Clinical trial NCT03108521
Conditions: Diabetes Mellitus
Keywords: DPP-4; GLP1R ; gene polymorphism research; diabetes
Sponsor: Sichuan Provincial People's Hospital
Start date: 2016-04-21; Completion date: 2018-10-08
Enrollment: 119 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Incretin-based therapy is currently one of the most popular diabetes treatment approaches. However, differences of response ware found in previous studies. We hypothesis that SNPs of DPP-4, GLP-1 and GLP-1R genes may play crucial roles in the response differences. Therefore, this study aims to investigate the correlation of incretin-related gene polymorphism and individual differences in the response of DPP-4 inhibators (take Sitagliptin as an example). In addition, The distribution differences of the SNPs in diabetics and non-diabetics are evaluated to study the relationships between the SNPs and diabetes onsets.

Detailed description:
Single Nucleotide Polymorphism (SNP) plays an important role in the differences of clinical manifestations and drug responses of diseases. The vast majority of SNP sites are located in the non-coding region of the gene (about 95%), which is called SNP(non-coding SNP (ncSNP), while the other part of SNP is located in the coding region of the gene, which is called coding SNP (cSNP). Furthermore, cSNP can be divided into two categories: SNP that does not change the encoded amino acid sequence is called synonymous SNP(synonymous SNP, SSNP); SNP that changes amino acid sequence is called SNP(non-synonymous SNP (NSNP). Although not involved in coding amino acid, some ncSNPs may also affect the regulation of protein expression. Therefore, it is of great significance to study the effects of NC SNP and cSNP on the occurrence and development of diseases and drugs.

DPP-4 enzyme inhibitor is combined with DPP-4 enzyme in human body to reduce hydrolysis of active GLP-1, thus increasing the level of endogenous active GLP-1. Active GLP-1 combines with its receptor GLP-1R to promote insulin release and inhibit glucagon release in hyperglycemia state, and produces opposite effect in hypoglycemia state.

Based on the above principles, we speculate that SNP of genes that may affect the hypoglycemic effect of DPP-4 enzyme inhibitor are:

1. SNP of DPP-4 enzyme gene. SNP of DPP-4 enzyme gene may affect the enzyme activity and/or protein expression level of DPP-4. Assuming that the effect of DPP-4 enzyme inhibitor is sufficient, patients with higher DPP-4 enzyme activity are more sensitive to DPP-4 enzyme inhibitor drugs; However, for patients with low DPP-4 enzyme activity, DPP-4 enzyme inhibitor drugs cannot play a stronger role in lowering blood sugar.
2. SNP of GLP-1 gene. SNP of GLP-1 gene may affect activity or expression level of GLP-1. Patients with high GLP-1 level are more sensitive to DPP-4 enzyme inhibitor drugs.
3. SNP of GLP-1R gene. SNP of GLP-1R gene may affect activity or expression level of GLP-1R. Patients with high GLP-1R level are also more susceptible to DPP-4 enzyme inhibitor drugs.

However, studies on the hypoglycemic effect of DPP-4, GLP-1 and their receptors on DPP-4 enzyme inhibitors in the treatment of T2DM are rare, which is not conducive to the evaluation of individualized treatment of such drugs. Therefore, this chapter intends to select SNP sites with high mutation frequencies of DPP-4, GLP-1 and GLP-1R genes to study the mutation frequencies of these SNPs in diabetic patients and non-diabetic patients and their effects on DPP-4 enzyme inhibitor sitagliptin's hypoglycemic effect on T2DM patients.

Arms: arm 1: Patients in this group will accept Sitagliptin phosphate tablets as their intervention. Specifications: Each tablet 100mg (with sitagliptin dollars). Regimen: The recommended dose is 100mg.QD for 3 months. arm 2: Subjects in this group are T2D free. We use their gene information to study SNP differences between T2D patients and non-T2DM people.
Interventions: intervention 1: Sitagliptin
Intervention descriptions: intervention 1: 100mg.QD for 3 months

Protocol documents:
Prot_SAP_000.pdf: 
Application number：15ZC0669
Project number：2015SZ0182
project name: Study on Polymorphism of DPP-4 and GLP-1R Genes in Chinese Population
and Its Empirical Study on Treatment of Diabetes
NCT number:NCT03108521
Document date：2017-03-01


Study protocol
1.1 Introduction
Single Nucleotide Polymorphism (SNP) plays an important role in the
differences of clinical manifestations and drug responses of diseases. The vast
majority of SNP sites are located in the non-coding region of the gene (about 95%),
which is called SNP(non-coding SNP (ncSNP), while the other part of SNP is located
in the coding region of the gene, which is called coding SNP (cSNP).
Furthermore,
cSNP can be divided into two categories: SNP that does not change the encoded
amino acid sequence is called synonymous SNP(synonymous SNP, SSNP); SNP that
changes amino acid sequence is called SNP(non-synonymous SNP (NSNP)[130] .
Although not involved in coding amino acid, some ncSNPs may also affect the
regulation of protein expression. Therefore, it is of great significance to study the
effects of NC SNP and cSNP on the occurrence and development of diseases and
drugs.
DPP-4 enzyme inhibitor is combined with DPP-4 enzyme in human body to
reduce hydrolysis of active GLP-1, thus increasing the level of endogenous active
GLP-1. Active GLP-1 combines with its receptor GLP-1R to promote insulin release
and inhibit glucagon release in hyperglycemia state, and produces opposite effect in
hypoglycemia state.
Based on the above principles, we speculate that SNP of genes that may affect
the hypoglycemic effect of DPP-4 enzyme inhibitor are:
(1) SNP of DPP-4 enzyme gene. SNP of DPP-4 enzyme gene may affect the
enzyme activity and/or protein expression level of DPP-4. Assuming that the effect of
DPP-4 enzyme inhibitor is sufficient, patients with higher DPP-4 enzyme activity are
more sensitive to DPP-4 enzyme inhibitor drugs; However, for patients with low
DPP-4 enzyme activity, DPP-4 enzyme inhibitor drugs cannot play a stronger role in
lowering blood sugar.
(2) SNP of GLP-1 gene. SNP of GLP-1 gene may affect activity or expression
level of GLP-1. Patients with high GLP-1 level are more sensitive to DPP-4 enzyme
inhibitor drugs.
(3) SNP of GLP-1R gene. SNP of GLP-1R gene may affect activity or expression
level of GLP-1R. Patients with high GLP-1R level are also more susceptible to DPP-4
enzyme inhibitor drugs.
However, studies on the hypoglycemic effect of DPP-4, GLP-1 and their
receptors on DPP-4 enzyme inhibitors in the treatment of T2DM are rare[131], which is


not conducive to the evaluation of individualized treatment of such drugs.
Therefore,
this study intends to select SNP sites with high mutation frequencies of DPP-4, GLP-1
and GLP-1R genes to study the mutation frequencies of these SNPs in diabetic
patients and non-diabetic patients and their effects on DPP-4 enzyme inhibitor
sitagliptin's hypoglycemic effect on T2DM patients (Fig. 1-1).
Figure 1-1 Study design
1.2 Research Design and Selection of Subjects
SNP Site Selection
The SNP site selection in this study is based on the following principles: (1) The
SNP site is DPP-4, GLP-1 or its receptor GLP-1R; (2) The mutation frequency of the
SNP site in Chinese Han population is greater than or equal to 30% (the query of
mutation frequency is based on NCBI database); (3) Studies have reported that the
SNP site may be related to the occurrence, development and treatment of T2DM.
Study on the Distribution Difference of SNP Sites between Diabetic Patients and
Non-diabetic Patients
Subject
screening
and
informed
Diabetes group
Non-diabetes group
Sitagliptin,
100mg/d,
observed for 12 weeks
Massarray SNP classification
Effect of SNP locus on the
efficacy of sitagliptin
Screening of Diabetes Mellitus
SNP Sites
SNP site mutation frequency
of DPP-4 and GLP-1R gene
Evaluation
of
HbAlc,
OGTT,
DPP-4
enzyme
activity
and
GLP-1 levels at baseline and at
the last follow-up


In order to study the distribution frequency of different genotypes of selected
SNP loci in diabetic patients and non-diabetic patients, inclusion exclusion criteria for
two types of subjects are specially set.
Diagnostic criteria for T2DM:
According to the diagnostic criteria for diabetes in the guidelines for the
prevention and treatment of T2DM in China[1](2013 edition) published by the
Diabetes Credit Association of the Chinese Medical Association in 2014: diabetic
symptoms (symptoms of acute metabolic disorders such as polydipsia, polyphagia,
polyuria, weight loss, skin pruritus, blurred vision, etc. caused by hyperglycemia),
patients diagnosed as T2DM with random blood glucose ≥11.1mmol/L, or fasting
blood glucose (FPG) ≥7.0 mmol/L, or 2h glucose ≥11.1mmol/L after glucose
loading.
The included non-diabetic patients have no major diseases such as tumor, no
chronic diseases such as dyslipidemia, hypertension, and non-prediabetes whose
blood sugar and glycosylated hemoglobin values cannot meet the criteria for
diagnosing T2DM, and are over 50 years old.
SNP of DPP-4, GLP-1 and Their Receptors on the Effect of Sitagliptin on T2DM
Inclusion criteria for patients
①According to the Chinese Medical Association, the diagnostic criteria for
diabetes in the Guidelines for the Prevention and Treatment of T2DM in China
published by the Diabetes Credit Association in 2014 are diagnosed as T2DM patients,
and glycosylated hemoglobin (calculated by HbA1c) is between 7% and 9%;
②age between 18 and 70 years old;
③BMI index ≥18.5;
④not receiving hypoglycemic treatment within 4 weeks, or receiving
hypoglycemic treatment within 4 weeks, but not changing the treatment plan
within 3 months;
⑤not participating in any clinical trial within 3 months;
⑥no serious heart, brain, liver, kidney and other diseases;
⑦signing the informed consent form.
Exclusion criteria for patients
①BMI is greater than 40;
②GLP-1 or DPP-4 drugs were taken orally in the past month;
③immunocompromised persons;


④patients with C peptide < 0.3ng/ml;
⑤being allergic to GLP-1 and DPP-4 enzyme inhibitor drugs;
⑥pregnant or lactating patients;
⑦patients taking drugs that may affect DPP-4 enzyme inhibitor metabolism;
⑧patients with severe cardiac, renal, liver and respiratory insufficiency,
patients with past or family history of medullary thyroid carcinoma (MTC), and
patients with type 2 multiple and endocrine tumor syndrome (MEN2);
⑨pancreatitis and drug abusers have occurred in the past year.
Exit criteria:
①Serious adverse reactions occurred during the implementation of clinical
research;
②compliance with medication＞120% or＜80%;
③patients unwilling to continue this clinical study.
Index Detection and Sample Collection
Subjects with T2DM will receive OGTT and HbA1c tests after entering the
group. OGTT tests will be carried out routinely in the nuclear medicine department of
Sichuan Academy of Medical Sciences and Sichuan People's Hospital, and HbA1c
tests will be carried out routinely in the laboratory of Sichuan Academy of Medical
Sciences and Sichuan People's Hospital Medical Center. The subjects collected blood
samples on an empty stomach in the morning. Professional nursing staff collected 8ml
of venous blood from the upper limbs of the patients using disposable venous blood
collection needles, and EDTA anticoagulation tube was used for preservation.
5ml
of whole blood was reserved for DNA extraction, centrifuged at 3000rpm for 10min,
and plasma and blood cells were separated and stored at -80℃.
Sitagliptin Treatment Scheme for T2DM Subjects
The diabetes patient was treated with sitagliptin phosphate 100mg qd for 12
weeks without changing the original treatment scheme. Sitagliptin Phosphate Tablets
were purchased by the research group from Mercadon (China) Investment Co., Ltd.
according to the scientific research and drug purchasing process of Sichuan Academy
of Medical Sciences and the Ministry of Science and Technology of Sichuan
Provincial People's Hospital.


Benefits and risks of subjects
For non-diabetic patients, they will receive diabetes knowledge education once
free of charge and take 5ml of venous blood from upper limbs. For diabetic patients,
they will receive 100 mg qd of sitagliptin phosphate for 12 weeks free of charge on
the basis of the original treatment, and will receive OGTT examination and
glycosylated hemoglobin examination free of charge at the time of admission and at
the end of the study. During OGTT examination, an additional 8ml venous blood was
drawn for SNP site, DPP-4 enzyme activity and GLP-1 level detection. Patients will
receive free professional consultation from clinicians with senior professional titles in
endocrinology when they leave the group, and the treatment plan will be readjusted
according to the blood glucose results of patients.
1.3 Statistical analysis
Microsoft EXCEL is used for data sorting and preprocessing. The HbA1c at
the end of clinical study minus HbA1c at baseline was used to obtain the HbA1c
reduction value, and the same was true for fasting blood glucose and postprandial
blood glucose. For each group of data difference (e.g. Δ HbA1c) data of each
genotype, the position test is performed, i.e. whether the difference is statistically
significant with 0 is tested. When the group of data meets the normal distribution, the
student t test is used for analysis, and when the normal distribution is not met, the
symbolic rank test (S test) is used for analysis. Continuous variables are described by
means of average standard deviation, and qualitative variables are described by means
of percentage. The following methods are used to test the difference of continuous
variables in patients with different genotypes: when the data of each group conform to
the normality and the homogeneity of variance, the hypothesis test is carried out by
variance analysis; when one group does not meet the normality or the homogeneity of
variance, wilcoxon rank sum test is used for analysis, Kruskal-Wallis Test is used for
non-parametric test, and the two comparisons between each group are carried out by T
test based on the estimation of the difference within the group of population samples
and LSmeans method. The difference test of blood glucose index control rate
(HbA1c<7%，FPG< 7mmol/L，PPG<10mmol/L) in patients with different genotypes
was conducted byχ2 test, and Fisher exact test was used for hypothesis test in case of
theoretical cell frequency< 5. SAS 9.21 is used to implement the statistical process.
Set the significance level at 0.05. If there are less than 3 data in a single group,


statistical analysis will not be conducted because intra-group differences cannot be
estimated.