Efficacy Study of Pioglitazone and Metformin and Association Between Pioglitazone Response and Peroxisome Proliferator-activated Receptor Gamma Gene Variants in Bangladeshi Type 2 Diabetes Mellitus Subjects
Condition: Type 2 Diabetes Mellitus
MeSH terms: Diabetes Mellitus; Diabetes Mellitus, Type 2
Study type: Interventional
Phase: Phase 4

Summary: 
      -  The present study was undertaken to assess the efficacy and safety of two different
           insulin sensitizers (namely Pioglitazone and Metformin) among subjects with type 2
           diabetes mellitus (T2DM) in Bangladesh.

        -  A prospective, double-blind, single group, 'within-subject' designed clinical trial of
           77 diagnosed T2DM patients out of 130 patients with glycosylated haemoglobin (HbA1c)
           ≥7.2±1.5%, aged 46±6.4 years and registered for diabetes treatment in Bangladesh
           Institute of Research and Rehabilitation in Diabetes Endocrine and Metabolic Disorders
           (BIRDEM) was carried out.

        -  The study was conducted between November 2008 and September 2010.

        -  Baseline data, included case history of the patients,anthropometric measurement,
           biomedical parameters psychosocial factors, were collected from each subject and then
           enrolled to receive treatment with 001 drug once daily for three months, then the
           patients were left for wash out with metformin 850mg once daily for one month; then they
           received 002 drug once daily for further three months.

        -  Dietary chart was remained as before.

        -  DNA was isolated by Chelex method using the primers and control DNA,restriction
           Digestion Enzyme Endonuclease Hae 111 for genotyping PPARγ-(Peroxisome Proliferator
           Activated Receptor gamma)Pro12Pro

             -  (Proline12Proline)/Pro12Ala-(Proline12 Alanine))/Ala12Ala-(Alanine12Alanine).

        -  The blinded drugs were decoded after analyzing results, 001 tablet was pioglitazone (30
           mg once daily) and 002 tablets was metformin (850mg once daily). Bio-medical outcomes
           were measured to assess the efficacy of both the drugs each month. After finishing the
           treatment period the effects of two drugs were compared using SPSS.And the association
           between the pioglitazone drug effects and genetic polymorphism was also assessed.

        -  The metformin effects was assessed also using the response rate of HbA1c <7.0% after 3
           months treatment to the patients.
    

Detailed description: 
      1. Introduction Thiazolidinediones and metformin are known drugs and especially metformin
           is widely used medicine to treat people with T2DM. Though pioglitazone has been
           suspended in many countries, its safety and efficacy is a matter of research for the
           drug investigators. Thiazolidinediones had been introduced in 1997 as an oral
           anti-diabetic drug (OAD). Metformin has been used as the key compound to treat T2DM for
           many years and is the most prescribed OAD worldwide. Both metformin and
           thiazolidinediones are considered as "insulin sensitizer". The ß-cell dysfunction and
           insulin resistance are the core defects in the progression of T2DM with associated
           metabolic syndrome. Evidence suggests that pioglitazone lowers glucose in blood by
           increasing glucose uptake into cell and metformin by decreasing glucose production.
           However, there are many controversies in clinical study specially about the improvement
           of insulin action of metformin. In contrast, a research recommended pioglitazone as the
           most appropriate OAD for the South Asian population. The key to this argument is that
           since pioglitazone is a proliferator-activated receptor gamma (PPAR-γ or PPARG) [a group
           of nuclear receptor proteins that function as transcription factors regulating the
           expression of genes] agonist and decreases insulin resistance, overall glycemic control
           seems to be better with this thiazolidinedione. However, there are very few published
           documents on the efficacy and safety of these drugs in South Asians. To the best of our
           knowledge, there is no published paper that examined and compared the efficacy and
           safety of these two drugs in Bangladeshi patients T2DM. This double-blind trail
           therefore examined the efficacy and safety of pioglitazone and metformin in T2DM
           individuals and also the association of the effects of pioglitazone and the variants of
           PPARG. We have compared the effects of pioglitazone with metformin on biomedical
           variables in T2DM patients who were eligible for oral hypoglycaemic therapy and then we
           also analyzed the association among the effects of pioglitazone and PPARG variants.

        2. Methodology

             1. Study Setting:

                The study was conducted between November 2008 and September 2010. The participants
                for this study were recruited from the outdoor, BIRDEM Hospital in
                Dhaka,Bangladesh.

             2. Subjects I.Individuals who had been diagnosed with T2DM treating with diet/exercise
                or Metformin 850 mg or Pioglitazone 30mg once daily, and were attending Outdoor
                Patient Department (OPD) of BIRDEM for consultation were approached by the
                researcher and invited to participate in the study. The patients were diagnosed
                already and registered in BIRDEM. T2DM was ascertained based on World Health
                Organization recommended criteria two repeated measures of fasting (plasma glucose
                ≥7.0 mmol ⁄ l or 2-h plasma glucose ≥11.1 mmol ⁄ l.)

                II.We screened a total of 130 patients for eligibility and selected 80 patients for
                enrollment based of our inclusion and exclusion criteria. But 77 T2DM patients with
                HbA1c level < 7.5 %, BMI kg/m2 ≥ 25, SGPT≤ 100 IU/L , creatinine ≤ 1.2 mg/dl) of
                both sexes, aged between 40-50, treated by monotherapy of pioglitazone or metformin
                received the drug for the trial according to inclusion and exclusion criteria. 53
                patients were screening failure as some did not match eligibility criteria (n=39),
                some refused to take part(n=11)and some were unable to take part (n=03)due to
                unknown cause.

             3. Patient Preparation:

                I.Written informed consent was obtained from all participants before study entry.
                Patients were instructed to follow adhere to a disease- and weight-orientated diet
                throughout the study as before.

                II.Each case history was documented in the case report form. The study was approved
                by the National Medical Ethics Review Boards (NMERB) of Bangladesh Medical Research
                Council (BMRC).The investigations were carried out in accordance with the
                principles of the Declaration of Helsinki as revised in 2000.

             4. Treatment:

                The patients were to receive treatment pioglitazone tablet 001 (30mg once per day)
                for 3 months followed by one month "metformin wash-out period", then to the
                alternative treatment regimen for further 3 months with metformin tablet 002 (850mg
                once per day). The drugs were supplied by the Aristopharma Pharmaceutical Ltd.,
                Bangladesh.

             5. Anthropometric Measurements I.Height: Standing height was measured using
                appropriate scales (Detect-Medic, Detect scales INC, USA) without shoes.

                II.Weight: Weight was measured with the balance was placed on a hard flat surface
                and checked for zero balance before measurement. The subjects were in the center of
                the platform wearing light cloths without shoes.

                III.BMI: Body mass indexes (BMI) of the subjects were calculated using standard
                formula: BMI= Weight (kg)/[Height (m)] 2.

                IV.Blood Pressure: Systemic and Diastolic pressure was measured according to
                WHO-IHS.

             6. Blood Sample Collection for Biochemical analysis :

                I. During the appointed date the patients came in the fasting condition. Fasting
                blood samples (10 ml) were drawn from the antecubital vein. The time was mentioned
                as 0 hour. Then the patients received drug. They were requested to swallow the
                tablet and have their breakfast according to their diet charts. Next blood sample
                had been drawn at 30 min and 2 hour and then they were provided the drugs for the
                whole month.

                II.The patients were requested to take the drug just before the breakfast every day
                for 29 days.

                III.From the fasting blood sample 1ml of blood was transferred in an EDTA
                containing tube for measurement of HbA1C and the rest of the blood was taken in an
                EDTA containing tube and centrifuged immediately.

                IV.Samples after processing were divided into two aliquots under sterile
                condition;one aliquot was sent for biochemical analysis for Fasting glucose, Lipid
                profile, Cholesterol, ALT, Insulin, Creatinine and another one was stored at -80 0C
                for further verification of results in case of any necessity.

             7. Biochemical Test Methods:

                I. Serum glucose (fasting, 1 and 2 hours) by Glucose Oxidase (GOD-PAP) method
                (Randox Laboratories Ltd., UK).

                II.Serum triglyceride by enzymatic colorimetric (GPO-PAP) method (Randox
                Laboratories Ltd., UK).

                III.Serum total cholesterol by enzymatic endpoint (Cholesterol Oxidase/ Peroxidase)
                method (Randox Laboratories Ltd., UK).

                IV.Serum HDL cholesterol by enzymatic colorimetric (Cholesterol CHOD-PAP) method
                (Randox Laboratories Ltd., UK) using micro-plate reader (Bio-Tec, ELISA).

                V.Serum creatinine by enzymatic colorimetric (GPO-PAP) method (Randox Laboratories
                Ltd., UK).

                VI.Serum alanine amino transferase (ALT) by UV method using ALT (GPT) opt.kit
                (Randox Laboratories Ltd., UK).

                VII.Serum insulin by enzyme linked immunosorbent assay (ELISA) method (Linco
                Research Inc., USA).

                VIII.Glycosylated Haemoglobin (HbA1c) by High Performance Liquid Chromatographic
                (HPLC) method.

             8. Blood sample collection for DNA analysis (PPARG gene):

           I.At the end of 3rd month of each treatment 1.5 ml of blood was taken in EDTA containing
           tube for genetic analysis and the whole blood specimen was collected in the vacuum
           collection tube containing EDTA, stored at -20 0C to - 80 0C.

           II.DNA was isolated by Chelex method, identified by electrophoresis method and amplified
           by using primers. We used a published document to select the primers for genotyping
           PPARγ Pro12Ala/Pro12Pro. The primers for the Pro12Ala SNP genotype, we amplified exon B
           using the reverse primer 5' CTG GAA GAC AAA CTA CAA GAG 3' and the forward primer 5' ACT
           CTG GGA GAT TCT CCT ATT GGC 3'. (Sigma product, Order No. SIGMA 03/11/09 4152936-F/185
           PPARG-R 8006875247-1).

           III.Control DNA: Professor Colin Palmer Laboratory, Biomedical Research Institute
           ,University of Dundee Medical School, University of Dundee, Scotland, UK sent six
           control samples of 3 types control DNA genotyped for PPARG SNP rs 1801282 (Pro12Ala).

           IV.Restriction Digestion Enzyme Endonuclease Hae 111 was used to identify the cutting
           site(Sigma Product No. R 5628).

        3. Response Rate:

      1. The response rate was defined by the decrease of ≥10% FBG or by the decrease of ≥1% HbA1c
      from the baseline values.

      ll.There was another response group was defined by the the HbA1c rate <7.0% after 3 months
      treatment with metformin only to find out the secondary failure rate of metformin.

      4) Statistical Analysis: I.Statistical analysis was performed using SPSS (Statistical Package
      for Social Science) software for Windows version 18 (SPSS Inc, Chicago, Illinois, USA). Data
      were expressed as mean + SD (Standard Deviation).

      II. Effects of drugs after 3 months treatment were analyzed using t pair tests. The groups
      were compared using one way ANOVA. If the p value was <0.05, the groups were compared using
      the student's t test for unpaired samples or χ2 test through univariate analysis for further
      verification. Correlation coefficient among the variables was tested using Pearson's test.
      Multivariate logistic regression analysis was performed to obtain the odds ratios and
      independent influencing factor for finding possible association between PPARγ genotypes and
      drug response in case of genetic analysis.

      III.A p value <0.05 was considered significant for all tests. lv. The statistical analysis
      for within group study like PPARG response group and metformin secondary failure group has
      not been displayed here.

      5) List of Abbreviations

      AEs Adverse Events

      ALT Alanine aminotransferase

      BMI Body Mass Index

      BMRC Bangladesh Medical Research Council

      BIRDEM Bangladesh Institute of Research and Rehabilitation in Diabetes,Endocrine and
      Metabolic Disorder

      BP Blood Pressure

      DNA Deoxynucleic Acid

      DBP Diastolic Blood Pressure

      DM Diabetes Mellitus

      EASD European Association for the Study of Diabetes

      EDTA Ethylene Diamine Tetra Acetic acid

      ELISA Enzyme Linked Immunosorbent Assay

      FBG/FSG Fasting Blood Glucose/Fasting Serum Glucose

      FSI Fasting Serum Insulin

      2hBG 2 hours Blood Glucose

      HbA1c Glycosylated Haemoglobin

      HOMA percent B Homeostasis Model Assessment percentage of beta cell function HOMA percent S
      Homeostasis Model Assessment percentage of sensitivity

      HOMA IR Homeostasis Model Assessment Insulin Resistance

      HDL-C High Density Lipid Cholesterol

      IU/L International Unit/Litre

      LDL-C Low Density Lipid Cholesterol

      ml millilitre

      mm millimetre

      mg/dl milligram/ decilitre

      MLR Multiple Logistic Regression

      OPD Outdoor Patient Department

      OMIM Online Mendelian Inheritance in Man

      OR Odds Ratio

      PPARγ Peroxisome Proliferator Activated Receptor gamma

      Pro12Pro Proline12Proline

      Pro12Ala Proline 12 Alanine

      Ala12Ala Alanine12Alanine

      PCR Polymerase Chain Reaction

      QUICKI Quantitative Insulin sensitivity Check Index

      SD Standard Deviation

      SPSS Statistical Package for Social Science

      SBP Systolic Blood Pressure

      TC Total Cholesterol

      TG Triglyceride

      T2DM Type 2 Diabetes Mellitus

      TZD Thiazolidinedione

      µl Microliter

      WHO World Health Organization
    

Intervention name(s): Pioglitazone hydrochloride; Metformin hydrochloride
Intervention type(s): Drug; Drug
Intervention description(s): 77 patients were treated with pioglitazone hydrochloride (B001) for 3 months.Biomedical parameters were measured each month.; After the treatment with pioglitazone(001) for 3 months first and then patients were on one month washout period;in the washout period they were given metformin tablet 850mg once daily,then treated with 002 (metformin) for further 3 months. The same biomedical measurements were assayed.
Other intervention name(s): GLUCOZON; 30 mg tablet, once, daily; Coded as B001(Preparation Date:Jun 08); Aristopharma LTD.,Bangladesh; GLUCOMET; 850 mg tablet, once, daily; Coded as B002 (Preparation date: Jun 08); Aristopharma LTD.,Bangladesh
Intervention MeSH terms: Metformin; Pioglitazone
Allocation: Non-Randomized
Intervention model: Single Group Assignment
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Treatment
Keywords: Bangladesh; T2DM; Pioglitazone; Metformin; Insulin secretion and Sensitivity