A randomized control trial to study remission of type 2 diabetes with intensive lifestyle modification versus a combination of oral semaglutide and dapagliflozin as an add-on to metformin monotherapy in comparison to standard medical management.A randomized control trial to study remission of type 2 diabetes with intensive lifestyle modification versus a combination of oral semaglutide and dapagliflozin as an add-on to metformin monotherapy in comparison to standard medical management.

Aims and Objectives

 Aim:

“To Compare Remission Approaches for Type 2 Diabetes

• Intensive Lifestyle Modification vs. Combination Therapy with Oral Semaglutide and Dapagliflozin as an addon to Metformin vs. a control arm receiving glimepride and vildagliptin in combination with Metformin”

Primary Objective:

1)    To determine the remission rates of Type 2 Diabetes in participants undergoing intensive lifestyle modification compared to those receiving combination therapy of oral Semaglutide and Dapagliflozin as an add-on to Metformin monotherapy, in comparison to control arm receiving glimepride and vildagliptin in combination with Metformin.

2)    Exploration of transcriptome and epigenome landscape and determine its correlation with the remission of diabetes.

3)    Determine the key pathways and proteins associated with diabetes.

Secondary Objectives:

1. To assess and compare the impact of intensive lifestyle modification, combination therapy of oral Semaglutide and Dapagliflozin, and standard medical management on the following parameters:

·                                  Glycemic control, as measured by HbA1c levels.

·                                  Weight loss and changes in body composition.

·                                  Blood pressure and lipid profile.

·                                  Quality of life and overall well-being.

2. To analyze the safety and tolerability profiles of intensive lifestyle modification, combination therapy of oral Semaglutide and Dapagliflozin, and standard medical management in participants with Type 2 Diabetes.

3. To assess the adherence and sustainability of the intensive lifestyle modification intervention and the pharmacotherapy regimens over the study duration.

4. To identify predictors of successful remission in participants undergoing each treatment approach.

5. To evaluate participant-reported outcomes, including satisfaction with treatment, treatment preferences, and perceived health improvements.

6. To assess the change in pancreatic fat pre and post intervention with MRI and MRS of Pancreas in all the groups.

7. To assess the change in level of gastrointestinal tract and Adipose tissue derived peptide levels viz. GLP1, Adiponectin, Leptin pre and post intervention among the study groups.

 To assess the change in quality of life by pre-validated SF-36 questionnaire pre and post intervention, in both the group

 To assess glycemic variability and risk hypoglycemia with the help of  CGMS in both intervention and control groups prior to intervention, during intervention phase and after intervention follow up period.

To study the body composition and metabolic vascular age prior to and after intervention.

Material and Methods

 This randomized control trial will be performed in PGIMER, a tertiary care health institute in Chandigarh UT, India. The study will be conducted in the department of Endocrinology. Ethical approval will be obtained from the ethics review committee of the institute. A written informed consent will be obtained from every participant.

 Duration of Study: 3 years

 **Sample Size:**This is is a pilot study using this pharmacotherapy to target remission of diabetes. therefore a sample size of convenience has been choosen and a  sample size 15 in each group will be enrolled.

 Study Subjects: This study will be conducted among type 2 diabetes mellitus patients visiting the Endocrinology and Medicine OPD of PGIMER, Chandigarh who will full fill the Inclusion and Exclusion criteria.

 Inclusion Criteria:

 Type 2 Diabetes Mellitus diagnosed within 2 years with

 HbA1c <9 %.

 Exclusion Criteria:

 Age <18 years or > 65 years.

 BMI<23kg/m2

 Current Insulin/ GLP1 analogue or SGLT-2 inhibitor therapy.

 Acute or Chronic Pancreatitis

 History of Ketoacidosis.

Objective evidence of Diabetic Neuropathy.

 Renal Failure defined as eGFR<60 ml/min/1.73m2.

 24hr Urinary protein >500 mg or Albumin to Creatinine ratio >300mg/g.

 Moderate to severe non-proliferative diabetic retinopathy, proliferative diabetic retinopathy, diabetic maculopathy.

Liver disease defined as ALT> 3 times ULN, Bilirubin > 2 mg/dl, USG evidence of Cirrhosis.

Any major chronic illness.

 Symptomatic Coronary artery disease/ Peripheral arterial disease/ cardiac failure/ diabetic foot/ Acute coronary syndrome <3 months duration

On steroid treatment.

 Pregnancy / Lactation

 Drug specific CI like MCT, MEN2.

 Positive GAD

 Fasting C -peptide <0.6 ng/ml

 Post COVID-Diabetes (onset of diabetes within 3 months of COVID)

 Methodology:

 All consecutive patients with type 2 DM will be recruited following fulfillment of Inclusion and exclusion criteria.

All patients will undergo baseline clinical and biochemical assessment as per proforma.

All patients will be advised lifestyle modification and dietary advice, statin therapy, blood pressure control.

All patients will be assessed for chronic diabetes related complications.

Baseline body fat composition, GLP-1 level, adiponectin, leptin level will be done for all patients.

Baseline Mixed meal tolerance test will be done for all patients.

All antidiabetic drug other than metformin will be stopped.

Metformin will be added or continued in all patients.

A run-in phase of two weeks will be given for drug washout.

Patient will be randomized in two groups by the help of random number generating software.

 In Study group A, patients will receive Dapagliflozin 5 mg and that will be escalated to 10 mg after one week,

Semaglutide will be titrated as 3 mg for 2 weeks then 7 mg for 2 weeks and then 14 mg. If anybody developed mild gastro-intestinal complications, same titration schedule will be followed. If severe gastro-intestinal intolerance occurs following dose escalation, a more gradual escalation over 4 weeks will be tried. The maximum tolerated dose will be continued.

Metformin will be titrated to 2 gm prior to intervention with other medication.

 Insulin will be added if glycemic target is not achieved and those patients will be included in final analysis in Intervention arm only.

Monitoring will be done every week.

 In Study Group B, will continue to receive metformin and intensive lifestyle modification will be advised as follows,

 Plan is to introduce raw food in their diet to achieve a ratio raw Diet (80) : Cooked Diet (20).

Fruits and salads are rich sources of raw food.

We will give the patients a diet having more of uncooked food like fruits and salads and less quantities of cooked food like chapati, dal tadka and cooked sabzi for 3 months and then we will follow the same diet with some variation for next 3 months. The dietary modifications will be such thatthese can be sustained for entire life.

The diet schedule will be as follows:

Juice Time

Sprout Time

Dryfruit Time

Coconut Water Time

Citrus Fruit Time

Lunch Time

Juice Time

Flax Seed Time

Dinner Time

 Diet Chart is prepared in such a way that the ratio of carbohydrates, proteins and fats is maintained. All vitamins and minerals are there in the diet. Fiber content is kept high in the diet.

As this diet is followed with a discipline, following time schedule is being given with the diet:

Time

Schedule

|05:30 AM

3 Glasses luke warm water

|06:00 AM to 07:00 AM

Bath and Exercise

|07:00 AM

Juice Time

|08:30 AM

Sprout Time

|10:00 AM

Dry Fruit Time

|11:00 AM

Coconut Water Time

|12:00 Noon

Citrus Fruit Time

|01:30 PM

Lunch Time

|04:00 PM

Juice Time

|05:30 PM

Flaxseed Time

|07:00 PM

Dinner Time

   

Quantity

Calories ( In Kcal)

|Juice Time – Bottle Gourd or Safed Petha, Juice of Karela Juice

300 ml

22

|Sprout time – Moong Sabut Sprouts

Salad

100 gm

300gm

30

61

|Dry Fruit Time

Almonds

Moong Phali

Walnut

Resins

Apricot

Fig

 Pumpkin Seeds

Sunflower Seeds

Melon

Water melon Seeds

 + Cinnamon Tea ( w/o milk or sugar)

• 10 Leaves each of Basil, Mint, Neem and Curry Patta

 6

6

1

5

1

1

 5g

5g

5g

5g

   42

 26          120

15

17

20

 27

28

27          112

30

  20

|Coconut Water Time

250 ml

44

|Citrus Fruit Time

Amla

and

Mausambi

 2

 1

 5

 43

|Lunch Time

Salad (cucumber, carrot, reddish, beetroot etc.)

Chapati

Boiled Daal

 700 gm

 Â½

1 Cup

 119

 53

147

|Juice Time

Bottle Gourd Juice

 300 ml

 22

|Makhane Time

Fox Nut Seeds

Flax Seeds

Apple

• 1 Cup cinnamon tea w/o milk or sugar

 20

10 gm

1

 18

53

90

|Dinner Time

Salad (cucumber, carrot, reddish, beetroot etc.)

Chapati (Small)

Boiled vegetable

 700 gm

 Â½

1 cup

 119

 53

45

Total Calorie Intake

Item Name

Calorie Intake

|Morning Juice Time

22 Kcal

|Sprout Time

91 Kcal

|Dry fruit Time

252 Kcal

|Coconut Water

44 Kcal

|Citrus Fruit

48 Kcal

|Lunch Time

319 Kcal

|Evening Juice Time

22 Kcal

|Makhana Time

151 Kcal

|Dinner Time

217 Kcal

|2 Lemons (at any time as per choice)

34 Kcal

|Total

1200 Kcal

It is necessary for the patient to adopt active life style. Patients will be adviced to do atleast one hour of physical activity which will include eith brisk walk, dance , yoga or anyother work out. We will give Yoga module to the patients consisting of Asanas, Pranayam and meditation.  We have adopted Intensive Indianized Life Style (IILS) using Yog as a tool for balancing the mind. ‘Yog’ is derived from Sanskrit word ‘YUJ’ which means ‘to connect’. So in every asna being performed by the practioner, his consciousness should remain aware of the body movements. While performing any asana, we have to visualize that body part which is being used in that asana. Gradually this will lead to perfection of the asana. As BMI of these patients is >25, participants are advised to do loosening exercises so as to make the body flexible.

After taking 3 glasses luke warm water, participants are advised to do

Tadasana = 10 Times

Tiryak Tadsasana = 10 Times left side

10 Times right side

Katichakrasana =  10 Times each side

10 times right side

If motion (potty) does not happen, then again the participant should take one more glass of luke warm water and practice.

Tadasana = 5 Times

Tiryak Tadsasana = 5 Times each side

Katichakrasana = 5 Times each side

 After this we start with following loosening practices :

Pivot Joint Movement:

Neck Up Down = 10

Neck Left Right = 10

Neck Rotation :

Clockwise = 10

Anticlockwise = 10

Ball and Socket Joint Movement (Shoulder)

Shoulder Up and Down = 10

Shoulder Rotation = 10

Hip Joint Movement

Hip Rotation Clockwise = 10

Hip Rotation Anticlockwise = 10

Knee Movement

Knee Bending Up and Down = 10

Ankle Movement

Malasana = 3 times with 10 second rest

 TRIKONASANA = 5 times from left side, 5 times from right side

Jumping on Toes (Feet Forward) = 30 Times | Pause | 30 Times | Pause | 30 Times

Jumping on Toes (Feet Backward) = 30 Times | Pause | 30 Times | Pause | 30 Times

Jumping on Toes (Feet Sidewise) =30 Times | Pause | 30 Times | Pause | 30 Times

Skipping = 30 Times | Pause | 30 Times | Pause | 30 Times

**Sr.
No**

Yoga Practice

Duration

|1

Prayer and Affirmation

1 Minute

|2

Loosening Practices

Neck bending

Shoulder Movement

Hip Movement

Knee Movement

5 minutes

|3

Yogasana

30 minutes

| |Standing Postures

Tadasana

Tiryak Tadasana

Kati Chakrasana

Hastttotaanasana

Pad Hastasana

Trikonasana

Agnisaar

Sitting Postures

Bhadrasana

Vajrasana

Ushtrasana

Shashank Asana

Bhu Naman Asana

Vakrasana

Ardha Matsyendra Asana

Janu Shirasana

Paschimottansana

|Prone Postures

Makrasana

Bhujangasana

Shalabhasana

Naukasana

Dhanurasana

Balasana

Supine Postures

Uttan Padasana

Paad Chakrasana

Kandharasana

Halasana

Chakrasana

Pawan muktasana

|4

Shatkriya

Kapaalbhati

3 minutes

|5

Pranayama

Nadi Shodhan

Seetkari or Sheetli

Bhramari

10 minutes

|6

Meditation or Yog Nidra

10 Minute

|7

Thanks to the Mother Nature

1 Minute

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           

Level of Glycemic off
target

Drug and Dose

|0-30 mg/dl

Vildagliptin 100 mg/day

|20-50 mg/dl

Vildagliptin 100 mg/day + Glimepiride 1mg

|50-75 mg/dl

Vildagliptin 100 mg/day + Glimepiride 2 mg

|75-100 mg/dl

Vildagliptin 100 mg/day + Glimepiride 3 mg

|>100 mg/dl

Vildagliptin 100 mg/day + Glimepiride 4mg

For control arm a prespecified formulation with Glimepiride and Vildagliptin will be added as per target and actual plasma glucose level.

Group C- Vildagliptin and glimepiride will be added according to the following titrate. Patients will be advised

        standard lifestyle modification i.e., 150 min of brisk walk or any other activity and 1200 Kcal diet as stated above.

Vildagliptin and Glimepiride will be started based upon the difference between the patient’s glucose values and the target values. If glucose levels are off target levels, dose titration will be done approximately as following,

Dose titration will then be done on each follow up based on glucose level. Further glycemic control if required will be done by alpha-glucosidase inhibitor. They will not be started on SGLT2 inhibitor and GLP-1 analogue. Monitoring and titration will be done each week.

Once glycemic targets i.e., FBS<100 and PPBS< 140 are achieved in both the groups, patients will be treated for a total of 24 weeks with the drugs and dose required to maintain glycemic targets.

After 24 weeks following achieving glycemic goal, therapy will be stopped in patients who have achieved HbA1c of <6.5%. All patients will undergo mixed meal challenge test, body fat composition, GLP-1 level, adiponectin, leptin level assessment.

MRI and MRS will be done in those patients who have undergone tests at baseline.

 Quality of life will be assessed pre and post intervention in both the group by SF-36 pre-validated questionnaire.

Symptomatic and biochemical hypoglycemia and glycemic variability will be assessed in both the groups by CGMS at base line during intervention at 3 months and during post intervention follow up period.

 Following discontinuation of drugs, patients who achieve glycemic target in both the arm will be followed monthly for 3 months and 3 monthly till the completion of this study or till FBS > 126 mg/dl or HbA1c> 6.5%, whichever come earlier.

Remission

Patients will be deemed to be in remission who will have HbA1C of <6.5% after 3 months of stopping all the medications.

 Patients will be called to out-patient department monthly in all the groups for dose titration and councelling.

Monthly Gpaq will be to assess adherence to lifestyle intervention. Global physical activity questionnaire scoring will be done at baseline.

In all the groups once glycemic targets are achieved, the patients will be treated for total of 24 weeks, including dose titration time, with the same drugs and dose required to achieve glycemic target.

At the end of the 24 weeks, all drugs will be stopped for those patients achieved HbA1c of < 6.5%. All patient will undergo Mixed meal test, GLP1, Adiponectin, Leptin level assessment and Pancreatic MRI and MRS at the base line, the same will be repeated in them.

 Target glycaemia during intervention:

Target glycaemia will be considered as Fasting Plasma Glucose < 100 mg/dl and PP Plasma Glucose <140 mg/dl.

If patient is on SMBG, patient will be asked to follow 7-point sugar profile for 3 days immediately prior to planned visit.

Average of 3 days fasting will be considered.

Average of 3 post meal glycaemic excursion will be considered for PPBG.

If on CGMS TIR 90%, will be considered as optimum.

Safety measures:

All patients will be counselled about the possibility of hypoglycaemia.

Corrective action will be taken.

Severely hypoglycaemic patients will be excluded from the study.

Those who will have adverse effect requiring drug discontinuation will be removed from the study.

Transcriptome and Epigenome

We will achieve genome-level correlations by performing deep multi-omics profiling to determine the transcriptome (i.e. genes) and epigenome(i.e. non-coding RNA) alterations using ultrasensitive RNA sequencing. The RNA sequencing approach allows measurement of expression of messenger RNA and non-coding RNA in a single assay with very high genome-wide coverage and great sensitivity. Further, systems biology analysis using interactive network analysis approach will be generated to identify the multi-layered network of interactive molecules that are altered. The pathways and modules with highest over-representation of dysregulated molecules will be considered a multi-omics T2DM derived pathogenesis signature.

Transcriptome and epigenome analysis of samples to understand mechanism and identify biomarkers:

In order to generate most comprehensive and global view of molecule alteration in T2DM. We will analyze the transcriptome including genes, miRNAs, and ncRNAs as well as their interactions across 4 groups (T2DM, healthy and three treatment groups). Transcriptome and epigenome analysis: Total RNA will be extracted with kit using manufacturer‘s protocols. RNase-free water will be added to resuspend RNA. Then a spectrophotometer will be used to detect the concentration, and agarose gel electrophoresis will be use to confirm the purity and integrity of the total RNA. Whole genome transcriptome (mRNA and non-coding RNA) profiling: Transcriptomes of blood from all four groups will be performed by next-generation sequencing (NGS) to generate deep coverage RNASeq data. NGS is a very sensitive and highly accurate technology for RNA quantification with no attenuation of signal at the high end of the dynamic range as happens with microarray platforms (Llorens et al., 2011). Transcriptome analysis will yield mRNA, splice variants, miRNA, and ncRNA expression levels, linked to T2DM. Sequencing libraries will be generated from the double-stranded cDNA using the Illumina TruSeq kit according to the manufacturer's protocol. Library quality control will be performed using the Agilent DNA High Sensitivity Chip and qRTPCR. Libraries will be sequenced on an Illumina X Ten.

Analysis of Transcriptome sequencing data:

RNA sequencing data will be analyzed using standard statistical algorithms after quality control filtering, alignment to reference genome and normalization to identify differentially expressed genes. We have developed an in-house package ―GEXPAS‖ for quality control, normalization and preprocessing of NGS and microarray data. Briefly, high quality reads from each sample will be mapped to the human genome using Hisat2 algorithm. The abundance of the mapped reads will be estimated using the exon-intersection approach in HT-SEQ. The transcript abundance data will be normalized for library complexity and sample sequencing depth to reduce technical noise and false positive results. Normalized data will be analyzed using unsupervised learning such as Principal Component Analysis (PCA) and hierarchical clustering. This should identify clusters of samples that are proximate in Euclidean or correlation space, and are mechanistically related. Standard statistical methods (e.g. baySeq, DEGseq and edgeR(HYPERLINK "E:\Projects\DHR_2019\DBT Allience grant

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with multiple test corrections (e.g. Benjamini and Hochberg method (HYPERLINK"E:\Projects\DHR_2019\DBT Allience grant 2019\correction&quot;l"_ENREF_4" o "Benjamini, 1995 #11"Benjamini and

Hochberg, 1995)) will be used to identify the sets of transcripts and splice variants that are significantly differentially expressed across various groups. The comparative analysis of results from Healthy, T2DM and Treatment groups will help in identifying transcripts that are specifically associated T2DM.

 STUDY OUTCOMES

Primary outcome:

To find out the remission rates in patients with Type 2 diabetes mellitus treated with a combination of oral Semaglutide and dapagliflozin vs lifestyle modification on top of metformin monotherapy, and to compare with the standard diabetes practice.

Secondary outcome:

To measure changes in beta- cell function following treatment, as assessed by mixed meal tolerance test.

STATISTICAL ANALYSIS

Statistical analysis will be performed according to the results obtained from parameters. Discrete categorical data will be represented in the form of either a number or percentage, continuous data assumed to be normally distributed, will be written as either in the form of its mean and standard deviation or in the form of its median and interquartile range, as per requirement. The normality of quantitative data will be checked by measure of Kolmogorov- Smirnov tests of normality. Student t-test or Mann Whitney U test will be applied to compare 2 groups at a time depending upon the normality of the data. Proportions will be compared using Chi-square or Fisher’s exact test, depending on their applicability for 2 groups. For comparison of time related variables Wilcoxon Signed rank test or paired t-test will be applied. ANOVA will be carried out when we will be comparing 3 groups at a time. All the statistical test will be two-sided and will be performed at a significant level of p<0.05.

Statistical analysis will be done by IBM SPSS statistics version 22.0 and Microsoft Excel.