mildred Alvarez-Canales MS 
Sara Stephania Salazar López MD 
Diana Farfán MD 
 
University of Guanajuato and Hospital Regional de Alta Especialidad del Bajío 
 
 
 
 
 
 
Last update: January 30, 2018 
 
 

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Introduction 
Type 2 diabetes (T2D) is a worldwide health problem, reaching estimated prevalence around 
18 % in countries like Mexico, and an unstoppable increase in the number of new cases, 
having a big economic impact due mainly to micro and macrovascular complications, and 
being the first cause of mortality in Mexico (1, 2) 
  
Physiopathology of hyperglycemia is complex and multifactorial in T2D. It is known that 
insulin resistance (IR) is one of the first observed abnormalities, leading to a decrease in the 
glucose uptake by muscle, increase in hepatic glucose output and lypolisis; different factors 
have been involved in IR (3-5).  IR stimulates insulin secretion from the pancreatic β cells, 
causing hyperinsulinemia to compensate IR and maintain normal glucose levels; however, if 
IR persist, β cell can be damaged by different factors; incretin defect, oxidative stress, and 
amyloid deposition (3, 6-8), causing a reduction on β-cell function and mass up to 50-80 % (3, 
4, 6, 9-12). Together with pancreatic β-cell dysfunction, α-cell dysfunction and 
hyperglucagonemia have been also involved in the development of hyperglycemia (6, 13-16); 
glucagon is one of the main regulators of hepatic glucose production, and together with the 
reduction on incretin hormones (GLP-1, GIP) and amyloid deposition are physiopathological 
defects that contribute to pancreatic islet dysfunction and hyperglycemia (17-21). 
 
Prediabetes is a condition with intermediate hyperglycemia, and based on the American 
Diabetes Association criteria(22) can be defined as: i) impaired fasting glucose (IFG): FPG 
≥5.5mmol/L [≥100mg/dl]; ii) impaired glucose tolerance (IGT): 2 h plasma glucose 
≥7.8mmol/L [140 - 199mg/dl]; and iii) IFG + IGT: FPG ≥5.5mmol/L [≥100mg/dl] and 2 h 
plasma glucose ≥7.8mmol/L [140 - 199mg/dl]. Most of the physiopathological abnormalities 
observed in T2D, have been already reported in prediabetes with a different severity (23), IR 
(24), pancreatic β-cell dysfunction (10, 11, 25, 26), pancreatic α-cells dysfunction and relative 
hyperglucagonemia (13, 15, 25), incretin defect, although it has been questioned whether 
incretin defect is a cause(27) or consequence of hyperglycemia in T2D (28). It has been 
previously shown that clinical and physiopathological characteristics are different between 
these three categories of prediabetes. IFG mainly presents hepatic insulin resistance and mild 
impairment on firs phase insulin secretion (β-cell dysfunction), IGT is more related to muscle 

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insulin resistance and first and second phase insulin secretion (β-cell dysfunction), while the 
combination of IFG+IGT present marked pancreatic β-cell dysfunction and whole body IR, 
with the greatest risk to develop T2D. 
 
Prevalence of prediabetes varies between 9.7 to 56.8 %; in Mexico it has been reported to be 
around 30 % (29). Eventually, most of the patients with prediabetes (70-80 %) will develop 
T2D during their life, and of note, the have a higher risk and may already present micro and 
macrovascular complications (30).   
Pancreatic β-cell function has been reported as one of the main factors involved in the 
progression from prediabetes to T2D in observational studies as well as in clinical trials (31-
33) 
 
Different pharmacologic and non-pharmacologic strategies have been used to reduces the 
progression risk from prediabetes to diabetes and prevent T2D. Lifestyle modification 
programs, based on physical activity and nutrition, have proved to be a useful strategy, 
reducing the risk up to 58 % in patients with IGT, in China and Finland (34) (35). The 
Diabetes Prevention Program Study (DPP) was designed to compare the risk reduction 
between an intensive lifestyle program vs placebo and metformin (36); DPP by the  first time 
evaluated the effect of metformin, an insulin sensitizer that mainly reduces hepatic glucose 
production(37); this study showed also a risk reduction of 58 % in the lifestyle group, and only 
31 % in the metformin group, in comparison to the placebo group; however, the main 
limitation of the lifestyle programs it´s the adherence to the program, and that besides of that, 
there is a progression from prediabetes to T2D and eventually most of the patients will 
develop T2D in the long  term follow-up, since in  the DPP Outcome Study(38) the 
cumulative T2D incidence at 15 years of follow-up was 55, 56 and 62 % in the lifestyle, 
metformin and placebo group, respectively.  
Other kind of drugs have been also used to prevent T2D; rosiglitazone in the DREAM study 
showed a 62 % risk reduction in patients with IGT to develop T2D (39), in the ACT-NOW 
Study, Pioglitazone showed a 72 % risk reduction in patients with IGT, but was associated 
with significant weight gain and edema, perhaps due to the used dose(40). 
 

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Few studies have been performed to evaluate the effect of incretin drugs to prevent T2D. If 
there is a reduction in the incretin effect in prediabetes, there is two ways of improving it, with 
GLP-1 receptor agonist or GLP-1 analogues, or with  the use of a DPP-IV inhibidor, that 
eventually will increase  endogenous GLP-1 levels two or three-fold. Most of the previous 
studies have been done with GLP-1 RA or agonist. Exenatide, a GLP-1 receptor agonist, after 
24-weeks, in patients with obesity and prediabetes, together with a lifestyle program showed 
reductions in calorie intake and glucose normalization, but without a difference in fasting, 2h 
glucose and T2D incidence, but with a mean body weight reduction of around 4.0 kg(41). 
Liraglutide 1.8 mg/d, also in patients with prediabetes or early T2D (<12 months), was 
compared to a lifestyle modification program in a small sample of patients (n=40), achieving 
in both groups a weight reduction of around 7.0 kg, and a slightly better improvement in 2h 
glucose and β-cell function in the liraglutide group(42); also in patients with prediabetes, 
liraglutide showed to improve glucose values during OGTT and β-cell function during 14 
weeks of treatment(43). At doses of 3.0 mg/d, liraglutide together with diet and exercise has 
shown a reduction in the incidence of T2D (HR 0.21 95% CI 0.13-0.34, p <0.0001), and 
improvement in glucose during OGTT in patients with overweight-obesity and prediabetes 
during a 3 year study (44). 
Endogenous GLP-1 is inactivated in 1-4 minutes by the DPP-IV, and with DPP-IV inhibitors 
the incretin effect gets improved; the use of DPP-IV inhibitors in prediabetes has been less 
explored, since there are few studies with short duration and small sample size; in an 8-week 
study, sitagliptin showed no effect in patients with IFG(45), but in patients with IGT it was 
reported a reduction in glucose levels as well as an improvement in insulin and glucagon 
levels during OGTT(46). In a 12-week study in patients with overweight and prediabetes, 
sitagliptin improved also glucose profile and β-cell function during OGTT (47).  Saxagliptin in 
a 24-week study (n = 5-7 per group), showed similar changes than metformin in fasting 
glucose and HbA1c and a slightly better improvement in 2 h glucose levels in patients with 
IFG or IGT(48). Linagliptin has been used only in 12-week study (n=8 per group) in 
comparison to metformin in overweight-obese patients with IGT, finding mainly a reduction 
in 2h glucose with linagliptin (49). 
Linagliptin is a potent and selective DPP-IV inhibitor with a long half-life to  be used once 
daily; it is the only DPP-IV inhibitor cleared mainly by the liver (˃80 %) and by consequence 

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it can be used without dose adjustment in patients with high risk or any kind of renal 
dysfunction (50); besides this, linagliptin has shown in recent clinical trials a safe profile 
regarding renal and cardiovascular function (51), and has proven efficacy in T2D treatment as 
a monotherapy or combined therapy (52) 
 
In human islets from non-diabetic and diabetic donors, linagliptine has shown to  better 
improve insulin secretion, β-cell function and α-cell function in non-diabetic islets, suggesting 
that this kind of drugs could have a better effect at very early stages of the disease, like 
prediabetes (53).  
Few studies have tried to evaluate integral and realistic interventions to prevent T2D, focusing 
these therapies to the group of patients with prediabetes at the highest risk to develop T2D, 
combining oral and practical medications with a realistic and real life feasible lifestyle 
program, and moreover, no study has deeply evaluated the physipathological effects of such 
therapies. 
The goal of this work was to evaluate the effect of linagliptin + metformin plus a lifestyle 
program vs metformin plus a lifestyle program on glucose metabolism, pancreatic β-cell 
function and T2D incidence in Mexican patients with IGT + IFG during 18 months. 
 
Methods 
 
Study design and participants 
The PRELLIM project (Diabetes Prevention with Linagliptin, Lifestyle and Metformin) is a 
single center double-blind randomized clinical trial performed in patients with IGT and at least 
two T2D risk factors.  Patients will  be selected from a metabolic cohort study performed at 
the Metabolic Research Laboratory in the University of Guanajuato, México, as part of the 
University Cohort Project CARE-In-DEEP Study (Cardiometabolic Risk Evaluation and 
Interdisciplinary Diabetes Education and Early Prevention). For this particular study, patients 
will be screened with anthropometrical, physical activity, nutritional, biochemical and 
metabolic evaluation, including oral glucose tolerance test and hyperglycemic clamp. Patients 
are eligible for enrollment in the study according to the following criteria: i) impaired glucose 
tolerance (2 h glucose 140-199mg/dl [7.8 to 11.0 mmol per liter]) during a single oral glucose 

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tolerance test, ii) age between 18 – 65 years, and iii) at least two T2D risk factors according to 
the American Diabetes Association; exclusion criteria: i) treatment with drugs affecting 
glucose levels during the previous 3 months, ii) previous pathological conditions affecting 
glucose metabolism or body weight (thyroid disease, Cushing´s syndrome, Acromegaly); iii) 
excesive alcohol intake (acute or chronic), iv) fasting plasma triglyceride >400mg/dl, v) 
pregnancy, vi) Systolic blood pressure >180mmHg or diastolic blood pressure >105mmHg 
(subjects could be re-screened after hypertension treatment).  Written informed consent will be 
obtained from all participants and the study was approved by the Ethical Council at the 
Hospital Regional de Alta Especialidad del Bajío. 
 
Procedures 
Anthropometrical and body composition measurements. Weight will be measured while 
participants are barefoot and wearing minimal clothing. Height is obtained while the 
participants are standing barefoot with their shoulders in a normal position. BMI (kg/m2) is 
obtained from standardized measurements of weight and height and is computed as a ratio of 
weight (kg):height squared (m2). Waist circumference will be measured at the high point of 
the iliac crest at the end of normal expiration to the nearest 0.1 cm. Body composition will be 
assessed with electrical bioimpedance through a Tanita Scale SC-240. All measurements will 
be performed by personal trained to use standardized procedures and reproducibility is 
evaluated, resulting in concordance coefficients between 0.88 and 0.94. Weight will be 
recorded at least every two months, while electrical bioimpedance every 6 months.    
 
Nutritional and Physical Activity Evaluation. A semi-quantitative food frequency 
questionnaire (FFQ) previously validated (54), will be applied to evaluate dietary intake. This 
questionnaire included data regarding the consumption of 116 food items. For each food, a 
commonly used portion size (e.g. 1 slice of bread or 1 cup of coffee) is specified on the FFQ 
and participants reported their frequency of consumption of each specific food over the 
previous year. Participants chose from 10 possible responses, ranging from “never” to “6 or 
more times per day.” For our analysis, the reported frequency for each food item is converted 
into a daily intake. Total energy intake is computed by summing the energy intakes from all 
foods. The PA level of participants is assessed using a self-administered questionnaire that is 

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verified when the patient assist for the metabolic evaluation. The questionnaire has a validated 
Spanish translation which has been adapted for use in the Mexican population (55). The 
questionnaire is self-administered and estimates the minutes devoted to the practice of 
different recreational physical activities during a typical week in the last year (including 
walking, running, cycling, aerobics, dancing, and swimming as well as playing football, 
volleyball, basketball, tennis, fronton, baseball, softball, and squash, among other activities). 
Each item includes time intervals that allow participants to detail the exact number of minutes 
or hours they dedicate to each form of recreational PA, as well as the intensity of each PA 
(light, moderate, vigorous). The total duration of each recreational PA is expressed in minutes 
per day. We calculated the number of hours per week devoted to each activity, which were 
then multiplied by the intensity of each activity, defined as multiples of the metabolic 
equivalent (MET) of sitting quietly. We used the Compendium of Physical Activities to assign 
METs to each activity (56). We then add the average weekly energy expenditure attributable 
to each activity to derive the total MET-hours per week. Energy intake and physical activity 
will be evaluated every 6 months. 
 
Metabolic evaluation.  
Oral glucose tolerance test (OGTT):  All subjects will be admitted to the Metabolic Research 
Laboratory of the Department of Medicine and Nutrition, Division of Health Sciences at the 
University of Guanajuato the day of the study between 7 and 8 AM, and a catheter will be 
placed into an antecubital vein for all blood withdrawal.  Subjects will not be allowed to eat or 
drink anything after 10 PM on the night before, until the study is completed.  After the 
intravenous catheter is placed and the first blood sample is drawn, the patients ingest 75 grams 
of glucose.  Serum samples for glucose and insulin measurement will be drawn at -15, and 0 
minutes and every 30 minutes thereafter for two hours. Prediabetes is diagnosed based on the 
OGTT; and only patients with IGT (2 h glucose 140-199 mg/dl), and ideally together, 
although not exclusively, with IFG (FG 100-125mg/dl) will be included in this study. Patients 
with newly diagnosed T2D will be excluded from the study. 
 
Hyperglycemic clamp. All patients will be invited to the hyperglycemic clamp, with the goal 
to have at least 10 to 20 % in both groups at 0, 6 and 12 months. Subjects are admitted to the 

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Metabolic Research Laboratory at University of Guanajuato on the day of the study at 7:00 
AM.  A catheter is placed in the antecubital vein for glucose infusion, and a second catheter is 
placed into a vein on the dorsum of the other hand and the hand will be inserted into a heated 
(65C) box.  At 8 AM, following the collection of two baseline blood samples (-30 and 0 
minutes), a single step hyperglycemic clamp will be performed. At the start of the 
hyperglycemic clamp (time zero), the plasma glucose concentration is acutely raised (glucose 
bolus with D50 %) and maintained at 125 mg/dl above baseline (i.e., approximately 225 
mg/dl) for 120 minutes, with a continuous D10% glucose infusion.  Serum glucose will be 
measured every 3 minutes during the first 15 minutes, and every 5 minutes thereafter; insulin 
will be measured every 3 minutes during the first 15 minutes and every 15 minutes thereafter; 
C-peptide will be measured every 3 minutes during the first 15 minutes and at 30, 60, 90 and 
120 minutes. Plasma glucagon is measured at 0, 15, 30, 60 and 120 minutes.  The 
hyperglycemic clamp will be performed at a 5-7 day interval after OGTT. 
 
Measurements. Glucose will be measured with an Analox glucoanalyzer GM9 (Analox 
Instruments) and by colorimetric glucose oxidase (Vitros 5600; Ortho Clinical Diagnostics). 
Lipid levels are measured by dry chemistry with colorimetric method (Vitros 5600; Ortho 
Clinical Diagnostics). Insulin and C-peptide will be measured by chemiluminiscent 
immunometric assay (IMMULITE 2000 Immunoassay system, Siemens), and Glucagon by 
Quantikine ELISA Immunoassay (R&D Systems Inc, Minneapolis, USA) with an intra-assay 
CV of 2.7 – 3.6 %. HbA1c will be determined using high-performance liquid chromatography 
with a DS-5 Analyzer (Drew Scientific, Inc. Miami, FL, USA). Renal function will be 
evaluated only at 12 and 24 months of follow-up with the 24 h creatinine clearance and the 
albumin urinary excretion. 
 
Calculations. The incremental and the AUC for glucose and insulin during the OGTT are 
calculated according to the trapezoidal rule. Insulin secretion is calculated dividing the 
AUCinsulin0_120 by the AUCglucose0_120 during the OGTT. The insulin secretion/insulin 
resistance (IS/IR) index (disposition index = DI) during OGTT is calculated as 
(AUCinsulin0_120 / AUCglucose0_120)*Matsuda index (57). Insulin sensitivity during OGTT is 
calculated from the Matsuda index (58), and at fasting with the homeostasis model assessment 

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(HOMA-IR). From the hyperglycemic clamp the incremental and AUC for glucose, insulin 
and C-peptide are also calculated according to the trapezoidal rule. Different measurements of 
pancreatic β-cell function will be derived from the hyperglycemic clamp: DI will be obtained 
as a product of insulin sensitivity (Matsuda index) and the incremental AUC for insulin and C-
peptide from 0 to 15 and from 0 to 120 minutes. 
 
Randomization and masking. Patients will be randomly assigned in a 1:1 ratio to receive 
linagliptin/metformin 2.5/850mg every 12 h + lifestyle modification program, or metformin 
850mg every 12 h + lifestyle modification program during 24 months. Randomization will be 
performed using an electronic random numbers table, ensuring to have a similar number of 
patients per group. Participants and investigators involved in the patients follow-up and 
outcome measurements will be masked to treatment allocation during the entire study.  
 
Interventions. i) Linagliptin + metformin + lifestyle: Patients allocated to this group will start 
linagliptin/metformin pills of 2.5/850mg once daily during the first month, and after that the 
dose was increased to 2.5/850mg twice daily from the second month until the end of the study 
(24 months). ii) Metformin + lifestyle: Patients on this group will start metformin pills of 
850mg once daily during the first month, and increased to 850mg twice daily thereafter until 
the end of the study (24 months). Both groups will receive the same lifestyle implementation 
program based on a prescribed diet every two months seeking to reduce their body weight at 
least by 5-7 %, adjusting their energy requirements based on their weight every two months, 
and composed from 55-60 % of carbohydrates, 25-30 % fat, and 10-15 % proteins. If the 
patient is sedentary, he or she will be advised to start with 45 min/week of mild to moderate 
exercise with a specific activity according to patients preference; patients will be counseled to 
increase the duration and frequency or intensity of exercise every two weeks until reaching 
150min/week of moderate activity or 75 min/week of intense activity. If the patient was 
already physically active, it will be recommended to continue like this and vary its exercise 
routines.  
 
Follow-up visits and outcome. Patients will have a follow-up visit every month during the first 
12 months, and every 2 months thereafter. Every appointment is about 30-45 minutes; 

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medications tolerance and side effects will be recorded in every patients´ visit. Nutritional and 
physical activity assessment according to the patient´s weight will be performed by a 
Nutritionist every month from 0 to 12 months, and every 2 months thereafter.  Monthly 
adherence to medications will be evaluated by pill counting; nutritional adherence and energy 
intake will be evaluated every 6 months by a food frequency questionnaire, and physical 
activity at 0 and 12 months. Every 6 months patients will have an OGTT. Primary objective is 
to evaluate at 6, 12, 18 and 24 months glucose profile during OGTT, insulin secretion and 
pancreatic β-cell function by the OGTT DI and the hyperglycemic clamp, and T2D incidence 
and regression to normoglycemia; T2D will be diagnosed and confirmed by at least two 
consecutive measurements of the same criteria: glycated haemoglobin ≥ 6.5 %, fasting glucose 
≥ 126mg/dl, or 2 h glucose ≥ 200 mg/dl. In a subgroup of patients, hyperglycemic clamp will 
be performed at 0, 6 and 12 months to complement the measurements of insulin secretion and 
pancreatic β-cell function (basal n = 19, 6 months n = 25, 12 months n = 32; total 76 
hyperglycemic clamps). Secondary endpoints include changes in body weight, insulin 
sensitivity and glucagon levels.  
 
Statistical analysis 
The sample size calculation was based on the fact that we expected an improvement of at least 
50 % on the pancreatic β-cell function and glucose profile during the OGTT in patients with 
the combined treatment and at least an improvement of 20% on the metformin group, based on 
previous reports about glucose levels and pancreatic β-cell function in prediabetic patients 
before and after treatment (40, 59); in order to be able to detect a minimum difference of 30 % 
between groups and assuming an alpha error of 5% and a beta error of 20%, we require 
approximately 41 patients per group.   
The primary analysis is the comparison of the change in glucose profile during OGTT as well 
as measurements of pancreatic β-cell function during OGTT and hyperglycemic clamp. 
Intergroup comparisons between absolute values and delta values will be performed with a t 
test for independent groups; intra-group comparisons will be performed using a paired t-test. 
Non-numerical variables will be compared between groups with the chi squared test. Relative 
risk and hazard ratio will be calculated to evaluate the TD2 risk in the groups of treatment. 

11 
 
The Log-rank (Mantel-Cox) test will be used to compare the incidente of T2D in the study 
groups. 
Statistical analyses and graphics will be performed using SPSS Version 21.0 (SPSS Inc) and 
GraphPad Prism 5.0. Statistical significance is considered when p value is less than 0.05. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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