Comparison of Efficacy and Safety of Epalrestat with Methylcobalamin in Patients with Diabetic Neuropathy”

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DISSERTATION ON “COMPARISON OF EFFICACY AND SAFETY OF EPALRESTAT WITH METHYLCOBALAMIN IN PATIENTS WITH DIABETIC NEUROPATHY”

Dissertation submitted to

THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY

In partial fulfillment of the requirement

For the award of the degree of

M.D. BRANCH-VI

PHARMACOLOGY

Submitted By

Registration Number: 201516452

KARPAGA VINAYAGA INSTITUTE OF MEDICAL SCIENCES

AND RESEARCH CENTRE, MADURANTHAGAM

THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY, CHENNAI

TAMILNADU

MAY-2018 CERTIFICATE

This is to certify that Dr. C. Preeth, a Post Graduate student in the

Department of Pharmacology has carried out the work titled “Comparison of efficacy and safety of Epalrestat with Methylcobalamin in patients with

Diabetic neuropathy” under the guidance of Dr.P.JACOB VERGHESE, M.D.,

PROFESSOR, Department of Pharmacology, towards the partial fulfillment of regulations laid down by The Tamilnadu Dr. M.G.R Medical University, Guindy,

Chennai, Tamilnadu, India for the award of Doctor of Medicine (M.D.,) in

Pharmacology.

Dr.P. JACOB VERGHESE, M.D., DR. R. KAVITHA, M.D., PROFESSOR, PROFESSOR & HOD, Karpaga Vinayaga Institute of Medical Karpaga Vinayaga Institute of Medical Sciences & Research Centre, Sciences & Research Centre, Chinnakolambakkam, Chinnakolambakkam, MaduranthagamTk, MaduranthagamTk, Kancheepuram District– 603 308, Kancheepuram District– 603 308, Tamilnadu, India. Tamilnadu, India.

DR. SUFALA SUNIL VISHWASRAO, PRINCIPAL, Karpaga Vinayaga Institute of Medical Sciences & Research Centre, Chinnakolambakkam, Maduranthagam Tk, Kancheepuram District– 603 308, Tamilnadu, India.

DECLARATION

I declare that dissertation entitled “Comparison of efficacy and safety of Epalrestat with Methylcobalamin in patients with Diabetic neuropathy” submitted by me for the degree of M.D., is the record work carried out by me under the guidance of Dr.P. JACOB VERGHESE, Professor Of

Pharmacology, Karpaga Vinayaga Institute of Medical Sciences and Research

Centre and has not formed the basis of any Degree, Diploma, Fellowship, titles in this or any other University or other similar Institution of Higher learning.

Place: Chinnakolambakkam Signature of the Candidate Date: DR. C. PREETH

Signature of the Guide Signature of HOD Dr.P. JACOB VERGHESE, M.D., DR. R. KAVITHA, M.D., PROFESSOR, PROFESSOR & HOD, KarpagaVinayaga Institute of Medical Karpaga Vinayaga Institute of Medical Sciences & Research Centre, Sciences & Research Centre, Chinnakolambakkam, Chinnakolambakkam, MaduranthagamTk, MaduranthagamTk, Kancheepuram District– 603 308, Kancheepuram District– 603 308, Tamilnadu, India. Tamilnadu, India.

ACKNOWLEDGEMENTS

At the outset I express my sincere thanks to my esteemed guide Dr.P.JACOB VERGHESE M.D., Professor in the Department of Pharmacology, Karpaga Vinayaga Institute of Medical Sciences and Research Centre for his encouragement and valuable guidance in the topic given from time to time for the successful completion of study.

I am extremely thankful to the Managing Director, Dr.R. ANNAMALAI, M.S., M.Ch., Principal Dr. SUFALA SUNIL VISHWASRAO, M.D., Medical Director Dr. SATHIYANARAYANAN, Karpaga Vinayaga Institute of Medical Sciences and Research Centre for providing me all the facilities to conduct this study.

I express my deep and sincere gratitude to Dr. R. KAVITHA, M.D., Professor and Head, Department of Pharmacology, Karpaga Vinayaga Institute of Medical Sciences and Research Centre for being my mentor and support at all levels.

I profusely thank my Co-guide Dr. V. GOPAL DASS, M.D., Professor and Head, Department of General Medicine, Karpaga Vinayaga Institute of Medical Sciences and Research Centre for having permitted to conduct this study and the constant support he extended throughout the study.

I thank Dr. D. SRINIVASAN, Ph.D., Professor in the Department of Pharmacology, KarpagaVinayaga Institute of Medical Sciences and Research Centre for his kind guidance and encouragement during the course of this study.

I express my deep and sincere gratitude to Dr. B. PRATHAP, M.D., Associate Professor, Department of Pharmacology, Karpaga Vinayaga Institute of Medical Sciences and Research Centre for his guidance and support at all levels.

My heartfelt thanks to my Assistant Professors, Dr. E. SESHATHRI, M.D., and Dr. SUNIL M VISHWASRAO, M.D., in guiding me through the course of the study.

I owe my sincere thanks to Dr. N. CHANDRAN, B.V.Sc., and AH for encouraging me towards this research.

I thank my Senior and Junior Post Graduate colleagues for their greatest help and support through the course.

I sincerely thank our bio-statistician GLADIUS JENIFER for her guidance during my dissertation.

I am immensely grateful to the staffs at the Department of Pharmacology and Department of General Medicine, Karpaga Vinayaga Institute of Medical Sciences and Research Centre for having provided me technical support throughout the study.

Last but no means the least, I am greatly indebted to all the patients who had taken part in this study without whom the study could not have been completed.

Finally my dissertation would have not been accomplished without the support of my parents Mr. Chandrasekaran& Mrs. Thiripurasundari, my husband Dr. Chakravarthi, my sons Sibi Aathyandha& Rishi Aathreya and my other family members.

Above all I thank my Almighty for his blessings.

PLAGIARISM CERTIFICATE

CONTENTS

CHAPTER PARTICULARS PAGE NO.

1 INTRODUCTION 1

2 AIMS AND OBJECTIVES 4

3 REVIEW OF LITERATURE 5

4 DRUG PROFILE 33

5 PLAN OF WORK 40

6 MATERIALS AND METHODS 41

7 OBSERVATION AND RESULTS 46

8 DISCUSSION 82

9 SUMMARY 86

10 CONCLUSION 88

11 BIBLIOGRAPHY 89

12 ANNEXURES 99

LIST OF TABLES

SL. TITLE PAGE NO NO.

Explains blood test levels for diagnosis of diabetes and 1 20 prediabetes.

2 Explains the study groups. 42

Explains the number of males and females among the study 3 49 groups A, B and C.

Shows the mean age (in years) of the patients belonging to 4 51 groups A, B and C.

Shows the mean BMI of all three groups at baseline and at 5 53 the end of 4th, 8th and 12th weeks.

Shows the mean fasting blood glucose level of all three 6 55 groups at baseline and at the end of 4th, 8th and 12th weeks.

Shows the mean post prandial blood glucose level of all 7 57 three groups at baseline and end of study.

Shows the mean glycosylated haemoglobin level of all three 8 59 groups at baseline and end of study.

9 Shows the mean AST concentration of groups A, B & C. 61 10 Shows the mean ALT concentration of groups A, B & C. 63

11 Shows the mean serum albumin level of groups A, B & C. 65

Explains the mean serum total bilirubin level of groups A, B 12 67 & C.

Explains the serum creatinine concentration of groups A, B 13 69 & C.

14 Shows the serum urea concentration of groups A, B & C. 71

15 Shows the urine albumin levels of groups A, B & C. 73

16 Explains the urine sugar levels of groups A, B & C. 74

Shows the frequency of various adverse drug reactions 17 75 between the study groups.

18 Shows the pain intensity scores (VAS Method). 76

Shows the MichiganNeuropathy Screening Instrument 19 78 (MNSI) score- Completed by the person with diabetes.

Shows the MichiganNeuropathy Screening Instrument 20 80 (MNSI) score - Completed by health professionals. LIST OF FIGURES

SL. TITLE PAGE NO NO.

1 Explains the synthesis, release and actions of insulin 17

2 Explains the pathogenesis of type 1 diabetes mellitus 18

3 Explains the pathogenesis of type 2 diabetes mellitus 19

4 Polyol (Sorbitol) Pathway 24

5 Polyol (Sorbitol) Pathway with accumulation of sorbitol 25 and fructose

6 Shows the percentage of males and females among the 50 study group A.

7 Shows the percentage of males and females among the 50 study group B.

8 Shows the percentage of males and females among the 50 study group C.

9 Illustration of the graphical representation of the mean age 52 of the study groups.

10 Shows the graphical representation of change in mean BMI 54 of all three groups during the visits.

11 Illustration of graphical representation of table 6. 56

12 Shows the graphical representation of table 7. 58

13 Illustration of graphical representation of table 8. 60

14 Illustration of graphical representation of table 9. 62 15 Shows the graphical representation of table 10. 64

16 Illustration of graphical representation of table 11. 66

17 Illustration of graphical representation of table 12. 68

18 Illustration of graphical representation of table 13. 70

19 Shows the graphical representation of table 14. 72

20 Shows the graphical representation of table 18. 77

21 Shows the graphical representation of table 19. 79

22 Illustration of graphical representation of table 20. 81

LIST OF ABBREVIATIONS

ACE Angiotensin-converting enzyme

ACR Albumin:Creatinine Ratio

ALA Alpha Lipoic Acid

ALT Alanine Amino Transferase-SGPT

ARBs Angiotensin Receptor Blockers

AST Aspartate Transaminase – SGOT

ATP Adenosine Triphosphate

BMI Body Mass Index

CBC Complete Blood Count

CDC Center for Disease Control

CHD Coronary heart disease

CNS Central Nervous System

DKA Diabetic Ketoacidosis

DM Diabetes Mellitus

FBS Fasting Blood Glucose

FPG Fasting Plasma Glucose

GAD Glutamic acid Decarboxylase

GFR Glomerular Filtration Rate

GIP Gastric Inhibitory Polypeptide

GLP Glucagon Like Peptide

GLUT2 Glucose Transporter 2 HbA1C Glycosylated Hemoglobin

HDL High Density Lipoprotein

Hyperosmolar hyperglycaemicnonketotic HHS coma

Human Leukocyte Antigen – antigen D HLA-DR Related

HNF Hepatocyte nuclear transcription factor

IDDM Insulin-Dependent Diabetes Mellitus

IDF International Diabetes Federation

IEC Institutional Ethical Committee

IM Intra Muscular

IRS Insulin Receptor Substrates

IV Intra Venous

LFT Liver Function Test

MC Methylcobalamin

MDRD Modification of Diet in Renal Disease

MI Myocardial Infarction

MNSI Michigan neuropathy screening instrument

NIDDM Noninsulin-Dependent Diabetes Mellitus

NPH Neutral Protamine Hagedorn

NRS Numeric Rating Scale

NS Non- Significant

OADs Oral Anti Diabetic Drugs OGTT Oral Glucose Tolerance Test

PKC Protein Kinase C

Peroxisome proliferator-activated receptor PPAR-γ gamma

PPBS Post Prandial Blood Glucose

PPG Post Prandial Glucose

PVD Peripheral Vascular Disease

PZI Protamine Zinc Insulin

RFT Renal Function Test

SC Subcutaneous

SD Standard Deviation

SEM Standard Error Mean

SIS Sleep Interference Scores

SUR Sulphonyl Urea Receptor

T2DM Type 2 Diabetes Mellitus

TZDs Thiazolidinediones

VAS Visual Analog Scale

INTRODUCTION

1. INTRODUCTION

DIABETES MELLITUS

Diabetes is a national as well as global epidemic disease in terms of incidence, healthcare costs and overall complications as reported by the Center for Disease Control (CDC)1. According to recent estimates, approximately 285 million people worldwide (6.6%) in the 20–79 year age group have diabetes in

2010 and by 2030, 438 million people (7.8%) of the adult population, is expected to have diabetes.

The International Diabetes Federation (IDF) estimates the total number of people in India with diabetes to be around 50.8 million in 2010, rising to

87.0 million by 20302. Globally, as of 2013, an estimated 382 million people have diabetes worldwide, with type 2 diabetes making up about 90% of the cases3,4. This is equal to 8.3% of the adult’s population, with equal rates in both women and men5. Worldwide in 2012 and 2013 diabetes resulted in 1.4 to 5.1 million deaths per year, making it the 8th leading cause of death6. The number of people with diabetes is expected to rise to 592 million by 20357,8. The number of people with diabetes has risen from 108 million in 1980 to 422 million in 20149,10. The global prevalence of diabetes among adults over 18 years of age has risen from 4.7% in 1980 to 8.5% in 20149. Diabetes prevalence has been rising more rapidly in middle- and low-income countries. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke and lower limb amputation. In 2014, 8.5% of adults aged 18 years and older had diabetes.

In 2015, diabetes was the direct cause of 1.6 million deaths and in 2012 high blood glucose was the cause of another 2.2 million deaths. In 2015, an estimated 1.6 million deaths were directly caused by diabetes. Another 2.2 million deaths were attributable to high blood glucose in 2012. Almost half of all deaths attributable to high blood glucose occur before the age of 70 years.

WHO projects that diabetes will be the seventh leading cause of death in

203011,12. Healthy diet, regular physical activity, maintaining a normal body weight and avoiding tobacco use are ways to prevent or delay the onset of type

2 diabetes. Diabetes can be treated and its consequences avoided or delayed with diet, physical activity, medication and regular screening and treatment for complications.

Methylcobalamin is one of the biologically active forms of vitamin B12.

Diabetic neuropathies can lead to a wide variety of sensory, motor and autonomic symptoms. The most common is the symmetrical distal sensory type, which is particularly evident in the feet and may slowly progress to a complete loss of feeling. It is most prevalent in elderly patients with type 2 diabetes but may be found with any type of diabetes, at any age beyond childhood.

Recently Epalrestat and Methylcobalamin are widely used in clinical practice to manage diabetic neuropathy. Hence, this study was undertaken to evaluate the efficacy and safety of Epalrestat with Methylcobalamin in patients with diabetic neuropathy.

AIMS AND OBJECTIVES

2. AIMS AND OBJECTIVES

AIM:

This study was aimed to assess and compare the efficacy and safety of

Epalrestat with Methylcobalamin in patients with diabetic neuropathy.

OBJECTIVES

1. To assess the efficacy and safety of individual effect of Epalrestat and

Methylcobalamin in patients with diabetic neuropathy.

2. To assess the efficacy and safety of combined effect of Epalrestat and

Methylcobalamin in patients with diabetic neuropathy and to compare

with individual effect of Epalrestat and Methylcobalamin in patients

with diabetic neuropathy.

REVIEW OF LITERATURE

3. REVIEW OF LITERATURES

A literature review is a summary of previous research on a topic. The review of literature aims to describe the ‘state of play’ in the area selected for study. Our literature review focused on the pharmacological potential of

Epalrestat, an aldose reductase inhibitor and Methylcobalamin. Such literature reviews describe what has been written about the area, the major research findings across studies, and the major debates in terms of substantive and methodological issues.

Diabetes mellitus (DM) is a group of metabolic disorders characterized by hyperglycemia and abnormalities in carbohydrate, fat, and protein metabolism. It results from defects in insulin secretion, insulin sensitivity, or both13. In this condition there are high blood sugar levels over a prolonged period14. This high blood sugar produces the symptoms of frequent urination, increased thirst, and increased hunger. Untreated, diabetes can cause many complications15. Acute complications include diabetic ketoacidosis and nonketotic hyperosmolar coma16. Serious long term complications include heart disease, kidney failure, foot ulcers and damage to eyes15.

CLASSIFICATION OF DIABETES MELLITUS

TYPE 1 DIABETES MELLITUS

It results from the body’s failure to produce insulin. This form was previously referred to as “insulin-dependent diabetes mellitus” (IDDM) or

“Juvenile diabetes”. The cause is unknown14. It is characterized by loss of the

5 insulin-producing beta cells of the islets of Langerhans in the pancreas, leading to insulin deficiency17. Type 1 DM is further divided into 2 subtypes:

Subtype 1A (immune-mediated) DM

It is characterized by autoimmune destruction of β-cells which usually leads to insulin deficiency.

Subtype 1B (idiopathic) DM

It is characterized by insulin deficiency with tendency to develop ketosis but these patients are negative for autoimmune markers18.

TYPE 2 DIABETES MELLITUS

It begins with insulin resistance, a condition in which cells fail to respond insulin properly14. As the disease progresses a lack of insulin may also develop19,20. This form was previously referred to as noninsulin-dependent diabetes mellitus (NIDDM) or “adult-onset diabetes”. The primary cause is excessive body weight and not enough exercise15. In the early stage of type 2, the predominant abnormality is reduced insulin sensitivity. At this stage, hyperglycemia can be reversed by a variety of measures and medications that improve insulin sensitivity or reduce glucose production by the liver.

GESTATIONAL DIABETES

It is the third main form and occurs when pregnant women without a previous diagnosis of diabetes develop a high blood glucose level11. About 4% pregnant women develop DM due to metabolic changes during pregnancy.

Although they revert back to normal glycaemia after delivery, these women are prone to develop DM later in their life18.

OTHER SPECIFIC TYPES OF DIABETES14 A. Genetic defect of β-cell function due to mutations in various enzymes (maturity-onset diabetes of the younger MODY)

E.g.: Hepatocyte nuclear transcription factor—HNF Glucokinase

B. Genetic defect in Insulin action

E.g.: Type A Insulin resistance

C. Diseases of exocrine pancreas

E.g.: Chronic pancreatitis, Pancreatic tumours, Post pancreatectomy

D. Endocrinopathies

E.g.: Acromegaly, Cushing’s syndrome, Pheochromocytoma

E. Drug or Chemical induced

E.g.: Steroids, Thyroid hormone, Thiazides, Beta blockers

F. Infections

E.g.: Congenital rubella, Cytomegalovirus

G. Uncommon forms of immune mediated DM

E.g.: Stiff man syndrome, Anti insulin receptor antibodies

H. Other genetic syndromes

E.g.: Down’s syndrome, Klinefelter’s syndrome, Turner’s syndrome

SIGNS AND SYMPTOMS16,21

Weight loss

Polyuria

Polyphagia

Headache

Polydipsia

Blurring of vision

Fatigue

Slow healing of cuts

Prolonged high blood glucose absorption in the lens of eyes, which lead to changes in the shape resulting in visual changes. Blurred vision is a common complaint leading to a diabetes diagnosis. A number of skin rashes that can occur in diabetes are collectively known as diabetic dermatomes.

RISK FACTORS FOR TYPE 2 DIABETES MELLITUS18, 19-24

1. Family history of type 2 DM

2. Obesity

3. Habitual physical inactivity

4. Race and ethnicity (Blacks, Asians, Pacific Islanders)

5. Previous identification of impaired fasting glucose or impaired

glucose tolerance

6. History of gestational DM or delivery of baby heavier than 4 kg

7. Hypertension

8. Dyslipidaemia (HDL level < 35 mg/dl or triglycerides > 250 mg/dl)

9. Polycystic ovary disease and acanthosis nigricans

10. History of vascular disease

COMPLICATIONS18, 25-30

As a consequence of hyperglycaemia of diabetes, every tissue and organ of the body undergoes biochemical and structural alterations which account for the major complications in diabetics which may be acute metabolic or chronic systemic. Both types of diabetes mellitus may develop complications which are broadly divided into 2 major groups:

I. Acute metabolic complications

II. Long-term diabetic complications

I. ACUTE METABOLIC COMPLICATIONS

These include diabetic ketoacidosis, hyperosmolar nonketotic coma, and hypoglycaemia. Metabolic complications develop acutely. While ketoacidosis and hypoglycaemic episodes are primarily complications of type 1 DM, hyperosmolar nonketotic coma is chiefly a complication of type 2 DM.

1. Diabetic ketoacidosis (DKA)

Ketoacidosis is almost exclusively a complication of type 1 DM. It can develop in patients with severe insulin deficiency combined with glucagon

9 excess. Failure to take insulin and exposure to stress are the usual precipitating causes. Severe lack of insulin causes lipolysis in the adipose tissues, resulting in release of free fatty acids into the plasma. These free fatty acids are taken up by the liver where they are oxidized through acetyl coenzyme-A to ketone bodies, principally acetoacetic acid and β-hydroxybutyric acid. Such free fatty acid oxidation to ketone bodies is accelerated in the presence of elevated level of glucagon. Once the rate of ketogenesis exceeds the rate at which the ketone bodies can be utilised by the muscles and other tissues, ketonaemia and ketonuria occur.

2. Hyperosmolar hyperglycaemic nonketotic coma (HHS)

Hyperosmolar hyperglycaemic nonketotic coma is usually a complication of type 2 DM. It is caused by severe dehydration resulting from sustained hyperglycaemic diuresis. The loss of glucose in urine is so intense that the patient is unable to drink sufficient water to maintain urinary fluid loss.

3. Hypoglycaemia

Hypoglycaemic episode may develop in patients of type 1 DM. It may result from excessive administration of insulin, missing a meal, or due to stress.

Hypoglycaemic episodes are harmful as they produce permanent brain damage, or may result in worsening of diabetic control and rebound hyperglycaemia, so called Somogyi’s effect.

II. LONG-TERM DIABETIC COMPLICATIONS

The long-term complications may occur in each type of diabetes, the spectrum of incidence is different. Many patients with type 2 diabetes have had their disease a longtime before the diagnosis, by which time many have developed diabetic complications. Hyperglycaemia and hypertension are the two major modifiable risk factors that influence the development of diabetic complications.

Diabetic complications are frequently divided into macrovascular and microvascular complications. Macrovascular complications arise from damage to large blood vessels and microvascular complications occur from damage to smaller vessels. The general aetiology of macro- and microvascular complications is the same and results from atherosclerosis of the vessels, which may lead to occlusion.

A. Macrovascular Complications

The risk of macrovascular complications, including cardiovascular disease (coronary heart disease and stroke) and PVD, is 2–4 times higher for people with diabetes.

1. Cardiovascular disease

The most common cause of death in people with type 2 diabetes is cardiovascular disease which accounts for an estimated 80% of deaths in this patient group. The risk of a person with diabetes having a myocardial infarction

(MI) is the same as someone without diabetes having a second myocardial

11 infarction. The risk of cardiovascular disease is increased further if nephropathy is present. Other cardiovascular disease risk factors are the same as in the non-diabetic population and include smoking, hypertension and dyslipidaemia.

2. Hypertension

Hypertension is twice as common amongst the diabetic population compared to the general population. It affects over 80% of those with type 2 diabetes. The treatment target ranges for people with diabetes are generally lower than for people without diabetes, as hypertension is associated with the development of macro- and microvascular complications.

3. Peripheral vascular disease (PVD)

PVD affects the blood vessels outside the heart. In people with diabetes, it often affects the arteries of the legs and may give rise to intermittent claudication, a cramping pain experienced on walking, due to reversible muscle ischaemia secondary to atherosclerosis. The iliac vessels can be affected, causing buttock pain and also erectile dysfunction. If PVD is present, the risk of cardiovascular disease increases.

B. Microvascular Complications

Microvascular complications include retinopathy, nephropathy and neuropathy.

1. Retinopathy

Diabetic retinopathy is the leading cause of blindness in people under the age of 60 in industrialized countries. Twenty years from the onset of diabetes, over 90% of people with type1, and over 60% of people with type 2, will have diabetic retinopathy. The main problem with diagnosing retinopathy is that it is symptom less until the disease is far advanced. Therefore, if regular screening is not undertaken, diagnosis may not be made early enough for successful treatment intervention. Tight glycaemic control has been shown to prevent and delay the progression of retinopathy in patients with type 1 disease.

2. Nephropathy

In diabetic renal disease, the kidneys become enlarged and the glomerular filtration rate (GFR) initially increases. However, if the nephropathy progresses, the GFR starts to decline. Serum creatinine used alone to estimate renal function has limitations. The GFR can be estimated (eGFR).

The most popular method is the modified Modification of Diet in Renal

Disease (MDRD) formula, which requires serum creatinine, age, sex and ethnicity. eGFR = 175× [plasma creatinine (µmol / L) 0.011312]1.154 × [age in years]0.203

× [1.212if black] × [0.742if female] eGFR = glomerular filtration rate (mL / minper1.73m2 )

The presence of nephropathy is indicated by the detection of microalbuminuria (small amounts of albumin present in urine). If higher

13 amounts of albumin are detected, this is termed proteinuria (or macroalbuminuria) and signifies more severe renal damage. Microalbuminuria is defined as an albumin:creatinine ratio (ACR) greater or equal to 2.5 mg/mmol (men) and 3.5 mg/mmol (women). Proteinuria may be defined as an albumin:creatinine ratio greater than 30 mg/mmol or albumin concentration greater than 200 mg/L. Proteinuria may progress to end-stage renal disease and require dialysis. Albumin in the urine increases the risk of cardiovascular disease, with microalbuminuria associated with 2–4 times the risk, proteinuria with nine times the risk and end-stage renal disease increasing risk by 50 times.

Tight control of both glycaemic levels and blood pressure reduces the risk of developing nephropathy.

3. Peripheral neuropathy30-37

Peripheral neuropathy is the progressive loss of peripheral nerve fibres resulting in nerve dysfunction. Diabetic neuropathies can lead to a wide variety of sensory, motor and autonomic symptoms. The most common is the symmetrical distal sensory type, which is particularly evident in the feet and may slowly progress to a complete loss of feeling. It is most prevalent in elderly patients with type 2 diabetes but may be found with any type of diabetes, at any age beyond childhood. Diabetic proximal motor neuropathy is rapid in onset and involves weakness and wasting, principally of the thigh muscles. Distal motor neuropathy can lead to symptoms of impaired fine co-ordination of the hands and/or foot slapping. Autonomic neuropathy may

14 also cause dry skin and lack of sweating, both of which may contribute to diabetic foot problems.

C. Macro- and microvascular disease combined

1. Diabetic foot problems

Infected diabetic foot ulcers account for the largest number of diabetes- related hospital bed-days and are the most common non-trauma cause of amputations. The rate of lower limb amputation in people with diabetes is 15 times higher than in the general population. The lifetime risk of a person with diabetes developing a foot ulcer is around 15%.

Diabetic foot ulcers are a costly problem and are associated with considerable morbidity. Foot problems often develop as a result of a combination of specific problems associated with having diabetes, which are sensory and autonomic neuropathy, PVD and hyperglycaemia.

Poor foot care and poorly controlled diabetes are also contributory factors. Development of foot ulcers may be partly preventable by patient education. People with diabetes learn that their feet are particularly vulnerable, and if problems arise, they must seek immediate professional advice.

There are three main types of foot ulcers: neuropathic, ischaemic and neuroischaemic.

Neuropathic ulcers occur when peripheral neuropathy causes loss of pain sensation. The ulcers can be deep but are usually painless and are caused by trauma to the foot which is not noticed until after significant damage has

15 occurred Ischaemic ulcers result from PVD and poor blood supply causing a reduction in available nutrients and oxygen required for healing. Ischaemic ulcers are painful and usually occur on the distal ends of the toes.

Most ulcers have elements of both neuropathy and ischaemia and are termed as neuroischaemic.

PATHOGENESIS18,38,39 PHYSIOLOGY OF NORMAL INSULIN SYNTHESIS AND SECRETION

The major stimulus for both synthesis and release of insulin is glucose.

The steps involved in biosynthesis, release and actions of insulin are as follows

Synthesis

Insulin is synthesized in the β-cells of pancreatic islets of Langerhans.

i) It is initially formed as pre-proinsulin which is single-chain 86-amino

acid precursor polypeptide.

ii) Subsequent proteolysis removes the amino terminal signal peptide,

forming proinsulin.

iii) Further cleavage of proinsulin gives rise to A (21 aminoacids) and B

(30 amino acids) chains of insulin, linked together by connecting

segment called C-peptide, all of which are stored in the secretory

granules in the β-cells.

Release

Glucose is the key regulator of insulin secretion from β-cells by a series of steps:

i) Hypoglycaemia stimulates transport into β-cells of a glucose transporter, GLUT2. Other stimuli influencing insulin release include nutrients in the meal, ketones, amino acids etc.

ii) An islet transcription factor, glucokinase, causes glucose phosphorylation.

iii) Metabolism of glucose to glucose-6-phosphate by glycolysis generates ATP.

iv) Generation of ATP alters the ion channel activity on the membrane. It causes inhibition of ATP-sensitive K+ channel on the cell membrane and opening up of calcium channel with resultant influx of calcium, which stimulates insulin release.

Action

Half of insulin secreted from β-cells into portal vein is degraded in the liver while the remaining half enters the systemic circulation for action on the target cells.

Figure 1: Explains the synthesis, release and actions of insulin.

PATHOGENESIS OF TYPE 1 DM

The basic phenomenon in type 1 DM is destruction of β-cell mass, usually leading to absolute insulin deficiency.

MECHANISMS OF TYPE 1A DM

1. Genetic susceptibility

2. Autoimmune factors

3. Environmental factors, are involved as basic mechanism.

Figure 2: Explains the pathogenesis of type 1 diabetes mellitus.

PATHOGENESIS OF TYPE 2 DM

The basic metabolic defect in type 2 DM is either a delayed insulin secretion relative to glucose load (impaired insulin secretion), or the peripheral tissues are unable to respond to insulin (insulin resistance).

The pathogenesis factors are,

1. Genetic factors

2. Constitutional factors

3. Insulin resistance

4. Impaired insulin secretion

5. Increased hepatic glucose synthesis

Figure 3: Explains the pathogenesis of type 2 diabetes mellitus.

DIAGNOSIS19,40,41

• A1C test, also called hemoglobin A1C, HbA1C, or glycohemoglobin

test.

• Fasting plasma glucose (FPG) test.

• Oral glucose tolerance test (OGTT).

TABLE 1: Explains blood test levels for diagnosis of diabetes and

prediabetes

Fasting Oral Glucose A1C Plasma Glucose Tolerance Test Diagnosis (Percent) (mg/dL) (mg/dL)

About 5 99 or below 139 or below Normal

5.7 to 6.4 100 to 125 140 to 199 Prediabetes

6.5 or above 126 or above 200 or above Diabetes

MANAGEMENT OF DIABETES MELLITUS42-45

For the management of diabetes mellitus life style modification is necessary along with pharmacological treatment

NONPHARMACOLOGICAL TREATMENT

Diet control : Avoid carbohydrates, sugar, reduction of salt

intake & to include more fiber containing

food, vegetables and fish oils

Moderate exercise : Include brisk walking, cycling, and restrict

alcohol consumption, avoid stress, avoid

smoking

PHARMACOLOGICAL TREATMENT

The treatment option for type 1 diabetes is insulin. Exogenous insulins are used to control blood sugar level.

TYPES OF INSULIN

A. Rapid Acting

 Insulin lispro, Insulin aspart, Insulin glulisine

B. Short Acting

 Regular (soluble) insulin

C. Intermediate Acting

 Insulin zinc suspension or Lente, Neutral protamine hagedorn

(NPH) or isophane insulin

D. Long Acting

 Protamine zinc insulin (PZI) , Insulin glargine

Insulin preparations:

 Soluble insulin : Human actrapid, Human velosulin

 Biphasic insulin : Human mixtard 10, Novomix 30

 Isophane insulin : Humulin 1, hypurinporcinisophane

 Insulinzinc suspension : Human monotard, Hypurin Bovine

Lente

ORAL HYPOGLYCAEMIC AGENTS

In type 2 diabetes glycaemic control cannot be achieved by only life style modification, along with pharmacological treatment also necessary to control blood sugar level. These agents are used as a monotherapy or combination therapy for the treatment of type 2 diabetes. The oral hypoglycemic agents are;

(1) Sulfonylureas:-

These drugs enhance release of insulin by a direct secretary mechanism.

Examples: Glibenclamide, Gliclazide, Glimepiride

(2) Biguanides:-

These drugs enhance the sensitivity of insulin in liver, and reduce gluconeogenesis and glycogenesis, and also enhance the glucose uptake.

Examples: Metformin

(3) α-glucosidase inhibitors:-

These drugs inhibit the activity of α-glucosidase enzyme which present in intestinal brush border, and these prevent the cleavage of polysaccharides to absorbable monosacharides. Examples: Acarbose, Voglibose, Miglitol

(4) Thiazolidinediones:-

These drugs increase the effect of insulin in skeletal muscle, adipose and hepatic tissues without enhancing pancreatic secretion of insulin. Examples:

Rosiglitazone, Pioglitazone

(5) Meglitinides:-

These drugs bind to ATP sensitive potassium channels and enhance the release of insulin from pancreatic cells. Examples: Repaglinide, Nateglinide

(6) Dipeptidyl peptidase four inhibitors:-

These drugs enhance the effect of GLP-1 and GIP, and increase glucose mediated insulin secretion and suppresses glucagon secretion, these drugs mostly commonly used as a combination therapy with metformin or TZDs.

Examples: Sitagliptin, Vildagliptin, Saxagliptin.

The present study mainly focused with diabetic neuropathy, which is a common complication that develops nearly 50% of over all diabetes population.

Diabetic neuropathy appears relatively early in the disease process. The prevalence of up to 7% is reported in South Indian patients even at the time of diagnosis of diabetes46. Diabetic neuropathy has widespread occurrence and devastating effects. The precise pathogenesis of diabetic neuropathy is unclear despite recent advances. Polyol pathway of glucose metabolism has been considered as one of the major mechanisms in the pathogenesis of diabetic neuropathy47-50. Conversion of glucose to sorbitol by the enzyme aldose

23 reductase is the rate limiting step of polyol pathway. Increased activity of polyol pathway due to hyperglycemia and subsequent accumulation of excess sorbitol explains the neuronal damage in diabetes48-50.

Figure 4: Polyol (Sorbitol) Pathway.

Figure 5: Polyol (Sorbitol) Pathway with accumulation of

sorbitol and fructose.

Methylcobalamin is one of the biologically active forms of vitamin B12.

It is used in the treatment of peripheral neuropathy, diabetic neuropathy, and as a preliminary treatment for amyotrophic lateral sclerosis. Unlike cyanocobalamin, Methylcobalamin is active in the spinal fluid. Due to this property, it is able to help heal the damaged nerve cells and restores normal functions. In clinical studies, Methylcobalamin showed improvement in the somatic and autonomic symptoms with regression of signs of diabetic neuropathy such as pain and paresthesia53,54. With this background, this study was undertaken to assess the efficacy and safety of Epalrestat and

Methylcobalamin alone and in combination, in diabetic patients with neuropathy condition.

Our literature review focused on the pharmacological potential of

 Ramirez MA and Boria NL., studied the effect of Epalrestat: an aldose

reductase inhibitor for the treatment of diabetic neuropathy. Unlike the

current treatment options for diabetic neuropathy, Epalrestat may affect

or delay progression of the underlying disease process. Data from six

clinical trials were evaluated, and it was determined that Epalrestat 50

mg 3 times/day may improve motor and sensory nerve conduction

velocity and subjective neuropathy symptoms as compared with baseline

and placebo. Epalrestat may serve as a new therapeutic option to prevent

or slow the progression of diabetic neuropathy. Long-term, comparative

studies in diverse patient populations are needed for clinical

application51.

 Yaqub BA., et al., studied the clinical and neurophysiological effects of

Methylcobalamin on patients with diabetic neuropathy. In a double-

blind study, the active group showed statistical improvement in the

somatic and autonomic symptoms with regression of signs of diabetic

neuropathy. Motor and sensory nerve conduction studies showed no

statistical improvement after 4 months. The drug was easily tolerated by

the patients and no side effects were encountered53.

 Hotta. N et al., evaluated and observed that the long term treatment

with Epalrestat was well tolerated and effectively delay the progression

of diabetic neuropathy in addition to that good glycemic control and

limited microangiopathy55.

 Epalrestat is an aldose reductase inhibitor (ARI), the author conducted a

questionnaire based study for the effectiveness of diabetic peripheral

neuropathy in Epalrestat treated patients and the author correlated the

therapeutic benefits and responses56.

 Karunanithi.P., et al., considered about the comparative study of

efficacy between Pregabalin and Epalrestat in diabetic peripheral

neuropathic pain and reported that there was rapid reduction of pain

scores in pain rating scale, biothesiometry scores and reduction of

plasma glucose post prandial (PGPP) levels are more in pregabalin

therapy when compared to Epalrestat therapy57.

 The author conducted a comparative trial namely evaluation of efficacy

and safety of Epalrestat and Epalrestat in combination with

Methylcobalamin in patient with diabetic neuropathy in a randomized

comparative trial. In which the author concluded that combination of

Epalrestat and Methylcobalamin is a better option for the treatment of

diabetic neuropathy than Epalrestat alone. Combination therapy was

associated with faster onset and better symptomatic relief58.

 Maladkar. M., et al., conducted a prospective, randomized single blind

comparative study of Epalrestat to Methylcobalamin in patient with

diabetic neuropathy. The author finally reported that Epalrestat, seems to

be a better alternative than Methylcobalamin in the treatment of diabetic

neuropathy. Epalrestat has a better efficacy and safety profile than

Methylcobalamin in the treatment of diabetic neuropathy59.

 The Chinese author conducted a randomized controlled trial along with

meta analysis, entitled effects of Epalrestat combined with

Methylcobalamin on diabetic peripheral neuropathy. The

methodological quality of the trials was evaluated and the valid data for

Meta-analysis were extracted. Six randomized controlled trials (496

cases) were included. In addition to conventional basic treatments, test

group was treated with additional Epalrestat combined with

Methylcobalamin, while control group was treated with additional B

vitamins. Meta analysis showed that the patient in test group was better

than those in control group in the improvement of clinical symptoms.

The author concluded that the meta analysis on this group shows that

Epalrestat combined with Methylcobalamin can improve the clinical

symptoms in the patients with diabetic peripheral neuropathy60.

 Ide H., Fujiya S., et al., conducted a study of Clinical utilization of

intrathecal injection of Methylcobalamin in patients with diabetic

neuropathy. Seven men and four women with symptomatic diabetic

neuropathy were treated with Methylcobalamin (2,500 micrograms in 10

ml of saline) injected intrathecally. Treatment was begun when patients

had good metabolic control, as determined by measurements of plasma

glucose and hemoglobin, and was repeated several times with a one-

month interval between injections. Three patients were re-treated one

year after the last intrathecal injection. Symptoms in the legs, such as

paresthesia, burning pains, and heaviness, dramatically improved. The

mean (+/- SD) concentration of Methylcobalamin in spinal fluid was

114 +/- 32 pg/ml before intrathecal injection (n = 5) and 4,752 +/- 2,504

pg/ml one month after intrathecal Methylcobalamin treatment (n = 11).

Methylcobalamin caused no side effects with respect to subjective

symptoms or characteristics of spinal fluid. These findings suggest that a

high concentration of Methylcobalamin in spinal fluid is highly effective

and safe for treating the symptoms of diabetic neuropathy61.

 Kuwabara S, et al., studied intravenous Methylcobalamin treatment for

uremic and diabetic neuropathy in chronic hemodialysis patients. The

main objective of the study was to study the effects of the intravenous

administration of Methylcobalamin, an analogue of vitamin B12, for

uremic or uremic-diabetic polyneuropathy in patients who are receiving

maintenance hemodialysis. An ultra-high dose of vitamin B12 has been

reported to promote peripheral nerve regeneration in experimental

neuropathy. Nine patients received a 500 microg Methylcobalamin

injection 3 times a week for 6 months. The effects were evaluated using

neuropathic pain grading and a nerve conduction study. The author

reported that Serum concentrations of vitamin B12 were ultra-high

during treatment due to the lack of urinary excretion. After 6 months of

treatment, the patients' pain or paresthesia had lessened, and the ulnar

motor and median sensory nerve conduction velocities showed

significant improvement. There were no side effects and finally

concluded that intravenous Methycobalamin treatment is a safe and

potentially beneficial therapy for neuropathy in chronic hemodialysis

patients62.

 Bhavani J and Lian LL studied a review article namely Vitamin B

supplementation for diabetic peripheral neuropathy. This review aims to

examine the existing evidence on the effectiveness of vitamin B12

supplementation for the treatment of diabetic peripheral neuropathy. A

search of PubMed and the Cochrane Central Register of Controlled

Trials for all relevant randomized controlled trials was conducted in

December 2014. Any type of therapy using vitamin B12 or its coenzyme

forms was assessed for efficacy and safety in diabetics with peripheral

neuropathy. Changes in vibration perception thresholds, neuropathic

symptoms and nerve conduction velocities, as well as the adverse effects

of vitamin B12 therapy, were assessed. Four studies comprising 363

patients met the inclusion criteria. This review found no evidence that

the use of oral vitamin B12 supplements is associated with improvement

in the clinical symptoms of diabetic neuropathy. Furthermore, the

majority of studies reported no improvement in the electrophysiological

markers of nerve conduction63.

 Mizukami H, et al., studied the effect of Methylcobalamin on diabetic

neuropathy and nerve protein kinase C in rats. The study conducted

with back ground of methyl-base-attached cobalamin

(Methylcobalamin) (MC) has a special affinity for nerve tissues to

promote myelination and transport of axonal cytoskeleton. It is not

known, however, how MC influences on peripheral nerve in

experimental diabetic neuropathy. This study suggested that correction

of impaired neural signalling of PKC and oxidative stress-induced

damage may be a major attribute to the beneficial effects of MC on

diabetic nerve64.

 Vasudevan D, et al., conducted a pilot study namely efficacy and safety

of Methylcobalamin, alpha lipoic acid and pregabalin combination

versus pregabalin monotherapy in improving pain and nerve conduction

velocity in type 2 diabetes associated impaired peripheral neuropathic

condition. The study aimed to assess whether Methylcobalamin and

alpha lipoic acid (ALA) added to pregabalin provide additional benefit

compared to pregabalin alone in type 2 diabetes mellitus associated

peripheral neuropathy. It was an open label, randomized, controlled

parallel-group pilot study. Thirty adult patients with type 2 diabetes

mellitus with symptoms of peripheral neuropathy for ≥6 months were

randomized to receive pregabalin 75 mg, Methylcobalamin 750 μg, and 31

ALA 100 mg (PMA, n = 15); or pregabalin 75 mg (PG, n = 15) for 12 weeks. Assessment variables were numeric rating scale (NRS), sleep interference scores (SIS), response rate to pain, and global assessment for the usefulness of therapy. Finally the author concluded that

Methylcobalamin, ALA and pregabalin combination provides pain relief and improves sleep interference. Addition of Methylcobalamin and ALA to pregabalin improves the nerve function65.

DRUG PROFILE

4. DRUG PROFILE

EPALRESTAT66-68

GENERIC NAME : EPALRESTAT

TRADE NAME :

 Aldonil (Zydus Medica), India

 Aldorin, Bangladesh

 Alrista (marketed and not manufactured by Macleods), India

 Epalrica-M (Ordain Global), India

 Eparel 50 (Microlabs Ltd), India

 Epimeth (Zaiva Lifesciences), India

 Eplistat 150 SR (Schem), India

 Letostat-SR (Amor Pharmaceuticals), India

 Listap-50 (Vivid Biotek), India

 Tanglin (Yangtze River Pharmaceutical Group), China

STRUCTURE :

MOLECULAR FORMULA : C15H13NO3S2

CATEGORY : Oral anti-diabetic agent: Aldose reductase

inhibitor

DOSE66 : 50mg, 150mg

DOSAGE FORM : Tablet

MECHANISM OF ACTION :

 Epalrestat is aldose reductase inhibitor. Aldose reductase reduces

glucose to sorbitol.

 Epalrestat suppresses accumulation of sorbitol in nerve by inhibiting

aldose reductase, and relieves numbness/pain of the hands and feet

and leg cramp, etc., which induced by diabetic peripheral neuropathy.

PHARMACOKINETICS:

 The absorption rate and elimination rate of Epalrestat were not

changed after multiple oral administrations.

 There was no accumulation of drug in plasma. No difference in

pharmacokinetics of Epalrestat in male and female gender.

 Plasma Epalrestat concentrations were measured using high-

performance liquid chromatography.

PRECAUTION :

 Precaution should be taken while patient with Diabetic neuropathy,

liver disease, renal impairment, pregnancy, nursing mothers, pediatric

and geriatric use.

 Before using this drug, inform your doctor about your current list of

medications, over the counter products (e.g. vitamins, herbal

supplements, etc.,), allergies, pre-existing diseases, and current health

conditions (e.g. pregnancy, upcoming surgery, etc.,).

SIDE EFFECT :

 Nausea

 vomiting

 diarrhea

 generalized gastric discomfort

 cutaneous reactions including erythema

 bullae and skin blistering.

CONTRAINDICATION :

Hypersensitivity & in severe hepatic insufficiency

DRUG INTERACTION : Not Known

METHYLCOBALAMIN69-73

GENERIC NAME : METHYLCOBALAMIN

TRADE NAME : TRINERGIC (UNICHEM)

PREGAB (TORRENT)

LYRICA ( PFIZER)

STRUCTURE :

MOLECULAR FORMULA : C63H91CoN13O14P

CATEGORY :

 Antianemic Preparations

 Blood and Blood Forming Organs

 Corrinoids

 Vitamin B12 (Cyanocobalamin and Analogues)

 Vitamin B12 and Folic Acid

DOSE AND DOSAGE FORMS72 :

TABLET

 100mcg

 250mcg

 500mcg

 1000mcg

 1500mcg

Tablet, Extended Release

 1000mcg

Tablet, Sublingual

 2500mcg

Injectable Solution

 1000mcg/mL

Nasal Spray

 500mcg/spray

MECHANISM OF ACTION :

 It's used as a dietary supplement and to treat certain anaemias.

 Vitamin B12 plays an important role in helping the body make red

blood cells

 Coenzyme; metabolic functions include protein synthesis and

carbohydrate metabolism

PHARMACOKINETICS :

 Mecobalamin is the neurologically active form of vitamin B12 and occurs as a water-soluble vitamin in the body.

 Bioavailability: 6.1% (intranasal solution relative to IM)

 Peak plasma concentrations after 3 hr (oral); 0.9 hr (IM); 3 min (IV).

 Distributed to liver, bone marrow, and other tissues.

 Protein bound: Binding to transcobalamins

 Excretion Via urine.

PRECAUTION :

 Intensive treatment of B12-deficient megaloblastic anemia may cause

hypokalemia and sudden death

 Use with caution in patients with leber optic nerve atrophy

 Injection is incompatible with some common drugs (eg, warfarin and

several phenothiazines) but is compatible with vitamins B and C

 Vitamin B12 deficiency for >3 months results in irreversible

degenerative CNS lesions

 Oral and intranasal route are not indicated for the treatment of

pernicious anemia until symptoms resolve

 IM and SC route used to treat pernicious anemia; oral and intranasal

not indicated until hematologic remission and no signs of nervous

system involvement.

 Use with caution in patients with leber’s disease; B12 treatment may

result in rapid optic atrophy

 Parenteral product may contain aluminum; toxic aluminum

concentrations may occur with renal dysfunction, high doses, or

prolonged use

SIDE EFFECT :

 Headache

 Itching

 Swelling

 Nervousness and anxiousness

 Involuntary or uncontrollable movements

CONTRAINDICATION:

Hypersensitivity to product; sensitivity to cobalt

DRUG-DRUG INTERACTION :

 Leukeran (chlorambucil)

 Prilosec (omeprazole)

 Colcrys and Mitigare (colchicine)

 The herbal supplement goldenseal

DRUG-FOOD INTERACTION :

 Alcohol actually decreases levels of B vitamins in the body especially if

you drink a lot.

PLAN OF WORK

5. PLAN OF WORK

The present study was planned to conduct a randomized comparative study on clinical efficacy of Epalrestat (150mg/day) versus Methylcobalamin

(1500 mcg/day) versus Epalrestat (150mg/day) with Methylcobalamin (1500 mcg/day) in diabetic neuropathy patients. The present study was conducted in

Karpaga Vinayaga Institute of Medical Sciences & Research Centre,

Chinnakolambakkam, Kanchipuram District- 603 308, Tamil Nadu.

The Plan of Work Includes:

I. Submission & design of protocol.

II. To get informed written consent from patient.

III. Select diabetic neuropathy patients for the study.

IV. Selection of efficacy and safety parameters.

V. Evaluation of collected data.

VI. Data analysis and statistical analysis was done with the help of Graph Pad Prism Instat Version 3 (USA). Basic statistical evaluation including Mean, Median, and Standard Deviation (SD) will be calculated from the raw data. Efficacy variables such as diabetic neuropathy symptoms, pain intensity were assessed by VAS (Visual Analog Scale) Pain scoring method. Muscle strength, muscle cramps, prickling feelings, burning pain, sensitive to touch, ulceration and ankle reflexes were assessed by Michigan Neuropathy Screening Instrument (MNSI) score method. Chi- square test and One way ANOVA followed by Tukey-Kramer multiple comparison test were used where ever applicable.

MATERIALS AND METHODS

6. MATERIALS AND METHODS

STUDY SITE

The study was carried out in Karpaga Vinayaga Institute of Medical

Sciences & Research Centre, Chinnakolambakkam, Kanchipuram District- 603

308, Tamil Nadu.

STUDY DESIGN

A prospective randomized controlled study carried out in diabetic neuropathy patients.

SAMPLE SIZE

A total number of 165 patients from the out- patient and in-patient department in Karpaga Vinayaga Institute of Medical Sciences & Research

Centre, Chinnakolambakkam, Kanchipuram District- 603 308, Tamil Nadu, who were diagnosed as Diabetic neuropathy were included.

STUDY PERIOD

The study was conducted from March 2016 to February 2017.

STUDY GROUP

For this study, about 165 diabetic patients with symptoms of neuropathy were randomly divided into three groups by block randomization method, i.e.,

Group A (55 patients) received 150 mg of Epalrestat per day, Group B (55 patients) received 1500 mcg of Methylcobalamin per day, Group C (55 patients) received 150 mg of Epalrestat + 1500 mcg Methylcobalamin per day.

They were treated with drugs for a period of 12 weeks and they were followed up on 4, 8 & 12 weeks.

TABLE 2: Explains the study groups

Group Drug treated Number of patients

A Epalrestat (150 mg/day) 55

B Methylcobalamin (1500 mcg/day) 55

Epalrestat (150 mg/day) C 55 + Methylcobalamin (1500 mcg/day)

A total of 165 diabetic patients with neuropathy symptoms were enrolled in our study program. The patients were observed for age, sex, family history, blood pressure, weight & height were recorded. The patients were monitored for their blood glucose level, both fasting and postprandial and glycosylated hemoglobin at the initial visit to the hospital and after 3 months of treatment.

INCLUSION CRITERIA:

1. Diabetic patients, both type 1 & type 2 with symptoms of neuropathy.

2. Age – 20 to 65 years.

3. HbA1C ≤ 9% ± 0.5% variation in the previous 3 months.

4. Patients on continued conventional therapy for Diabetes Mellitus.

EXCLUSION CRITERIA:

1. Patients having alcoholic neuropathy.

2. Patients having Foot ulcer.

3. Patients having Carpal tunnel syndrome.

4. Patients having Cerebrovascular sequelae.

5. Patients having hepatic and renal impairment.

6. Patients taking antiepileptic and anti- depressant drugs.

7. Patients on any other medications that affects symptoms of neuropathy.

ETHICAL CONSIDERATION:

All the investigational procedures and protocols used in this study were reviewed & approved by the Institutional Ethical Committee (IEC Reference

No: 22/2016) and were in accordance with the CONSORT guidelines.

STUDY EVALUATION:

Informed consent was obtained after a detailed explanation of the study purpose and methods. Patients were received Epalrestat or Methylcobalamin or

Epalrestat with Methylcobalamin combination. The treatment period was 12 weeks and both the medications were administered once daily. This is a single blind study; therefore patients were unaware of treatment given to them.

Patients were monitored on 4th, 8th and 12th weeks of the study. Patient’s demographic data and medical history were recorded at screening visit.

Physical examinations were recorded at screening and 12th week of follow up

43 visit. Systemic examination and vital examination were recorded at every follow up visit.

EFFICACY PARAMETERS:

1. Visual Analog Scale of pain intensity based on 10 point scoring method - mild, moderate, severe, very severe, worst.

Score Symptoms 0 No pain 1-2 Mild pain 3-4 Moderate pain 5-6 Severe pain 7-8 Very severe pain 8-10 Worst pain

2. Loss of sensation, burning sensation, numbness, muscle cramps, spontaneous pain, weakness, dizziness, loss of sensation of heat & cold assessed by Michigan neuropathy screening instrument score method. ( Patient Version and Physician Version)

3. HbA1C at baseline & at the end of treatment.

SAFETY PARAMETERS:

1. Adverse events like skin rash, hot flushes, etc.,

2. FBS at baseline & at the end of treatment.

3. PPBS at baseline & at the end of treatment.

4. LFT at baseline & at the end of treatment.

5. RFT at baseline & at the end of treatment.

6. CBC at baseline & at the end of treatment.

STATISTICAL ANALYSIS:

Statistical analysis was done with the help of Graph Pad Prism Instat

Version 3 (USA). Basic statistical evaluation including Mean, Median, and

Standard Deviation (SD) were calculated from the raw data. Efficacy variables such as diabetic neuropathy symptoms, pain intensity were assessed by VAS

Pain scoring method. Muscle strength, muscle cramps, prickling feelings, burning pain, sensitive to touch, ulceration and ankle reflexes were assessed by

Michigan neuropathy screening instrument (MNSI) score method. Chi-square test and one way ANOVA followed by Tukey-Kramer multiple comparison test were used where ever applicable.

OBSERVATION AND RESULTS

7. OBSERVATION AND RESULTS

Out of 267 patients screened, 102 were excluded and 165 were enrolled based on the inclusion and exclusion criteria. The reasons for exclusion were:

a. Patients with Foot ulcer (31)

b. Patients having alcoholic neuropathy (33)

c. Patients having hepatic impairment (29)

d. Patients taking antidepressants (09)

The 165 recruited patients were randomized into 3 groups A, B and C consisting of 55 patients each. Patients of group A received Epalrestat, group B received Methylcobalamin and group C received Epalrestat combined with

Methylcobalamin.

Patients withdrawn from the study:

Group A : 3

a. 2 patients had diabetic foot ulcer complication with standard therapy which required additional care and therapy.

b. 1 patient had abnormal laboratory parameters especially HbA1C.

Group B : 2

a. 2 patients had developed symptoms of chronic kidney failure which requiring additional care and therapy.

Group C : 3

a. 3 patients had abnormal laboratory parameters especially HbA1C.

Patient lost to follow-up :

Group A : 3 patients were failed to follow up.

Group B : 2 patients were failed to follow up.

Group C : 2 patients were failed to follow up.

Patient who completed the study:

Group A : 49

Group B : 51

Group C : 50

The results at the end of the study are as follows.

TRAIL FLOW CHART

SEX DISTRIBUTION

TABLE 3: Explains the number of males and females among the

study groups A, B and C.

Chi-Square

GROUP MALES FEMALES TOTAL test

independence

A 32 65% 17 35% 49 p-value B 28 55% 23 45% 51 0.775 (NS)

C 34 68% 16 32% 50

(NS-Not Significant)

There was no statistically significant difference in sex distribution between all three groups.

Figure 6,7 & 8 :Shows the percentage of males and females

among the study groups.

Group A

35% MALES FEMALES 65%

Group B

45% MALES

55% FEMALES

Group C

32% MALES FEMALES 68%

AGE DISTRIBUTION

TABLE 4: Shows the mean age (in years) of the patients belonging

to groups A, B and C.

Chi-Square

MEAN Age test GROUP N SD in Years independence

p-value

A 49 53.87 14.14

B 51 53.61 11.24 0.846 (NS)

C 50 57.62 11.70

(NS-Not Significant)

There was no statistically significant difference between the study groups with respect to age.

Figure 9 : Shows the graphical representation of the mean age

of the study groups.

MEAN AGE IN YEARS

58 57.62

Mean Age in Years

53.87

Age Years in 54 53.61

51 Group A Group B Group C Study Groups

BODY MASS INDEX (BMI)

TABLE 5: Shows the mean BMI of all three groups at baseline and

at the end of 4th, 8th and 12th weeks.

Group C Chi-Square test Group A (n=49) Group B (n=51) (n=50) Visit independence

Mean S.D Mean S.D Mean S.D p-value BMI BMI BMI Baseline 25.49 2.34 25.65 2.52 25.78 2.89 0.750 (NS) (1)

2 25.57 2.47 25.71 2.49 25.82 2.97 0.767 (NS)

3 25.51 2.38 25.63 2.76 25.69 2.67 0.762 (NS)

4 25.50 2.65 25.59 2.65 25.62 3.12 0.625 (NS)

(NS-Not Significant)

There was no statistically significant difference between the groups as

well within the groups at each visit.

Figure 10 : Shows the graphical representation of change in mean BMI of study

groups during the visits.

BODY MASS INDEX

25.9

25.82

25.8 25.78

25.71 25.7 25.69 25.65 25.63 25.62 Baseline 2nd Visit 25.6 25.59 25.57 3rd Visit 4th Visit

Mean BMI Mean

25.51 25.5 25.5 25.49

25.4

25.3 Group A Group B Group C Study Groups

FASTING BLOOD SUGAR

TABLE 6: Shows the mean fasting blood glucose level of all three

groups at baseline and at the end of 4th, 8th and 12th weeks.

Group A Group B Group C One way

(n=49) (n=51) (n=50) ANOVA – Visit Tukey- Mean Mean Mean S.D S.D S.D Kramer (mg/dl) (mg/dl) (mg/dl) Multiple

Baseline Comparison 161.17 14.76 165.53 13.51 164.18 12.37 0.263 (NS) (1) Test P Value

2 160.76 13.34 165.37 14.52 163.41 14.28 0.262 (NS)

3 159.89 15.21 163.16 15.26 163.56 14.72 0.416 (NS)

4 157.87 14.29 161.59 14.47 162.892 13.67 0.191 (NS)

(NS-Not Significant)

There was no statistically significant difference between the groups or within the groups at each visit.

Figure 11 : Shows the graphical representation of table 6.

FASTING BLOOD SUGAR

168

166 165.53 165.37

164.18 164 163.56

162.892 163.41 163.16 162 161.17 161.59 160.76

160 159.89

Fasting Blood (mg/dl) Sugar 158 157.87

156

154 Baseline 2nd Visit 3rd Visit 4th Visit Visits Fasting Blood Sugar Group A Fasting Blood Sugar Group B Fasting Blood Sugar Group C

POST PRANDIAL BLOOD SUGAR

TABLE 7: Shows the mean post prandial blood glucose level of all

three groups at baseline and end of study.

Group A Group B Group C One way

(n=49) (n=51) (n=50) ANOVA – Visit Tukey- Kramer Mean Mean Mean S.D S.D S.D Multiple (mg/dl) (mg/dl) (mg/dl) Comparison

Baseline 271.22 21.34 275.24 13.82 274.18 12.19 Test0.442 P Value(NS)

End of 270.34 18.41 273.39 14.84 270.45 14.25 0.552(NS) Study

(NS-Not Significant)

There was no statistically significant difference between the groups or within the groups at baseline and end of study.

Figure 12 : Shows the graphical representation of table 7.

GLYCOSYLATED HEMOGLOBIN (HbA1C)

TABLE 8: Shows the mean glycosylated haemoglobin level of all

three groups at baseline and end of study.

Group A Group B Group C One way

(n=49) (n=51) (n=50) ANOVA – Visit Tukey- Kramer Mean Mean Mean S.D S.D S.D Multiple (%) (%) (%) Comparison Test

Baseline 7.67 0.34 7.85 0.62 7.76 0.69 0.293P Value (NS)

End of 7.56 0.51 7.78 0.74 7.56 0.69 0.154 (NS) Study

(NS-Not Significant)

There was no statistically significant difference between all the groups at baseline and end of study.

Figure 13 : Shows the graphical representation of table 8.

Glycosylated Haemoglobin (HbA1C) 7.85

7.85 7.78 7.76 7.8

7.67 7.75

7.7

7.65 7.56 7.56 7.6

HbA1C (%)

7.55

7.5

7.45

7.4 Baseline End of Study Visits

Group A Group B Group C

ASPARTATE TRANSAMINASE (AST) CONCENTRATION

TABLE 9: Shows the mean AST concentration of groups A, B & C.

One way ANOVA, Group followed by Group A Group B Tukey- Visit C (n=49) (n=51) Kramer (n=50) Multi comparison test P-Value

Mean 33.78 32.28 33.36 (IU/L) Baseline 0.902 (NS) S.D 19.27 17.74 13.73

Mean 33.13 32.02 33.13 End of (IU/L) 0.695 (NS) Study S.D 11.65 12.89 12.46

(NS-Not Significant)

There was no statistically significant difference between and with in the groups at baseline and at the end of study.

Figure 14 : Shows the graphical representation of table 9.

ASPARTATE TRANSAMINASE (AST) CONCENTRATION

34 33.78

33.36 33.5 33.13 33.13

Group A 32.5 32.28 Group B Group C 32.02

31.5

31 Baseline End of Study Visits

ALANINE AMINOTRANSFERASE (ALT) CONCENTRATION

TABLE 10: Shows the mean ALT concentration of groups A, B & C.

One way ANOVA, Group followed by Group A Group B Tukey- Visit C (n=49) (n=51) Kramer (n=50) Multi comparison test P-Value

Mean 34.56 35.41 35.73 (IU/L) Baseline 0.851 (NS) S.D 9.37 10.64 11.54

Mean 33.13 35.12 34.59 End of (IU/L) 0.669(NS) Study S.D 13.65 10.89 9.46

(NS-Not Significant)

There was no statistically significant difference between and with in the groups at baseline and at the end of study.

Figure 15 : Shows the graphical representation of table 10.

ALANINE AMINOTRANSFERASE (ALT) CONCENTRATION

36 35.73

35.41 35.5 35.12

35 34.56 34.59

34.5

34 Group A Group B 33.5

33.13 Group C ALT IU/L Conc. ALT

32.5

31.5 Baseline End of Study Visits

SERUM ALBUMIN

TABLE 11: Shows the mean serum albumin level of groups A, B & C.

One way Group ANOVA, Group A Group B followed by Visit C (n=49) (n=51) Tukey-Kramer (n=50) Multi comparison test P-Value

Mean 3.26 3.18 3.2 (gm/dl) Baseline 0.569(NS) S.D 0.37 0.45 0.34

Mean 3.29 3.22 3.25 End of (gm/dl) 0.802(NS) Study S.D 0.52 0.49 0.57

(NS-Not Significant)

There was no statistically significant difference between and with in the

groups at baseline and at the end of study.

Figure 16 : Shows the graphical representation of table 11.

SERUM ALBUMIN

3.29 3.3

3.28 3.26 3.25

3.26

3.22 3.24

3.22 3.2 Group A Group B 3.18 3.2 Group C

Albumin (gm/dl)

3.18

3.16

3.14

3.12 Baseline End of Study Visits

SERUM TOTAL BILIRUBIN

TABLE 12: Explains the mean serum total bilirubin

level of groups A, B & C.

Group A Group B Group C Visit (n=49) (n=51) (n=50)

Mean 0.76 0.73 0.74 (mg/dl) Baseline S.D 0.08 0.07 0.07

Mean 0.75 0.74 0.75 (mg/dl) End of Study S.D 0.07 0.06 0.05

Unpaired t test, two tail P- 0.512(NS) 0.125(NS) 0.413(NS) value

(NS-Not Significant)

There was no statistical significance difference was observed with in the groups at baseline and at the end of study.

Figure 17 : Shows the graphical representation of table 12.

SERUM TOTAL BILIRUBIN

0.76

0.755 0.75 0.75

0.75

0.745 0.74 0.74

0.74 Group A Group B 0.735 Group C 0.73

S. Total Bilirubin (mg/dl) Bilirubin Total S. 0.73

0.725

0.72

0.715 Baseline End of Study Visits

SERUM CREATININE

TABLE 13: Explains the serum creatinine

concentration of groups A, B & C.

Group A Group B Group C Visit (n=49) (n=51) (n=50)

Mean 0.89 0.91 0.93 (mg/dl) Baseline S.D 0.08 0.10 0.18

Mean 0.86 0.89 0.91 (mg/dl) End of Study S.D 0.09 0.07 0.08

Unpaired t test, two tail P- 0.084 (NS) 0.245 (NS) 0.475(NS) value

(NS-Not Significant)

There was no statistical significance difference was observed with in the groups at baseline and at the end of study.

Figure 18 : Shows the graphical representation of table 13.

SERUM CREATININE

0.93 0.94

0.91 0.91 0.92

0.89 0.89 0.9

Group A 0.88 Group B 0.86 Group C

Sr. Creatinine (mg/dl) Creatinine Sr. 0.86

0.84

0.82 Baseline End of Study Visits

SERUM UREA

TABLE 14: Shows the serum urea concentration of groups A, B & C.

Group A Group B Group C Visit (n=49) (n=51) (n=50)

Mean 16.23 16.75 16.54 (mg/dl) Baseline S.D 0.17 0.12 0.18

Mean 16.10 16.70 16.48 (mg/dl) End of Study S.D 0.9 0.13 0.17

Unpaired t test, two tail P- 0.332 (NS) 0.066 (NS) 0.065(NS) value

(NS-Not Significant)

There was no statistical significance difference was observed with in the groups at baseline and at the end of study.

Figure 19: Shows the graphical representation of table 14.

SERUM UREA

16.8 16.75 16.7

16.6 16.54 16.48

16.4

16.23 Group A 16.2 Group B

16.1 Group C S. Urea (mg/dl) Urea S.

15.8

15.6 Baseline End of Study Visits

URINE ALBUMIN

TABLE 15: Shows the urine albumin levels of groups A, B & C.

Group Group Group A Chi-square Visit B C (n=49) Test P-Value (n=51) (n=50)

nil 43 45 42

Baseline trace 6 6 8 0.801(NS)

present 0 0 0

nil 44 46 45 End of trace 4 5 4 0.630(NS) Study present 1 0 1

(NS-Not Significant)

There was no statistically significant difference between all the groups at baseline and at the end of study.

URINE SUGAR

TABLE 16: Explains the urine sugar levels of groups A, B & C.

Group Group A Group B Chi-square Visit C (n=49) (n=51) Test P-Value (n=50)

nil 43 45 40

trace 4 6 9 Baseline 0.054(NS) P 2 0 0

PP 0 0 1

nil 43 43 46

trace 5 7 3 End of 0.240(NS) Study P 1 0 1

PP 0 1 0

P- One Plus; PP- two plus by urine dipstick

(NS-Not Significant)

There was no statistically significant difference between all the groups at baseline and at the end of study.

ADVERSE DRUG REACTIONS

TABLE 17: Shows the frequency of various adverse drug reactions

between the study groups.

Group Group Adverse drug Group A S.No % B % C % reaction (n=49) (n=51) (n=50)

01 Diarrhoea - 0 1 1.9 - 0

02 Erythema - 0 - 0 - 0

03 Gastric 2 4 4 7.8 2 4 Discomfort

04 Head ache 1 2 - 0 - 0

05 Hepatic - 0 - 0 - 0 Dysfunction

06 Hot flush 1 2 1 1.9 2 4

07 Itching - 0 1 1.9 - 0

08 Nausea & 1 2 3 5.9 3 6 Vomiting

09 Skin rash 1 2 - 0 1 2

10 Swelling - 0 1 1.9 - 0

In group B, 7.8% of patients suffered with gastric discomfort and 5.9% of patients suffered with nausea and vomiting, in group A, 4% of patients suffered with gastric discomfort. In group C, 6% of patients suffered with nausea and vomiting.

VISUAL ANALOG PAIN SCORE

TABLE 18: Shows the pain intensity scores (VAS Method)

Group A Group B Group C Visit (n=49) (n=51) (n=50)

Mean Score 6.39∗∗∗ 6.28∗∗∗ 6.34∗∗∗ Baseline/ Visit 1 S.D 0.60 0.59 0.47

Mean Score 4.65 ΔΔΔ 4.75 ΔΔΔ 4.89 ΔΔΔ

Visit 2 S.D 0.43 0.45 0.39

Mean Score 4.12 ΨΨΨ 3.98 ΨΨΨ 3.09 ΨΨΨ

Visit 3 S.D 0.42 0.38 0.28

Mean Score 1.92 1.84 1.49

Visit 4 S.D 0.11 0.13 0.10

Data represented as mean with S.D (n=49, 51 & 50), which represents mean VAS pain Score. ∗∗∗ denotes p<0.001, Base line (Visit 1st) compared with visit 2nd, 3rd and 4th visit, ΔΔΔ denotes p<0.001, visit 2nd compared with visit 3rd and 4th. ΨΨΨ denotes p<0.001, visit 3rd compared with visit 4th.

(One-way ANOVA followed by Tukey-Kramer multiple comparisons test).

Figure 20: Shows the graphical representation of table 18.

MEAN VAS PAIN SCORE

7 6.39 6.28 6.34

4.89 4.65 4.75 5

4.12 3.98

3.09 Group A Group B 3 Group C Mean Pain Score Pain Mean 1.92 1.84 2 1.49

0 Baseline 2nd Visit 3rd Visit 4th Visit Visits

MICHIGAN NEUROPATHY SCREENING INSTRUMENT (MNSI)

SCORE

TABLE 19: Shows the michigan neuropathy screening instrument (MNSI)

score- Completed by the person with diabetes.

Group A Group B Group C Visit (n=49) (n=51) (n=50)

Mean Points 11∗∗∗ 10∗∗∗ 11∗∗∗ Baseline/ Visit 1 S.D 1.63 1.19 1.77

Mean Points 07 06 05 End of 4th week S.D 0.58 0.52 0.61

Data represented as mean with S.D (n=49, 51 & 50), which represents mean points of MNSI. ∗∗∗ denotes p<0.001, Base line (Visit 1st) compared with visit 4th visit (One tail P value with Unpaired t test).

Figure 21: Shows the graphical representation of table 19.

MNSI Score - Patient Version

12 11 11 10

8 7

6 6 Baseline 5 4th Visit

Mean Score of MNSI Score Mean 4

0 Group A Group B Group C Groups

TABLE 20: Shows the Michigan Neuropathy Screening Instrument

(MNSI) score - Completed by health professionals

Group A Group B Group C Visit (n=49) (n=51) (n=50)

Mean Score 7.0∗∗∗ 7.5∗∗∗ 7.0∗∗∗ Baseline/ Visit 1 S.D 0.68 0.78 0.64

Mean Score 2.0 2.5 1.0 End of 4th week S.D 0.17 0.19 0.92

Data represented as mean with S.D (n=49, 51 & 50), which represents mean score of MNSI- Completed by health professional. ∗∗∗ denotes p<0.001,

Base line (Visit 1st) compared with visit 4th visit (One tail P value with

Unpaired t test).

Figure 22: Shows the graphical representation of table 20.

Mean Score- Physician Version

7.5 8 7 7

4 Baseline 4th Visit 2.5 3

Mean Score of MNSI Score Mean 2

2 1

0 Group A Group B Group C Groups

DISCUSSION

8. DISCUSSION

Diabetic neuropathies are nerve disorder; it is a common complication of diabetes caused by hyperglycaemia which can damage nerve fibers to whole body. Depends upon the types of nerves involved, which is categorized as peripheral, autonomic, proximal and focal neuropathies. The exact mechanism of diabetic neuropathy remains unknown. Several reports suggested that a variety of molecules are involved in the development of diabetic neuropathy, such as protein kinase C, polyol, aldose reductase, advanced glycation end products, reactive oxygen species, cytokines. More over, some risk factors like metabolite, autoimmune, inherited traits and life style, may contribute to the development of diabetic neuropathy. Methylcobalamin has an extended record as a nerve and it has been used in the treatment of neuropathy for a long time.

Epalrestat is a relatively newer addition in this category that has gained the acceptance of the healthcare society as an effective treatment option for diabetic neuropathy, potentially preventing or ameliorating long term diabetic complications51.

Present study was conducted to compare the efficacy and safety of epalrestat, methylcobalamin alone and epalrestat in combination with methylcobalamin in treatment of patients with diabetic neuropathy. We have evaluated most common diabetic neuropathy complications including pain assessed by VAS scale of pain intensity based on 10 point scoring method, loss of sensation, burning sensation, numbness, muscle cramps, spontaneous pain, weakness, dizziness, loss of sensation of heat & cold assessed by Michigan

82 neuropathy screening instrument score method and HbA1C at baseline & at the end of treatment.

The results of the present study reveal that combination group and

Epalrestat monotherapy group showed a very good improvement and comparably moderate improvement respectively in diabetic neuropathy symptoms over the baseline. All the evaluated neuropathy symptoms showed statistically significant (P<0.001) and (P<0.01) in both groups. Despite the fact that, in combination group symptomatic relief was achieved much earlier with reduction in score values and was better compared to Epalrestat alone.

Improvement in diabetic neuropathy patients was investigated in terms of VAS (Visual analog scale) pain intensity score, MNSI (Michigan neuropathy screening instrument) score and HbA1C levels. In group C significant (p<0.001) reduction in pain score (3.09) was observed at 8th week onwards while compared to baseline and very good reduction in pain score

(1.49) was observed at 12th week of therapy. All three groups had significant

(p<0.001) reduction in MNSI score by patient version, especially group C were shown to have very good reduction (Mean Score Value: 5) compared to rest other groups. Similarly, all three groups had significant (p<0.001) reduction in

MNSI score by health professional version, especially group C were shown to have very good reduction (Mean Score Value: 1.0) compared to rest other groups. The MNSI diabetic patient version score was evaluated on the basis of higher score out of maximum 13 points indicates more neuropathic symptoms as well as MNSI physician version score grater than 2 points out of 10 point

83 scale were considered neuropathic74. With respect to HbA1C, both group A &

C were shown very slight amount (7.56%) of reductions compared to baseline, which is not merely significant. The demographic profiles were not statistically significant. Blood glucose profiles like FBS, PPBS, HbA1C and BMI were measured before and after study, which also not significant. The serum profiles like AST, ALT, serum albumin and urine albumin & urine sugar were measured before and after study period, but which were not observed any significant difference. Serum urea, creatinine and total bilirubin were measured baseline and end of study, which were showed statistically not significant difference between and with in the groups at baseline and at the end of study.

The synergistic effect of Epalrestat and Methylcobalamin may be linked to their complementary mechanisms of neuroprotection. Epalrestat helps to prevent neuronal degeneration by reducing the accumulation of toxic sorbitol and decreasing the oxidative stress while Methylcobalamin helps to recover neuronal injury. Methylcobalamin is one of the biologically active forms of vitamin B12. It is used in the treatment of peripheral neuropathy, diabetic neuropathy, and as a preliminary treatment for amyotrophic lateral sclerosis.

Unlike cyanocobalamin, methylcobalamin is active in the spinal fluid. Due to this property, it is able to help heal the damaged nerve cells and restores normal functions. In clinical studies, Methylcobalamin showed improvement in the somatic and autonomic symptoms with regression of signs of diabetic neuropathy such as pain and paresthesia53,54.

Epalrestat is a carboxylic acid derivative that acts as aldose reductase inhibitor. Epalrestat is proven to have beneficial effects in diabetic neuropathy in many controlled clinical trials. In hyperglycemia, Epalrestat significantly reduces intracellular sorbitol accumulation by an uncompetitive aldose reductase inhibition. Epalrestat improves motor and sensory nerve conduction velocity and subjective neuropathy symptoms in patients with diabetic neuropathy49-51. So this combination of Epalrestat and Methylcobalamin treated patients were more efficacious and well tolerated with safety for the management of diabetic neuropathy than monotherapy of Epalrestat and

Methylcobalamin. The combination therapy showed faster onset and quick relief of symptoms. Apart from this as per our observation, in general people with diabetes who smoke and drink alcohol are more likely to develop neuropathy. Hyperglycemia promotes the synthesis of an endogenous protein kinase C activator, diacylglycerol. This excess protein kinase C activation induces ischemia and promotes vascular permeability and thickening of the basement memberane and causes neuropathy75-77. So, inactivation of protein kinase C indirectly reduces the risk of diabetic neuropathy.

The wellbeing and safety of both drugs was assessed based on the occurrence of adverse events reported by the patients who received the medicine. From our study, it was observed that nausea and vomiting (5.9%), gastric discomfort (7.8%) and diarrhoea (1.9%) was reported as ADR. All three groups were well tolerated and do not generate any safety concern.

SUMMARY

9. SUMMARY

The present study outcome focuses mainly with the following highlights,

 The proportion of male diabetic neuropathy patients were high

[group A (65%), group B (55%), group C (68%)] compared to

female population in our study.

 The over all mean age group of patients were in group A- 53.87yrs,

group B- 53.61 yrs and group C- 57.62 yrs respectively.

 There was no statistically significant difference of BMI between

three groups of patients as well as within the groups at each visit.

 There was no statistically significant difference of the other

laboratory parameters like FBS, PPBS, AST, ALT, serum albumin,

serum total bilirubin, serum creatinine, serum urea, urine albumin,

urine sugar between three groups of patients as well as within the

groups at each visit.

 There was no statistically significant difference of HbA1C level

between three groups of patients as well as within the groups at each

visit. The mean percentage of HbA1C was 7.78% at the end of study.

 Nausea and vomiting (5.9%), gastric discomfort (7.8%), diarrhoea

(1.9%), swelling (1.9%), itching (1.9%) was reported as ADR from

our patients.

 From VAS method, in group C patients had significant (p<0.001)

reduction in pain score (3.09) at 8th week of therapy while compared

to baseline and very good reduction in pain score (1.49) was

observed at 12th week of therapy.

 All three groups had significant (p<0.001) reduction in MNSI score

by patient version, especially group C were shown to have very good

reduction (Mean Score Value: 5) compared to rest of other groups.

 Similarly, all three groups had significant (p<0.001) reduction in

MNSI score by health professional version, especially group C were

shown to have very good reduction (Mean Score Value: 1.0)

compared to rest of other groups.

 In combination therapy (group C), the majority of the patients had

absence of muscle cramps, prickling feelings, burning pain

especially at 12 weeks of therapy, though they were having presence

of the same neurological problems at base line of therapy.

CONCLUSION

10. CONCLUSION

From our study, it was revealed that

 Combination of Epalrestat and Methylcobalamin was more

efficacious, well tolerated and safer when compared to

monotherapy of Epalrestat and Methylcobalamin. for the

management of diabetic neuropathy.

 The combination therapy showed better improvement of

symptoms with respect to MNSI patient version followed by

physician version score and VAS pain intensity score.

 All the three groups had no significant difference between

baseline and end of study with respect to HbA1C levels.

 There was no statistically significant change between the groups

and with in groups at base line and end of study with respect to

BMI, FBS, PPBS, AST, ALT, serum albumin, urea, creatinine,

total bilirubin, urine albumin and urine sugar.

BIBLIOGRAPHY

11. BIBLIOGRAPHY

1. Keecia DK, Jocelyn DJ, Jessica W. Micro vascular and macro vascular

complications of diabetes mellitus. American Journal of

Pharmaceutical education, 2005; 69: 1-8.

2. Ramachandran A, Das AK, Joshi SR, et al., Current status of diabetes in

India and need for novel therapeutic agents. Journal of the Association

of Physicians of India, 2010; 58: 7-9.

3. Melmed, Shlomo, Williams, et al., Textbook of endocrinology (12th

ed.). Philadelphia: Elsevier/Saunders: 1371–1435.

4. Shi Y, Hu FB. The global implications of diabetes and cancer. The

Lancet, 2014; 383: 1947–1948.

5. Vos T, Flaxman AD, Naghavi M, et al., Years lived with disability

(YLDs) for 1160 sequelae of 289 diseases and injuries 1990-2010: a

systematic analysis for the Global Burden of Disease Study. TheLancet,

2010; 380: 2163–96.

6. The top 10 causes of death Fact sheet N°310, World Health

Organization, October 2013.

7. IDF DIABETES ATLAS (6 ed.), International Diabetes Federation, 2013.

8. International Diabetes Federation: Diabetes Atlas, Retrieved 4 April

2014.

9. John WS, Diana F, Karen LG. Projections of global mortality and

burden of disease from 2002 to 2030. PLoS Medicine, 2006;

3(11):e442.

10. Sarwar N, Gao P, Seshasai SR, Gobin R, et al., Diabetes mellitus,

fasting blood glucose concentration, and risk of vascular disease: a

collaborative meta-analysis of 102 prospective studies. Lancet, 2010; 26

(375):2215-2222.

11. Bourne RR, Stevens GA, White RA, Smith JL, et al., Causes of vision

loss worldwide, 1990-2010: a systematic analysis. Lancet, 2013; 1: 339-

349.

12. USRDS annual data report: Epidemiology of kidney disease in the

United States. United States Renal Data System. National Institutes of

Health, National Institute of Diabetes and Digestive and Kidney

Diseases, 2014:188–210.

13. Barbara GW, Joseph TD, Terry LS, et al., Pharmacotherapy Handbook,

Seventh edition: 210.

14. About diabetes, World Health Organization, Retrieved 4 April 2014.

15. Diabetes Fact sheet N°312, WHO, October 2013, Retrieved 25 March

2014.

16. Kitabchi AE, Umpierrez GE, Miles JM, et al., Hyperglycemic crises in

adult patients with diabetes. Diabetes care, 2009; 32: 1335–43.

17. Rother KI, Diabetes treatment—bridging the divide. The New England

Journal of Medicine, 2007; 356:1499-501.

18. Harsh Mohan, Textbook of Pathology. 2010; sixth edition: 818-25.

19. RSSDI textbook of diabetes mellitus. (Rev. 2nd ed.). New Delhi: Jaypee

Brothers Medical Publishers, 2012: 235.

20. Riserus U, Willett WC, Hu FB. Dietary fats and prevention of type 2

diabetes. Progress in Lipid Research, 2009; 48: 44-51.

21. Cooke DW, Plotnick L. Type 1 diabetes mellitus in pediatrics.

Pediatrics in Review, 2008; 29: 374–84.

22. John W.S., Charles R.C., Gerald JM, Joseph RE., et al., Prevalence,

Risk Factors, and Complications of Diabetes in the Kilimanjaro Region:

A Population-Based Study from Tanzania. PLOS ONE, October 6th ,

2016.

23. Yanping D, Yoshimasa T, Wen Z and Yanling Wu. Review Risk Factors

Contributing to Type 2 Diabetes and Recent Advances in the Treatment

and Prevention. International Journal of Medical Sciences 2014;

11(11): 1185-1200.

24. Prediabetes, Canadian diabetes association clinical practice guidelines,

2013, Chronic disease burden update, state of illinosis, illinosis

department of public health, volume 1, issue 2, Nov 2012.

25. Sumanth M. M., Bala S, Gautam R, Ashok Kumar D. Risk Factors of

Diabetes Mellitus in Rural Puducherry. Online Journal of Health and

Allied Sciences 2012; 11(1): 1-7.

26. Clinical Pharmacy and Therapeutics by Roger Walker, fifth edition:

691-694.

27. Manouk B and Charles A. Prevalence and complications of diabetes

mellitus in Northern Africa, a systematic review. BMC Public health

2013; 13: 387. 201313:387.

28. Loewen SL, Haas LB. Complications of diabetes: acute and chronic.

Nurse Practitioner Forum, 1991; 2(3):181-7.

29. Huzar JG. Diabetes now. Preventing acute complications. RN. 1989;

52(8):34-40.

30. Sun J, et al., Protection from Retinopathy and Other Complications in

Patients with Type 1 Diabetes of Extreme Duration. Diabetes Care,

2011; 34: 968.

31. Bonnefont RD. The role of antioxidant micronutrients in the prevention

of diabetic complications. Treatments in Endocrinology. 2004; 3 (1):

41–52.

32. Juster SK, Smith AG. Updates in diabetic peripheral neuropathy.

Faculty Review, 2016;5: F1000 -738.

33. Callaghan BC, Cheng HT, Stables CL, et al., Diabetic neuropathy:

clinical manifestations and current treatments. Lancet Neurology,

2012;11(6):521–34.

34. Tesfaye S, Boulton AJ, Dyck PJ, et al., Diabetic neuropathies: update on

definitions, diagnostic criteria, estimation of severity, and

treatments. Diabetes Care, 2010; 33(10):2285–93.

35. Tesfaye S, Vileikyte L, Rayman G, et al., Painful diabetic peripheral

neuropathy: consensus recommendations on diagnosis, assessment and

management. Diabetes Metabolism Research and Reviews,

2011;27(7):629–38.

36. Sadosky A, Mardekian J, Parsons B, et al., Healthcare utilization and

costs in diabetes relative to the clinical spectrum of painful diabetic

peripheral neuropathy. Journal of Diabetes Complications, 2015;

29(2):212–17.

37. Gordois A, Scuffham P, Shearer A, et al., The health care costs of

diabetic peripheral neuropathy in the US. Diabetes Care, 2003;

26(6):1790–5.

38. Scheen AJ. Pathophysiology of insulin secretion. Annals of

Endocrinology. 2004; 65(1):29-36.

39. Howard R, Kathleen CZ, Shridar G, Roberto C and Walter SZ.

Physiology and Pathophysiology of Insulin Secretion, Diabetes Care,

1990; 13(6): 655-666.

40. National Diabetes Information Clearing house, Diagnosis of Diabetes

and Prediabetes, 2014: 4-7.

41. Classification and Diagnosis of Diabetes, American Diabetes Association,

Diabetes Care, 2015; 38(1): S8-S16.

42. Tripathi KD, Essentials of Medical Pharmacology, Jaypee brothers

medical publishers, 6th edition: 254-274.

43. Bosenberg LH, Vanzyl DG. The mechanism of action of oral anti-

diabetic drugs. 2008: 80-88.

44. Harrigan RA, Nathan MS, Beettie P. Oral agents for the treatment of

type 2 diabetes mellitus: pharmacology, toxicology and treatment,

Annals Of Emergency Medicine. 2001: 68-78.

45. David MN, John BB, Davidson MB. Medical Management of

Hyperglycemia in Type 2 Diabetes: A Consensus Algorithm for the

Initiation and Adjustment of Therapy. Diabetes care, 2009; 32: 193-203.

46. Guideline for the management in primary health care of diabetes: 06-17.

47. Ramachandran, A., Snehalatha, C., Vijay, V. and Vishwanathan, M.

(1996) Diabetic retinopathy at the time of diagnosis of NIDDM in South

Indian subjects. Diabetes Research and Clinical Practice, 32, 111-114.

48. Chihiro, Y.N. (1998) Aldose reductase in glucose toxicity: A potential

target for the prevention of diabetic complications. Pharmacological

Reviews, 50, 21-33.

49. Itagaki I, Shimizu K, Kamanaka Y, Ebata K, et al., The effect of an

aldose reductase inhibitor (epalrestat) on diabetic nephropathy in rats.

Diabetes Research and Clinical Practice, 1994; 25:147-154.

50. Hotta N, Kakuta H, Koh N, Fukasawa H, et al., In-vitro retinal and

erythrocyte polyol pathway regulation by hormones and an aldose

reductase inhibitor. Diabetes Research and Clinical Practice, 1991; 14 :

29-35.

51. Ramirez MA and Boria NL. Epalrestat: an aldose reductase inhibitor for

the treatment of diabetic neuropathy. Pharmacotherapy 2008; 28: 646-

55.

52. Sharma S.R. and Sharma N. Epalrestat, an aldose reductase inhibitor, in

diabetic neuropathy: An Indian perspective. Annals of Indian Academy

Neurology, 2008; 11: 231- 235.

53. Yaqub BA, Siddique A, Sulimani R. Effects of methylcobalamin on

diabetic neuropathy. Clinical Neurology and Neurosurgery. 1992;

94(2):105-11.

54. Sun Y, Lai M.S and Lu CJ. Effectiveness of vitamin B12 on diabetic

neuropathy: Systematic review of clinical controlled trials. Acta

Neurologica Taiwanica, 2005; 14: 48-54.

55. Hotta N, Akanum Y, Kawamori R, Matsuoka K, et al., Long term

clinical effects of epalrestat, an aldose reductase inhibitor, on diabetic

peripheral neuropathy. Diabetes Care. 2006; 29: 1538-44.

56. Ito A, Nozawa RI, Ibuki C, Atarashi H, et al., Examination of

questionnaire items regarding diabetic peripheral neuropathy in

epalrestat- treated patients and their usefulness in the treatment of this

disorder – Influence on Treatment course. Yakugaku Zasshi 2009; 129:

1239-1247.

57. Karunanithi P, Mallayasamy M, Manikandan S, Rajasoundara P., et al.,

Comparative study of efficacy between pregabalin and epalrestat in

diabetic neuropathic Pain. Asian Journal of biomedical and

pharmaceutical sciences 2014; 04(35): 49-54.

58. Maladkar M, Saggu NJS, Moralwar P, Aziz A., et al., Evaluation of

efficacy and safety of epalrestat and epalrestat in combination with

methylcobalamin in patients with diabetic neuropathy in a randomized,

comparative trial. Journal of diabetes Mellitus 2013; 3(1): 22-26.

59. Maladkar M., Rajadhyaksha G, Vengataswamy N, Hariharan S., et al.,

Efficacy, safety, and tolerability of epalrestat compared to

methylcobalamine in patients with diabetic neuropathy. International

Journal of diabetes in developing countries 2009; 29(1); 28-34.

60. Ruan Y, Jianhua MA., Xiaojing Xie., et al., Effects of epalrestat

combined with methylcobalamin on diabetic peripheral neuropathy.

Jiangsu Medical Journal. 2012; 03.

61. Ide H, Fujiya S, Asanuma Y, Tsuji M, Sakai H, Agishi Y. Clinical

usefulness of intrathecal injection of methylcobalamin in patients with

diabetic neuropathy. Clinical Therapeutics 1987; 9(2):183-92.

62. Kuwabara S, Nakazawa R, Azuma N, et al., Intravenous

methylcobalamin treatment for uremic and diabetic neuropathy in

chronic hemodialysis patients. Internal Medicine 1999 Jun; 38(6):

472-5.

63. Bhavani J and Lian LL. Vitamin B supplementation for diabetic

peripheral neuropathy. Singapore Medical Journal, 2016; 57:55-59.

64. Mizukami H, Ogasawara S, Yamagishi S, Takahashi K, et al.,

Methylcobalamin effects on diabetic neuropathy and nerve protein

kinase C in rats. European Journal of Clinical Investigation 2011 ;

41(4):442-50.

65. Vasudevan D, Manoj M N, Qayum I M. Efficacy and safety of

methylcobalamin, alpha lipoic acid and pregabalin combination versus

pregabalin monotherapy in improving pain and nerve conduction

velocity in type 2 diabetes associated impaired peripheral neuropathic

condition. Annals Indian Academy of Neurology 2014;17:19-24.

66. Terashima H, Hama K. Effects of a new aldose reductase inhibitor on

various tissue in-vitro. Journal of Pharamacology and Experimental

Therapeutics, 1984; 229: 226–230.

67. Sharma SR, and Sharma N. Epalrestat, an aldose reductase inhibitor, in

diabetic neuropathy: An Indian perspective. Annals of Indian academy

of neurology, 2008; 11(4); 231-235.

68. John WS, Diana Faulds, Karen LG. A Review of its Pharmacology, and

Therapeutic Potential in Late-Onset Complications of Diabetes

Mellitus. Drugs & Aging, 1993; 3 (6): 532–555.

69. Thakkar K, Billa G. Treatment of vitamin B12 deficiency-

methylcobalamine? Cyancobalamine? Hydroxocobalamin?-clearing the

confusion. European Journal of Clinical Nutrition, 2015; 69: 1–2.

70. Ram P, Atul P and Rajiv R. Efficacy and Tolerability of a Fixed Dose

Combination of Methylcobalamin and Pregabalin in the Management of

Painful Neuropathy. North American Journal of Medical Sciences,

2012; 4(11): 605-607.

71. Gupta JK and Qureshi SS. Potential Benefits of Methylcobalamin: A

Review. Austin Journal of Pharmacology and Therapeutics, 2015;

3(3): 1-4.

72. Manish M, Chitra T, Urja D. Post-Marketing Surveillance of Fixed Dose

Combination of Methylcobalamin, Alpha Lipoic Acid, Folic Acid,

Biotin, Benfotiamine & Vitamin B6-Nutripathy for the Management of

Peripheral Neuropathy. Journal of Diabetes Mellitus, 2014; 4: 124-132.

73. Jacqueline CD, Arlene RN, Ludwig FD. A prospective, open label, 24-

week trial of methylcobalamin in the treatment of diabetic

polyneuropathy. Journal of Diabetes Mellitus, 2012, Vol.2, No.4, 408-

412.

74. Dyck PJ, Karnes J, O'Brien PC, Swanson CJ. Neuropathy Symptom

Profile in health, motor neuron disease, diabetic neuropathy, and

amyloidosis. Neurology, 1986;36:1300-8.

75. Borghini I, Ania LA, Regazzi R, et al., Alpha, beta II,delta and epsilon

protein kinase C isoforms and compound activity in the sciatic nerve of

normal and diabetic rats. Journal of Neurochemistry, 1994; 62: 686-96.

76. Hempel A, Maasch C, Heintze U, et al., High glucose concentration

increase endothelial cell permeability via activation of protein kinase C

alpha. Circulation Research, 1997;81:363-71.

77. Mara L. The Polyol Pathway as a Mechanism for Diabetic Retinopathy:

Attractive, Elusive, and Resilient. Experimental Diabetes Research,

2007, 1-31.

ANNEXURES

(Annexure I A)

INFORMATION SHEET

You are invited to participate in this study titled “Comparison of efficacy and safety of Epalrestat with Methylcobalamin in patients with diabetic neuropathy.”

Study Purpose:

The purpose of this study is to evaluate the comparison of efficacy and safety of Epalrestat with Methylcobalamin in patients with diabetic neuropathy.

Study Details:

 Along with you total 165 patients will be included in this study.

 You will be enrolled only after giving your voluntary consent.

 The study involves administration of epalrestat or methylcobalamin or

combination of epalrestat with methylcobalamin for the period of 12

weeks. For analytical purpose, 5 ml of blood sample will be colleted from

your vein by expert for routine baseline investigations like FBS, PPBS,

HbA1C, ALT, AST, serum albumin, serum total bilirubin, serum

creatinine, serum urea, urine albumin, urine sugar at first week of therapy

and at each follow-up visit like 4th, 8th and 12th weeks of therapy.

 You will be asked questions to assess pain intensity by VAS scoring

method and to assess your loss of sensation, burning sensation, numbness,

muscle cramps, spontaneous pain, weakness, dizziness, loss of sensation

of heat & cold assessed by Michigan neuropathy screening instrument

score method as mentioned in the proforma. Benefit of participating this study:

By participation in this study you will be recovered from the problem of diabetic neuropathic related complications.

Risks involved in participation of this study:

As these epalrestat, aldose reductase inhibitor and methylcobalamin have produce adverse effects like head ache, nausea, vomiting, skin rash, swelling, hot flush, gastric discomfort and diarrhoea. If any unwanted side effects occur, it will be treated free of cost and your health will be safe guarded.

Rights:

Participation in this study is purely voluntary. If you do not want to participate you can withdraw from study at any time.

Confidentiality:

Your participation will be kept confidential. The investigators and other authorized personnel will only have access to the data. The information will be used for publication and further research without revealing your identity.

Annexure : I (B)

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Annexure II (A)

CONSENT FORM

I, the undersigned confirmed that…

 I have read and understood the information about the study titled

“Comparison of efficacy and safety of Epalrestat with Methylcobalamin

in patients with diabetic neuropathy” as provided in the information sheet.

 I have been given opportunity to ask questions about the project and my

participation.

 I understand that I can withdraw myself from study at any time without

giving any reason.

 Procedures regarding confidentiality have been very well explained to

me.

 The use of data for publication, research sharing and archiving has been

explained to me.

 I voluntarily agree to participate in the study research.

 I along with the researcher agree to sign and date this informed consent

form.

Signature Signature

Name of the Participant: Name of the Researcher:

Date: Date:

Annexure : II (B)

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ANNEXURE – III

IEC APPROVAL CERTIFICATE

ANNEXURE – IV

Comparison of efficacy and safety of Epalrestat with

Methylcobalamin in patients with Diabetic neuropathy

PROFORMA

NAME:

Age : OP. No:

Sex : M / F ID No:

Marital Status: M / Single Edu. Status : L / IL

ADDRESS: Phone No :

Randomised Group : Group A [ ] Group B [ ] Group C [ ]

Whether Informed consent

Obtained : Yes [ ] No [ ]

Chief Complaints / Reason for admission:

History of Present illness:

Past Medical History:

Past Medication History:

Personal History:

Smoker : Y/N Alcoholic : Y/N

Tobacco : Y/N Known Allergies : Y/N

-1- VISIT 1-BASELINE General Physical Examination: Ht: Wt: BMI: Pallor : Edema: Icterus :

Cyanosis: Clubbing: Lymphadenopathy :

Vital Signs : Temp. : ……. BP : …….. Pulse : ……. RR : ……..

SYSTEMIC EXAMINATION: CVS : RS : Abdomen ; NERVOUS SYSTEM: 1. VAS Pain Scoring Method: [V/ U] 0 1-2 3-4 5-6 7-8 8-10 Moderate Very No Pain Mild Pain Severe Pain Worst Pain Pain Severe Pain

2. Michigan Neuropathy Screening Instrument : [V/ U]

Refer Page No : 5& 6

LABORATORY INVESTIGATION: 1. Hemogram: Hb : (g/dl) CBC : 2. FBS: (60-90) PPBS : (80-150) 3. HbA1C : 4. URINE EXAMINATION: Colour: ….. Albumin: …… Sugar : …….. Deposits: 5. Liver Function Test: AST:…… ALT: ……. Albumin: ……… Total Bilirubin: … 6. Renal Function Test: Urea: ……… Sr. Cr : ……. Uric acid: ……. BUN: ……..

-2- VISIT 2 - END OF 4 WEEKS

Wt: BP : …….. ADR : ………

FBS : ……… Empty foils returned : Yes [ ] No [ ]

NERVOUS SYSTEM: 1. VAS Pain Scoring Method: [V/ U]

0 1-2 3-4 5-6 7-8 8-10 Moderate Very No Pain Mild Pain Severe Pain Worst Pain Pain Severe Pain

VISIT 3 - END OF 8 WEEKS

Wt: BP : …….. ADR : ………

FBS : ……… Empty foils returned : Yes [ ] No [ ]

NERVOUS SYSTEM: 1. VAS Pain Scoring Method: [V/ U]

0 1-2 3-4 5-6 7-8 8-10 Moderate Very No Pain Mild Pain Severe Pain Worst Pain Pain Severe Pain

-3- VISIT 4 - END OF 12 WEEKS

General Physical Examination:

Ht: Wt: BMI:

Pallor : Edema: Icterus :

Cyanosis: Clubbing: Lymphadenopathy :

Vital Signs :

Temp. : ……. BP : …….. Pulse : ……. RR : ……..

SYSTEMIC EXAMINATION: CVS : RS : Abdomen ; NERVOUS SYSTEM: 1. VAS Pain Scoring Method: [V/ U] 0 1-2 3-4 5-6 7-8 8-10 Moderate Very No Pain Mild Pain Severe Pain Worst Pain Pain Severe Pain

2. Michigan Neuropathy Screening Instrument : [V/ U]

Refer Page No : 5 & 6 LABORATORY INVESTIGATION: 1. Hemogram: Hb : (g/dl) CBC : 2. FBS: (60-90) PPBS : (80-150) 3. HbA1C : 4. URINE EXAMINATION: Colour: ….. Albumin: …… Sugar : …….. Deposits: 5. Liver Function Test: AST:…… ALT: ……. Albumin: ……… Total Bilirubin: … 6. Renal Function Test: Urea: ……… Sr. Cr : ……. Uric acid: ……. BUN: …….. Adverse Drug Reactions : …………

-4-

MICHIGAN NEUROPATHY SCREENING INSTRUMENT

PATIENT VERSION

A. History (To be completed by the person with diabetes)

Please take a few minutes to answer the following questions about the feeling in your legs and feet. Check yes or no based on how you usually feel. Thank you.

1. Are you legs and/or feet numb? Yes No

2. Do you ever have any burning pain in your legs and/or feet? Yes No

3. Are your feet too sensitive to touch? Yes No 4. Do you get muscle cramps in your legs and/or feet? Yes No 5. Do you ever have any prickling feelings in your legs or feet? Yes No

6. Does it hurt when the bed covers touch your skin? Yes No

7. When you get into the tub or shower, are you able to tell the

hot water from the cold water? Yes No

8. Have you ever had an open sore on your foot? Yes No

9. Has your doctor ever told you that you have diabetic neuropathy? Yes No 10. Do you feel weak all over most of the time? Yes No

11. Are your symptoms worse at night? Yes No

12. Do your legs hurt when you walk? Yes No

13. Are you able to sense your feet when you walk? Yes No

14. Is the skin on your feet so dry that it cracks open? Yes No 15. Have you ever had an amputation? Yes No

Total:

-5-

MICHIGAN NEUROPATHY SCREENING INSTRUMENT

B. Physical Assessment (To be completed by health professional)

1. Appearance of Feet

Right Left a. Normal 0 Yes 1 No Normal 0 Yes 1 No b. If no, check all that apply: If no, check all that apply:

Deformities Deformities Dry skin, callus Dry skin, callus Infection Infection Fissure Fissure Other Other specify: specify: Right Left Absent Present Absent Present 2. Ulceration 0 1 0 1

Present/ Present/ Present Reinforcement Absent Present Reinforcement Absent 3. Ankle Reflexes 0 0.5 1 0 0.5 1

Present Decreased Absent Present Decreased Absent 4. Vibration 0 0.5 1 0 0.5 1 perception at great toe

5. Monofilament Normal Reduced Absent Normal Reduced Absent 0 0.5 1 0 0.5 1

Signature: Total Score /10 Points

-6-

MASTER CHART

Annexure V – MASTER CHART Group- A MNSI BMI HbA1C FBS PPBS Urine Sugar S.No Age (Yrs) Sex (M/F) (Physician (Kg/m2) (%) (mg/dl) (mg/dl) (Nil/Trace/P/PP) Version) 1 65 F 25.45 7.65 150.26 270.25 Nil 7.0 2 62 M 25.54 7.45 165.56 265.63 Nil 7.5 3 58 F 25.75 7.63 153.34 272.28 Nil 7.0 4 55 F 25.32 7.78 166.26 274.78 T 7.5 5 30 M 24.98 7.27 154.65 256.90 Nil 7.0 6 63 M 24.89 7.28 165.15 269.45 Nil 7.5 7 44 F 26.15 7.55 153.34 290.28 P 7.5 8 58 M 25.23 7.45 172.56 250.56 T 6.5 9 35 F 25.65 7.63 168.27 265.43 Nil 7.0 10 35 F 25.65 7.48 157.56 275.23 Nil 6.5 11 61 M 25.87 7.77 171.78 276.45 P 7.0 12 45 F 25.35 7.28 152.15 269.34 Nil 7.5 13 45 F 25.75 7.27 157.34 274.78 Nil 7.0 14 64 M 25.32 7.28 168.56 257.90 Nil 7.5 15 47 M 24.98 7.55 158.27 249.45 Nil 7.0 16 40 F 26.15 7.45 167.56 289.28 Nil 7.5 17 62 F 25.23 7.63 168.78 250.56 Nil 7.5 18 50 F 25.65 7.48 158.78 290.43 Nil 6.5 19 40 F 25.65 7.77 150.15 270.23 Nil 7.0 20 65 M 25.87 7.28 170.34 276.45 Nil 6.5 21 61 F 25.35 7.84 167.56 269.34 Nil 6.5 22 28 F 25.75 7.98 161.27 265.32 Nil 7.0 23 65 F 25.32 7.46 152.56 275.45 Nil 6.5

MNSI BMI HbA1C FBS PPBS Urine Sugar S.No Age (Yrs) Sex (M/F) (Physician (Kg/m2) (%) (mg/dl) (mg/dl) (Nil/Trace/P/PP) Version) 24 64 F 25.23 7.63 170.15 274.78 Nil 6.5 25 53 F 25.65 7.78 154.34 256.90 Nil 7.0 26 63 M 25.65 7.27 171.56 269.45 T 6.5 27 32 M 25.87 7.38 166.27 290.28 Nil 7.0 28 60 F 25.35 7.55 159.56 250.56 Nil 7.5 29 47 F 25.75 7.45 166.78 276.43 Nil 7.0 30 28 F 25.32 7.53 157.15 264.23 Nil 7.5 31 43 F 24.98 7.48 160.34 276.45 Nil 7.0 32 63 M 26.15 7.97 164.56 269.34 Nil 7.5 33 55 F 25.23 7.28 173.27 274.78 Nil 7.5 34 63 M 25.65 7.27 152.56 257.90 Nil 6.5 35 55 F 25.65 7.28 183.78 279.45 Nil 7.0 36 63 M 25.87 7.55 143.78 259.28 Nil 6.5 37 40 F 25.35 7.45 165.15 255.56 T 6.5 38 38 M 25.75 7.63 155.34 285.43 Nil 7.0 39 60 F 25.32 7.48 165.56 270.23 Nil 6.5 40 42 F 24.65 7.77 163.27 276.45 Nil 7.0 41 60 F 26.10 7.28 162.56 269.34 Nil 7.5 42 50 F 25.27 7.84 160.34 245.32 Nil 7.0 43 60 F 25.34 7.98 164.56 295.45 Nil 7.5 43 58 M 25.65 7.46 163.27 279.45 Nil 7.5 45 35 F 25.65 7.48 162.56 259.28 Nil 6.5 46 63 F 25.87 7.77 168.78 232.56 Nil 7.0 47 50 F 25.35 7.28 158.24 290.43 Nil 6.5 48 65 F 25.75 7.84 172.45 281.23 Nil 6.5 49 45 M 25.32 7.98 153.15 279.16 Nil 7.0 50 34 M 24.65 7.46 183.52 276.34 Nil 6.5 MNSI BMI HbA1C FBS PPBS Urine Sugar S.No Age (Yrs) Sex (M/F) (Physician (Kg/m2) (%) (mg/dl) (mg/dl) (Nil/Trace/P/PP) Version) 51 60 M 25.23 7.46 142.17 271.26 Nil 7.0 52 51 F 24.65 7.45 140.34 269.28 Nil 7.0 53 63 F 26.10 7.53 184.56 272.56 Nil 7.5 54 64 M 25.27 7.48 168.27 270.43 Nil 7.0 55 62 M 25.34 7.37 157.56 271.23 Nil 7.5

Group- B

Urine MNSI BMI PPBS Sugar (Physician S.No Age (Yrs) Sex (M/F) HbA1C (%) FBS (mg/dl) (Kg/m2) (mg/dl) (Nil/Trace/ Version) P/PP) 1 45 M 25.27 7.84 140.15 255.28 Nil 7.5 2 42 F 25.34 7.56 180.34 292.56 Nil 8.0 3 55 M 25.65 7.56 155.56 280.43 Nil 7.0 4 62 M 25.65 7.45 173.27 261.23 Nil 7.0 5 40 F 25.87 7.45 157.56 289.16 Nil 7.5 6 62 M 25.35 7.53 165.34 256.34 T 7.5 7 40 F 25.75 7.48 144.56 271.26 Nil 7.5 8 36 M 25.32 7.97 183.27 275.67 Nil 7.0 9 60 F 24.65 7.28 142.56 278.56 Nil 7.0 10 31 M 25.23 7.27 173.78 279.16 Nil 7.5 11 39 M 24.56 7.28 168.24 275.45 T 7.5 12 43 M 25.31 7.55 167.45 280.45 Nil 7.5 13 64 M 26.01 7.45 183.15 255.15 Nil 7.5 14 62 F 25.53 7.63 143.52 295.19 Nil 7.5 15 62 M 25.01 7.48 167.17 278.56 Nil 8.0 16 42 M 25.25 7.77 160.78 279.16 Nil 7.0 17 47 F 24.78 7.28 145.34 275.45 T 7.0 18 59 F 25.75 7.84 184.34 280.45 Nil 7.5 19 38 M 25.32 7.98 144.67 285.15 Nil 7.5 20 60 F 24.65 7.46 184.98 265.19 Nil 7.5 21 59 M 25.23 7.48 154.87 294.18 Nil 7.0 22 55 M 24.56 7.77 175.27 254.39 Nil 7.0 23 57 F 25.31 7.28 155.27 295.76 Nil 7.5

Urine MNSI BMI PPBS Sugar (Physician S.No Age (Yrs) Sex (M/F) HbA1C (%) FBS (mg/dl) (Kg/m2) (mg/dl) (Nil/Trace/ Version) P/PP) 24 51 M 25.53 7.98 167.56 255.45 Nil 7.5 25 65 M 25.01 7.46 165.34 280.45 Nil 7.0 26 38 M 25.29 7.46 144.56 274.15 T 7.5 27 60 F 25.16 7.45 183.27 278.19 Nil 7.5 28 59 M 24.85 7.53 168.56 284.18 Nil 7.5 29 60 F 25.67 7.48 157.78 264.39 Nil 7.0 30 26 F 26.02 7.97 168.24 285.76 Nil 7.0 31 34 M 25.36 7.28 157.45 265.45 Nil 7.5 32 40 F 25.45 7.27 183.15 280.45 Nil 7.5 33 60 F 25.53 7.28 143.52 279.15 Nil 7.5 34 43 M 25.01 7.38 182.17 271.19 Nil 7.5 35 63 F 25.29 7.46 155.78 272.18 Nil 7.5 36 60 F 25.16 7.46 160.34 276.31 Nil 8.0 37 43 M 24.85 7.45 159.34 277.76 T 7.0 38 58 F 25.67 7.53 184.67 273.45 Nil 7.0 39 60 F 26.02 7.48 159.98 280.45 Nil 7.5 40 43 M 25.36 7.27 160.87 255.15 Nil 7.5 41 62 F 25.45 7.28 155.27 295.19 Nil 7.5 42 60 F 26.02 7.67 160.27 294.18 T 7.0 43 43 M 25.36 7.28 168.78 254.39 Nil 7.0 43 64 F 25.45 7.45 163.34 255.76 Nil 7.5 45 60 F 25.53 7.45 166.24 294.89 Nil 8.0 46 55 F 25.01 7.73 168.67 270.56 Nil 7.0 47 62 M 25.16 7.53 160.98 281.67 Nil 7.0 48 50 F 24.85 7.39 162.85 275.67 Nil 7.5 49 50 M 25.67 7.46 167.27 270.45 Nil 7.5 Urine MNSI BMI PPBS Sugar (Physician S.No Age (Yrs) Sex (M/F) HbA1C (%) FBS (mg/dl) (Kg/m2) (mg/dl) (Nil/Trace/ Version) P/PP) 50 55 F 26.02 7.23 185.27 274.56 Nil 7.5 51 47 F 25.36 7.45 145.32 282.53 Nil 7.0 52 63 M 25.65 7.98 185.27 275.45 Nil 7.5 53 63 M 25.65 7.46 145.27 280.45 Nil 7.0 54 53 M 25.87 7.46 168.78 276.15 Nil 7.0 55 40 F 25.35 7.45 160.34 275.19 Nil 7.5

Group- C

Urine MNSI BMI HbA1C FBS PPBS Sugar (Physician S.No Age (Yrs) Sex (M/F) (Kg/m2) (%) (mg/dl) (mg/dl) (Nil/Trace/ Version) P/PP) 1 64 M 25.45 7.75 144.43 254.34 Nil 7.0 2 52 F 23.56 7.98 185.27 290.45 Nil 7.5 3 63 M 26.54 7.46 145.27 286.15 Nil 7.5 4 65 F 24.87 7.46 183.78 265.19 Nil 7.5 5 63 F 25.10 7.45 169.34 294.18 Nil 7.0 6 63 M 24.78 7.53 160.24 279.39 Nil 6.5 7 45 F 25.40 7.48 166.67 274.76 Nil 6.5 8 40 F 26.1 7.77 162.98 284.89 Nil 6.5 9 62 M 25.16 7.28 144.85 278.56 Nil 6.5 10 60 F 25.34 7.27 185.27 276.67 Nil 7.0 11 61 M 25.89 7.28 185.27 275.67 Nil 7.0 12 34 M 24.78 7.78 160.32 279.45 PP 7.0 13 63 M 25.40 7.46 159.27 271.56 Nil 7.0 14 44 F 26.1 7.46 154.27 276.53 Nil 6.5 15 48 M 25.16 7.45 163.78 280.45 Nil 6.5 16 62 M 25.34 7.73 165.34 276.15 Nil 6.5 17 56 F 25.89 7.48 169.24 275.19 T 6.5 18 36 M 25.45 7.27 159.67 294.18 Nil 7.0 19 63 F 23.56 7.78 166.98 254.39 Nil 7.0 20 49 F 26.54 7.27 162.85 293.76 Nil 7.0 21 50 F 24.87 7.28 160.27 254.89 Nil 7.0 22 45 M 25.10 7.45 170.27 275.56 Nil 7.0 23 35 M 24.78 7.45 165.32 276.67 T 6.5

Urine MNSI BMI HbA1C FBS PPBS Sugar (Physician S.No Age (Yrs) Sex (M/F) (Kg/m2) (%) (mg/dl) (mg/dl) (Nil/Trace/ Version) P/PP) 24 55 F 26.1 7.53 175.27 276.45 Nil 7.0 25 64 M 24.87 7.48 155.27 274.56 Nil 7.0 26 51 F 25.10 7.77 173.78 278.53 T 7.0 27 56 F 24.78 7.28 155.34 274.45 Nil 7.0 28 38 M 25.40 7.27 166.24 274.56 Nil 6.5 29 64 F 26.1 7.28 162.67 276.53 Nil 6.5 30 40 M 25.16 7.78 167.98 280.45 Nil 6.5 31 50 F 25.34 7.46 161.85 276.15 Nil 6.5 32 63 F 25.89 7.46 168.27 275.19 Nil 7.0 33 50 F 24.78 7.45 162.27 294.18 T 7.0 34 60 F 25.40 7.73 170.32 254.39 Nil 7.0 35 50 F 26.1 7.48 160.27 283.76 Nil 7.0 36 64 F 25.16 7.27 166.27 264.89 Nil 7.0 37 63 F 25.34 7.78 162.78 285.56 Nil 6.5 38 47 F 25.89 7.27 167.34 266.67 T 6.5 39 63 M 25.45 7.28 162.24 276.45 T 7.0 40 54 M 23.56 7.45 168.67 274.56 Nil 7.0 41 42 F 25.16 7.45 160.98 276.53 Nil 7.0 42 52 F 25.34 7.46 168.85 280.45 Nil 7.0 43 40 M 25.89 7.46 161.27 276.15 Nil 6.5 43 50 F 24.78 7.45 166.27 275.19 Nil 6.5 45 50 M 25.40 7.73 164.32 284.18 Nil 6.5 46 26 F 25.89 7.48 160.45 264.39 Nil 6.5 47 58 M 25.45 7.27 168.78 273.76 Nil 7.0 48 65 M 23.56 7.78 174.26 274.89 T 7.0 49 60 F 26.54 7.56 154.65 275.56 Nil 7.0 Urine Sugar MNSI BMI HbA1C FBS PPBS S.No Age (Yrs) Sex (M/F) (Nil/Trace/P (Physician (Kg/m2) (%) (mg/dl) (mg/dl) /PP) Version) 50 54 F 24.87 7.84 184.27 276.67 Nil 7.0 51 53 M 25.10 7.46 143.27 275.20 Nil 7.0 52 64 F 25.34 7.46 166.27 276.25 T 6.5 53 58 F 26.1 7.46 164.32 275.56 Nil 6.5 54 62 F 24.87 7.95 160.45 277.53 Nil 7.0 55 47 M 25.10 7.73 168.78 273.45 Nil 7.0