Pioglitazone is a PPAR gamma agonist that is a useful armamentarium in management of insulin resistant states in diabetes. PPAR gamma nuclear receptors have been isolated from various tissues like adipose tissue, proximal tubular cells of kidney, colon, bone, heart, muscle pancreas, spleen and macrophages. Recently there has been increasing concern regarding detrimental effects of pioglitazone on bone health. It has been hypothesized that these effects are due to direct action on bone leading to differentiation of mesenchymal stem cells in the bone towards an adipocyte lineage rather than an osteoblast lineage. A few articles also suggest contributory role of hypercalciruria as a potential mechanism of increased bone loss due to pioglitazone. 1 alpha hydroxylase enzyme is present on proximal renal tubular cells and is a mitochondrial enzyme also known as   cytochrome P450 17B1. Pioglitazone is known to inhibit many other P450 enzymes like CYP 2C8 and CYP 3A4. Whether piogltazone interferes with renal 1-alpha hydroxylase activity has not been studied so far; we plan to assess the effect of pioglitazone on renal calcium handling and serum vitamin D levels in patients with Type 2 diabetes attending Diabetic Clinic of the Dept of Endocrinology & Metabolism of our hospital.  If  a significant  effect on Vitamin D is found it would not only elucidate another mechanism of adverse effect of pioglitazone on bone , but would also guide us to undertake studies where supplementation of calcitriol may prevent pioglitazone associated bone loss and thus help us preserve the sanctity of this unique drug in the anti-diabetic drug armamentarium.

BACKGROUND INFORMATION & RATIONALE

Diabetes mellitus is one of the most common endocrine diseases, characterized by an increase in plasma glucose. The most prevalent form of diabetes is type 2 diabetes (T2D), currently affecting more than 300 million people worldwide. (1) T2D is characterized by the combination of insulin resistance and failing β-cell function.(2) 

 PPAR gamma agonists are a novel approach to tackling insulin resistance in diabetic subjects. Pioglitazone is a PPAR gamma agonist that is a useful armamentarium in management of insulin resistant states in diabetes. Other glitazones like troglitazone and rosiglitazones which have already been withdrawn from the market due to concerns of hepatotoxicity and cardiotoxicity. Three types of PPARs have been identified: alpha, gamma, and delta (beta).α (alpha)  are expressed in liver, kidney, heart, muscle, adipose tissue. �’/δ (beta/delta) is expressed in many tissues but markedly in brain, adipose tissue, and skin.  γ (gamma) receptors although transcribed by the same gene, this PPAR through alternative splicing is expressed in three forms: γ1 - expressed in virtually all tissues, including heart, muscle, colon, kidney, pancreas, and spleen, γ2 - expressed mainly in adipose tissue , γ3 - expressed in macrophages, large intestine, white adipose tissue.(3)

 Recently there has been increasing concern regarding detrimental effects of pioglitazone on bone health. It has been hypothesized that these effects are due to direct action on bone leading to differentiation of mesenchymal stem cells in the bone towards an adipocyte lineage rather than an osteoblast lineage. However a few articles also suggest contributory role of hypercalciruria as a potential mechanism of increase bone loss due to pioglitazone.(4) However effect of piogltazone on Vitamin-D status is unknown. No study has been done in this regards. 1 alpha hydroxylase enzyme is present on proximal renal tubular cells and is a mitochondrial enzyme also known as   cytochrome P450 17B1. Pioglitazone is known to inhibit many other P450 enzymes like CYP 2C8 and CYP 3A4.(5) Pioglitazone has also been found to have inhibitory effect on estradiol synthesis by inhibiting aromatase activity.(6) Pioglitazone also has been found to inhibit androgen production by inhibition of CYP 17 and HSD3beta 2 enzymes. (7)We plan to assess the effect of pioglitazone on renal calcium handling, serum levels of 25(OH) vitamin D and 1,25(OH) Vitamin D among patients with diabetes attending Diabetic Clinic of Dept of Endocrinology & Metabolism of our hospital.  If  a significant  effect on Vitamin D is found it would elucidate another mechanism of adverse effect of pioglitazone on bone , but would also guide us to undertake studies where supplementation of calcitriol or cholecalciferol  may prevent pioglitazone associated bone loss and thus help us preserve the sanctity of this unique drug in the anti-diabetic drug armamentarium.

Study goals & Objectives

The aim of this study is to study the effect of pioglitazone on renal handling of calcium and the effect on vitamin D status of the patients.

Methodology and study design

Patients attending diabetic clinic or endocrine outpatient department (OPD) services of SSKM Hospital and IPGMER will be considered. Patients of type 2 diabetes not controlled (fasting sugar >110, Post prandial > 180 or HbA1C >6.5) on medical nutrition therapy and metformin would be undertaken for the study. Patients with very poorly controlled sugars i.e. HbA1C >8 will not be enrolled since glycosuria is associated with hypercaiciuria and control of glycemic status with pioglitazone can decrease calcium excretion and confound results. Fasting serum calcium, phosphate, albumin, alkaline phosphatase, intact PTH, 25(OH) Vitamin D, 1, 25(OH) Vitamin D and spot urinary calcium creatinine ratio and urinary phosphate concentrations would be measured at baseline and then the same measurements shall be made after 12 weeks of pioglitazone 45mg/day. Dietary intake of calcium, phosphate, sodium and protein would be estimated for each patient through the prescribed diet charts. Compliance to diet would be assessed by measurement of spot urinary sodium concentrations both before and after the study periods. Patients taking insulin and/or other anti-diabetic drugs will be excluded. Patients on ACE inhibitors and diuretics will be excluded as will patients having blood pressures above 180/110 mm Hg. Also patients with associated disorders like primary hyperparathyroidism, chronic kidney disease, liver disease, any chronic illness, malignancy, chronic drug use like anti-epileptic agents, oral contraceptive pills, steroids which are likely to interfere with vitamin-D metabolism will be excluded. Patients with pregnancy would be excluded. Patients on calcium or vitamin-D supplementation would also be excluded.

Laboratory tests used would be as follows;

VITAMIN-D3, 25(OH) by CMIA

VITAMIN-D, 1, 25-DIHYDROXY by RIA

PTH, INTACT by CLIA

CALCIUM, Biochemical

PHOSPHORUS,Biochemical

ALBUMIN,Serum, Biochemical

ALKALINE PHOSPHATASE,Spectrophotometry

CALCIUM/CREATININE RATIO-URINE,Biochemical

PHOSPHORUS-URINE,Biochemical

SODIUM, URINE by ICT

Patients will be kept on a normal sodium diet and diet charts shall be provided to all subjects.

 The patients would be explained about the study and only those who gave informed written consent would be included in the final study. Clearance from the Institutional Ethics committee will be obtained before the study initiation. The finally included patients in the study would be given an appointment to attend the OPD services after a 12 hour fast.  Data on age, sex, and smoking status (never, past, or current), duration of diabetes, glycemic control will be estimated at baseline. All patients would undergo detailed clinical examination. Blood samples would then be collected. Lipid profile, serum creatinine, HbA1c, electrolytes would be estimated in all patients.

Proposed Statistical Analysis

Paired t test will be used for analysis of continuous Quantitative variables to compare parameters before and after pioglitazone therapy. All results of continuous variables are expressed as mean ± SD. No study has seen the effects of pioglitazones on vitamin D status of diabetic subjects. A single study on effect of pioglitazones on renal handling of calcium recruited only 8 diabetic subjects. We plan to recruit 30 patients for our study.

References in recent Literature

1)      IDF diabetes atlas. 4th edition. International Diabetes Federation; 2009. Available at: http://www.diabetesatlas.org/.

2)      Sadikot SM, Nigam A, Das S, Bajaj S, Zargar AH, Prasannakumar KM, Sosale A, Munichoodappa C, Seshiah V, Singh SK, Jamal A, Sai K, Sadasivrao Y, Murthy SS, Hazra DK, Jain S, Mukherjee S, Bandyopadhay S, Sinha NK, Mishra R, Dora M, Jena B, Patra P, Goenka K; DiabetesIndiaThe burden of diabetes and impaired glucose tolerance in India using the WHO 1999 criteria: prevalence of diabetes in India study (PODIS) Diabetes Res Clin Pract. 2004 Dec;66(3):301-7

3)      PPAR Gamma and Metabolism: Insights from the Study of Human Genetic Variants; Clin Endocrinol. 2003;59(3)

4)      Lauri I. Kajosaari, Tiina Jaakkola, Pertti J. Neuvonen and Janne T. Backman. Pioglitazone, an in vitro inhibitor of CYP2C8 and CYP3A4, does not increase the plasma concentrations of the CYP2C8 and CYP3A4 substrate repaglinide.  European Journal of Clinical Pharmacology. Volume 62, Number 3, 217-223

5)      Anne Zanchi, Antoinette Peche` re-Bertschi, Michel Burnier, and Olivier Bonny. Effects of Pioglitazone on Renal Calcium Excretion. (J Clin Endocrinol Metab 96:E1482–E1485, 2011)

6)      Rosiglitazone and pioglitazone inhibit estrogen synthesis in human granulosa cells by interfering with androgen binding to aromatase. Seto-Young D, Avtanski D, Parikh G, Suwandhi P, Strizhevsky M, Araki T, Rosenwaks Z, Poretsky L. Horm Metab Res. 2011 Apr;43(4):250-6

7)      Petra Kempná, Gaby Hofer, Primus E. Mullis and Christa E. Flück. Pioglitazone Inhibits Androgen Production in NCI-H295R Cells by Regulating Gene Expression of CYP17 and HSD3B2. Molecular Pharmacology March 2007 vol. 71 no. 3 787-798.

Results( Interim)

A total of 14 patients finished trial period as off May 2013 and follow up data of 13 patients was collected, 1 patient was lost to follow up. The Interim analysis of 13 patients has been computed which shows a statiscally significant decrease in 1 alpha hydroxylase activity as evidenced by a mean decrease of  13.6% in ratio of 1,25 di hydroxyl vitamin D to 25 hydroxyl vitamin D at the end of study period. There was no significant effect on serum calcium, phosphorous, iPTH, urine calcium or phosphate excretions.

None of the patient had any serious adverse effects of the drug.

The drug was banned briefly by government of India, and hence the trial was terminated during that period, and 3 patients who were still to complete the trial study period were called prematurely and the drug was stopped. The study samples were collected on first visit thereof, the sample data for these 3 patients is yet to be computed in the final analysis.

After this although the government revoked the ban on the drug no patient has been recruited thence, for most patients have refused consent to participate in the trial.