Effect of Low Dose Combination of Linagliptin and Metformin to Improve Pancreatic Beta Cell Function, Insulin Resistance and Cardiovascular Function in Patients With Prediabetes and Overweight/Obesity: Randomizer Clinical Trial
Phase: PHASE3
Conditions: Prediabetic State, Insulin Resistance
Sponsor: Universidad de Guanajuato

 
 
 
 
Effect of low dose combination of  linagliptin + metformin on the function of pancreatic 
beta cells, resistance to insulin and cardiovascular function on patients with prediabetes and 
overweight/obesity. Randomized Clinical trial 
 
 
 
 
 
 
 
 
 
Research Protocol 
 
 
 
 
Rodolfo Guardado-Mendoza MD PhD 
Principal Investigator 
University of Guanajuato 
Hospital Regional de Alta Especialidad del Bajío, León, Guanajuato, México 
 
Elizabeth Rodríguez-Guzmán MD 
Master in Clinical Research Student, University of Guanajuato 
 
 
 
 
Collaborators 
Alberto Aguilar-García MD 
University of Guanajuato and Hospital Regional de Alta Especialidad del Bajío 
 
 
 
 
 
 
 
 
 
 

BACKGROUND 
 
Epidemiological impact of type 2 diabetes 
 Diabetes mellitus type 2 (DM2) one of the most frequent endocrine diseases in the general 
population, being in Mexico the main cause of death.  The DM2 is considered a syndrome 
with an altered intermediate metabolism, caused by inadequate insulin secretion, resistance 
to the effect of the same or the combination of both, which as a consequence cause 
hyperglycemia in both fasting and postprandial .(1) 
 
 
The risk factors for DM2 are: overweight and obesity, first-degree relatives with DM2, ≥ 45 
years of age, women with a history of macrosomic products (>4kg) and/or with a history of 
gestational diabetes, women with a history of polycystic ovaries, arterial hypertension 
≥140/90mmhg, dyslipidemia (cholesterol HDL ≤40mg/dl, triglycerides ≥250mg/dl, and 
cardiovascular disease. 
 
It is estimated that around 11.7 million Mexicans will have DM2 for the year 2025, which 
means an increase of 207.8% occupying 7th place with more places worldwide.(2)  
According to the national health survey conducted in the year 2000, found a prevalence of 
DM2 of 8.2%, reporting that this increase is partly due to the greater number of patients 
with DM2 under 40 years, and the high prevalence of risk factors that contribute to the 
development of chronic complications is what explains why it is the main cause of 
death.(3)  However, a global prevalence of DM2 was reported in the 2006 National Health 
and Nutrition Survey (NHNUTS) with greater prevalence in the center and center-west of 
the country.(4); Likewise, this survey found a significant increase in the number of people 
<40 years of age with a diagnosis of  DM2.(5) 
 
In countries like Mexico, DM2 is a pathology that has marked an epidemiological 
transition. The impact of this pathology is very important since more than 180,000 new 
cases are registered annually and cause around 36,000 deaths. It is estimated that the annual 
cost of diabetes for Mexicans is 430 million dollars per year, figures that represent an 
approximate cost of 45 dollars per person per year and this corresponds to three quarters of 
the entire budget granted for Health.(6) 

 
 
The complications of DM2 can be microvascular: retinopathy, neuropathy and 
nephropathy, or macrovascular: heart disease, cerebral vascular disease and peripheral 
vascular disease, with cardiovascular disease being the main cause of death in more than 
75% of patients with diabetes, since they have a high risk of coronary artery disease, with a 
higher incidence of ischemic events and death after an acute myocardial infarction; In the 
absence of ischemic heart disease, the risk of presenting it is the same as those without 
diabetes but with a history of ischemic heart disease. 
 
This implies that prevention measures for asymptomatic diabetic patients should be similar 
to secondary prevention measures for non-diabetic patients with ischemic heart disease.(7) 
Prediabetes has also been associated with increased cardiovascular morbidity and mortality, 
so it is suggested that endotheliopathy also begins in the early stages of the disease, also 
affecting renal function(8–10). 
 
 
PHYSIOPATHOLOGY OF TYPE 2 DIABETES 
 
The pathogenesis of DM2 is multifactorial. The knowledge established so far indicates a 
strong environmental influence, with an important contribution of the genetic factor(11).   
The two most important factors that contribute to the pathophysiology of DM2 are insulin 
resistance and beta cell dysfunction. There is a complex interrelation between the different 
components involved in the process of production, consumption and storage of energy. The 
sequence of pathophysiological events seems to indicate that the first thing to appear is 
insulin resistance, conditioned by several factors such as lack of physical activity, the 
westernized diet high in simple carbohydrates and saturated fatty acids, the ethnic group 
and perhaps a genetic influence so far not well determined(12). 
 
Several pathophysiological mechanisms have been described in insulin resistance. At the 
liver level it has been observed that there is a decrease in the number and activity of insulin 
receptors, a decrease in glucose uptake due to a decrease also in the number and activity of 
the glucotransporters and due to the effect of the glycolytic pathway that secondarily 

prevent the entry of glucose, as well as an effect of free fatty acids at this level interfering 
with insulin signaling. On the other hand, at the muscular level this insulin resistance is also 
very important, because as previously mentioned this is the place where the highest insulin-
dependent glucose uptake is carried out (80%).  Here there has also been a decrease in 
glucotransporters (in this case GLUT4), accumulation of triglycerides and excess free fatty 
acids that compete with glucose as an energy substrate and increase levels of Malonyl-CoA 
it has the ability to inhibit carnitine palmitoyltransferase type 1, an important enzyme for 
beta oxidation of fatty acids. In addition, both at the liver, muscle level and mainly in 
adipose tissue, an interrelation between different cytokines has been found, such as tumor 
necrosis factor alpha (TNFA) which determines insulin resistance by causing 
phosphorylation of the intracellular part of the insulin receptor substrate 1 (IRS-1), and its 
counterpart, adiponectin which is the only protective cytokine against insulin resistance. 
 
Due to this insulin resistance, glucose (muscle) uptake decreases at the peripheral level and 
at the liver level it is not able to suppress glucose production, therefore, as a compensatory 
mechanism the beta cell increases insulin secretion and thanks to this hyperinsulinemia, 
equilibrium is achieved and euglycemia is maintained, so hyperinsulinemia is considered as 
an indirect marker of insulin resistance. 
 
With the passage of time and the persistence of insulin resistance and the increase of some 
toxic factors for the beta cell (glucose and lipids), it begins to deplete and decrease insulin 
secretion, so that ability to maintain suppressed the hepatic glucose production is lost and 
fasting hyperglycemia appears, until reaching diagnostic levels of DM2 where we have a 
50-80% reduction in functional capacity and in the volume of beta cells(11–14) 
 
DYSFUNCTION OF PANCREATIC ISLOTS 
 
When insulin resistance appears, there are many factors that determine whether or not the 
dysfunction of the beta cell and pancreatic islets appear as a functional unit that lead the 
beta cell to that inability to maintain normoglycemia and normotolerance to glucose(15).  
The factors are many, genetic factors not well established, alteration in the cell cycle 

regeneration-apoptosis with decrease in the first and increase in the last, a toxic effect of the 
same glucose (glucotoxicity), prolonged exposure to free fatty acids(16), deficiency or 
resistance to the effect of incretins(17), oxidative stress and increased production of 
superoxide at the beta cell level(18,19),  and a factor identified for many decades but 
perhaps undervalued, abnormalities in the secretion of amylin or IAPP (islet amyloid 
plypeptide), conditioning amyloid formation and deposition at the level of pancreatic islets 
as well as dysfunctional remodeling thereof(20–23). 
All pathophysiological alterations in the natural history of diabetes mellitus, occur 
progressively during the course of the disease, its identification and characterization being 
vital, as well as knowing the risk factors associated and involved with each of them that we 
allowed to focus prevention strategies and timely diagnosis towards stages prior to the 
onset of DM2 and thus be able to impact on the occurrence of chronic complications and 
morbidity and mortality in these. 
 
PREDIABETES 
 
Since the natural history of the disease is a continuous spectrum, and as such, DM2 is only 
a limited part of that spectrum, something known as prediabetes was created, which is 
identified with fasting glucose levels between 100 and 125mg/dl, and is known as impaired 
fasting glucose, or with glucose levels between 140_199mg/dl at 2 hours after an oral load 
of 75g of glucose, known as glucose intolerance; In both entities the presence of 
complications, both macro and microvascular, have been described. 
 
In patients with prediabetes, the presence of pathophysiological phenomena similar to those 
occurring in DM2 but in less severity have been described(24); Patients with prediabetes 
have insulin and muscular resistance to insulin(23,25–27), pancreatic beta cell 
dysfunction(23,26,28–30), pancreatic alpha cell dysfunction with the consequent absolute 
or relative hyperglycagonemia(30–32), reduction of the incretin effect(33,34); Adipose 
tissue is an active endocrine organ that secretes several adipocytokines (leptin, adiponectin, 
resistin, visfatin) and cytokines (TNF Alfa, IL-1, IL-6) which are also involved in insulin 
resistance and inflammation present in subjects with obesity and prediabetes(35,36).  We 

could, then, summarize the pathophysiology of prediabetes as follows: subjects with 
impaired fasting glucose predominantly have insulin resistance at the liver level and 
decrease in the first phase of insulin resistance at the peripheral level, specifically at the 
muscle level, and deterioration in the first and second phase of insulin secretion(37,38). 
 
Both entities, impaired fasting glucose and glucose intolerance, identify different 
populations, since fasting glucose reflects the rate of hepatic glucose secretion and in 
varying degrees the secretion of insulin by the pancreas, since one of its main effects is to 
inhibit hepatic glucose production; On the other hand, glucose intolerance evaluates the rate 
of glucose uptake (in muscle) and the ability of the pancreas to compensate for this defect. 
 
The prevalence of prediabtes in Mexico, according to ENSANUT 2006, refers to a 
prevalence of impaired fasting glucose greater than 30%(39). 
 
The transition from prediabetes (GAA and ITG) to DM2 can take a long time, however, the 
existing information indicates that the majority of these patients (70-80%) will develop at 
some point in their DM2 life. People with GAA + ITG and who are also overweight and 
other risk factors for DM2 are the ones most likely to have this disease progression, and 
factors such as reduced insulin secretion and severe insulin resistance, serve as markers to 
identify these types of patients. These patients have a slightly elevated cardiovascular risk 
(relative risk 1.1-1.4), which rises up to 2 to 4 times more with progression to DM2(40), in 
the same way, in patients with prediabtes  a prevalence of retinopathy of 8%has been 
observed, which rises to 13% when progressing to DM2(41).   
 
With the above, and considering again the continuous spectrum of the disease, we can 
realize that there is another stage that also belongs to a prediabetic state, this is when there 
is only insulin resistance without fasting or postprandial hyperglycemia, which in fact is the 
initial stage of the disease, and that commonly occurs in patients with obesity(42).   
 
A chronological sequence has been proposed in the appearance of the different types of 
prediabetes(27,42), Which would be: I) insulin resistance isolated without fasting 

hyperglycemia or postprandial hyperglycemia, II) isolated GAA, III) isolated ITG, and IV) 
GAA + ITG, for the appearance of these 4 states the presence of resistance to the insulin (in 
liver, muscle and adipose tissue) and pancreatic beta cell dysfunction; It is clear, by 
definition, that the diagnosis o the last three is made with the fasting glucose measurement 
and the performance of the CTOG with an oral load of 75g of glucose, on the other hand, 
for the diagnosis of the first (resistance to isolated insulin), there are several methods. The 
hyperinsulinemic-euglycenic clamp is the most accurate method to measure insulin 
resistance, while the hyperglycemic clamp is mainly used to evaluate the function of the 
pancreatic beta cell, but it has the advantage that it can also be used to evaluate the function 
of the pancreatic islet, when glucagon levels are also measured, and also allows a reliable 
assessment of insulin resistance at the same time(43). 
 
 
TREATMENT OF PREDIABETES 
 
Different pharmacological and non-pharmacological strategies have been used to try to 
prevent the progression of prediabetes to DM2. In general, there are two types of preventive 
strategies, those that are carried out at the population level and whose objective is to avoid 
the presence of modifiable risk factors and that which is carried out in high-risk population, 
such as patients with GAA, ITG and gestational diabetes, this strategy aims to improve 
insulin resistance and pancreatic beta cell dysfunction. 
 
One of the investigations that best show the impact of lifestyle modifications and the use of 
metformin on the prevention of DM2, is the Diabetes Prevention Program (DPP); This 
study was a randomized clinical trial with an average follow-up of 2.8 years, which 
included 3,234 patients with BMI> 24 and GAA and/or ITG that were randomized in 3 
study groups: 1) Metformin 850mg daily treated during the first month and then 850mg 
twice a daily plus routine recommendations (orally and written) annually on  lifestyle, 2) 
placebo treated group plus routine lifestyle recommendations, and 3) group treated with an 
intensive lifestyle modification program, which consisted of performing 150min of physical 
activity per week and reducing fat intake and calories in the diet, in order to lose 7% of 

body weight. This group had 16 personalized sessions during the first 24 weeks, and then 
every month. Is worth mentioning that in this study, only 50% of the participants in the 
intervention group to modify the lifestyle managed to lose 7% of body weight within 24 
weeks of initiation, and only 38% maintained it until the end of the study; 74% maintained 
the required physical activity 24 weeks after the study and only 58% until the end of the 
study; at 24 weeks of the study, the intervention group achieved a reduction in energy 
intake of 450 kcal/day compared to approximately 250 kcal/ day in the other two groups 
The results showed that with intensive changes in lifestyle, the risk of DM2 was reduced by 
58%, and with metformin by 31%; even so, the cumulative incidence of DM2 in 3 years 
was 14.4% in the intervention group, 21.7% in the metformin group, and 28.9% in the 
placebo group(44). However, the long-term results (15 years) of this type of interventions 
have shown that the effect is lost over time and that eventually most patients will develop 
DM2. 
 
Other antidiabetic medications that have been used are: acarbose, which in a cohort of 1429 
patients with ITG showed a reduction in the risk of DM2 of 25% with a necessary number 
to treat of 11 to avoid a case of DM2, however, more than a quarter of patients did not 
complete the follow-up due to side effects of the medication(45); rosiglitazone in the 
DREAM study that included 5269 patients with GAA and/or ITG randomized 4-8mg of 
rosiglitazone or placebo, at an average follow-up of 3 years, the rosiglitazone-treated group 
showed a DM2 risk reduction of 62%(46); The ACT NOW study that used pioglitazone 
45mg a day in subjects with GAA and/or ITG shows a risk reduction of DM2 of about 80% 
compared to placebo(47). 
 
Currently, the pharmacological interventions used in patients with DM2 , are directed 
towards the pathophysiological alterations, while in prediabetes, if we try to avoid the 
progression of the disease, it is clear that the options that can greatly impact each of the 
pathophysiological alterations are those that could have greater effect to prevent disease 
progression(28,29); within these pharmacological interventions, those that could be more 
effective, due to the pharmacological profile, are: 

1) METFORMIN:  Metformin is the most widely used antidiabetic drug in the world. 
Although its main effect is exerted at the liver level, it also has an effect on the 
tissue such as muscle, adipose and endothelial tissue; most of its effects are related 
to the control of insulin resistance and/or hyperinsulinemia. At the liver level, 
metformin activates an enzyme known as AMP kinase (AMPK), which causes an 
inhibition of lipogenic and gluconeogenesis enzymes, decreasing lipolysis, 
increasing 
fatty 
acid 
oxidation, 
reducing 
hepatic 
glucose 
production 
(gluconeogenesis), increasing hepatic glucose uptake and glycolysis, all this is 
because increases the sensitivity to the effect of insulin. At the muscle level, 
metformin also works by improving muscle glucose uptake, among other effects. It 
is the only medication approved for use in people with prediabetes; it is associated 
with weight reduction in a variable percentage, as monotherapy is very unlikely to 
cause hypoglycemia. The disadvantage of metformin is that it has been observed in 
long-term studies in prediabtes (15 years of follow-up) that the effect is almost 
equated to a placebo and that eventually the majority of treated patients will develop 
DM2.  
  
2) PIOGLITAZONE: Pioglitazone belongs to a group of medicines known as 
thiazolidinediones; this type of drug activates the peroxisome proliferator receptor 
(PPAR), by binding to this receptor they form a heterodimer together with the 
receptor of retinoic acid and other molecules, through this mechanism regulate the 
expression of several genes that affect the oxidation of fatty acids, increases the 
transcription of the gene encoding for glucotransporter 4 (Glut4), affects the 
production of cytokines (increasing the secretion of adiponectin and reducing that of 
alpha TNF), and improves mitochondrial function; it is through these mechanisms 
that pioglitazone increases insulin sensitivity in muscle, adipose tissue and liver. 
(48) Pioglitazone is the only medicine in this family that persists on the market as 
an antidiabetic.  Pioglitazone manages to reduce glycosylated Hb in patients with 
DM2 by 0.5 – 1.4% and has shown great utility in delaying (47) 
3) INCRETINES: As already mentioned, patients with DM2 and prediabetes have 
dysfunction of the pancreatic islets (beta cell dysfunction with consequent reduction 

in insulin secretion, alpha cell dysfunction with increased glucagon production, and 
amyloid deposits) and this is a crucial event for the progression of prediabetes 
disease to DM2, so medications that can improve the functionality of this islet, 
increasing insulin secretion and reducing glucagon, could  have a major impact on 
the disease.  There are two incretins, GLP-1 and GIP, of these two, the first being 
found reduced in patients with DM2 and with prediabetes(33,34,49,50) and both are 
produced and released by intestinal cells.  The pharmacological effects of incretins 
are: increased insulin secretion, decreased glucagon secretion, delay gastric 
emptying, and reduced appetite leading to weight reduction.  In general there are 
two ways to increase the incretin effect, by applying incretin analogues, which are 
injectable, and by inhibiting the enzyme that degrades to endogenous GLP-1, which 
is dipeptidil-peptidase 4.  As the incretin effect is reduced in patients with DM2 and 
with prediabetes, and knowing that the effect of incretinomimetic medicinal 
products improves pancreatic islet function and could have some effect on the 
peripheral action of insulin(51–54), it is interesting to know the impact they could 
have on DM2 prevention, especially when combined with insulin sensitizers.  There 
are few studies that evaluate the effect of incretins on people with prediabetes; with 
GLP-1 analogues, one study randomized 38 patients with GAA or ITG to receive 
exenatid 10 g every 12 hrs subcutaneously for 24 weeks, finding that in addition to 
the weight-reducing effect, a large percentage of patients with prediabetes treated 
with exenatide reverted to normal (77% vs. 56% for the placebo group)(55).  
Another randomized clinical trial compared the effect of liraglutide, another LPG-1 
analogue, on glucose metabolism, insulin resistance and insulin secretion in 24 
patients treated with liraglutide and 27 with placebo, of which 10 and 11, 
respectively, they had altered fasting glucose + glucose intolerance; achieved a 29% 
reduction in peripheral insulin resistance and a 21% increase in insulin 
secretion(56).  In another study, 22 patients with GAA were randomized to receive 
100 mg of sitagliptin, DPP-IV inhibitor, for 8 weeks reporting no effect on glucose 
metabolism, although reports of some clinical cases mention important effect 
glucose in patients with prediabetes. 
 

Other non-diabetic drugs have also been used and shown variable results on the prevention 
of DM2 (valsartan, statins, orlistat, fibrates, estrogens, etc.)(57). 
 
DIFFERENCES BETWEEN DPP-IV INHIBITORS  
Power and Efficacy to Inhibit the Enzyme DPP-IV 
 It is important to mention that all DPP-IV inhibitors are competitive reversible inhibitors 
and it is difficult to compare their effects when analyzing studies, experimental conditions 
may be different; however, there is a study in which the inhibitors were compared under 
identical conditions showing similar efficacy (maximum effect) to inhibit DPP-IV in-vitro, 
although there were differences in potency, the most potent being linagliptin(58).    
 
Half Life  
As for the half-life, vildagliptin and saxagliptin (59) have shorter half-lives, on the other 
hand, linagliptin (60) and sitagliptin(61) have longer duration of effect, which gives the 
latter two the ability to inhibit the DPP-IV enzyme for 24h at 80%; although it should be 
noted that in the original study that evaluated the half-life and potency of sitagliptin to 
inhibit DPP-IV was performed on healthy volunteers(62), unlike the original study 
evaluating the half-life and potency of linagliptin to inhibit DPP-IV that was performed in 
patients with type diabetes mellitus 2 (DM2)(60), scenario in which medicines are usually 
used.  However, it is important to mention that the activity in these trials is evaluated ex 
vivo and is generally not corrected by the dilution of the sample, so the actual inhibition of 
DPP-IV is expected to be even greater than the measure. 
 
Selectivity and disposal pathway 
The selectivity of the different DPP-IV inhibitors by this enzyme has been evaluated in in-
vitro studies and has been reported to have both linagliptin (58) like sitagliptin(62) are the 
ones who have the most selectivity by the enzyme DPP-IV; linagliptine has a selectivity of 
10,000 per DPP-IV than for the DPP-8/9 compared to sitagliptin which is ˃5550; this is 
important because inhibition of these two DPP-8/9 enzymes is the one that has theoretically 
been thought to be associated with side effects of inhibition of lymphocyte activity, 
although this effect has not been observed in the clinic, as these 2 enzymes are at the 

intracellular level (63–65).  On the other hand, linagliptin only has less selectivity over 
fibroblast activation protein α (FAPα) which is a protein not found in adult tissue so the 
implications of this data are even smaller, as DPP-IV inhibitors are only indicated in adults.  
Sitagliptin, vildagliptin and saxagliptin are eliminated by more than 80% renally, instead, 
linagliptin is eliminated by more than 80% by biliary route, so it can be used in patients 
with any degree of renal impairment without the need to adjust the dose and without loss of 
the pharmacological effect.(66). 
 
CLINICAL ASPECTS OF DPP-IV INHIBITORS 
Clinical efficiency 
In terms of the clinical efficacy and ability of the different DPP-IV inhibitors to reduce 
HbA1c, fasting glucose and postprandial glucose, various meta-analyses and clinical 
studies have shown similar efficacy, achieving a reduction in HbA1c of 0.5 – 1.0 % (-
0.8%), with larger reductions when baseline values are higher(63–65,67,68). 
 
Side effects and safety 
So far no higher rate of side effects have been found with DPP-IV inhibitors compared to 
control groups, and likewise, no differences in side effects have been reported between the 
various DPP-IV inhibitors(63,64,67,68); In addition, it´s safe in terms of pancreatitis and 
pancreatic cancer risk has been validated by the FDA and the European Medicines Agency 
recently (69). 
 
TABLE 1. Differential characteristics between DPP-IV inhibitors  
NAME 
POTENCY 
(IC50) 
DOSIS 
EFICIENC
Y 
TO 
INHIBIT   
DPP-IV 
x 
24h 
HALF 
LIFE 
(hrs) 
ELIMINATION 
SELECTIVITY 
USE IN RENAL 
INEFFICIENCY 
CLINIAL 
EFFICIEN
CY ON HB 
A1C 
Linagliptin(6
0) 
1Nm 
5mg/2
4h 
˃ 80 % 
10 – 40 
Bile ˃ 80 % 
High 
Recommended 
0.5 – 1.0 
% 
Sitagliptin(61
) 
19nM 
100mg
/24h 
˃ 80 % 
8 – 24 
Renal ˃ 80 % 
High 
Not 
recommended 
or adjust doses 
0.5 – 1.0 
% 
Vildagliptin(
59) 
62nM 
50mg/
12h 
˃ 80 % 
1.5 
– 
4.5 
Renal ˃ 80 % 
Half 
Not 
recommended 
0.5 – 1.0 
% 
Sitagliptin 
50nM 
5mg/2
4h 
˃ 70 % 
2 – 4 
Renal ˃ 80 % 
Half 
Not 
recommended 
or adjust doses 
0.5 – 1.0 
% 
 
 

LINAGLIPTIN 
From the above we can summarize that the mechanism of action of linagliptin is to inhibit 
the enzyme DPP-IV (dipeptidil peptidase type IV); this enzyme (DPP-IV) has the 
biological effect of inactivating the peptide similar to glucagon type 1 (GLP-1), so by being 
inhibited by linagliptin this favors endogenous GLP-1 levels to rise up to 3.2 times above 
the previous values (same as being reduced in patients with type 2 diabetes mellitus, which 
partly explains that these patients have a reduction in the incretin effect), which conditions 
the biological effects of GLP-1 as stimulation of insulin secretion by beta cells pancreatic 
and inhibition of glucagon secretion by pancreatic alpha cells. This effect on stimulation in 
insulin secretion is totally dependent on glucose levels, so it does not cause hypoglycemia. 
Linagliptin has a half-life of 12 hrs so it can be used every 12 or 24 hrs, it is eliminated by 
biliary by more than 70% so it can be used in patients with nephropathy without the need to 
adjust the dose, offers a power to achieve a sustained inhibition 90% in DPP-IV for 24 hrs 
and is highly selective for inhibiting DPP-IV compared to other enzymes such as DPP-8 
and DPP-9.  Linagliptin is indicated in the treatment of patients with uncontrolled type 2 
diabetes mellitus, which does not cover control targets such as: B glucosyl less than 7%, 
fasting glucose less than 110mg/dl and postprandial glucose less than 140mg/dl. It can be 
used as therapy in combination with metformin, sulfonylureas, thiazolidinedionas and 
insulins, either as double or triple therapies. 
 
 
It is clear that the use of a placebo to compare any drug is no longer justifiable, in all 
studies the comparison has been made based on a control group with or without placebo, 
and moreover, some studies have only used as a point of comparison to the placebo group, 
which obviously increases the chances that the drug in comparison may show some 
beneficial effect; on the other hand in Mexico, until today, there are no studies on primary 
prevention of DM2, and if pathophysiological alterations occur from prediabetes stages, it 
is important to evaluate the effect of drugs that combined may have the greatest impact on 
alterations and therefore on the progression of the disease; It would also be useful to assess 
the impact that the combination of medications, with different mechanisms of action and at 

low doses, can have a greater impact on the progression of the disease and reduce the 
cardiovascular risk that occurs since prediabetes. 
 
JUSTIFICATION 
 
DM2 is a chronic disease that has reached the proportions of a global epidemic from the 
increasing number of patients in all countries; has become the disease that causes the most 
chronic and acute complications to patients, consuming a large percentage of health 
spending in countries such as Mexico, it generates a large number of disabilities in 
productive ages and is one of the main causes of cardiovascular morbidity. The 
pathophysiology of the disease is complex and multifactorial, with great influence of the 
environment that the appearance of pathophysiological phenomena that as manifestation 
intermediation have hyperglycemia, and as a final manifestation, complications; and it is 
precisely the sequence of pathophysiological events that gives the disease a continuous 
spectrum in the natural history of it, unfortunately, when the diagnosis of DM2 is made we 
are identifying patients in very advanced stages of the disease, at which point up to 50% of 
them may have any of the known complications, and this together with the fact that we do 
not have a good diagnostic tool and that most patients with DM2 are outside the goals of 
cardiometabolic control, explains the great significance, exponential growth and difficulty 
that has conferred on health systems to control it.  For all of the above, it seems clear that 
the best strategies will be those that are aimed at the early stages of the disease, because it 
will involve more awareness in the population and the impact on the stability and/or 
reversibility of the pathophysiological alterations, which will eventually translate into a 
lower incidence DM2.  The studies on prevention of DM2 that have been conducted have 
not compared the effect of some of the drugs that have so far been more effective for this 
purpose and in some of these studies the doses used are frequently associated with the 
appearance of side effects; of these studies, none have been conducted in Mexico, and we 
are convinced that if we use the combination of drugs with additive pathophysiological 
impact plus cardiovascular protection in early stages, we can obtain better, longer-lasting 
and more impactful results on the natural history of the disease with a view to long-term 
follow-up that shows measures that may have an applicability in clinical practice, in order 

to contribute to the control of this pathology. . Therefore, combining medications with 
different mechanisms of action, in low doses, could be a useful strategy not only to prevent 
DM2, but also to prevent macro and microvascular complications early. 
 
PROBLEM STATEMENT  
 
¿What is the effect of low-dose combination of linagliptin + metformin compared to 
metformin alone on pancreatic beta cell function, insulin resistance and cardiovascular 
function in patients with prediabetes and overweight/obesity? 
 
HYPOTHESIS 
 
Of Work 
 
The low-dose combination of linagliptin + metformin has a greater effect on 
pancreatic beta cell function, insulin resistance and cardiovascular function compared to 
metformin alone in patients with prediabetes and overweight/ Obesity. 
 
Null 
 
The low-dose combination of linagliptin + metformin has no greater effect on 
pancreatic beta cell function, insulin resistance and cardiovascular function compared to 
metformin alone in patients with prediabetes and overweight/ Obesity. 
 
 
OBJETIVES 
 
General: Evaluate the effect of low-dose combination of linagliptin + metformin against 
metformin alone on pancreatic beta cell as function, insulin resistance and cardiovascular 
function in patients with prediabetes and overweight or overweight or Obesity. 
 
 
 

Specific 
- 
Evaluate insulin resistance, pancreatic beta cell function and cardiovascular function 
in a group of patients with prediabetes, treated with Metformin 1700 mg/day for 6 
months, at the start of surgery and at 6 months. 
- 
Evaluate insulin resistance, pancreatic beta cell function, and cardiovascular 
function in a group of patients with prediabetes treated with the combination of 
linagliptin 2.5mg every 24 h + metformin 1700 mg every 24 h for 6 months, at the 
start of intervention and at 6 months. 
- 
Compare markers of cardiovascular risk before and after treatments in the 2 study 
groups. 
- 
Compare insulin resistance and pancreatic beta cell function between study groups 
at the end of the intervention. 
- 
Know glucose metabolism by oral glucose tolerance curve at baseline and end of 
treatment in the study groups. 
- 
Determine changes in body composition. 
 
Secondary objectives 
 
- 
Evaluate therapeutic adherence in study groups. 
- 
Know the incidence of type 2 diabetes mellitus in each study group during follow-
up to this project. 
- 
Know renal function by depuration of creatiline and albuminuria at the beginning 
and end of the study in the study groups. 
 
 
STUDY DESIGN. 
Type of Study: Clinical trial (randomized, controlled and triple-blind). 
MATERIAL AND METHODS. 
Study universe: Mexican patients with prediabetes and overweight/obesity. 

Study population: Mexican patients with prediabetes and overweight/obesity, from the State 
of Guanajuato. 
Place of realization: Hospital Metabolism Laboratory of Division of Health Sciences of the 
University of Guanajuato, León, Gto. 
Type of sampling: Non-probabilistic, of consecutive cases. 
Sample size: The calculation of the sample size was based on the formula for comparing 
proportions.  We hope that the low-dose combination of linagliptin + metformin will 
improve the outcome variables (insulin resistance, pancreatic beta cell function and 
cardiovascular function) by at least 75%, metformin use alone achieves this only by 
30%(44.46), so the study is designed to detect a minimum difference of 45% between study 
groups with a type I (alpha) error of 0.05 and a sample power of 80% , and according to the 
following formula(70): 
 
n = (Zα/2+Zβ)2 p¯ (1-p¯ )(r + 1) 
                       (d)2r 
Where,  
(Zα/2+Zβ)2 = 7.9 
p1 = proportion of individuals in the best treatment who failed to achieve improvement = 
0.25 
p2 = proportion of individuals in the worst treatment who failed to achieve improvement = 
0.70  
r = reason for individuals in treatment groups = 1 
d = difference you're expected to find = 0.45  
n = 14 + 20% of losses = 17 patients per group 
 
 
 SELECTION CRITERIA TO PARTICIPATE 
 
- 
Women and men aged 18 to 65 overweight or obese (BMI 25 kg/m2) 
- 
Patients with prediabetes, defined by: 
1) Glucose intolerance (ITG = glucose at 2h en la CTOG between 140-199mg/dL). 

 
2) The coexistence of fasting impaired glucose (GAA = fasting glucose between 
100 and 125mg/dl) and glucose intolerance (ITG = glucose at 2h in CTOG 
between 140-199mg/dL). 
 
- 
Those who agree to participate in the study sign an informed consent letter. 
 
NOT SELECTED TO PARTICIPATE 
- 
Patients with DM2 previously diagnosed or detected during sampling and CTOG. 
- 
People currently being treated or for the past 3 months with metformin, 
pioglitazone, or any other antidiabetic drug, including insulin. 
- 
Serum creatinine >1.6mg/dl. 
- 
Very high hipertriglyceridemia (>500mg/dl). 
- 
Pregnant women. 
- 
Uncontrolled arterial hypertension (systólic >180mmhg o diastolic >105mmHg). 
- 
Excessive alcohol intake, acute or chronic. 
- 
Medications or medical conditions affecting glucose homeostasis (thiazides, beta 
blockers, systemic glucocorticoids, weight-reducing or anorexigenic drugs, Cushing 
syndrome, thyrotoxicosis) 
 
ELIMINATION 
- 
Intolerance to the medicine used. 
- 
Absence to more than 20 % of follow-up appointments. 
- 
That they do not finalize the study and monitoring protocol. 
 
 
 
VARIABLES 
INDEPENDENT VARIABLES  
1) Intervention 
 
DEPENDENT VARIABLES  

1) Glucose homeostasis. 
2) Sensitivity to insulin. 
3) Pancreatic beta cell function. 
4) Cardiovascular function. 
 
 
 
 
OPERATION 
OF 
VARIABLES, 
MEASUREMENT 
SCALES 
AND 
MEASUREMENT UNITS. 
 
DEPENDENT 
VARIABLE  
CONCEPTUAL 
DEFINITION 
OPERATIONAL 
DEFINITION 
CLASIFICATION 
MEASURE-
MENT 
LEVEL 
INDICATOR 
INTERVENTION 
It is the 
established 
therapeutic 
strategy to 
prevent the 
progression of 
Prediabetes to 
type 2 diabetes 
mellitus and its 
complications. 
A. Administration of 
1 tablet of 
linagliptin 2.5 mg 
/metformin 850 
mg every 24 
hours + 1 tablet 
of metformin 850 
mg every 24 
hours.  
B. Administration of 
1 tablet of 
metformin 850 
mg every 12 
hours. 
Qualitative 
Nominal 
A. Group with 
low dose 
treatment of 
Linagliptin/ 
Metformin. 
B. Metformin 
treatment 
group. 
 
 
 
 
 
 
 
 
 

DEPENDENT 
VARIABLE 
CONCEPTUA
L 
DEFINITION 
OPERATIONAL 
DEFINITION 
CLASIFICATION 
MEASUREMEN
T LEVEL 
INDICATOR 
FUNCTION OF 
THE PANCRETIC 
BETA CELL 
Pancreatic 
beta cell's 
ability to 
secrete 
enough 
Insulin to 
keep blood 
glucose 
levels at 
normal 
levels.  
Value obtained by 
the CTOG, when 
applying the 
formula 
(ΔI0-30)/(ΔG0-30),  
(ΔI0-30) = 
difference in 
insulin value 
measured at 0 
and 30 minutes. 
(ΔG0-30) = 
difference in 
glucose value 
measured at 0 
and 30 minutes. 
Quantitative 
Reason 
Pmol/mmol, 
HOMEOSTASIS 
OF GLUCOSE 
Ability of 
therapeutic 
interventions 
to reverse 
glucose 
intolerance. 
It shall be 
considered as 
YES  when the 
fasting glucose 
level at 6 months 
after the 
intervention 
begins, either at 2 
h during CTOG is 
<140mg/dL, and 
NO when glucose 
levels at 2h 
during CTOG are 
140mg/dL. 
Qualitative 
Dichotomous 
Nominal 
Yes 
No 
SENSITIVITY 
TO INSULIN 
 
Insulin's 
ability to 
promote 
glucose 
uptake by 
muscle 
tissue, and 
suppress 
glucose 
production 
by the liver 
and avoid 
the lipolysis 
in adipose 
tissue. 
It will be 
evaluated using 
Matsuda's index 
with glucose and 
insulin values 
during CTOG 
using the 
following 
formula: Insulin 
sensitivity =  
  
10000 /  
√(G0 x I0 )  x (͠G0-
120 x ͠I0-120 ) 
 
 
Quantitative 
Reason 
Natural numbers 

CARDIO-
VASCULAR 
FUNCTION 
Function of 
the system 
made up of 
heart and 
blood vessels 
for transport 
activities. 
Measurement of 
ejection fraction, 
diastolic and 
systolic preloads 
and ejection 
fraction. 
Quantitative 
Continues 
Percentage % 
 
 
METHODS. 
Identification of patients at risk 
During this initial phase, overweight or obese patients will be identified with and at least 2 
risk factors for DM 2, who will be invited to the screening of the study and as a first step 
will answer a survey of nutrition and physical activity. 
 
Recruitment of patients 
Metabolic evaluation and classification of patients: Once obtaining weight and size, we  
identify overweight or obese patients and risk factors for DM2, they will be invited to 
continue the counting phase; The next step will be to cite patients for fast glucose 
measurement, and only patients who have a glucose level of 100mg/dL will have CTOG 
performed to determine the presence of prediabetes. 
 
Glucose tolerance curve: The tolerance curve consists of citing the patient with an 8-12 hrs 
fast, the vein is channeled to the ulnar with 0.9% 250cc saline solution, basal blood samples 
are taken and 75g of glucose is given to drink and subsequently take blood samples every 
30 minutes for the next two hours (0, 30, 60, 90 and 120min), for glucose determination at 
that time and for subsequent measurement of insulin, glucagon and GLP-1.  As mentioned 
in the selection criteria, patients who are diagnosed with DM2 during screening (CTOG) 
will not be included in the study. 
 
According to glucose values within 2 hours of oral loading, patients will be classified as:  
            1) Glucose-tolerant norms when glucose at 2hrs during CTOG is <140mg/dl,  
2) Patients with glucose intolerance when glucose at 2hrs of CTOG is between 140 
and 199mg/dL, 

3) Recently diagnosed DM2 patients when glucose is 2 hrs. CTOG is 200mg/dL. 
 
Patients with CTOG glucose intolerance are those who will be invited to participate in the 
trial and those who accept will continue with a more thorough metabolic evaluation.  For 
this purpose, patients will have completed a medical history and the following studies: 
 
1) Fasting basal blood samples for measurement of: 
 
- Blood count  
 
- Lipid profile 
 
- Insulin for homa-IR determination, HOMA-B, insulin sensitivity, insulin secretion 
and pancreatic beta cell function. 
 
2) Determination of body composition (% visceral fat, % total body water, bone 
mineral density, etc.) using DEXA analysis. Here the patient scrutiny ends and if at 
this time the patient agrees to participate in the phase of intervention, the following 
will be carried out, after explanation and signature of informed consent letter. 
1) Cardiovascular function assessment: This will be done through standard 
echocardiography area of the High Specialty Regional Hospital. 
2) Evaluation of renal function: Through 24-hour urine collection, which will measure 
creatinine clearance and 24-hour albuminuria. 
3) Randomization: It will be done by using a table of random numbers generated by a 
computer program. Patients will be randomized to receive, for 6 months, one of the 
following 2 interventions: 1) Dosis of linagliptin 2.5 mg every 24 h + metformin 1700 
mg every 24 h y 2) 1 tablet every 12 h of Metformin 850mg. 
 
THIRD PHASE: Intervention and follow-up 
 
All patients will receive nutritional guidance and physical activation during the study. Once 
patients have all baseline measurements and have met the selection criteria, they will be 
randomized to one of the two interventions. All patients will have monthly follow-up and 
global clinical evaluation and treatment attachment, and anthropometry. 

 
FOURTH PHASE: Ending of the study (final measurements). 
 
At 6 months of treatment, patients will be quoted again in fasting, on two different 
occasions, 
1) FIRST APPOINTMENT: For measurement of glucose and fasting insulin, 
determination of body composition by imdoanciometry and performance of 
CTOG. 
2) SECOND APPOINTMENT: Evaluation of cardiovascular function by 
echocardiography, and renal function by depurating creatiline and albumin in 
urine of 24h. 
 
 
STATISTIC ANALYSIS 
 
The data will be presented with descriptive statistics, mean and standard or median 
deviation and interquartile travel, as well as percentages and incidence.  For comparisons of 
numeric variables between study groups, we will use Student t if the distribution of the data 
follows a normality pattern, otherwise the Mann-whitney U test will be used; to compare 
qualitative variables we'll use square Chi.  Analysis by intent to be treated and analyzed by 
protocol shall be performed and considered as significant when the p-value is <0.05.  For 
the analysis and presentation of the data we will use the sPSS Version 15 and GraphPad 
Prism program 5. 
 
 
ETHICAL ASPECTS 
 
Ethical standards, Regulations of the General Health Law on Health Research and the 
Helsinki Declaration of the World Medical Association of the 64th General Assembly, 
StrongHold, Brazil in 2013 and current international codes and standards of good clinical 
research practices.  Special attention will be given to taking care of the privacy and 

autonomy of the patient.  Once the patient has been informed about the study and agrees to 
participate in it, and since it involves a greater risk to a minimum, the signature of informed 
consent letter will be requested. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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