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Differences in Effect of Insulin Detemir and NPH Insulin on Glucose and Lipid Metabolism

by

Sara Victoria Margaret Hordern MBBS BSc MRCP

Submitted for the degree of Doctor of Medicine

Postgraduate Medical School University of Surrey

August 2004

Sara Victoria Margaret Hordern 2004

ProQuest N um ber: 27605345

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Summary

Insulin detemir has a 14 C fatty acid chain, which associates with free fatty acid binding

sites on albumin. Capillary endothelial cells in adipose tissue and muscle may limit transfer

of insulin detemir from the circulation into the extravascular extracellular space. In the

liver, the open sinusoids may expose hepatocytes to insulin detemir enabling it to have

greater effect on the liver than on the periphery.

We investigated the effects of two doses of insulin detemir and one dose of NPH insulin on

hepatic glucose output (Ra), peripheral glucose uptake (Rd), glycerol Ra using stable isotope

techniques and on NEFA, IGF-1, IGFBP-1, C-peptide, and glucagon concentrations in 7

healthy volunteers, 6 volunteers with type 1 diabetes and 6 volunteers with type 2 diabetes

during a 16h euglycaemic clamp.

In healthy volunteers we demonstrated that 9nmol/kg insulin detemir had greater effect on

suppression of glucose Ra than an equipotent dose of NPH (0.3iu/kg or 1.8nmol/kg). In

subjects with type 1 and type 2 diabetes 18nmol/kg insulin detemir was equipotent to 0.6

iu/kg (3.6 nmol/kg) NPH, with a similar time action profile. In subjects with type 1 diabetes

we were unable to demonstrate differences in the effect of insulin detemir on glucose Ra but

demonstrated small but significant differences in effect on glucose Rd compared to the

equipotent dose of NPH. The suppression of NEFA concentrations by insulin detemir was

less than with an equipotent dose of NPH, both in healthy subjects and to a lesser extent in

those with type 1 diabetes. We were unable to demonstrate a statistical difference in effect

on glucose or lipid metabolism in subjects with type 2 diabetes. There were no differences

in the effect of these two insulins on glycerol Ra, IGF-1, IGFBP-1, glucagon or c-peptide

concentrations in any of our three subject groups. We demonstrated a dose response effect

for insulin detemir.

This study suggests that when equipotent doses of insulin detemir are compared to NPH

insulin, detemir has greater effect on the liver than peripheral tissues and thus the potential

to restore the normal insulin gradient.

Contents

Summary 1

Contents 2

Acknowledgements 6

List of Figures 7

List of Tables 9

Abbreviations 10

Chapter 1. Introduction

Diabetes 13

Definition of diabetes 13

Epidemiology 14

History of diabetes 15

The insulin molecule 20

Structure of the insulin molecule 20

Biosynthesis of insulin 21

The insulin receptor 22

Glucose metabolism 24

Endogenous glucose production 25

Glucose utilization 25

Glucose homeostasis 28

Fat and Linid Metabolism 32

The Endothelial Barrier 33

Henatic Insulinisation 36

Lone Actine Insulin 40

Background 40

Insulin detemir 43

Physical and Chemical Properties 43

Pharmacology 43

Formulation 45

Efficacy pharmacology 46

Pharmacokinetics and protein binding 46

Mitogenicity 48

Aims of this study 48

Chapter 2. Clinical Methodology

Introduction to the glucose damn technique 49

Isotope dilution 49

Calculations 51

Clamp Protocol 52

Preparation of Tracer solutions 56

Laboratory assessments 56

Glucose concentration and enrichment 56

Glycerol concentration and enrichment 57

Non-Esterified Fatty Acids (NEFA) 58

Insulin and insulin detemir, C-peptide and Glucagon59

IGF-1 59

IGFBP-1 60

Trial Design 61

Type of trial 61

Rationale for trial design 62

Rationale for dosage regime 62

Trial population 63

Number of subjects in each group 63

Inclusion Criteria 63

Exclusion criteria 64

Safety visit 65

Statistical analyses 66

Chapters. Results

Healthy Subiects 67

Subject demographics 67

Plasma glucose 67

Glucose infusion rate 68


Glycerol metabolism 75

NEFA 77

IGF-1 78

IGFBP-1 79

Insulin and insulin detemir 80

C-peptide 81

Glucagon 82

Tvne 1 Subiects 83


Plasma glucose 83




NEFA 91

IGF-1 93

IGFBP-1 94


C-peptide 96

Glucagon 96

Type 2 subiects 97


Plasma glucose 97




NEFA 104

IGF-1 106

IGFBP-1 107


C-peptide 108

Glucagon 109

Combined Results 110

Plasma Glucose 110

Glucose Infusion Rate 111

Glucose Metabolism 112

Chapter 4. Discussion 116

Chapter 5. References and Bibliography 133

Acknowledgments

I would like to thank Sarah Hills for her assistance performing the clamps, but more

importantly Juliet Wright for clamping in extreme circumstances, for all her support while I

was entering data and doing the calculations, without her help, support and fantastic sense of

humour at the lowest times, this study would never have been completed.

I would like to thank Jo Bullen for helping me order the equipment I needed for the studies.

I would like to thank Margot Umpleby for all her help with the stable isotope data, for

teaching me how to do the calculations, for setting up the spreadsheets for the calculations

and for her support in the papers that we have written.

I would like to thank Fariba Shojaee-Moradie for teaching me how to clamp, for helping us

to do the first clamp and for her help along with Nicola Jackson, Premilla Croos and

William Jefferson in measuring glucose, glycerol and NEFA.

I would like to thank Gwen Wark for measuring IGF-land IGFBP-1 samples. I would also

like to thank Steven Halloran and Derek Teale in the biochemistry department at The Royal

Surrey County Hospital for their help with obtaining our YSI instrument and providing

samples for its calibration.

I would like to thank the late Ken McRae for all his help with the statistical analysis for this

study, and latterly Julie Amiss and David Lovell for their statistical help.

I would like to thank Guildford Pharmacology Unit for allowing us to use their centrifuge

during the clamps.

I would like to thank Lisbeth Jacobsen, Lars Endahl and Eberhard Draeger from Novo

Nordisk for their help in the study design.

I would like to thank Novo Nordisk and the Dunhill Medical Trust for sponsorship and

funding of the study.

I would like to thank Professor Gordon Ferns for being one of my educational supervisors.

Finally I would like to thank David Russell-Jones for all his invaluable support throughout.

For the concept and design of the study, for helping with arranging the funding, for keeping

our morale high during clamping, and for his persistence in encouraging me to finish writing

up.

List of figures

1. Schematic representation of the three dimensional structure of insulin 20

2. The glycolysis pathway 27

3. Schematic representation of albumin binding of insulin detemir, and potential

mechanism for differences in effect on the liver and periphery. 36

4. Schematic representation of the Physiological Insulin Production. 37

5. Schematic Representaion of Pharmacological Replacement of Insulin. 37

6. Schematic structure of insulin detemir. 42

7. Structure of insulin detemir dimer. 44

8. Di-hexamerisation of insulin detemir. 45

9. Distinct binding sites on albumin. 47

10. Graph of healthy subjects mean plasma glucose. 68

11. Graph of healthy subjects mean glucose infusion rates 69

12. Graph of healthy subjects mean glucose rate of appearance. 70

13. Graph of healthy subjects mean glucose rate of disappearance. 71

14. Graph of healthy subjects mean glucose Ra and Rd. 71

15. Bar charts showing the relative proportion of glucose lowering effect from effect on

glucose Ra and glucose Rd of each insulin. 72

16. Pie chart showing the overall mean proportion of effect of each insulin on glucose

Ra and Rd. 73

17. Graph of Healthy Subjects Glucose TraceriTracee ratio during the clamp studies 75

18. Graph of healthy subjects mean rate of appearance of glycerol. 76

19. Graph of healthy subjects mean plasma NEFA concentrations. 78

20. Graph of healthy subjects mean plasma IGF-1 concentrations. 79

21. Graph of healthy subjects mean plasma IGFBP-1 concentrations. 80

22. Graph of healthy subjects mean plasma insulin and insulin detemir concentrations.

81

23. Graph of healthy subjects mean plasma c-peptide concentrations. 82

24. Graph of Type 1 Subjects Mean Plasma Glucose. 84

25. Graph of Type 1 Subjects Mean Glucose Infusion Rates. 85

26. Graph of Type 1 Subjects Mean Glucose Rate of appearance. 86

27. Graph of Type 1 Subjects Mean Glucose Rate of Disappearance. 87

28. Graph of Type 1 Subjects Mean Glucose Ra and Rd 87

29. Graph of Type 1 Subjects Mean Glucose Ra and Rd Shown as change from 60

minutes. 89

30. Graph of Type 1 Subjects Mean Glucose Tracer:Tracee Ratio. 89

31. Graph of Type 1 Subjects Mean Gycerol Rate of Appearance. 91

32. Graph of Type 1 Subjects Mean Plasma Non Esterifred Fatty Acid Concentrations.92

33. Graph of Type 1 Subjects Mean Plasma Insulin Like Growth Factor-1

Concentrations. 94

34. Graph of Type 1 Subjects Mean Plasma Insulin Like Growth Factor Binding Protein-

1 Concentrations. 95

35. Graph of Type 2 Subjects Mean Plasma Glucose Concentrations. 98

36. Graph of Type 2 Subjects Mean Glucose Infusion Rates. 99

37. Graph of Type 2 Subjects Mean Glucose Rate of Appearance. 100

38. Graph of Type 2 Subjects Mean Glucose Rate of Disappearance. 101

39. Graph of Type 2 Subjects Mean Glucose Ra and Rd. 101

40. Graph of Type 2 Subjects Mean Glucose Ra and Rd Expressed as a change from 60

minutes. 102

41. Graph of Type 2 Subjects Mean Plasma Glucose Tracer:Tracee Ratio. 102

42. Graph of Type 2 Subjects Mean Glycerol Rate of Appearance. 104

43. Graph of Type 2 Subjects Mean Plasma Non Esterifred Fatty Acid Concentrations.

105

44. Graph of Type 2 Subjects Mean Plasma Insulin Like Growth Factor-1

Concentrations. 106

45. Graph of Type 2 Subjects Mean Plasma Insulin Like Growth Factor Binding Protein-

1 Concentrations. 108

46. Graph of type 2 Subjects Mean Plasma C-Peptide Concentrations. 109

47. Graph of Mean Glucose Infusion Rates for Each Subject Group. 110

48. Graph of Mean Glucose Infusion Rates for Each Insulin from the Combined Data

from each Subject Group. 112

49. Graph of Mean Glucose Ra and Rd for each insulin from the combined data from

each subject group. 113

50. Bar charts showing the relative proportion of glucose lowering effect from effect on

glucose Ra and glucose Rd of each insulin using data combined from the three

subject groups. 114

51. Pie chart showing the overall mean proportion of effect of each insulin on glucose

Ra and Rd using data combined from the three subject groups. 115

52. Diagram of the Physiological Plasma Insulin Secretion Profile. 117

List of tables

1. Blood Sampling Times 55

2. Healthy Subjects Plasma Glucose and Glucose Infusion Rate. 69

3. Healthy Subjects Glucose Metabolism 74

4. Healthy Subjects Glycerol Metabolism. 76

5. Healthy Subjects NEFA results. 77

6. Healthy Subjects IGF-1 results. 78

7. Healthy Subjects IGFBP-1 results. 80

8. Type 1 Subjects Plasma Glucose and Glucose Infusion Rate. 85

9. Type 1 Subjects Glucose Metabolism. 90

10. Type 1 Subjects Glycerol Metabolism. 91

11. Type 1 Subjects NEFA results. 92

12. Type 1 Subjects IGF-1 Results. 93

13. Type 1 Subjects IGFBP-1 Results. 95

14. Type 2 Subjects Plasma Glucose and Glucose Infusion Rate. 99

15. Type 2 Subjects Glucose Metabolism. 103

16. Type 2 Subjects Glycerol Metabolism. 104

17. Type 2 Subj ects NEFA Results. 105

18. Type 2 Subjects IGF-1 Results. 106

19. Type 2 Subjects IGFBP-1 Results. 107

20. Combined Glucose and Glucose Infusion Rate Data. I l l

21. Combined Glucose Metabolism Data. 113

Abbreviations used in thesis

Degrees Celsius

ACTH Adrenocortrophic Hormone

AD Ano Domini

ADA American Diabetes Association

ANOVA Analysis of variance

AOC Area over curve

APE Atoms percent excess

ATP Adenosine tri-phosphate

AUC Area under curve

a-v arterio-venous

BC Before Christ

BMI Body Mass Index

cAMP cyclic adenosinemono-phosphate

cDNA complimentary deoxyribonucleic acid

Cl confidence interval

Cmax Maximum concentration

C-Peptide Connecting peptide

CV Coefficent of variation

Da Daltons

DCCT Diabetes control and complications trial

DNA deoxyribonucleic acid

g grams

GH Growth Hormone

GIR Glucose Infiision Rate

GLUT Glucose transporter

GTP Guanine Tri-Phosphate

H^ Hydrogen ion

HbAic Glycosylated Haemoglobin

10

HD High dose detemir (18nmol/kg)

HDL High density lipoprotein

IDL Intermediate density lipoprotein

lEMA Immunoenzymometric assay

IGF-1 Insulin like growth factor-1

IGFBP-1 Insulin like growth factor binding protein-1

IGF-II Insulin like growth factor-II

IRS-1 Insulin receptor substrate

IRTK Insulin receptor tyrosine kinase

lU International Units

kDa Kilo Daltons

Kg Kilogram

1 Litre

lb pounds

LD Low dose detemir (9nmol/kg)

LDL Low density lipoprotein

m metre

mg milligram

min minutes

ml millilitres

mmol millimol

mRNAmessenger ribonucleic acid

MTBSTFA N-Methyl-N-(t-butyldimethylsilyl)trifluoroacetamide

NEFA Non esterified fatty acid

nm nanometre

NMR Nuclear magnetic resonance

NPH Neutral Protamine Hagedom

OGTT Oral glucose tolerance test

pH pondus hydrogenii (potential hydrogen)

PEPCK Phosphoenolpyruvate Carboxykinase

Ra Rate of appearance

11

Rd Rate of Disappearance

RER Rough endoplasmic reticulum

rpm revolutions per minute

SD Standard deviation

SEM Standard Error of the Mean

SH Src Homology

SIM Selected ion monitoring

Src Sarcoma

SSA Sulphosalicylic acid

Tmax Time to reach maximum concentration

UK United Kingdom

UKPDS United Kingdom prospective diabetes study

USA United States of America

VEI Vesicle encapsulated insulin

VLDL Very low density lipoprotein

WHO World Health Organisation

a Alpha

p Beta

p micro

12

Chapter 1

Introduction

Diabetes

Definition

Diabetes mellitus is a state of chronic hyperglycaemia, classically associated with symptoms

of excessive thirst, increased urine volume and weight loss. It results from the insufficiency

of or diminished effectiveness of endogenous insulin.

Classification of diabetes (American Diabetes Association 1999)

I Type 1 diabetes (P-cell destruction, usually leading to absolute insulin deficiency)

A. Immune mediated

B. Idiopathic

II Type 2 diabetes (ranging from predominantly insulin resistance with relative insulin

deficiency to predominantly an insulin secretory defect with insulin resistance).

III Other specific types of diabetes

A. Genetic defects of P-cell function

B. Genetic defects of insulin action

C. Diseases of the exocrine pancreas

D. Endocrinopathies

E. Drug or chemical induced

F. Infections

G. Uncommon forms of immune mediated diabetes

H. Other genetic syndromes sometimes associated with diabetes.

IV Gestational diabetes

Criteria for the diagnosis of diabetes mellitus

I. Symptoms of diabetes plus casual plasma glucose concentration >200mg/dl

(11.1 mmol/1). Casual is defined as any time of day without regard to time since last meal.

The classic symptoms of diabetes include polyuria, polydipsia and unexplained weight loss.

13

Or

2. Fasting plasma glucose > 126mg/dl (7.0mmol/l). Fasting is defined as no calorie intake

for at least 8 hours.

Or

2-hour plasma glucose >200mg/dl (11.1 mmol/1) during an OGTT. The test should be

performed as described by WHO, using a glucose load containing the equivalent of 75mg

anhydrous glucose dissolved in water.

In the absence of unequivocal hyperglycaemia with acute metabolic decompensation, these

criteria should be confirmed by repeat testing on a different day.

Epidemiology

The world-wide incidence of type 1 diabetes varies greatly from as high as 36 per 100 000

per year in some Scandinavian countries while in countries such as Japan it is only 2 per 100

000 per year, Venezuela has the lowest recorded incidence of 0.1 per 100 000 per year

(Wareham, 2002). The disease is predominantly one of white Caucasian populations and is

relatively rare in both oriental and black populations (Bell and Hockaday, 1996). The

incidence of type 1 diabetes appears to be increasing. Increases have been reported in

Scotland of about 2 % per annum during the period 1984-1993 (Rangasami et al, 1997).

The overall pooled incidence in 37 countries was 3% per year, the increase was relatively

greater in populations with the lowest incidence. The incidence of type 1 diabetes also

varies with season, being highest in autumn and winter. Type 1 diabetes incidence varies by

age and sex, with in the UK the peak incidence in boys being at 12-13 years and the peak

incidence in girls being 9-12 years. The incidence is slightly higher in boys than girls (Ratio

1.08: 1). (Wareham, 2002)

Type 2 diabetes is far more common than type 1. In some populations of Polynesian-

Micronesian stock the prevalence may be as high as 40-50%, whether on affluent Pacific

islands (eg Nauru) or in reservations in Arizona (Pima Indians). The prevalence in the

United Kingdom was believed to be 2%. However recent studies have suggested values

14

ranging from 8 to 12% depending on ethnic mix (Bell and Hockaday, 1996). There are

currently believed to be 1 million people in the UK with undiagnosed diabetes (Diabetes

UK). The prevalence of type 2 diabetes is lowest in rural areas of developing countries, is

generally intermediate in developed countries and is highest in some ethnic groups who have

adopted Western lifestyle patterns. The populations with the highest prevalence (Pima

Indians and Nauruans) have a high prevalence of obesity. It is hypothesized that genetic

susceptibility to obesity in these populations would be disadvantageous in times of food

abundance, but would give evolutionary advantages at times when food is scarce, giving

rise to increased prevalence of the gene by natural selection. This “thrifty genotype”

hypothesis is supported by evidence of gene environment interaction - individuals who

migrate from low prevalence areas to the West are at increased risk of type 2 diabetes. The

prevalence of type 2 diabetes is also increasing with figures from the USA showing a 33%

increase in prevalence from 4.9% in 1990 to 6.5% in 1998 (Mokdad et al, 2000). This

increase in prevalence mirrors the increasing prevalence of obesity. The main aetiological

risk factors for type 2 diabetes are age, obesity, family history, physical inactivity and

dietary factors such as a high proportion of energy consumed as saturated fat and low intake

of finit and vegetables. (Wareham, 2002)

History of diabetes.

Early Hindu writings attributed to Susrata in 400BC, describe people with a mysterious and

deadly disease that caused increased thirst, enormous urine output and wasting away of the

body whose urine was attractive to black ants. (MacCracken, 1997, Owens, 1986). Areteus

of Cappadocia a contempory of Nero (30-90 AD) wrote “diabetes is a strange disease,

which fortunately is not very frequent. It consists o f the flesh and bones running together

into the urine.... The illness develops very slowly. Its final outcome is death... The patients

are tortured by an unquenchable thirst; they never cease drinking and urinating.... The

disease was called diabetes, as though it were a siphon because it converts the human body

into a pipe for the transflux o f liquid humors. Now since the patient goes on drinking and

urinating while only the smallest portion o f what he drinks is assimilated in the body, life

naturally cannot be preserved fo r very long, fo r a portion o f the flesh is also excreted

15

through the urine.” (Hoskins, 1941). The term diabetes (from Greek Dia, through and

baino, I flow and mellita, a bee) had actually been coined earlier by Apollonius of Memphis

and Demetrius of Apamea around 250 BC.

It wasn’t until 1674 that Thomas Willis re-described the presence of glycosuria and

concluded that this must be preceeded by presence of sweetness in the blood. In 1774

Matthew Dobson demonstrated that the sweetness of urine and serum was due to sugar. In

1849 Claude Bernard working on glucagon metabolism in the liver linked diabetes with

glycogen metabolism. In 1869 Paul Langerhans, then 22 described small bodies in the

pancreas in his thesis in Berlin. Unfortunately he was unable to continue his work,

developing tuberculosis, he had to leave Germany to live in Madeira, where he died at the

age of 40 (Andreani, 1999).

In the late 19 Century Minkowski and von Mehring were interested in the study of the

pancreas in relation to digestion. However it was a kennel worker who was caring for the

experimental animals of the two doctors who noticed that their urine was attracting great

swarms of flies, he drew the doctor’s attention to this whereupon it was discovered the urine

had large amounts of sugar (Minkowski later denied this version of events). Thus

experimental diabetes had been produced. Minkowski recognised the significance of the

discovery and tried to make an extract of dog’s pancreas that would have a beneficial effect

on the experimental diabetes he had produced but without success. Caparelli however

anticipated him and discovered a simple saline extract of pancreas injected directly into the

abdominal cavity of dogs had a marked lessening or even disappearance of glucose in the

urine.

Clinicians immediately attempted to use the knowledge, some administered fresh pancreas

by mouth, some gave juice expressed from the gland, and some administered extracts

rectally. Bormann administered roast pancreas by mouth, later infusions by rectum and

finally subcutaneous injections of raw pancreas extract. One of his patients who received

the latter treatment gained eight pounds in six weeks. Others were finding similar results

however these pancreatic extract contained digestive enzymes and patients were unable to

16

tolerate the injections for long (Hoskins, 1941). In 1898 Blumenthal squeezed the juice

from raw pancreas and treated it with alcohol to remove the protein. When administered

intravenously it killed his experimental animals, and when injected subcutaneously caused

ulcers however he was able to show that subcutaneous injection of his preparation increased

glucose utilization by 40%. Blumenthal was actually using a crude preparation o f insulin,

and had his side effects been less severe he may have enjoyed some of the glory given to

workers a quarter of a century later.

The next ten years revealed little hope for the treatment of diabetes, indeed “The Household

Physician” “A family guide to the preservation of health and to the domestic treatment of

ailments and disease” (McGregor-Robertson, 1898) wrote that the chief treatment consisted

of regulation of the diet. It gives two tables of permitted and not permitted foods and drinks

and also recommended that the patient “take regular moderate exercise, flannels should be

worn, and warm baths taken frequently”. Of pharmacological approaches to treatment its

author wrote, “many medicines have been tried, but none have been very successful. Opium

and ergot are probably the best, and may be taken in pill containing % grain powdered

opium and 2 grains ergotine, one thrice daily. In many cases the use o f opium ought be

pushed but not without medical supervision.” In 1913 Frederick Allen wrote “all

authorities are agreed upon the failure ofpancreatic opotherapy in diabetes ...injections o f

pancreatic preparations have both proved useless and harmful. The failure began with

Minkowski and has continued to the present without interruption... The negative reports

have been numerous and trustworthy. ” (Allen, 1913).

Frederick Madison Allen (bom in 1876) working at Harvard Medical school, carried out

extensive work on diabetes and carbohydrate metabolism. He concluded that while diabetes

was mostly a problem of carbohydrate metabolism, it was not just the carbohydatres but the

proteins and fats as well. Diets that involved cutting back sharply on carbohydrates and

increasing proteins and fats achieved little and if anything caused a deterioration in the

condition with a higher rate of acidosis and death resulting from the metabolism of fats.

Described as a stem, cold, tireless scientist (Bliss 1982) Allen had a ward of diabetic

patients at the Rockefeller Institute in New York. Here he applied his diets to an assortment

17

of diabetic patients restricting some to as little as 400 calories per day. He published his

findings of 76 of the 100 patients he treated in 1919 (Total dietary regulation in the

treatment of diabetes), some patients survived for a few years others only for a few months.

Allen’s diets were generally thought to be the best therapy at the time and were adopted by

medical schools and up to date practitioners.

During this time many were dying from diabetes, but Eugene Opie (an American at Johns

Hopkins University in Baltimore) in 1901 described that in a high proportion of autopsies

destructive changes could be seen in the pancreatic “islands of Langerhans”.

In 1908 Georg Ludwig Zuelzer a young German physician collected pancreas glands from

animals at the height of digestion. He pressed out the juice and treated it with alcohol to

precipitate the proteins. The material was then freed from the alcohol and injected into

dogs. He managed to reduce the urine sugar output in these dogs with the substance he

called “acomatrol”. He treated 8 patients and successfully reduced the amount of sugar in

their urine. Indeed he injected acomatrol into a fifty year old diabetic in a private clinic in

Berlin. The patient was comatose, but after receiving the extract his condition improved,

only to deteriorate and die when supplies of Zuelzer’s acomatrol dried up (MacCracken,

1997). His extract needed purifyiiig further to decrease the side effects of vomiting, high

fevers and convulsions that his patients were suffering. Zuelzer continued to attempt to

make a pancreas preparation suitable for the treatment o f diabetes. He visited many

pharmaceutical companies to try to gain backing without success. Finally in 1911 he was

given funding by one, however he had little success and in 1914 his hospital was taken over

by the military. The presence of digestive enzymes in the pancreatic products was causing

great difficulties. Several others including Scott, Starling, Knowlten and Macleod continued

research along the lines of extracting and purifying the pancreatic juices, but their work was

also interrupted by the outbreak of World War 1.

In 1921 Banting (an orthopaedic surgeon) returned from the war, and reading an article by

Barron written in 1920 describing the old experiment of ligating the pancreatic ducts which

results in atrophy of the exocrine pancreas leaving the islets intact, it occurred to him that

18

the troublesome digestive enzymes may be circumvented using this technique. Under

Professor Macleod at the University of Toronto he was given the assistance of Best, a

medical student. They started ligating ducts in the pancreas of dogs and after the pancreas

had degenerated injected extracts of it into depancreatised dogs. They recorded a lowering

of the urine sugar output, however they soon ran out of pancreatic tissue. At this point they

too were confronted with the problem of preparation and refinement of the extracts of

pancreatic tissue. Collip was then added to the group and brought to bear the experience

gained in several years of devotion to this type of chemistry. They moved on to using the

foetal calf pancreas to avoid the activity of trypsin, and then onto ox pancreas, with which

they discovered that if the whole gland was immediately placed in acid aqueous alcohol

yielded adequate amounts of insulin. The use of alcohol and acid in the extraction process

circumvented the enzyme action. The resulting extract in concentrated form was first given

to the investigators themselves.

In January 1922 its first use in patients was begun. Leonard Thompson a 14 year old

weighing only 641b was the first patient to be given insulin. He received 7.5 ml in each

buttock of “thick brown muck”. Although initially his blood sugar and glycosuria fell,

unfortunately he developed abscesses at the injection sites and became more unwell. The

injections were stopped. Six weeks later he was given Collip’s more refined extract, his

blood sugar fell and he quickly began to gain weight. Collip’s extract was then given to a

number of patients with diabetes at Toronto General hospital, they responded well. During

the summer of 1922 insulin began being produced commercially by Eli Lilly in Indianapolis,

USA. The first insulin complementary deoxyribonucleic acid (cDNA) was cloned and

sequenced from rat islet messenger ribonucleic acid (mRNA) by Ullrich et al., (1977). The

human insulin gene was later fully characterised (Bell et al., 1980). Owerbach et al., 1981,

located the human insulin gene to the short arm of chromosome 11. Recombinant DNA

techniques have allowed biosynthetic human insulin to be produced by Escherichia coli

(Keefer et al., 1981).

19

The Insulin molecule

Structure of the insulin molecule.

Sanger and co-workers determined the complete amino acid sequence of insulin and its

primary structure in 1955 (Ryle et al 1955). Schlichchtkrull (1956) demonstrated that the

rhombohedral crystals of insulin contain two zinc atoms and consisted of six molecules of

insulin. Extensive crystallographic studies of the three dimensional model of the insulin

molecule demonstrated that the hexamer of insulin with a molecular weight of 36000 Da (at

neutral pH in the presence of zinc) consisted of three slightly asymmetric dimers each with a

molecular weight of 12000Da (Adams et al 1969, Blundell et al 1972, Hodgkin, 1972) (see

figure 1).

Figure 1 (after Hodgkin, 1972)

Schematic representation of the three dimensional structure of insulin

20

The secondary structure of the monomers includes three a-helical regions (Blundell et al

1971 & 1972), of which two exist on the A chain and one on the B chain. The folding of the

B chain around the A chain core is also brought about with disulphide bridges between

cysteine residues. The globular structure achieved in this way provides the molecule with a

hydrophobic core which is essential for its structural stability, all polar residues are on the

surface and the non polar residues, mainly on the B chain, are buried during the formation of

the dimers and the 2 zinc-insulin hexamers. Insulin aggregation in solution depends on

concentration, pH, ionic strength and the presence of divalent cations and anions (Blundell

et al., 1972, Bradbury and Brown, 1977). X-ray studies demonstrate that most mammalian

and fish insulins form similar hexamers, (except the hagfish, guinea pig and coypu (Cutfield

et al., 1974, Peterson et al., 1974, Wood et a l, 1975).

Biosynthesis of insulin

Insulin is first synthesized as preproinsulin (molecular weight 11 500Da) on the rough

endoplasmic reticulum (RER) in the P-cells of the islets of Langerhans in the pancreas

(Patzelt et a l, 1978, Steiner et a l ,1974). The islets of Langerhans comprise 1-3% of the

pancreatic mass and are probably derived from the ectoderm (Bell and Hockaday, 1996).

The N terminus of preproinsulin has a hydrophobic extension of 23 amino acid residues

which directs release of the nascent polypeptide chain to the cisternal space of the RER

(Chan et a l, 1976). The signal sequence is rapidly removed by proteolytic cleavage and the

resulting proinsulin molecule (molecular weight 9390Da and 86 amino acid residues) is

transferred via the vesicles of the RER to the Golgi apparatus. This transfer is an energy

dependent process (Howell and Tyhurst, 1972) and by pulse labelling technique has been

shown to occur in about 15 minutes.

In the Golgi apparatus proinsulin is packed into clathrin coated trans-Golgi vesicles

containing an ATP-dependent proton pump (Orci et a l, 1986 & 1984). As the vesicles

mature, H+ ions are transported inwards, via the proton pump, causing a reduction in pH and

thus increasing the activity of the proteolytic enzymes and ensuring the conversion of

21

proinsulin to insulin within maturing vesicles. This process takes 60-90 minutes (Orci et al.,

1973)

The connecting peptide (C-peptide) is joined to the insulin molecule by basic amino acids;

two arginines at the amino end and a lysine and arginine at the carboxyl end. These are the

points of trypsin and carboxypeptidase B-like enzymatic cleavage which convert proinsulin

into insulin, consequently releasing these basic residues (Orci et al, 1986, Steiner and Oyer,

1967, Chance et al, 1968).

The conversion of proinsulin to insulin takes place in non-coated mature secretory granules

(Orci et al., 1984) and the removal of the C-peptide chain decreases its solubility. The

resulting insulin is crystallized with 2 zinc ions per 6 insulin molecules and is stored in

equimolar ratio with C-peptide in secretory granules where it is available for secretion

through exocytosis (Orci et al., 1984). A small amount of proinsulin co-crystallizes with

insulin (Steiner, 1973) and constitutes approximately 5% of insulin secretion.

Insulin secretion is stimulated primarily by a rise in glucose concentration. Glucose is

metabolised through the glycolytic pathway generating ATP within p-cells (Randle, 1993).

The increase in intracellular ATP concentration (Ashcroft et al., 1984) or ATP/ADP ratio

(Ashcroft and Ashcroft, 1990) leads to closure of the ATP sensitive potassium channels and

depolarisation of the p-cell membrane thus causing the entry of calcium through calcium

channels (Sussman et al., 1987). Calcium then activates Ca-calmodulin dependent protein

kinase which in turn leads to phosphorylation of specific protein substrates and the release

of insulin from the P cell.

The Insulin Receptor

The insulin receptor consists of a membrane spanning glycoprotein complex lodged in the

plasma membranes of cells, with an apparent molecular weight of 450 kDa and consisting of

two distinct glycosylated subunits (Yip et al, 1978 and 1980). There are two a-subunits and

two p-subunits linked covalently by disulphide bonds. The two a-subunits are located at the

22

extracellular face of the plasma membrane and contain the insulin binding domain. The two

p-subunits are held together by disulphide bonds, they consist of three regions: an

extracellular domain of 194 amino acid residues with a segment rich in cysteine residues and

forming disulphide bridges with the a-subunits, a hydrophobic transmembrane domain of 23

amino acid residues, and an intracellular domain of 403 amino acid residues that contains a

catalytic site and several sites of autophosphorylation (Kasuga et al, 1982 a and b, White et

al, 1985).

Following binding of insulin to its receptor a series of events is triggered: internalisation of

insulin, processing of insulin and/or the insulin receptor comlex and initiation of a wide

spectrum of metabolic processes. Experimental evidence suggests that insulin binds to its

receptor with high affinity in a ratio of 1:1 at physiological concentrations. Carpentier et al

(1979 a & b) using radio-iodinated (^ I) insulin demonstrated that the insulin receptor

complex is internalised (endocytosis), generating coated vesicles containing the complex in

the subplasmalemmal cytoplasm. The acidic pH within the endosomes causes dissociation

of insulin from the receptor (Farquhar, 1983; Tyko and Maxfield, 1982). Insulin is then

degraded within the secondary lysosomes. The internalised receptor (Fehlmann et al 1982 a

& b; Juul et al 1986) is either recycled to the cell surface from endosomes (Marshall et al

1981; Carpentier et al, 1986) or degraded (Heindreich et al, 1983; Berhanu et al, 1982).

Insulin affects many cellular systems including: transport of molecules across the plasma

membrane, levels of cyclic nucleotides, activities of key enzymes in intermediaiy

metabolism, rates of protein synthesis and degradation, rates of DNA and RNA synthesis,

including specific gene expression and cellular growth and differentiation. However, it was

not understood how the rapid effects of insulin (observed within seconds) could be

explained by the process of internalisation (Lamer et al., 1981).

Kasuga et al., (1982a) found that the addition of insulin to cells led to rapid phosphorylation

of the P-subunit on serine and threonine residues. Subsequently, the (3-subunit was shown to

be phosphorylated on tryrosine residues in cell free systems which led to the idea of tyrosine

kinase activity associated with the insulin receptor itself (Kasuga et al., 1982b).

23

It was then shown that immediately after insulin binds to the receptor a cytoplasmic protein,

is phosphorylated on tyrosine reidues (White et a l, 1985). This protein which is now

referred to as insulin receptor substrate 1 (IRS-1) has been purified from rat liver by affinity

chromatography and the cDNA of this protein has been isolated (Rosenberg et a l, 1991; Sun

et a l, 1991). This protein has 10 tyrosine phosphorylation sites. After phosphorylation, 6 of

these tyrosine-containing regions can interact with other proteins which have Src homology

(SH2) domains (Koch et a l, 1991). One of the cytoplasmic proteins which has the SH2

domain is phosphotidylinositol 3’-kinase. It has been demonstrated that

phosphotidylinositol 3’-kinase is associated with the IRS-1 (Sun et a l, 1991). Several other

signalling proteins containing SH2 domains eg phosphotyrosine phosphatase Syp (Kuhne et

a l, 1993) and growth factor receptor bound-2 (grb-2) (Baltensperger et a l, 1993) have been

shown to couple with IRS-1. This appears to connect the insulin receptor to a signalling

chain which involves GTP-binding proteins such as ras which interact with a cascade of

serine/threonine specific kinases (MAP-kinase cascade, mitten-activated protein

kinase)(Gammeltoft and van Obberghen, 1986).

In vitro studies with adipocytes treated with a tyrosine phosphatase inhibitor (okadaic acid)

demonstrated that IRS-1 was phosphorylated on serine and threonine residues, but in these

cells the stimulation by insulin of glucose transport was inhibited (Tanti et al, 1994).

Further studies involving microinjection of anti-IRS-1 antibody into rat fibroblasts have

suggested that IRS-1 initiates mitogenic activity of IGF-1 and insulin receptors (Rose et al,

1994). These findings have led to the suggestion that IRS-1 has a major role in insulin

mediated metabolic and mitogenic signalling and glucose transport.

Glucose Metabolism

Usually when the concentration of glucose is elevated (fed state) and other nutrients are

adequate, the liver, muscle and adipose tissues are geared to anabolic process and storage.

When dietary glucose is not available, ie in the catabolic state, glycogen stores from the liver

and fat and protein stores are mobilized to provide an alternative energy source.

24

Endogenous Glucose Production

Circulating plasma glucose, in the fed state, is mainly derived from the splanchnic bed.

However, during a short fast, liver alone, and in a prolonged fast both liver and kidney

(Flock et al., 1969) are geared towards glucose production. These two tissues contain

glucose 6 phosphatase activity which catalyses the conversion of glucose 6 phosphate to

glucose. Glucose can then be transported out of the cell by glucose transporters.

Glucose from the liver is produced by two processes: glycogenolysis and gluconeogenesis.

Initially glycogen stores in the liver are mobilized. Studies of serial liver biopsies in human

subjects (Nilson and Hultman, 1973) and nuclear magnetic resonance (NMR) studies to

measure the rate of glycogen depletion from the liver (Rothman et al., 1991) have suggested

that after an overnight fast, glycogenolysis contributes to 40-50% to the hepatic glucose

production. After depletion of glycogen stores (20 hours of fast) gluconeogenesis maintains

hepatic glucose output (Consoli et al., 1987). A variety of substrates are used in this

process. In muscle, during a prolonged fast glycogen stores and protein are released as

lactate and alanine which are then transported and used in the liver for glucose production.

Glycerol from adipose tissue during a prolonged fast is another gluconeogenic source and

provides 50% of the hepatic glucose output (Bortz et al, 1972 and Nurjhan et al, 1986).

Insulin affects glucose homeostasis by inhibiting hepatic glucose output and stimulating

glucose disposal.

Glucose Utilization

In most tissues glucose is taken up via transport systems that display saturation kinetics.

The intracellular glucose concentration is normally lower than the extracellular glucose

concentration, and glucose is transported down its concentration gradient by facilitative

diffusion via glucose transporters across the cell membrane which is thought to be the rate

limiting step (Britton, 1964) in glucose disposal.

25

Glucose taken up by the cells is either stored as glycogen or lipid, oxidised to CO2 and H2O

or converted to lactate and alanine. The Cori (lactate) and Glucose-alanine cycles account

for 20-25% of glucose utilization (Felig, 1973).

Various factors can influence the fate of circulating glucose, including the degree of fasting,

the availability of substrates such as free fatty acids and various endocrine factors. The

tissues involved in glucose utilization include splanchnic tissues, muscle, brain and adipose

tissue. Brain and muscle are the major tissues for glucose oxidation whereas splanchnic

tissues, adipose tissues and under certain circumstances muscle are geared for storage.

Both glucose production and utilization are affected by insulin and glucagon. The

glycolysis pathway where glucose is broken down to pyruvate and lactate is illustrated in fig

1. The steps where insulin or glucagon has an effect by causing phosphorylation or

dephosphorylation of the enzyme has been shown.

26

GLUCOSE

/GLYCOGEN

^ ^ glycogen

phosphoryase -GLUCOSE-1- PHOSPHATE -----

GLUT ' GLUCOSE* * c T I glucokinaseÎ 1Glucose-6-

- phosphstase | T hexokinase +

GLUCOSE-6-PHOSPHATEGlucose phosphate isomerase

Loss of 2- H

FRUCTOSE-6-PHOSPHATE

î l 6-phosphofructokinase 4"fiructose 1,6-

- biphosphatase

FRUCTOSE-l,6-PHOSPHATE

aldolase

DIHYDROXYACTONEPHOSPHATE T

Loss of 3-^H t iî i

+ Stimulation by Insulin - Inhibition by Insulin * * Stimulation by glucagon

OXALOCETATE ^

^ GLYCERALDEHYDE-3-PHOSPHATEglyceraldehydes-phosphatedehydrogenase

3-PHOSPHOGYCEROYLPHOSPHATEphosphoglycerate

3-PHOSPHOGLYCERATE

^ ^ phosphoglyceromutase

2-PHOSPHOGLYCERATE

enolase

PHOSPHOENOLPYRUVATE (PEP)

Pyruvatekinase +

PYRUVATEpryruvate carboxylase

Loss of 6- H

lactatedehydrogenase

LACTATE

Pyruvatedehydrogenase

ACETYL-CoA

Figure [ 2]- The glycolysis pathway showing the effects of insulin and glucagon on the enzymes involved and the steps at which 2-[^H], 3-[^H] and 6-[^H]glucose lose their label. PEPCK (phosphoenolpymvate carboxykinase) (Umpleby and Sonksen, 1987). GLUT is representing glucose transporters.

27

Glucose Homeostasis

The regulation of plasma glucose levels is of central importance. Tissues such as muscle

and liver are able to metabolise substrates other than glucose, however the brain almost

exclusively utilizes glucose as a source of energy. Some researchers have demonstrated that

the brain is able to metabolise ketones and lactate (Veneman et al, 1994, Maran et al, 1994,

Amiel et al, 1991). The brain has high energy requirements estimated at approximately

Img/kg/min glucose corresponding to about lOOg glucose per 24 hours, however it has low

energy reserves (enough for only a few minutes), and is unable to synthesize glucose (Bolli,

1998). The energy utilised is required to maintain the ionic gradient across nerve

membranes critical for maintenance of brain activity, and as a result of brainstem functions

critical in maintaining life. The brain is therefore mainly dependent on the maintenance of

a constant supply of glucose from the blood.

Intricate mechanisms to protect the supply of glucose to the brain have therefore evolved.

Only the liver and the kidney are able to release glucose. These organs contain glucose-6 -

phosphatase which liberates glucose into the bloodstream (Bolli and Fanelli, 1999). The

liver and the kidney do not contribute equally to postabsorptive glucose homeostasis. It is

generally believed that the liver makes the major quantitative contribution during this state,

whereas the kidney has a minor role, except during prolonged starvation, when it is

estimated that the kidney may contribute almost half of glucose production (Bolli 1998,

Cahill et al,1966,Cahill 1970).

In normal subjects the plasma (extracellular) glucose concentration remains between 3.5 and

7 mmol/1 despite intermittent glucose loads entering the body from the gastrointestinal tract

following meals. The renal tubule absorbs all glucose from the glomerular filtrate up to

concentrations of around lOmmol/1, so that in the non-diabetic individual with a normal

renal threshold, urine is glucose free. Blood glucose is sensed in the brain (Frohman and

Nagai, 1976, Ritter et al, 1981, Borg et al, 1995), pancreas (Taborsky et al, 1998) and liver

(Connolly et al, 1996), each of these organs is involved in the counter-regulation response to

low blood glucose in healthy subjects.

28

The release of insulin, glucagon, growth hormone, catecholamines, glucocorticoids and

insulin like growth-factor 1 (IGF-1) have all been shown to be sensitive to changes in

glucose concentration (Garber et ah, 1976, DeFronzo et ah, 1977, Cryer, 1991, Bolli and

Fanelli, 1999).

Both insulin (secreted from the (3-cells of the pancreas) and glucagon (secreted from the a

cells) play an important role in the regulation of glucose homeostasis. A fall in blood sugar '

concentration that may occur in the postabsorptive state leads initially to a reduction in

insulin secretion, by direct action of glucose on p-cells of the islets of Langerhans (Cryer

1997, Bolli, 1998). This leads to a reduction in the plasma insulin concentration in the

portal circulation, and therefore a reduction in the suppression of hepatic glucose output (or

a rise in hepatic glucose output). If the blood glucose continues to drop counter-regulatory

hormones are stimulated and released, oc-cells of the islets of Langehans in the pancreas are

stimulated to release glucagon, this is reversed in hyperglycaemia (Matschinksy and

Bedoya, 1989). Like insulin, glucagon is also released into the portal circulation, it has no

effect on peripheral glucose uptake, but increases hepatic glucose production. The increase

in hepatic glucose output is early and sustained probably because of the sustained

stimulation of hepatic gluconeogenesis, the main mechanism of increased hepatic glucose

output. The kidney does not appear to be affected by glucagon stimulation.

However, hyperglycaemia per se (with suppression of insulin and glucagons secretion) has

an inhibitory effect on hepatic glucose production in man (Liljenquist et al., 1979). In

addition, during hypoglycaemia induced by insulin in humans, Bolli et al (1985) found that

when glucose concentrations decreased below 2.5mmol/l there was a residual hepatic

glucose output independent of neurohormonal influences or an autoregulatory process by the

liver. In dogs also, it has been shown that this residual hepatic glucose output is maintained

by glycogenolysis and gluconeogenesis after a prolonged hypoglycaemia induced by insulin

infusion (Tyler Frizzell et al., 1988).

29

ACTH is released from the pituitary and stimulates a rise in cortisol several hours after

hypoglycaemia. Short term infusion of cortisol (the main glucocorticoid in man) affects

glucose homeostasis by increasing hepatic glucose output and decreasing peripheral glucose

disposal (Rizza et al, 1982). However studies of patients with Cushing’s syndrome (ie with

long term effects of excess glucocorticoids) using an intravenous bolus of D-[3-^H] glucose

have shown that the hyperglycaemia sometimes found in these patients is primarily due to

decreased glucose disposal rather than increased glucose production (Bowes et al., 1991).

Furthermore, both basal (Bowes et al., 1991) and glucose stimulated insulin secretion

(Kitabchi et al., 1973) are increased in the presence of excess glucocorticoids. In contrast, in

the absence of glucocorticoids, insulin secretion is suppressed, for example following

adrenalectomy in man, although the absence of the catecholamines (adrenaline and

noradrenaline) may contribute to the effect of this example (Lenzen and Bailey 1984;

Marker et al., 1991).

Adrenaline is released from the adrenal medulla as a result of stimulation of the sympathetic

nervous system (Bolli and Fanelli, 1999). Catecholamines exhibit multiorgan effects.

Adrenaline reduces secretion of insulin from the pancreas, lowering portal plasma insulin

concentration further, and thus reducing the suppression of hepatic glucose output. It also

increases hepatic and renal glucose output, probably secondary to increased

gluconeogenesis. Adrenaline reduces peripheral glucose uptake and stimulates lipolysis.

The stimulation of lipolysis contributes to counter regulation via non esterified fatty acids

which stimulate hepatic gluconeogenesis and suppress glucose oxidation and use. The effect

of free fatty acids on the production of glucose is greater than that on the use of glucose.

Noradrenaline concentration in plasma rises less, but it acts though the same pathways as

adrenaline with similar effects. It also inhibits insulin secretion and stimulates glucagon

synthesis.

Growth hormone is released from the pituitary as a result of hypoglycaemia, it also increases

hepatic glucose output and suppresses peripheral glucose uptake. It stimulates lipolysis and

therefore increases plasma concentration of non esterified fatty acids and glycerol.

30

Hepatic autoregulation occurs when hypoglycaemia is severe. Investigators have

demonstrated that endogenous glucose production increases despite complete neurohumoral

counter regulatory blockade. (As reviewed by Bolli 1998 and Bolli & Fanelli 1999).

A large part o f the counter regulation hormones’ effect, at least the ones that possess

peripheral effects are mediated by the common pathway of increased lipolysis and greater

plasma levels of free fatty acids. As described above these fatty acids exert their counter

regulatory effect mainly at the liver. Thus all counter regulatory responses to

hypoglycaemia increase hepatic glucose output either directly alone or in combination with

an indirect effect by the stimulation of lipolysis, some also decrease peripheral glucose

uptake. In normal physiology these defence mechanisms are appropriate as insulin is

released into the portal circulation and has its first effects at the liver, and the liver plays a

vital role in the production of glucose to support brain metabolism.

In insulin dependent diabetes no insulin is released by the pancreas in most cases exogenous

insulin is administered by subcutaneously delivery, it circulates peripherally first, and is

controlled by its pharmacokinetics, with peak actions occurring 1 to 8 hours after injection

(Bolli, 1998). The insulin profile does not resemble physiological insulin profiles of a

normal 24 hour day, and is unable to respond to changing insulin need (Amiel, 1998, Cryer,

2003). In this situation the liver is relatively underinsulinised and peripheral tissue over

insulinised. In the event of hypoglycaemia, the first line of counter regulation (that is a

decrease in insulin release) is not possible, insulin levels remain inappropriately high

suppressing hepatic glucose output, increasing peripheral glucose uptake and inhibiting

lipolysis. Further counter regulation has evolved to exert itself primarily at the liver,

suppression of hepatic glucose output is thus easily overcome since in these circumstances

the liver is relatively underinsulinsed, however the peripheral over insulinisation presents a

more difficult obstacle, and therefore it could be argued that greater counter regulation has

to occur to prevent a further fall in plasma glucose and maintain the brain’s glucose supply.

This may cause later hyperglycaemia as a result of the sustained release of counter

regulatory hormones and their prolonged duration of action may be a factor in the overall

deterioration in glycaemic control seen in patients with frequent hypoglycaemia. Some have

31

proposed that intraperitoneal insulin would be a more physiological replacement (see hepatic

insulinisation page 36)

Fat and Lipid metabolism

Lipids in the plasma exist as free cholesterol, cholesterol ester, triglycerides (glycerol and

fatty acids) and phospholipids (triglycerides and a phosphate derived group).

Plasma lipids are transported as lipid protein complexes. These lipoproteins are classified as

chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins

(IDL), low density lipoproteins (LDL) and high density lipoprotein (HDL) according to their

densities. Insulin stimulates fatty acid synthesis from pyruvate and lactate (Denton et al.,

1975) by increasing the activity of acetyl CoA carboxylase and pyruvate dehydrogenase and

increases the estérification of fatty-acyl CoA and glycerol to form triglycerides. Acetyl CoA

carboxylase is inactivated by reversible phosphorylation which is catalysed by the enzyme

cyclic adenosinemono-phosphate (cAMP) dependent protein kinase. Hormones that

decrease the concentration of cAMP (for example insulin) will activate the enzyme whereas

those that elevate the concentration of cAMP (for example adrenaline or glucagon) will

cause the inactivation of acetyl CoA carboxylase (Brownsey and Denton, 1982; Holland et

al., 1985).

Insulin also inhibits lipolysis (Garratt et al., 1979) in adipose tissue by dephosphorylation of

the enzyme triacylglycerol lipase to an inactive form via lowering cAMP concentrations

hence decreasing the activity of the protein kinase. The activity of lipoprotein lipase which

hydrolyses chylomicrons and other lipoproteins to fatty acids and glycerol is increased by

insulin in a dose dependent manner. The increase in activity is caused by cleaving the

enzyme from the cell membrane via stimulation of a phosphatidylinositol-specific

phospholipase C (Chan et al., 1998).

32

When the concentrations of fatty acids and ketone bodies are elevated as during starvation or

exercise there is an increased rate of their oxidation. This reduces glucose oxidation and

utilization and increases the flux through gluconeogenesis (Randle et al., 1963). In contrast,

in the presence of an elevated glucose concentration lipid oxidation is suppressed due to

inhibition of lipolysis and increased fatty acid re-esteriflcation (Sugden et al., 1989).

The Endothelial Barrier

The capillaries are the sites of exchange of substances between flowing blood, extracellular

fluid and tissue cells. The endothelial barrier provides a membrane across which substances

move both actively and passively in response to pressure and concentration gradients. For

small molecules (for example water, gases and small ions) equilibration is rapid (Hill 1928)

but for macromolecules (for example yglobulins, albumin and probably insulin)the process

is slower (Renkin, 1964). In general, there are two continuous layers in the walls of the

capillaries: the endothelium and its underlying basement membrane or basal lamina. The

permeability of the capillaries is dependent on the porosity of these layers.

Endothelial cells of some capillaries have been shown to have a large proportion of small

vesicles and fenestrae (Wissig and Charonis, 1984). Vesicles have been shown to shuttle

fluid across endothelial cells (Palade, 1961). Fenestrae are openings throughout the

endothelial plasma membrane with a range of pore diameters around 30-80nm closed by a

delicate unilaminar diaphragm 2-4nm thick with a nodal swelling 15nm in diameter at its

centre (Clementi and Palade, 1969).

Endothélia of different types of capillaries differ in their permeability (Renkin 1964). The

tightest capillaries are those of the central nervous system, retina, iris, and larger peripheral

nerves. The endothelial cells of these capillaries have tight junctions, lack fenestrae and

have minimal vesicle activity (Kamovsky et al 1967). Capillaries of muscle and skin have

small openings in the junctions between endothelial cells or nonrestrictive gaps between

cells.

33

A bidirectional shuttle of pinocytic vesicles has been described for transport of

macromolecules across their walls independent of fluid volume movement (Garlick and

Renkin, 1970, and Renkin 1979). Data from studies with various sized dextrans,

polyvinylpyrrolidone and proteins ranging from l - 1 2 nm radius from skeletal muscle

demonstrated that solutes measuring 4-12nm in radius drained from lymphatics and did not

easily cross through the capillary wall (reviewed by Parker et al 1984). Wissig, 1979 and

Williams and Wissig, 1975 showed after intravenous injection of a variety of molecules with

peroxidase activity (size range 1900-40000 Da or molecular diameter 2-5nm) that the

smaller molecules leave the capillary lumen more rapidly and reach a higher concentration

in the pericapillary interstitium of the diaphragm muscle than do larger molecules. It was

shown that ferritin (an electron dense protein probe) with Mr 750 000 Da and molecular

diameter lln m was unable to leave the capillary lumen (Bums and Palade, 1968b). The

behaviour of these molecules in the size range 1,900-750 OOODa suggests that they use a

different diffusion pathway with some restrictions to enter the interstitium. Two structures

have been proposed to serve this pathway, 1 , the transendothelial vesicular channels and 2 ,

the cleft between endothelial cells (Wissig and Williams, 1975).

The most permeable capillaries are found in the liver. In the liver, sinusoids are lined by

highly fenestrated epithelial cells, between which there are large gaps. The sinusoids also

have no basal lamina (Wisse 1970). Because of the large gaps in the endothelial cell layer

and the absence of a continuous basal lamina, no significant barrier exists between blood

plasma in the sinusoid and the hepatocyte plasma membrane (Ross et al, 2003). This is

known as the “space of Disse” (the perisinusoidal space)(Gray, 1973, Reichen, 1999).

Measurement of circulating chylomicrons in the space of Disse in the rat liver revealed a

substantial filtration effect of endothelial fenestrations only on chylomicrons greater than

250nm in diameter (Naito and Wisse, 1978).

Early infusion studies with pork proinsulin in dogs (Tomkins et al 1981) and

hyperinsulinaemic-euglycaemic clamp studies with human proinsulin in healthy volunteers

(Revers et al, 1984, Glauber et al, 1986, Galloway et al 1992) suggested that proinsulin was

34

more active in the liver (glucose Ra) than in the periphery compared to (glucose Rd) insulin.

The reasons for the differential effect of proinsulin on glucose Ra and Rd could be attributed

to differences in local distribution or metabolism, to differences in binding or activation of

the receptors or to differences in the post-receptor cascade. Although proinsulin had 8-9%

biopotency of that of insulin, the postreceptor activities of insulin and proinsulin were

shown to be the same in cultured rat hepatocytes (Agius et al, 1986). Furthermore, Ciraldi et

al (1986) showed that glucose transport was affected similarly for insulin and proinsulin in

rat adipocytes in vitro.

The in vitro results suggested that differential observations on glucose Ra and glucose Rd

from the in vivo studies could simply be due to the differences in distribution of proinsulin

in the liver compared to the periphery in comparison to insulin. The observation that

proinsulin exhibited apparent hepatoselectivity promoted the suggestion that the mechanism

of this differential effect could relate to the effect of the increased molecular size (proinsulin

9390 Da versus an insulin 5807 Da) on the rate of passage through the capillary walls and

hence to insulin receptors in intact tissues. Low receptor affinity per se does not convey

relative hepatoselectivity (Chap et al, 1987) In support of the hypothesis for the existence of

a capillary endothelial barrier is the observation that monomeric insulin analogues (eg B25

Asp, B9 Asp and B27Glu)(Brange et al, 1988, Kang et al, 1991) are absorbed more rapidly

from subcutaneous tissue in the reverse direction through the capillary endothelial barrier

than native insulin which at concentrations used for treatment subcutaneously are mostly

dimeric and hexameric. Hyperinsulinaemic euglycaemic clamp studies performed in pigs

demonstrated that monomeric insulin analogues (BIO Asp and B9Asp and B27 Glu) were

equipotent in stimulating glucose Rd and inhibiting glucose Ra in comparison to human

insulin (Ribel et al, 1990).

35

Albumin

pores

liver sinusoid

periphery

endothelium

no pores

Figure 3. Schematic representation of albumin binding of insulin detemir, and potential

mechanism for differences in effect on the liver and periphery.

Hepatic insulinisation

In normal physiology the pancreas releases its hormones into the hepatic vein these

hormones must pass the liver before reaching the systemic circulation (Figure 4.). 40-60%

of the insulin presented to the liver is removed in a single passage (Field et al 1973, Ishida et

al 1983, Chap et al 1987). Flepatic extraction of insulin is therefore potentially important for

control of the circulating concentration of the hormone. Using insulin pharmacologically in

the treatment of Type 1 diabetes, insulin is administered subcutaneously and absorbed

directly into the systemic circulation (Figure 5). In the absence of the portal-peripheral

gradient, glycaemic control is only achieved by relative portal-peripheral hyperinsulinaemia.

Many factors will influence hepatic exposure to subcutaneously delivered insulin including,

rate of absorption from the injection site into the venous system, the volume of distribution,

possible insulin extraction in the pulmonary and mesenteric capillary beds before reaching

36

Figure 4. Schematic Representation of Physiological Insulin Production.

food

Peripheral Tissuesi'oticrms

liisuiln

atllpos Glucose

Figure 5. Schematic Representaion of Pharmacological Replacement of Insulin.

food

Peripheral lissuKiPancreas

muscle

Insulin

adipose

Liver

Glucose

Insulin

37

the hepatic portal vein and the blood flow in the hepatic artery. However, concentration of

insulin after intraportal administration is affected only by portal vein flow (from the gut).

The effects of insulin on glucose and fat metabolism are not fully understood. Hormonal

control of glucose metabolism may be either direct by inhibition of glycogenolysis and or

gluconeogenesis in the liver (Hers and Ave, 1983; Chiasson et al 1976), or indirect by

restriction of flux of gluconeogenic substrates or rate of lipolysis from peripheral tissues,

(Giaccaetal, 1992).

Many studies have shown the importance of intraportal delivery of insulin. Intraportal

delivery of insulin to the liver has been shown to have an important role in the regulation of

hepatic glucose output in normal and diabetic dogs (Stevenson et al 1978, 1981 and 1983)).

Ishida et al (1984) demonstrated that in depancreatized diabetic dogs the liver is more

sensitive than peripheral tissue to small changes (a rise from less than 2|uU/ml to 9 and 16

pU/ml) in the plasma insulin concentration. Griffen et al (1987) performed a study

catheterising either the jugular vein or the portal vein (ie systemic versus portal insulin

delivery) in diabetic rats. They demonstrated that insulin exerts its metabolic and mitogenic

effects by direct action on the liver and established that the insulin delivery via the portal

vein is important for glucose homeostasis. Spangler, 1985 and Spangler et al, 1986)

demonstated that peripherally delivered vesicle encapsulated insulin preparations (VEI) and

insulin had an equivalent capacity to suppress hepatic glucose output but VEI was less

effective in stimulating glucose disposal.

Whilst these studies suggest that intraportal delivery of insulin is important in the control of

glucose Ra other data have suggested that systemic delivery of insulin may be more

effective in the suppression of glucose Ra. Glucose turnover studies with hyperinsulinaemic

euglycaemic clamping, in depancreatised dogs (Giacci et al 1992) or in normal dogs

combined with an infusion of somatostatin (Ader and Bergman 1990) demonstrated that

systemic delivery of insulin was more effective in the suppression of hepatic glucose output

than portal delivery.

38

In kidney pancreas transplanted patients, the surgical anastomoses are such that hormones

released from the transplanted pancreas drain into the systemic circulation. This surgical

method abolishes the normal portal-peripheral insulin gradient and also results in relative

hyperinsulinaemia in the periphery. Data from metabolic studies in patients with such

transplants compared to a control group using infusion of D[6 -^H] glucose and

bicarbonate and a meal enriched with D-[2-^H] glucose, suggest that systemic delivery of

insulin normalised the basal glucose concentrations to a similar extent as the control group

and there was a tendency for a more effective suppression of hepatic glucose Ra than in the

control group (Katz et al 1991).

Other studies find portal and systemic delivery of insulin to be equally effective in the

suppression of glucose Ra. A comparative study of glucose turnover performed in diabetic

rats which had received transplanted islets either under the kidney capsule (peripheral

delivery) or in the splenic pulp (portal delivery) (Kruszynska et al, 1985) indicated that

glucose turnover and recycling were not different between the groups. In another study the

direct and indirect action of insulin on carbohydrate metabolism during fasting and after an

oral glucose load has been investigated in a group of dogs in which the portal vein was

anastomosed to the inferior vena cava (peripheral delivery) (Kryshak et al 1990). This

resulted in insulin sectreted by the intact pancreas passing directly into the systemic

circulation, bypassing extraction by the liver and thereby producing hyperinsulinaemia in

the periphery. Glucose Ra and Rd, measured with D-[2-^H]glucose and D-[3-^H]glucose,

after a meal ingestion in these dogs, was not different from a control group suggesting that

glucose Ra may be controlled by a peripheral signal.

Cherrington in his review “control of glucose uptake and release by the liver in vivo”

(1997), discusses glucose homeostasis when fasting, as well as the effects of glucoregulatory

feedback loops in the regulation of hepatic glucose output. In particular the direct and

indirect role of insulin on hepatic glucose output is discussed. Evidence for indirect

mechanisms of insulin action on hepatic glucose output demonstrated by Bergman and

Giacca are presented as well as further studies by the author. Cherrington concludes that

39

“insulin thus appears to acutely inhibit liver glucose output through a direct effect on

glycogenolysis and an indirect effect on glycolysis/gluconeogenesis. When one considers

that the sinusoidal insulin level in a normal individual is 2-2.5 times the arterial insulin level

and that the liver is extremely sensitive to alterations in the portal insulin concentration, it

becomes evident that changes in insulin secretion must bring about the majority of their

effects via a direct action on the liver. It is also obvious, however, that when insulin is

administered peripherally, such as occurs in insulin-requiring diabetic individuals, the

indirect effects of the hormone on the liver will take on greater significance.”

One explanation for the conflicting conclusions of the experiments described in this section

could be the choice of mathematical model used for calculation of glucose Ra and Rd. The

results from most of these studies were calculated using Steele’s model (a non-physiological

mono-compartment model; Steele, 1959 and Steele et al, 1965) which has been shown to

underestimate glucose Ra and may introduce errors in the conclusions reached. More

experiments and better mathematical models of glucose kinetics are required to establish the

effects of insulin on glucose metabolism.

Long Acting insulin

Background

Initially insulin treatment consisted of subcutaneous injections of insulin shortly before

meals. In order to avoid multiple injections a number of attempts were made to prolong the

duration of action of insulin or to slow its absorption. Substances used were gum arabic

(1923), lecithin (1923) and oil suspensions (1925) (Rosskamp and Park 1999). These

attempts were mostly unsuccessful because of poor stability or side effects at the local

injection site.

40

The first successful insulin preparation with a prolonged action was protamine insulin

(Hagedom et al 1936). Here the principle of protracting the insulin action was to make the

insulin less soluble at the neutral pH of tissue fluid. When injected as a suspension, the

duration of action was roughly twice as long as that of regular unmodified insulin.

Protamine insulin is a complex of the fish protein protamine and insulin. When injected

subcutaneously proteolytic enzymes degrade the protamine and the insulin is absorbed. A

further way of protracting the action of insulin was the so-called lente series. These were

preformed amorphous or crystalline suspensions obtained by addition of small amounts of

zinc ions (Hallas-Moller 1956). Since their introduction in the 50s the technology of

prolonging insulin action has remained relatively unchanged, until now.

In the 1990s the link between metabolic control and development of micro vascular

complications became firmly established (DCCT). This has led to a renewed interest in

long-acting insulins on the basis of a new therapeutic concept. Now the objective is not

avoidance of multiple injections but the imitation of the physiological secretion of basal

insulin in healthy subjects, with additional mealtime insulin requirements taken care of by

short acting insulin bolus injections before meals. This basal-bolus concept is the

cornerstone of any kind of intensified insulin treatment, which leads to significantly better

metabolic control than one or two insulin injections per day can provide (Rosskamp and

Park 1999). However this therapy is not without its problems, currently available long

acting insulins were recognised not to be good at imitating physiological secretion of basal

insulin. They had peaks of action, and they were variable in absorption (Barnett, 2003).

Therefore renewed interest in prolonging the duration of action of insulin led to

developments along different strategic routes.

It was hoped that proinsulin would increase basal insulin concentrations, but concerns on

cardiovascular safety that arose from clinical studies meant this work was abandoned

(Owens et al, 2001). Proinsulin’s metabolite, des (64,65)-human proinsulin had an even

shorter period of action than NPH insulin and was discontinued (Owens et al, 2001).

Slowing absorption and clearance by strengthening hexameric interactions in the form of

cobalt (Ill)-insulin complex also proved unsuccessful (Burge and Schadel, 1997). However,

41

increase of the isoelectric point of insulin from 5.4 towards neutrality thereby causing

precipitation or crystallisation at the site of injection, delays absorption, and prolongs the

effect of insulin (Markussen et al, 1990). The first acid soluble extended action insulin

analogue made by this method was Novosol Basal (GlyA21, ArgB27, ThrBSO-NHz-insulin)

(Jorgenssen et al, 1989), but escalating dose requirements (reduced bioavailability) possibly

associated with local inflammatory reactions at the site of injection, meant that this insulin

was also abandoned.

On the basis of the same concept, a di-arginylinsulin analogue known as insulin glargine

was developed that had a much slower and more reproducible rate of subcutaneous

absorption than NPH insulin (Owens and Barnett, 2000), resulting in slower onset and

extended effect. An additional phenol molecule bound to the hexamer of crystalline insulin

glargine might further contribute to its protracted action. Plasma glucose and insulin

concentrations vary less between patients when insulin glargine is used compared to

ultralente insulin (Lepore et al, 2000, Heineman et al, 2000).

42

Insulin detemir

Physical and chemical properties

Insulin detemir is produced by a process including recombinant DNA technology using

Saccharomyces cerivisiae and chemical synthesis. The molecular formula is C267H402N 6 4S6

and the molecular weight is 5916.9 Da. In comparison to human insulin, the amino acid

residue at position B30 has been omitted, and a 14-C fatty acid chain has been attached to

position B29. The molecule is lipophilic due to the fatty acid chain. By adding zinc to the

formulation, the molecule is stabilized and hexamers are formed making it hydrophilic and

thereby soluble in water (Kurtzhals et al, 1995, Markussen et al, 1996).

Figure 6 .

ScheinniatDC Stractyire of InisylDim DetemSr

H2 0

H,N

Amino Acid R esidues A l - A21 COOH

Amino Acid R esidues B1 - B29 Lys® ® COOH

C =0I

(CH2)i2I

CH3

Pharmacology

Like native insulin, insulin detemir exists predominantly as hexamers in the prescence of

zinc and phenol. The fatty acid side-chains contribute to the aggregation of hexamers.

43

which results in delay in dissociation into monomers, and thereby may delay systemic

absorption (Havelund et al, 2004, Brunner et al, 2000), In monomeric state, the 14-C fatty

acid chain attached to position B29 binds to the fatty acid binding sites of albumin in the

subcutis, which further delays absorption into the bloodstream (Havelund et al, 2004,

Brunner et al, 2000).

Figure 7.

Structure of the insulin detemir dimer

Myristic acid

Myristic acidOlsen HB and Kaarsholm NC. Biochemistry 2000;39:11893-11900

44

Figure 8.

Di-hexamerisation of insulin detemir

Size exclusion chromatography of insulin detemir after exclusion of phenol (as expected in subcutis) reveals formation of dihexamers ->

hexameridihexamerequilibrium

Myristicacid

Olsen HB and Kaarsholm NC. Biochemistry 2000;39:11893-11900; Havelund S et al. Submitted 2003

Formulation

There have been three formulations of insulin detemir used during its clinical development

program. Formulation A was the first and contained 600 nmol/ml insulin detemir, and

glycerol as an isotonic agent. One dosing unit was defined as 6 nmol, the same as in current

human insulin products. Formulation B contained 1200 nmol/ml insulin detemir and had

mannitol as isotonic agent. One dosing unit was defined as 12 nmol. The final formulation

of insulin detemir formulation C contains 2400 nmol/ml, one dosing unit being defined as 24

nmol.

The constituents in formulations B and C are insulin detemir, phenol, m-Cresol, mannitol,

disodium phosphate, sodium chloride, zinc (as zinc acetate), hydrochloric acid and water for

injections. Both formulations are clear and colourless and are at pH 7.4. In this study we

have used formulation B. (Investigator’s Brochure, Soluble, basal insulin analogue Insulin

Detemir (NN304))

45

Efficacy pharmacology

The receptor binding properties of insulin detemir have been studied using soluble human

insulin receptors and the human hepatoma cell line HepG2. The affinity of insulin detemir

for the insulin receptor was about 18-46% relative to human insulin using soluble receptors,

(Markussen et al, 1996 and Kurtzalls et al, 2000) and 27% (when corrected for albumin

binding) using HepG2 cells. Correction for albumin binding is necessary to get a measure

for the receptor affinity, when assay buffers containing albumin are used, as only free (non

albumin bound) insulin detemir is available for binding to the receptor.

Insulin binding/dissociation kinetics were studied using Chinese Hamster ovary cells over

expressing the insulin receptor (CHO-hIR cells). Insulin detemir dissociates about 2 times

faster from the insulin receptor than human insulin (Kurtzhals et al, 2000).

Activation of the insulin receptor tyrosine kinase (IRTK), was studied using CHO-hIR cells.

Insulin detemir was approximately 9.5% as potent as human insulin in stimulating IRTK

activity (corrected for albumin binding) (Kurtzhals et al, 2000).

The IGF-1 receptor binding properties of insulin detemir were examined using soluble

human IGF-1 receptors, and using HepG2 cells. The affinity of insulin detemir for the IGF-

1 receptor was determined to 16% relative to human insulin using soluble receptors and to

41% (corrected for albumin binding) using HepG2 cells (Kurtzhals et al, 2000).

Pharmacokinetics and protein binding

The albumin binding properties of insulin detemir have been extensively studied using an

immobilised human serum albumin preparation (Kurtzalls et al, 1995 & 1997). The binding

constant of insulin detemir for HAS was determined to be 2.4 x 10-5 M-1 at 23 *C and 1.0 x

10-5 at 37®C in this assay system. Based on this binding constant, insulin detemir is

estimated to be 98.4 % albumin bound in plasma (37°C, 0.6mM albumin). This estimate is

46

consistent with in vitro studies using ^^^I-labelled insulin detemir in human plasma which

determined the plasma protein binding (98.8%) of insulin detemir.

The interaction between albumin binding of insulin detemir, fatty acids and selected drug

substances including warfarin, acetylsalicylic acid, diazepam, phenylbutazone, valproic acid,

ibuprofen, tolbutamide and glibenclamide was also examined in vitro using the immobilised

human serum albumin preparation (Kurtzalls et al, 1997). Insulin detemir binds to the fatty

acid binding sites of the albumin molecule. The binding of insulin detemir to the

immobilised human serum albumin preparation was independent of the binding of drugs in

the two major binding pockets located in domains HA and IIIA of the albumin molecule.

None of the tested drugs competed with the albumin binding of insulin detemir at clinically

relevant drug to albumin concentration ratios. In conclusion the in vitro studies do not

suggest any clinically relevant albumin binding interactions.

Figure 9.

Distinct binding sites on albumin

Nfvr2

Curry S et al. Nature Structural Biology 1998:5:827-835

47

Mitogenicity

The biological effects of insulin are both metabolic and mitogenic. The metabolic potency

was evaluated by measuring stimulation of lipogenesis in primary mouse adipocytes.

Insulin detemir stimulated lipogenesis with a potency of approximately 29% of human

insulin (corrected for albumin binding) (Kurtzalls et al, 2000). This is consistent with the

relative affinity to the insulin receptor.

The mitogenic potency of insulin detemir was evaluated using Chinese Hamster ovary cells,

K1 strain (CHO-Kl), human mammary cancer fibroblasts (MCF-7 cells) and human

osteosarcoma cells BIO (Saos/BlO cells), respectively. The mitogenic potency of insulin

detemir was determinined to be approximately 9% (CHO-Kl), 14.6% (MCF-7) and 11%

(Saos/BlO) relative to human insulin after correction for albumin binding (Kurtzhals et al,

2000).

Taken together the in vitro pharmacology studies indicate that insulin detemir is less potent

than human insulin in binding to insulin and IGF-1 receptors and in stimulating metabolic

and mitogenic responses.

Aims of this study

To determine whether insulin detemir by the nature of being a larger protein bound insulin

molecule has different effect on glucose and lipid metabolism when compared to NPH

insulin.

48

Chapter 2

Clinical methodology

Introduction to the glucose clamp technique

A particular problem encountered in studies investigating glucose metabolism is the

interrelationship between glucose concentration and insulin. Initial attempts were made to

make comparisons during a standard glucose tolerance test but both insulin and glucose

concentrations are in a state of flux, thus rendering it difficult to separate cause and effect. The

feedback loop between glucose and insulin is broken by controlling blood glucose

concentration by frequent measurement and altering an exogenous glucose infusion. Two

types of clamp can be performed:

i. Hyperglycaemic clamp in which blood glucose is held at hyperglycaemic levels and the

insulin response measured.

ii. Hyperinsulinaemic euglycaemic clamp in which an insulin infusion is given and blood

glucose is maintained with an exogenous infusion of glucose.

These two methods measure beta cell responsiveness and insulin action respectively. The latter

was used in the studies reported here to compare the actions of insulin detemir and NPH.

Isotope dilution

The tracer dilution technique was introduced by Stetton et al in 1951 for measuring glucose

turnover using a constant infusion of labelled glucose. Later, administration of a priming

injection of labelled glucose followed immediately by a constant infusion of the labelled

glucose was found to shorten the time required for mixing the labelled glucose in the body

49

glucose pool (Steel et al., 1956; Searle et al., 1954). Constant isotope infusions were also

used to measure the glucose appearance rate in non-steady state conditions during

hyperinsulinaemia (Steele et al., 1956). The tracer dilution technique in combination with a

constant insulin infusion and a euglycaemic clamp was used by Steele et al. (1965). This

method provided a means for the assessment of the differential effects of insulin on glucose

metabolism in the absence of any fluctuations in the concentration of glucose or counter-

regulatory hormones. Using these techniques in most circumstances the endogenous rate of

glucose appearance (Ra) equals the hepatic glucose production rate as apart from a small

contribution by the kidney, the liver is the only organ responsible for de novo glucose

production (Dmry et al\95V).

There are potential inaccuracies in these methods. For example during hyperinsulinaemic

euglycaemic clamps many workers have reported negative values for hepatic glucose

production, which is clearly impossible (Defronzo et al 1985, Butler et aï 1990, Ward et al

1990). Everyone has assumed when negative values are reached that hepatic glucose output is

zero but this calls into question the methodology used. The source of the errors in such studies

has stimulated much discussion and investigation. Potential sources of error include:

a) Inability of the simple model to cope with the complex in-vivo physiology of glucose

metabolism. The Steele model is a simple one compartment model with a fixed pool volume

which does not mirror the complex multicompartment physiology of glucose metabolism.

More sophisticated models have been proposed. However the original Steele model is still

used widely as it is easily understood and suited to individual clinical studies (Insel et al 1975,

Radzuik et al 1974, Steele et al 1974). The errors generated are proportional to the rate of

change of tracer enrichment and this can be offset by “spiking” the exogenous glucose infusion

with tracer to ensure uniform plasma enrichment during the study (Finegood et al 1987).

b) Tracer recycling. The isotope dilution technique assumes that once labelled tracer

has entered a metabolic pathway it does not return to the pool in its original molecular form. If

a tracer does return to the pool it is “recycling”. Different tracers lose their labels at different

positions in their metabolic pathways (Wolf 1984) and it is therefore possible for recycling to

50

occur leading to erroneous estimates for Ra and Rd (Bell et al 1986). For example glucose

enters the glycolytic pathway and is broken down to lactate, pyruvate or alanine which can be

converted back to glucose in the Cori cycle. This has been estimated to account for up to 20%

of the hepatic glucose production (Reichard et al 1963). Thus glucose labelled with deuterium

or tritium on carbon atoms 2, 3 or 6 are now the most commonly used tracers (See Figure 2 for

the steps at which labels are lost). [6 ,6 -^H2] glucose was used in the studies below. It loses one

of its labels in the pyruvate carboxylase step and one between malate and lumarate.

c)Isotope discrimination and tracer impurities. An assumption is made that the isotope

tracer is metabolised in an identical manner to unlabelled tracer. There is evidence in vitro to

suggest that some isotopes are metabolised in different ways (Argoud et al 1987). Finegood et

al (1988) have suggested that different arterio-venous specific activity of [ tf] glucose

suggested differential metabolism but others have shown that impurities in the tracer which are

metabolised several fold more slowly may have accounted for the reported a-v differences

(Butler et al 1990, Schwenk et al 1990). Thus highly purified tracers are essential for accurate

metabolic studies. In comparison to radioactively labelled glucose the stable isotope labelled

glucose is theoretically less likely to be subject to isotope discrimination as there is less of a

mass difference between it and the native glucose (Wolfe 1984).

Calculations

Rate of appearance (Ra), and Rate of disappearance (Rd) were calculated using the model

originally proposed by Steele (1959) and adapted by Finegood et al (1987) to account for the

addition of stable labelled tracer to the exogenous glucose infusate.

51

where Ey is the enrichment of the basal continuous [6 ,6 -^H2] glucose infusion in APE, Ep(t) and

Evar are the APE of the plasma and enriched glucose infusate respectively, ly is the continuous

tracer infusion rate (mg/kg/min, p is the pool fraction (ratio of effective glucose to actual space)

which was assumed to be 0.65, V is the glucose space assumed to be 220ml/kg of the body

weight, G(t) is the plasma glucose and Ivar is the exogenous variable glucose infusion rate.

These data were subjected to a smoothing procedure. The glucose concentration and the APE

of the enriched 2 0 % glucose infusâtes and the glucose concentrations of the tracer infusate

were all measured and these values used in the calculations.

Clamp protocol

Following screening subjects were assigned the lowest available randomisation number and

were randomised to a specific treatment sequence in a randomised block design which was

unknown to the investigator until the day before each study. The study was not blinded.

Subjects were studied on three separate occasions 7 to 28 days apart. Subjects received 1]

detemir 9nmol/kg(LD), 2] detemir 18nmol/kg (HD) or 3] human NPH insulin 0.3 lU/kg

(1.8nmol/kg) (NPH) (Novo-Nordisk) in random order. Subjects with diabetes received a

higher dose of NPH insulin 0.6IU/kg (3.6nmol/kg) instead of the 0.3 lU/kg (1.8nmol/kg)

dose. Subjects were asked to have a moderate carbohydrate rich meal the night before the

clamp. They were not allowed to perform strenuous exercise in the 48 hours preceding the

study, and were not allowed to consume alcohol for 24 hours before the study. They were

52

asked if they had taken any other medication that might interfere with the study drug, or

suffered any adverse events since the previous visit.

Subjects arrived at 8 in the morning following an overnight (10 hour) fast, they were

allowed to drink water freely throughout. Those subjects with Type 1 diabetes were asked

to omit their long/intermediate acting insulin for 24 hours prior to the study day. They were

given soluble human insulin (actrapid) to take the evening before the study but were asked

not to take any insulin after midnight the night before the study. Subjects with type 2

diabetes taking insulin were managed as those with type 1 diabetes. Those on oral

hypoglycaemic agents were asked to omit their oral hypoglycaemic medication for 48 hours

prior to the study.

On arrival their blood pressure and pulse were taken supine and standing, they were weighed

and body fat percentage estimated using electrical impedance (Tanita body fat scales, Tanita

UK Ltd, Yiewsley, Middlessex). They were then asked to lie or sit on a bed and an 18-

gauge cannula was inserted into each antecubital fossa if possible (other large veins in the

forearm or hand were used if there was no suitable vein in the antecubital fossa). One

cannula was used for blood sampling and one for the administration of the various infusâtes.

Baseline blood samples were taken, then a priming dose of 170mg [6 ,6 -^H2 ] glucose and

0.14 mg/kg D5 glycerol was given, followed by a continuous infusion 1.7 mg/min [6 ,6 -^H2 ]

glucose and 0.5 mg/kg/h D5 glycerol. Subjects with diabetes were given a tapering infusion

of human soluble insulin to bring their plasma glucose into the normal range (4-7mmol/l).

The infusion of human soluble insulin was tapered off by 25% from t = -10 to -5 min and by

a further 50% from t = -5 to 0 min and stopped at t = 0 min.

An 120 minute equilibration period was allowed to reach steady state tracer enrichment

before taking samples at -30, -20, -10 and time zero. For subjects with diabetes a 130

minute equilibration period was allowed and baseline blood samples were taken at -40, -30,

-20, -10 and time zero. Immediately after the blood sample was taken at time zero, subjects

were dosed with subcutaneous injection of trial insulin, either detemir 9 nmol/kg (LD),

detemir 18 nmol/kg HD or human NPH insulin 0.3 lU/kg (1.8 nmol/kg) (or 0.6IU/kg for

53

subjects with diabetes) (NPH) according to randomisation. Blood samples were taken at 15

minute intervals for measurement of plasma glucose, this was done at the bedside using a

blood glucose analyser (YSI 2300), the infusion rate of 20% dextrose (spiked with 400 mg

[6 ,6 -^H2 ] glucose per 500 ml 20% dextrose) was altered accordingly to clamp blood

glucose. The blood glucose was clamped between 4.0 mmoll'^ and 5.5 mmoll'^ for 16 hours

after subcutaneous insulin injection. In healthy subjects blood glucose was clamped to their

presenting blood glucose on their first study day.

Blood samples for glucose enrichment, insulin, c-peptide, glucagon, IGFBP-1, IGF-1,

NEFA and glycerol enrichment analyses were taken at regular intervals during the clamp

(see table 1). Following the 16 hour period the infusions were stopped, cannulae removed

and the subject was fed a carbohydrate rich meal and allowed to sleep until the following

morning, when they were allowed home. The clamp studies were then repeated on two

further dosing days separated by at least 7 days and not more than 28 days. On these dosing

days the subjects received the other doses according to the randomisation schedule.

34 (35 for subjects with diabetes) samples each requiring 2 ml of blood were taken at regular

intervals throughout the study for insulin, C-peptide and glucagon analyses. 34 (35 for

subjects with diabetes) samples each requiring 3 ml of blood were taken for glucose and

glycerol enrichment. 21 samples each requiring 2 ml blood were taken for IGFBP-1 and

IGF-1 analyses. A further 15 samples each requiring 1 ml of blood were taken for NEFA

analysis. Samples for measurement of blood glucose were midway between samples for

other assays at 15 minute intervals. In total 41 samples requiring 1 ml blood were taken for

glucose analysis. This is a total of 268 ml blood taken during each clamp study.

All blood samples were put immediately into bottles once the blood was drawn. The bottles

were then kept on ice. Every hour all the samples on ice were transferred to a refrigerated

centrifuge and centrifuged at 3060 revolutions per minute (2000g) for 10 minutes. The

samples were then immediately removed from the centrifuge, pippetted into the appropriate

aliquots and stored in a freezer at - 2 0 ®C.

54

Blood samples for insulin, insulin detemir, C-peptide and glucagon analysis were sent in

batches to Medi-lab in Copenhagen where the analyses were made. They were packed in

dry ice and sent by overnight courier service. Confirmation was received when the samples

arrived that they were still frozen and that the packing cases were intact.

Blood samples for glucose and glycerol enrichment as well as those for NEFA assay were

transferred in dry ice to St Thomas’ hospital by private transfer. All samples remained

frozen and were immediately placed in the freezer to await analysis.

Blood samples for analysis of IGFBP-1 and IGF-1 were kept at the Royal Surrey County

Hospital where the assays took place.

Table 1.

Blood Sampling TimesTime

Insulin,C-peptide SGIucagon

Glucose and Glycerol Concentration and enrichment NEFA

IGF-1&IGFBP-1

-160 X X X X-40 X X-30 X X-20 X X-10 X X X X0 X X X X30 X X X X60 X X X X90 X X X X120 X X X X150 X X X X180 X X X210 X X240 X X X X270 X X300 X X X X330 X X360 X X X X390 X X420 X X X450 X X480 X X X510 X X540 X X X X570 X X600 X X X630 X X660 X X X690 X X720 X X X X750 X X780 X X X X840 X X X X960 X X X X

55

Preparation of tracer solutions

Tracer solutions of [6 ,6 -^H2] glucose and [^Hs] glycerol (both 99% enrichment, sterile and

pyogen free from Phenome Siences Inc, Woburn, M.A., U.S.A. were used.

Laboratory assessments

Glucose concentration and enrichment

Blood samples were taken in fluoride oxalate tubes and whole blood initially and then

plasma glucose concentrations were measured on YSI 2300 STAT plus glucose analyser

(Yellow Springs instruments. Yellow Springs, Ohio). This method is based on glucose

oxidation. 25 pi of blood or plasma is aspirated by a sipper, which then transfers and injects

the sample into a buffer filled sample chamber. Some of the substrate diffuses through the

membrane where it contacts the immobilized oxidase enzyme, it is rapidly oxidised

producing hydrogen peroxide (reaction 1 )

Reaction 1

P-D-glucose + O2 glucose oxidase Glucono-Ô-lactone + H2O2

The hydrogen peroxide (H2O2) is in turn oxidised at the platinum anode producing electrons

(reaction 2 )

Reaction 2

H2O2 2 H^ + O2 + 2 e

A dynamic equilibrium is achieved when the rate of H2O2 production and the rate at which

H2O2 leaves the immobilised enzyme layer are constant and is indicated by a steady state

response. The electron flow is linearly proportional to the steady state concentration and

56

therefore the concentration of glucose. The platinum electrode is held at anionic potential

and is capable of oxidising many substances other than H2O2 . To prevent these reducing

agents from contributing to the sensor current the membrane contains an inner layer

consisting of a very thin film of cellulose acetate. The film readily passes H2O2 but excludes

chemical compounds with molecular weights above approximately 200. The analyser was

calibrated before use using glucose standard solution 25 mmol/1, and standard normal and

pathological serum with glucose concentrations of 4.5 mmol/1 and 13 mmol/1 respectively.

[6 ,6 -^H2] Glucose analysis

Glucose isotopic enrichment was determined by gas chromatography-mass spectrometry

(GC-MS) on a VG Trio-2 5890 (Hewlet-Packard, Woking, UK) using selected ion

monitoring of a glucose acetate boronate derivative (Bier et al, 1977). The samples were

first deproteinised by adding 50 pi of plasma sample to 0.5ml of ethyl alcohol and

centrifuging at 1000 rpm for 5 min. The supernatant was then removed and dried under a

continuous stream of N 2 at 50 °C. The glucose acetate boronate derivative was produced by

adding butylboronic acid in pyridine (10 mg/ml), heating this solution at 95°C for 30 min

and then cooling at room temperature. Acetic anhydride (100 pi) was then added and the

mixture left for 1 hour again at room temperature. This solution was dried under N2 and

dissolved in 500 pi of decane before being introduced into the GC-MS. The ions monitored

were of mass 297 and 299 representing [M-C4H9] and the corresponding fragment enriched

with two deuterium atoms respectively. The within assay coefficient of variation of

measurement of isotopic enrichment was < 2 %.

Glycerol concentration and enrichment

0.5 ml plasma was pipetted into plastic tubes and 1ml 3.5%SSA was added. Samples were

mixed and then allowed to stand for 15 to 30 minutes. They were then centrifuged at 2000

rpm for 20 minutes at 4®C. Meanwhile the ion exchange columns were prepared; 0.5 ml of

57

cation resin was added first to the column, then rinsed with 1 ml of distilled water, then 0.5

ml of anion resin was added and rinsed with 1 ml of distilled water. The supernatant from

the centrifuged tubes was then added to the columns and the elute collected in labelled vials.

Each column was rinsed with 3 ml distilled water. The vials were covered with parafilm and

frozen at -20®C. The samples were dried under a stream of dried compressed air at room

temperature until approximately 200pl of liquid remained (around 15 hours). At that point,

the sample temperature was increased to 50°C while continuing the air stream until the

sample completely dried (around 2 hours). lOOpl of pyridine and lOOpl of MTBSTFA were

added to the dried residue, and the mixture was vortexed. The solution was allowed to

remain at room temperature for 30 minutes allowing derivatisation to reach completion. The

derivatised sample was transferred into a plastic micro-injection autosampler vial and was

sealed.

After a 2- pi splitless injection of the derivatised sample onto the column, the oven was held

at the initial temperature of 100°C for 0.5 minutes, increased to 300°C at 25 °C /min and

maintained at 300 °C for 1.5 min. Thus the total run time was equal to 10 minutes with

glycerol and 5D glycerol eluting at 6 minutes. Using SIM, the major fragments monitored

for the tBDMS derivatives of glycerol and d5-glycerol were the [M-57] ion fragments,

377m/z and 382m/z respectively. With d5-glycerol used as the tracer, the enrichment

quantitated in plasma as the ratio of d5-glycerol:glycerol (ion abundance o f 377/382 m/z).

NEFA

NEFA was measured using the Wako NEFA C assay on the Cobas Fara II (Roche,

Welwyn Garden City, UK.) the method of which relies upon the acylation of coenzyme

A(CoA) by the fatty acids in the presence of added acyl-CoA synthetase (ACS). The acyl-

CoA produced is oxidized by added acyl-CoA oxidase (ACOD) with the generation of

hydrogen peroxide. Hydrogen peroxide, in the presence of peroxidase (POD) permits the

oxidative condensation of 3-methyl-N-ethyl-N-(b-hydroxyethyl)-aniline (MEHA) with 4-

aminoantipyrine to form a purple color which can be measured spectrophotometrically at '

550nm.

58

The within-run precision for this assay is 2.7%CV, 1.1 %CV and 1.1%CV for mean

concentrations of 0.33mmol/L, 0.62mmol/L and 0.99mmol/L, respectively. The linear

range of the assay is 0-2.0 mmol/L.

Insulin & insulin detemir, C-Peptide and Glucagon

The following assays were done at Medilab by the request of Novo Nordisk. Insulin detemir

was measured using the ELISA specific insulin detemir assay (Heineman et al 1999).

Human insulin was measured using the DAKO insulin ELISA assay (Inter-assay CV 4.2-

9.3%). C-peptide and glucagon were measured using DAKO ELISA assays (Inter-assay CV

3.3-5.7% for C-Peptide and 6 % for glucagon).

IGF-1

lGF-1 was measured using Nichols institute Diagnostics chemiluminescence immunoassay.

This assay is a two site chemiluminescence assay for the measurement of lGF-1 in human

serum. Acridium esters emit light upon treatment with hydrogen peroxide and an alkaline

solution. The Trigger 1 solution contains hydrogen peroxide in dilute acid and Trigger 2

solution contains dilute sodium hydroxide. The system automatically injects Trigger

solutions 1 and 2 into the wells which oxidse the acridium ester. The oxidised product is in

an excited state. The subsequent return to the ground state results in emission of light which

is quantified in 2 seconds and is expressed in relative light units (RLU) by the intergrated

system luminometer. The antibody to the C-terminal with the amino acid sequences of 62-

70 is biotinylated for capture and the antibody to the amino acid sequences of 1-23 and 42-

61 is labelled with acridium ester for detection.

The patient sample is acidified to separate lGF-1 from IGFBPs, then excess lGF-11 is added

in the assay to block IGFBP binding sites from recombining with the released lGF-1. The

acidified patient sample is incubated simultaneously with the biotinylated capture antibody,

excess lGF-11, and the acridium ester labelled tag antibody. During the first incubation.

59

IGF-1 in the sample forms a sandwich complex with the capture antibody and the acridium

labelled antibody. After the initial incubation period streptavidin coated magnetic particles

are added to the reaction mixture and a second incubation follows. The streptavidin coated

magnetic particles allow for a highly specific and efficient means of binding the sandwich

complex to the solid phase via the high affinity interaction between biotin and streptavidin.

Free labelled antibody is separated from the labelled antibody bound to the magnetic

particles by aspiration of the reaction mixture and subsequent washing. The wells

containing the washed magnetic particles are transported to the system luminometer, which

automatically injects Trigger 1 and Trigger 2, initiating the chemiluminescence reaction.

The light is quantitated by the luminometer and expressed as RLU. The amount of bound

labelled antibody is directly proportional to the concentration of IGF-1 in the sample.

The assay runs on the Nichols advantage speciality system. The sample is placed in the

designated sample racks and these racks placed in the sample loading area. The system

performs an assay specific on board dilution by first pipetting 13 pi of the sample and 400

pi of the acidification buffer into one well of the cuvette. 50 pi of the acidified sample

mixture is then pipetted and added to a mixture of 1 0 0 pi of of biotinylated goat polyclonal

antibody, 50 pi of the IGF-II solution and 50 pi of the acridium labelled goat polyclonal

antibody in the next well of the cuvette. The cuvette strip is transported to the incubation

chamber and incubated for 20 minutes at 37®C. After this initial incubation, 25 pi of

streptevidin coated magnetic particles are added to each well. The incubation is prolonged

for a further 10 minutes at 37®C. After the entire incubation the wells are washed and

measured in the measuring chamber. The coefficient of variation for this assay was 4.8%.

IGFBP-1

IGFBP-1 was measured using Oy Medix Biochemica Ab IGFBP-1 lEMA test. The

principle of this test is as follows. A monoclonal antibody specific to IGFBP-1 is

immobilized on microwell plates, and another monoclonal antibody, also specific to IGFBP-

1, is conjugated with horse-radish peroxidase (HRP). IGFBP-1 from the sample is bound to

the plates. After a washing step, HRP is added. After a second washing step, enzyme

60

substrate is added. The enzymatic reaction is proportional to the amount of IGFBP-1 in the

sample. The reaction is terminated by adding stopping solution. Absorbance is measured

on a plate reader.

80 pi of assay buffer was pipetted into each well. 2 0 pi of the serum sample was then

pipetted into the well. The plate was covered and incubated for 30 minutes at room

temperature on a plate shaker. The wells were then aspirated and washed 3 times with 300

pi of washing solution. 1 0 0 pi of enzyme conjugate was then pipetted into the wells and the

plate covered and incubated for a further 30 minutes at room temperature on the plate

shaker. The wells were then washed again as above and 100 pi of HRP solution was added

to each of the wells. The plate was covered again and incubated for 10 minutes at room

temperature on the plate shaker. The reaction was stopped by adding 50 pi of stopping

solution to each of the wells. The plate was shaken gently to mix the solutions. The

absorbance at 414 nm was then measured using a plate reader. The inter-assay CV was

6.4%.

Trial design

Type of trial

We performed a single centre randomised open label three period crossover trial with

subcutaneous injections of insulin detemir and NPH insulin using a euglycaemic clamp with

labelled glucose in healthy subjects and in subjects with Type 1 and Type 2 diabetes

mellitus. Insulin detemir was given in doses that were expected to cause the same blood

glucose lowering effect as the dose of NPH administered. An additional dose o f insulin

detemir was also studied. In healthy subjects this additional detemir dose was higher than

the comparative doses, but in the diabetic subjects the additional detemir dose was lower.

61

Rationale for trial design

The trial was open labelled. The insulins have different appearances and it was therefore not

possible to blind the trial. The crossover design was chosen to minimise the number of

subjects needed as each subject then becomes their own control and intra-subject variation is

smaller than inter-subject variation.

A clamp procedure with no pre-dose insulin infusion was used for healthy subjects. Type 1

and Type 2 diabetic subjects were expected to enter the trial sessions with high blood

glucose levels and thus the main issue was to lower their fasting blood glucose to predefined

clamp level. In order to obtain a clamp level as normal as possible, an insulin infusion was

started at the same time as the glucose and glycerol infusion. This insulin infusion was

tapered off before administration of the trial drug. Intravenous administered insulin is

rapidly eliminated and the effect on the endpoints of the trial was considered minimal. The

effect has been evaluated. Each subject was clamped at the same level at each visit.

Free fatty acid lipogenesis generally takes place immediately following an increase in

insulin concentration and stops when insulin levels are low. Measurements of NEFA were

made during the first hours after trial drug administration, at the maximum concentration

and during the last hours of the study. The concentrations of IGF-1 and IGFBP-1 were

measured hourly.

Rationale for dosage regimen

The effect of two dose levels of insulin detemir was investigated in order to allow for a dose

response relationship to be generated. One dose level of NPH insulin was administered for

comparison, and it was hoped that this dose would be equipotent to one of the detemir doses.

Pharmacodynamic trials with insulin detemir in healthy subjects indicated a difference in

blood glucose lowering potency of approximately a factor of five between insulin detemir

and NPH insulin. Thus the dose of insulin detemir expected to give the same blood glucose

lowering effect as NPH 0.3 lU/kg (1.8 nmol/kg) was 9 nmol/kg. The second dose of insulin

62

detemir was 18 nmol/kg and expected to give a similar blood glucose lowering effect as

NPH 0.6 lU/kg (3.6 nmol/kg).

Trial population

Number of subjects in each group

Sample size calculation was based on the peripheral glucose uptake (PGU) in a two-period

two-treatment sub-design of the three periods. Intrasubject coefficient of variation (CV) was

assumed to be maximum 25%. Considering a relative difference of 50%, and using a

significance level of 5%, the power would be 85% if eight subjects completed the trial and

70% if six subjects completed the trial.) Based on this sample size calculation eight subjects

were included in the trial. If more than two subjects were withdrawn, replacement subjects

were to be enrolled in order to obtain at least six completing subjects in each group (healthy

subjects and subjects with Type 1 and Type 2 diabetes). Therefore 18 subjects completing 3

studies.

Subjects were recruited based on the following inclusion and exclusion criteria:

Inclusion criteria

Healthy subjects

Signed informed consent obtained before any trial related activity.

Healthy male and female subjects between 19 and 60 years inclusive.

Considered generally healthy upon completion of medical history and physical examination.

Body mass index <30kg/m^

Fasting blood glucose < 6 mmol/1

Subjects with type 1 diabetes as above and/or

Age> 18 years

63

Type 1 diabetes according to ADA criteria (see introduction)

Duration of diabetes >12 months

HbAic < 12 %

C-peptide negative

Subjects with type 2 diabetes

Type 2 diabetes according to ADA criteria (see introduction)

Duration of diabetes >12 months

Age >35 years

BMI < 35 kg/m2

H bA ic<12%

Exclusion criteria

Healthy subjects

Participation in any other clinical trial involving other investigational products within the

last 3 months

Clinically significant abnormal haematology or biochemistry screening tests.

Any serious systemic infectious disease that occurred during the four weeks prior to the first

dose of the trial drug.

Any intercurrent illness that may effect blood glucose

Subject with a history of drug or alcohol dependence.

Use of prescription drugs except oral contraceptives within 3 weeks prior to the first dose of

the trial drug.

Use of non prescription drugs, except ordinary vitamin preparations within 3 weeks of the

first dose of the trial drug. Occasional use of analgesics was allowed up to 48 hours prior to

entrance in the clinical research centre.

Mental incapacity, unwillingness or language barriers precluding adequate understanding or

cooperation.

Blood donation of more than 500ml within the last 12 weeks.

64

Subjects with a first degree relative with diabetes mellitus.

History or presence of diabetes, cancer or any clinically significant cardiac, respiratory,

metabolic, renal, hepatic, gastrointestinal, endocrinological, dermatological, venereal

haematological, neurological or psychiatric disorder.

Previous anaphylactic reaction

History of significant drug allergy or hypersensitivity.

Females of child bearing potential not using an acceptable method of contraception which

include an intrauterine device (lUD) which has been in place for at least 3 months, or

sterilisation, or the oral contraceptive pill which should have been taken without difficulty

for at least 3 months or an approved hormonal implant or barrier methods.

Subjects with type 1 diabetes above and/or

Laser therapy for retinopathy within the last two months

Current treatment with systemic corticosteroids

Impaired hepatic fiinction measured as alanine aminotransferase (ALAT) > two times the

upper reference limit

Impaired renal function measure as creatinine >150 pM (1.7 mg/dL)

Cardiac problems defined as: 1) decompensated heart failure (New York Heart Association

class III and IV) and/or 2) angina pectoris and/or 3) myocardial infarction at any time

Uncontrolled treated/untreated hypertension as judged by the investigator

Subjects with type 2 diabetes as above and

Total insulin dosage >120 lU/day

Safety visit

Before screening took place the subjects were provided with written and oral information

about the study and the procedures involved. They were asked to sign an informed consent

form. All subjects were then asked a series of questions to determine their eligibility for the

trial with respect to the inclusion and exclusion criteria. The subject’s demographics (date

of birth, sex, ethnic origin and smoking habits) were recorded. A full medical history was

65

taken and the subject examined. The subject’s height and weight were recorded and their

body mass index calculated. Blood pressure and pulse were also measured and an

electrocardiograph (ECG) recorded. Blood was then taken for haematology and clinical

chemistry, and a urine sample sent for urinalysis and pregnancy test if relevant.

If the subject fulfilled all of the inclusion and exclusion criteria they were accepted into the

trial and an appointment was made for visit two within 5-21 days. Screen failures were

logged.

Subjects passing screening went on to perform the study days, which were held 7 to 21 days

apart from each other and were as described above.

Statistical analyses

Glucose Ra and Rd were calculated using a modified form of the Steele equation (Steele,

1959) adapted by Finegood et al, (1987) to account for the addition of stable labelled tracer

to the exogenous glucose infusate. The volume of distribution was 220 ml/kg and ratio of

effective glucose to actual space was 0.65. Glycerol Ra was calculated using the Steele

equation with a volume of distribution of 230ml/kg. Glucose Ra and Rd expressed as a

percentage of GIR at each time point was averaged over 960 minutes to determine the

proportion of the glucose lowering effect of each insulin due to a decrease in Ra and the

proportion due to an increase in Rd. All values are expressed as mean ± SEM. Statistical

analysis of the results was either by a 2 way ANOVA with subjects and treatment as factors

using baseline as a covariate or a paired students t-test.

66

Chapter 3

Results

Healthy Subjects

Subject demographics

10 healthy subjects 5 female and 5 male were screened. 4 female subjects and 4 male

subjects were randomised for the study. One male subject failed screening because he was

taking regular inhaled corticosteroids for asthma. One female subject passed screening, but

withdrew from the study before the first clamp. One female subject completed one clamp

study and then withdrew from further studies after finding the clamp procedure too arduous,

her results have not been included in the analysis as the data were not paired. 3 female

subjects and 4 male subjects completed all three clamps, they were aged 29.9 ± 3.4 years

with BMI 22.9 ± 0.72 kg/m^, they weighed 68.01 ± 2.88 kg and a body fat percentage of

24.71 ± 4.14 %. One of the subjects completing all three study days was not fasting for the

high dose study day. The results for this study have therefore not been included in our

analysis. The results presented therefore are for six subjects for high dose, and seven for

low dose and NPH.

Plasma Glucose

Euglycaemia was maintained throughout all clamp studies, there was no difference between

the plasma glucose during the three studies. Mean plasma glucose was clamped at

4.92+0.11, 4.95+0.08 and 5.01±0.14mmol/l in the LD, NPH and HD studies respectively.

The mean and standard differences of the individual coefficient of variation for glucose

67

during the clamp studies was 4.95+1.82 % for LD, 6.32±2.34 % for NPH and 7.82±3.76 %

for HD.

Figure 10. Graph of healthy subjects mean plasma glucose.

Healthy Subjects Plasma Glucose

I85

-180 -60 60 180 300 420 540 660 780 900 1020

Mean LD ■ Mean NPH Mean HD

Time mins

Glucose Infusion Rate

The total amount of glucose infused to maintain euglycaemia was similar for LD (2019 ±

374 mg/kg) and NPH (2102 ± 384 mg/kg)) (p=NS) demonstrating their equipotency (Fig

11). There was no difference between LD and NPH in the time to starting the glucose

infusion, peak glucose infusion rate or glucose infusion rate at 960 minutes. Glucose was

infused earlier following HD (40+7.5 min) than LD (61 ±9.6 min, p<0.03). The peak

glucose infusion rate following HD was higher (5.7±0.82 mg/kg/min) than LD (3.68+0.72

mg/kg/min, p<0.03). Total glucose infused was significantly higher following HD

(3267±388mg/kg) than LD (p<0.02). (Table 2)

68

Table 2. Healthy Subjects Plasma Glucose and Glucose Infusion Rate.

LD NPH HD

Plasma Glucose

mmol/14.92 ±0.11 4.95 1 0.08 5.0110.14

Total Amount of Glucose Infused,

mg/kg20191374* 21021384 32671388

Time to Start of Glucose Infusion,

minutes61 ±9.45* 6417.18 4017.35

Peak Glucose Infusion Rate,

mg/kg/min3.68 ± 0.72 * 4.0410.73 5.710.82

Glucose Infusion Rate At 960

minutes, mg/kg/min1.6410.23 * 1.4210.15 2.92 1 0.44

* Indicates a statistically significant difference between LD and HD.

** Indicates a statistically significant difference between LD and NPH.

***Indicates a statistically significant difference between NPH and HD.

Figure 11. Graph of healthy subjects mean glucose infusion rates

Healthy Subjects Mean Glucose infusion Rates (GIR)

6

5

4

2

1

00 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960

Tim e m ins

■ Mean GIR LD■ Mean GIR NPH Mean GIR HD

69

Glucose Metabolism

The minimum glucose Ra was significantly lower (p<0.02) with LD (-0.10±0.15

mg/kg/min) than NPH (0.17±0.10 mg/kg/min) (Fig 12). Using ANOVA, glucose Ra with

LD was significantly lower than with NPH (mean difference 0.24mg.kg'\min'^)(CI 0.09,

0.39)(p<0.01). This difference was still significant (p<0.05) when corrected for baseline

(mean difference 0.25 mg.kg'\min"^ Cl 0.05, 0.44) using analysis of covariance.

Figure 12. Graph of healthy subjects mean glucose rate of appearance.

Healthy Subjects Mean Glucose Rate of Appearance (Ra)

2.5

MEAN LD Ra MEAN NPH Ra MEAN EŒ) Ra

S - h

S ^

> M 1 1 1 ^ ,3èO 420^4801540 600 660 720 780 840 900 960

Time mins

The mean of the maximum change from baseline of glucose Rd (for each individual) was

lower with LD (1.52±0.49 mg/kg/min) than NPH (1.97+0.52 mg/kg/min) but this did not

reach statistical significance.

70

Figure 13. Graph of healthy subjects mean glucose rate of disappearance.

Healthy Subjects Mean Glucose Rate of Disappearance (Rd)

,S

-60 0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960

■MEANNPH Rd MEAN HD Rd MEANLDRd

Time mins

Figure 14. Graph of healthy subjects mean glucose Ra and Rd.

Healthy subjects Mean Glucose Ra & Rd

Glucose Ra and Rd mg/kg/m in

Time mins

-M E A N L D R d -M E A N N PH Rd

M EA N HD Rd- M EA N LD Ra- M EA N N PH Rii

M EA N HD Ra

Of the mean glucose lowering effect of insulin detemir over the 960 minutes (i.e. the

proportion of glucose infused needed to overcome each of Ra and Rd) 71.3±4% was due to

the decrease in Ra and 28.7±4% was due to the increase in glucose Rd, whereas for NPH

this was 63.3+3.9% from the decrease in Ra and 36.7+3.9% from the increase in Rd

(p<0 .0 1 ).

71

Figure 14. Bar charts showing the relative proportion of glucose lowering effect of each

insulin from effect on glucose Ra and glucose Rd.

Bar Chart to demonstrate the effects of 9nmol/kg detemir on glucose Ra&Rd

B Mean LD Rd

□ Mean LD Ra

3.5

c

0.5

-0.5

Time mins

Bar Chart to demonstrate the effects of 0.3IU/kg NPH on glucose Ra &Rd

4 - ___

r f l3.5 3

1.51 —

0.5 — 0 %

-0.5

_L

□ Mean NPH Rd□ Mean NPH Ra

_o o o c > > o o c > . o o o o o o o

Time mins

Iw

72

Figure 15. Pie chart showing the overall mean proportion of effect of each insulin on

glucose Ra and Rd.

Pie chart showing the mean proportion o f effect on glucose Ra and Rd following LD

29%

□ Ra ■ Rd

71%

Pie chart showing mean proportion o f effect on glucose Ra and Rd following NPH.

37%□ Ra ■ Rd

63%

The maximum Rd for HD was 5.21±0.71 mg/kg/min which was significantly greater than

with LD (p<0.03). HD appeared to suppress glycerol Ra more than LD but this difference

did not reach statistical significance. The maximum change from baseline was -1.31+0.20

pmol/kg/min with HD.

73

Table 3. Healthy Subjects Glucose Metabolism

LD NPH HD

Baseline Ra mg/kg/min 2.01 ±0.12 2.17 ±0.09 1.68 ±0.15

Minimum Ra mg/kg/min -0.10 ±0.15** 0.17±0.10 -0.69 ±0.16

Maximum change from

baseline R a mg/kg/min

-2.10 ±0.24 -2.00 ±0.18 -2.37 ± 0.26

AOC Ra mg/kg 1401.48 ±147.99 1328.69 ±135.80 1515.81 ±213.29

Baseline Rd mg/kg/min 2.06 ±0.12 2.18 ±0.08 1.75 ±0.18

Maximum Rd

mg/kg/min

3.58 ±0.54* 4.15 ±0.58 5.25 ±0.74

Maximum change from

baseline Rd mg/kg/min

1.52 ±0.49 1.97 ±0.52 3.51 ±0.75

AUC Rd mg/kg 564.42 ± 252.93 760.46 ± 247.27 1767.85 ±431.14

* Indicates a statistica ly significant difference between LD and HD.



74

Figure 16. Graph of Healthy Subjects Glucose Tracer:Tracee ratio during the clamp studies

Healthy Subjects mean Plasma Glucose TraceriTracee ratio

5I

Mean LD ■ Mean NPH Mean HD

■180 -60 60 180 300 420 540 660 780 900 1020

Time mins

Glycerol metabolism

Mean glycerol Ra at baseline was similar with the three study doses at 1.75 ± 0.15

pmol/kg/min for LD, 1.91 ±0.33 pmol/kg/min for NPH and 1.94 ± 0.41 pmol/kg/min for

HD (p=NS). The point of maximum suppression following dosing was 0.93 ± 0.18

pmol/kg/min for LD, 0.81 ±0.18 pmol/kg/min for NPH and 0.63 ± 0.07 pmol/kg/min for

HD (p=NS). The maximum change from baseline for glycerol Ra was -0.82 ± 0.17

pmol/kg/min with LD, -1.10 ± 0.24 pmol/kg/min with NPH and 1.31 ± 0.20 pmol/kg/min

with HD. The area over the curve (AOC) effect on glycerol Ra was less for low dose than

NPH or high dose detemir, although not statistically significant. Following dosing with LD

AOC was -366.1 ± 145.52, following NPH it was -691.0 ± 215.44 and following HD it was

-945.6 ± 172.28.

75

Table 4. Healthy Subjects Glycerol Metabolism.

LD NPH HD

Baseline Ra pmol/kg/min 1.75 ±0.15 1.91 ±0.33 1.94 ±0.41

Minimum Ra pmol/kg/min 0.93 ±0.18 0.81 ±0.18 0.63 ±0.07

Maximum change from

baseline Ra pmol/kg/min

-0.82 ±0.17 -I.10±0.24 1.31 ±0.20

AOC Ra pmol/kg -366.1 ±145.52 -691.0 ±215.44 -945.6 ± 172.28

* Indicates a statistically significant difference between LD and I ID.



Figure 17. Graph of healthy subjects mean rate of appearance of glycerol.

Healthy Subjects Mean Rate of apparence o f glycerol (Ra)

tI

0:&

-30 30 90 150 210 270 330 390 450 510 570 630 690 750 810 870 930

Mean HD ■ Mean LD - Mean NPH

76

NEFA

All three insulin doses suppressed NEFA concentrations. Plamsa NEFA concentrations at

baseline were 0.65 ± 0.08 mmol/1 for LD, 0.52 ±0.11 mmol/1 for NPH and 0.52 ± 0.12

mmol/1 for HD (p=NS). There was significantly more suppression of NEFA concentration

with NPH than with LD (mean difference O.lOmmol/1 Cl 0.03, 0.17)(ANOVA, p<0.02).

However there was no significant difference in the minimum NEFA concentrations

following LD (0.12 ± 0.04 pmol) or NPH (0.09 ± 0.03 pmol). Plasma NEFA concentration

following HD was significantly lower than with LD (ANOVA, p<0.01).

Table 5. Healthy Subjects NEFA results.

LD NPH HD

Baseline NEFA pmol/l 0.65 ± 0.08 0.52 ±0.11 0.52 ±0.12

Maximum change from

baseline NEFA pmol/1

0.54 ± 0.08 0.43 ±0.11 0.48 ±0.11

AOC NEFA pmol/1 -305.83 ± 59.8 -252.79 ± 89.9 -340.32 ± 90.73



***lndicates a statistically significant difference between NPH and HD.

77

Figure 19. Graph of healthy subjects mean plasma NEFA concentrations.

Healthy Subjects Mean Plasma Non Esterified Fatty Acid Concentrations

i 8:e

-------------- ^

II

-180 -60 300 42060 180 540 660 780 900 1020

Mean LD • Mean NPH Mean HD

Time mins

IGF-1

IGF-1 levels did not alter during the studies. Mean IGF-1 levels at baseline were 24.15 ±

3.43 nmol/1 for LD, 24.97 ± 2.68 nmol/1 for NPH and 26.5 ± 2.98 nmol/1 for HD. Mean

IGF-1 level following injection with LD was 23.01 ± 2.46 nmol/1, 25.02 ± 2.53 nmol/1 for

HD and following NPH injection it was 24.13 ± 2.46 nmol/1, there was no difference

between these three groups.

Table 6. Healthy Subjects IGF-1 results.

LD NPH HD

Baseline IGF-1 concentration nmol/1 24.15+3.43 26J±:L98 24.97 ±2.68

Mean IGF-1 concentration nmol/1 23.01 ±2.46 24.13 ±2.46 25^ 2± 2^ 3




Figure 20. Graph of healthy subjects mean plasma IGF-1 concentrations.

78

Healthy Subjects Mean Plasma Insulin Like Growth Factor Concentrations (IGF-1)

35 1

i

-180 -60 60 180 300 420 540 660 780 900 1020

■ MEAN LD •MEAN NPH MEAN HD

Time mins

IGFBP-1

IGFBP-1 levels were suppressed by insulin. Mean IGFBP-1 levels at baseline were 7.25 ±

3.7 pg/l for LD, 3.08 ± 0.41 pg/1 for HD and 5.96 ± 3.48 pg/l for NPH study day. There

was no significant difference between these baseline values. They fell to a mean minimum

value of 1.76 ± 0.87 pg/1 following LD, 0.9 ± 0.65 pg/1 following HD and 1.57 ± 0.60 pg/1

following NPH insulin. The mean minimum was significantly lower following high dose

detemir than following low dose detemir (p<0.03), however it was not significantly different

to the mean minimum following NPH. There was no difference between the mean

minimums following NPH and low dose detemir. Using analysis of variance there was no

difference between the three insulins on effect on IGFBP-1 levels.

79

Table 7. Healthy Subjects IGFBP-1 results.

LD NPH HD

Baseline IGFBP-1 concentration pg/l 7.25 + 3.7 5.96 ±3.48 3.08 ±0.41

Minimum IGFBP-1 Plasma

concentration pg/1

1.76 ±0.87* 1.57 ±0.60 0.9 ±0.65




Figure 21. Graph of healthy subjects mean plasma IGFBP-1 concentrations.

Healthy subjects mean IGFBP-1 concentratrion

mo

-60-180 60 180 300 420 540 660 780 900 1020

■MEANLD ■MEAN NPH MEAN HD

Time mins

Insulin and insulin detemir

Mean AUC (0-960mins) was 1350.8±125.8, 2220.4+417.5 and 81.4±11.7 nmol.F^ x min for

LD, HD and NPH respectively. There was no individual difference in the time to reach

maximum concentration (Tmax) for the three insulins being 617+85 mins, 520±79 mins and

420±60 mins for LD, HD and NPH respectively. The maximum concentration (C max) was

greater for HD (4478±190 pmolf^) than LD (2345+222 pmolF*) (p<0.01). C max for NPH

was 122±14 pmolf \ A doubling in insulin detemir dose resulted in an approximately two

fold increase in AUC and Cmax

80

Figure 22. Graph of healthy subjects mean plasma insulin and insulin detemir

concentrations.

Healthy Subjects Plasma Insulin and Insulin Detemir Concentrations

4500

4000

3500

3000

I 2500cxI 2000I

1500

1000

500

0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960

Mean LD Mean HD

■ Mean NPH

Time mins

C-Peptide

C-peptide at baseline was similar in each insulin study being 0.47±0.04 nmol/1, 0.55±0.08

nmol/1 and 0.31 ±0.13 nmol/1 for LD. NPH and HD respectively. C-peptide suppressed

following each insulin and there was no difference in this suppression between the insulins

with minimum c-peptide 0.25+0.05 nmol/1, 0.27+0.05 nmol/1 and 0.15+0.06 nmol/1

following LD, NPH and HD respectively.

81

Figure 23. Graph of healthy subjects mean plasma c-peptide concentrations.

Healthy Subjects Mean Plasma C-Peptide concentrations

0. ?

0.6

o 0.5-

-o 0.4

Iu 0.3

0.3

-40 360160 560 760 960

■ Mean LD • Mean NPH Mean HD

Time mins

Glucagon

The glucagon plasma concentrations did not change after dosing with insulin detemir and

NPH insulin, and there was no difference between insulin detemir and NPH insulin.

82

Type 1 Subjects


5 male subjects and one female subject passed screening. The female subject suffered a

serious adverse event (cellulitis from infected cannulation site) following her second clamp

and did not complete her third clamp (the low dose study). Mean age of subjects 29.5 ± 2.39

years, they had had diabetes for 15.3 ± 3.86 years, they had reasonable diabetes control with

HbAlc of 8.45 ± 0.43 %. Their weight was 75.5 ± 1.83 kg and body fat percentage 21.5 ±

349%^

Plasma Glucose

Subjects arrived for the study day with high blood glucose concentrations having omitted

their long acting insulin for the previous 24 hours. However there was no difference in

plasma glucose levels on arrival for the study days. Plasma glucose levels were brought into

the clamp range using a tapering dose of soluble insulin. There was no difference in the

soluble insulin infusion rates on the separate dosing days. There was no difference in

plasma glucose concentrations following the subcutaneous administration of insulin (ie

during the clamp period) being 6.56 ± 0.21 mmol/1 following HD, 6.64 ± 0.2 mmol/1

following NPH and 6.55 ±0.19 mmol/1 following LD. The mean and standard differences of

the individual coefficient of variation for glucose during the clamp studies was 8.77±2.55 %

forLD, 13.35±5.31 % for NPH and 9.14±1.65 % for HD.

83

Figure 24, Graph of Type 1 Subjects Mean Plasma Glucose.

Type 1 subjects Plasma Glucose

60-180 -60 180 300 420 540 660 780 900 1020

Mean HD n=6 • Mean NPH n=6 ■ Mean LDn=5

Time mins

Glucose infusion rate

There was no difference in the time to starting glucose infusion between the 3 insulins with

time to start at 89 ± 26.5 mins for HD, 95.3 ± 24.0 for NPH and 136.4 ± 34.5 for LD. The

maximum glucose infusion rate was similar for the two equipotent doses at 2.9 ± 0.54

mg/kg/min for HD and 4.64 ± 0.79 mg/kg/min for NPH (p=NS), but lower following LD at

2.24 ± 0.47 mg/kg/min than NPH (p<0.04). There was no difference in the maximum GIR

following HD and LD. There was no difference in the time to reach maximum GIR for the

three insulins, nor was there a difference between the areas under the curve of GIR . AUC

for HD was 1537.04 ± 279.2 mg/kg, for NPH it was 2015.14 ± 407.7 mg/kg and for LD it

was 1019.4 ± 266.9 mg/kg. The GIR at the end of the studies was significantly lower

following LD administration at 0.11 ± 0.11 mg/kg/min than following HD at 1.45 ± 0.51

mg/kg/min (p<0.05), but not different to NPH at 0.91 ± 0.50 mg/kg/min.

84

Table 8. Type 1 Subjects Plasma Glucose and Glucose Infusion Rate.

LD NPH HD

Plasma Glucose mmoI/1 6.55 ±0.19 6.64 ± 0.2 6.56 ±0.21

Time to start of glucose

infusion mins

136.4 ±34.5 95.3 ±24.0 89 ± 26.5

Maximum glucose infusion

rate mg/kg/min

2.24 ±0.47 ** 4.64 ± 0.79 2.9 ±0.54

Area under the curve mg/kg 1019.4 ±266.9 2015.14 ±407.7 1537.04 ±279.2

Glucose infusion rate at 960

mins

0.11 ±0.11 * 0.91 ±0.50 1.45 ±0.51

* Indicates a statistically significant difference between LD and LID.


* Revindicates a statistically significant difference between NPH and HD.

Figure 25. Graph of Type 1 Subjects Mean Glucose Infusion Rates.

Type 1 subjects Glucose Infusion Rates (GIR)

aE 2.5

I0.5

0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960

Mean HD n=6 • Mean NPH n=6 Mean LD n=5

Time mins

85

Glucose Metabolism

There was no differenee in hepatic glucose output (Ra) at baseline for the 3 insulins being

2.14±0.40 mg/kg/min for LD, 1.94±0.19 mg/kg/min for HD and 2.14± 0.18 mg/kg/min for

NPH. These insulins had similar effects suppressing glucose Ra with a mean change from

baseline o f -2.19+0.45 mg/kg/min for LD, -2.20 ± 0.30 mg/kg/min for HD and -2.53 ± 0.36

mg/kg/min for NPH (p=NS). Areas over the curve for suppression of Ra was also similar

between the 3 insulins being -1452.33± 401.61 mg/kg for LD, -1452.71 ± 268.83 mg/kg for

HD and-1461.18 ± 288.18 mg/kg for NPH (p=NS).

Figure 26. Graph of Type 1 Subjects Mean Glucose Rate of appearance.

Type 1 Subjects Glucose Rate of Appearance (Ra)

SIE

I-60 60 540 660 780 900 1020180 300 420

- Mean LD Ra (n=5) HD Ra (n=6)

- NPH Ra (n=6)

Time mins

There was no difference in peripheral glucose uptake (Rd) at baseline for the 3 insulins

being 1.93± 0.42 mg/kg/min for LD, 2.02 ± 0 .17 mg/kg/min for HD and 1.99 ± 0.20

mg/kg/min for NPH (p=NS).

86

Figure 27. Graph of Type 1 Subjects Mean Glucose Rate of Disappearance.

Ia:

aÜ

-60

Type 1 subjects Glucose Rate of Disappearance (Rd)

54t5 ■■

4T T T A T T T T

3.5

T Jr j T T T T T + M T

1.51

0.5rO-

- Mean LD Rd (n=5) HD Rd (n=6)

-NPH Rd(n=6)

60 180 300 420 540

Time mins

660 780 900 1020

There was significantly greater effect on change of Rd from baseline following NPH than

either HD (p<0.03) or LD (p<0.01), with the mean maximum change from baseline being

0.29 ± 0.46 mg/kg/min for LD, 0.83 ± 0.31 for HD and 2.22 ± 0.41 mg/kg/min for NPH.

There was no difference between HD and LD for change from baseline. There was also

significantly greater areas under the curve of Rd following NPH than HD (p<0.01) being

-234.32 ± 350 mg/kg for LD, 55.74 ± 197.5 mg/kg for HD and 678 ± 138.14 mg/kg for

NPH. There was no difference between the AUC for HD and LD nor LD and NPH (there

was great inter-subject variability in the AUC for LD).

87

Figure 28. Graph of Type 1 Subjects Mean Glucose Ra and Rd.

Type 1 subjects Mean Glucose Ra & Rd

5.5

60 180 300 420 540 660 780 900 1020

- Mean LD Ra (n=5) HD Ra (n=6)

- NPH Ra (n=6)■ Mean LD Rd (n=5) HDRd(n=6)

•NPHRd(n=6)

Time mins

Blood glucose was controlled before the injection of subcutaneous insulin by using

intravenous soluble insulin. The infusion was stopped just before the subcutaneous insulin

was injected. This technique led to poor maintenance of euglycaemia in the first 60 minutes

of the clamp. We have therefore calculated areas under/over the curve for Ra and Rd using

a baseline of 60 minutes post injection (ie calculating change from the 60 minute value).

The area over the curve (calculated in this way) of suppression of Ra was not significantly

different for insulin detemir (1057.8 ± 129.6 mg/kg) compared to NPH (1060.5 ± 188.8

mg/kg (p=NS). The area under the glucose Rd curve (from T=60) was also not significantly

different for the two insulins at 322.2 ± 197.1 mg/kg for detemir and 930.4 ± 290.7mg/kg

for NPH (p=0.06).

Figure 29. Graph of Type 1 Subjects Mean Glucose Ra and Rd Shown as change from 60

minutes

Type 1 subjects Mean Ra & Rd Shown as Change from 60 minutes

JLT

-0.5 6p-X nO 180 240 300 360 420 480 540 600 660 720 780-840 900 960

-2.5

Time mins

HDRa-NPHRaHDRd

■NPHRd

Figure 30. Graph of Type 1 Subjects Mean Glucose Tracer;Tracee Ratio.

Type 1 subjects M ean Plasm a G lucose Traceritracee R atio

■ M ean N P H

• M ean LD

M ean H D

-1 8 0 -120 -60 0 60 120 180 240 300 360 42 0 4 8 0 540 600 66 0 720 780 840 900 960

T im e m ins

89

Table 9. Type 1 Subjects Glucose Metabolism.

LD NPH HD

Baseline Ra

mg/kg/min

2.15±0.40 2.14±0.18 1.94±0.19

Maximum change

from baseline Ra

mg/kg/min

-2.19±0.45 2.53 ±0.36 -2.20 ±0.30

AOC Ra mg/kg 1452.33± 401.61 1461.18 ±288.18 1452.71 ±268.83

Baseline Rd

mg/kg/min

1.93± 0.42 1.99 ±0.20 2.02 ±0.17

Maximum change

from baseline Rd

mg/kg/min

0.29 ±0.46 ** 2.22 ± 0.41 0.83 ±0.31 ***

AUC Rd mg/kg -234.32 ± 350 678 ±138.14 55.74 ±197.5 ***




Glycerol Metabolism

There was no difference in baseline glycerol Ra between the 3 insulins with 1.47 ± 0.26

mg/kg/min for LD, 2.61 ±0.72 mg/kg/min for HD and 3.27 ± 0.85 for NPH. The maximum

change in glycerol Ra from baseline was significantly greater with NPH than HD (p<0.05)

but not different from LD, nor was LD different from HD with maximum change in glycerol

Ra being -0.55 ± 0.22 mg/kg/min for LD, -1.09 ± 0.39 mg/kg/min for HD and -2.07 ± 0.67

mg/kg/min for NPH. There was no difference in the areas over the curve between the 3

insulins being -143.7 ±211.1 mg/kg/min for LD, -374.7 ± 277.6 mg/kg/min for HD and

-828.3 ± 439.5 mg/kg/min for NPH.

90

Table 10. Type 1 Subjects Glycerol Metabolism,

LD NPH HD

Baseline Ra pmol/kg/min 1.47 ±0.26 3.27 ±0.85 2.61 ±0.72

Maximum change from


-0.55 ± 0.22 -2.07 ± 0.67 -1.09 ±0.39***

AOC Ra pmol/kg -143.7 ±211.1 -828.3 ±439.5 -374.7 ± 277.6

* Indicates a statistically significant difference between LD and FID.


***Indieates a statistically significant difference between NPH and HD.

Figure 31. Graph of Type 1 Subjects Mean Gycerol Rate of Appearance.

Type 1 Subjects Mean Glycerol Rate of Appearance (Ra)

c5'SbII6ai

O

-60 60 180 300 420 540 660 780 900 1020

Mean H ■ Mean N • Mean L

T im e m in s

NEFA Metabolism

There was no difference in baseline plasma NEFA concentrations between LD and NPH or

LD and HD, with baseline values for LD and NPH being 1.03 ± 0.25 pmol/1 and 1.03 ± 0.23

pmol/1 respectively. Baseline concentrations for HD were significantly lower than NPH at

0.57 ± 0.20 pmol/1 (p<0.04). The maximum change from baseline was greater with NPH

than HD (p<0.004) but not different between LD and NPH with maximum decrease in

91

plasma concentration from baseline being 0.62 ± 0.17 pmol/l, 0.90 ± 0.20 pmol/1 and 0.36 ±

0.14 pmol/l, for LD, NPH and HD respectively. There was significantly lower area over the

curve for HD than NPH (p<0.02), but no difference in area over the curve between LD and

NPH or LD and HD, with values being 262.3 ± 167.0 pmol/1, 537.4 ± 208.9 pmol/1 and

110.5 ± 126.2 pmol/l for LD, NPH and HD respectively.

Table 11. Type 1 Subjects NEFA results.

LD NPH HD

Baseline NEFA pmol/1 1.03 ±0.25 1.03 ±0.23 0.57 ±0.20 ***

Maximum change from

baseline NEFA pmol/I

0.62 ±0.17 0.90 ± 0.20 0.36 ±0.14 ***

AOC NEFA pmol/l 262.3 ± 167.0 537.4 ±208.9 110.5 ±126.2***




Figure 32. Graph of Type 1 Subjects Mean Plasma Non Esterified Fatty Acid

Concentrations.

Type 1 subjects M ean Plasma Non Esterified Fatty Acid (NEFA) concentrations

- Mean LD- M ean NPH M ean HD

-180 -60 60 180 300 420 540 660 780 900 1020

Time mins

92

IGF-1 Plasma Concentrations

There was a significant difference in the baseline between HD and NPH (p<0.02), but no

difference in baseline between LD and HD or NPH and LD, baseline values being 16.92 ±

2.32 nmol/1,19.24 ± 2.59 nmol/1 and 16.56 ± 2.51 nmol/1 for LD, NPH and HD respectively.

There was a significant rise from baseline after dosing with all three insulins (p<0.02 for

all), with a maximum IGF-1 plasma concentration of 22.36 ± 2.69 nmol/1, 22.72 ± 3.29

nmol/1 and 22.11 ±2.86 nmol/1 for LD, NPH and HD respectively. However there was no

difference in plasma IGF-1 concentrations between the three insulins.

Table 12. Type 1 Subjects IGF-1 Results.

LD NPH HD

Baseline IGF-1 concentration nmol/I 16.92 ±2.32 19.24 ±2.59 16.56 ±2.51

Mean IGF-1 concentration nmol/1 17.86 ±2.03 19.52 ±2.84 18.23 ±2.36

Maximum IGF-1 concentration nmol/1 22.36 ±2.69 22.72 ±3.29 22.11 ±2.86

* Indicates a statistically significant cifference between LD and HD.



93

Figure 33. Graph of Type 1 Subjects Mean Plasma Insulin Like Growth Factor-1

Concentrations.

Type 1 Subjects Mean Plasma Insulin Like Growth Factor-1 (IGF-1) concentrations

30 1

----------- 2 5 -

-T Î I f i l l [ Î I lJL 1 1

■ T

40

- 0-

■ Mean NPH - Mean LD Mean HD

-180 -60 60 180 300 420 540 660 780 900 1020Time mins

IGFBP-1 Plasma Concentrations

All three insulins suppressed IGFBP-1 concentrations. There was no significant difference

in the baseline or minimum concentrations, nor was there a difference in the change for

baseline or the areas over the curve. These results are shown in the table below.

94

Table 13. Type 1 Subjeets IGFBP-1 Results.

LD NPH HD

Baseline IGFBP-1

concentration pg/l

36.03 ± 8.07 28.99 ±9.00 31.91 ± 3.87

Minimum IGFBP-1

Plasma

concentration pg/1

4.14 ±0.64 4.38 ±0.80 3.26 ± 0.66

Maximum change from

baseline pg/1

31.89 ±7.65 24.61 ±8.52 28.64 ± 3.39

AOC 20528.4 ±4933.6 15842.3 ±7045.4 19244.0 ± 1912.1

* Indicates a statistica ly significant difference between LD and HD.

** Indieates a statistically significant difference between LD and NPH.


Figure 34. Graph of Type 1 Subjects Mean Plasma Insulin Like Growth Factor Binding

Protein-1 Concentrations.

Type 1 Subjects Mean Plasm Insulin Like Growth Factor Binding Protein-1 (IGFBP-1)Concentrations

140 1

ffla

-60 420-180 60 180 300 540 780660 900 1020

- Mean LD • Mean NPH Mean HD

Time mins

95


Mean AUC (0-960mins) was 2301.8 ± 505 (nmol/l)*min, 3703.2 ± 230 (nmol/l)*min and

306.7 ± 94.26(nmol/l)*min for LD, HD and NPH respectively. There was no individual

difference in the time to reach maximum concentration (Tmax) for the three insulins being

390 ± 32.8 mins, 515 ± 67.2 mins and 374 ± 74 mins for LD, HD and NPH respectively.

The maximum concentration (C max) was greater for HD (6160 ± 231.4 pmolf^) than LD

(4182 ± 1013.4 pmoir^). C max for NPH was 517.7 ± 117.1 pm olf\ The increase in AUC

and Cmax was less than two-fold however, one subject (Subject 205) dosed with 9 nmol/kg

insulin detemir had a very high insulin detemir plasma concentration profile.

C-Peptide

All subjects with Type 1 diabetes were c-peptide negative.

Glucagon



96

Type 2 Subjects


5 male subjeets and 6 female subjects were screened. 3 male subjects and 3 female subjects

passed screening. Of the 5 subjects who failed screening, 4 failed because their body mass

index was too high, and 1 failed because of general ill health.

The mean age of subjects was 58.7 ± 2.85 years, they had diabetes for an average of 6.88 ±

0.41 years and had good control with a mean HbAlc of 6.57 ± 0.35 %. Their weight was

82.8 ± 8 kg and body fat percentage 33.4 ± 3.59 %.

Plasma Glucose

Subjects arrived for the study day with high blood glucose concentrations having omitted

their long acting insulin for the previous 24 hours or oral hypoglyeaemic agents for the

preceding 48 hours. However there was no difference in plasma glucose levels on arrival

for the study days (9.74 ± 1.79 mmol/1 for LD, 9.09 ± 1.09 mmol/1 for HD and 10.15 ± 2.33

for NPH). Plasma glucose levels were brought into the clamp range using a tapering dose of

soluble insulin. There was no difference in the soluble insulin infusion rates on the separate

dosing days nor was there a differenee in baseline glucose levels at 6.51 ± 0.33 mmol/1 for

LD, 6.26 ± 0.33 for HD and 6.28 ± 0.37 for NPH. There was no difference in plasma

glucose concentrations following the administration of insulin being 6.46 ± 0.25 mmol/1 for

LD, 6.27 ± 0.27 mmol/1 for HD and 6.23 ± 0.35 mmol/1 for NPH. The mean and standard

differences of the individual coefficient of variation for glucose during the clamp studies

was 5.19+1.32 % for LD, 6.8+3.08 % for NPH and 5.5+1.26 % for HD.

97

Figure 35. Graph of Type 2 Subjects Mean Plasma Glucose Concentrations.

Type 2 Subjects Mean Plasma Glucose Concetnrations

I%I

-240 -120 0 120 240 360 480 600 720 840 960

■ Mean LD■ Mean NPH Mean HD

Time mins

Glucose infusion rate

There was no difference in the time to start the glucose infusion, maximum glucose infusion

rate, area under the curve of the glucose infusion or glucose infusion rate after 960 minutes

between HD and NPH. Comparing HD and LD there was no significant difference in time

to start the glucose infusion, but the maximum glucose infusion rate was lower with LD

(p<0.05), the area under the curve was lower with LD (p<0.03) and the glucose infusion rate

at the end of the study (960 mins) was lower with LD (p<0.01). Comparing LD and NPH

the glucose infusion rate at 960 minutes was lower with LD (p<0.02), but the differences in

the other measurement parameters did not reach statistical significance. The time to the start

of the glucose infusion was 81.5 ± 10.82 mins for LD, 50.8 ± 7.54 mins for HD and 46.0 ±

10.82 mins for NPH. The maximum glucose infusion rate was 1.93 ± 0.76 mg/kg/min for

LD, 2.96 ± 0.51 mg/kg/min for HD and 3.29 ± 0.76 mg/kg/min for NPH. The area under the

curve (ie total glucose infused) was 1130.2 ± 211.2 mg for LD, 1868.5 ± 348.6 mg for HD

and 1674 ± 211.2 mg for NPH. The glucose infusion rates at the end of the study were 0.89

98

±0.13 mg/kg/min for LD, 2,18 ± 0.39 mg/kg/min for HD and 1.79 ± 0.13 mg/kg/min for

NPH.

Table 14. Type 2 Subjects Plasma Glucose and Glucose Infusion Rate.

LD NPH HD

Mean Plasma Glucose mmol/I 6.51 ±0.33 6 2 8 ± 0 3 7 6 2 6 ± 0 3 3

Time to start of glucose

infusion mins

81.5 ± 10.82 46.0 ± 10.82 50.8 ±7.54

Maximum glucose infusion rate

mg/kg/min1.93 ±0.76* 3.29 ±0.76 2.96 ±0.51

Area under the curve mg/kg 1130.2 ±211.2* 1674±211.2 1868.5 ±348.6

Glucose infusion rate at 960

mins

0.89 ±0.13 *, ** 1.79 ±0.13 2.18 ±0.39




Figure 36 Graph of Type 2 Subjects Mean Glucose Infusion Rates.

Type 2 Subjects Mean Glucose Infusion Rates (GIR)

3.5

0.5

-40 160 360 560 760 960

■ Mean LD Mean NPH Mean HD

Time mins

99

Glucose Metabolism

There were no statistically significant differences found between LD, HD and NPH when

analysed for baseline Ra, maximum change in Ra, AOC Ra, baseline Rd, maximum change

in Rd or area over the curve Rd, except the maximum change in glucose Ra for LD was 1.58

±0.13 mg/kg/min which was lower than that for NPH (which was 2.07 ±0.19 mg/kg/min)

(p<0.04), the maximum change in Ra from baseline for HD was 2.01 ± 0.28 mg/kg/min.

There were also no differences found using ANOVA to compare the data sets.

Figure 37. Graph of Type 2 Subjects Mean Glucose Rate of Appearance.

Type 2 subjects Mean Glucose Rate of Appearance (Ra)

i j

! -I ^a

300-60-0.5

180 420 540 660 780 900 1020

Time mins

■ Mean Ra LD■ Mean Ra NPH Mean Ra HD

100

Figure 38. Graph of Type 2 Subjects Mean Glucose Rate of Disappearance.

Type 2 Subjects Mean Glucose Rate of Disappearance (Rd)

-60 60 180 300 420 540 660 780 900 1020

• Mean Rd LD ■ Mean Rd NPH Mean Rd HD

Time mins

Figure 39. Graph of Type 2 Subjects Mean Glucose Ra and Rd.

Type 2 Subjects Mean Glucose Ra & Rd

3.5 1

-3 -

2:5-T T T t i l - Mean Ra NPH

■ Mean Rd NPH Mean Ra HD Mean Rd HD

■ Mean Ra LD • Mean Rd LD

420 5io^

Time mins

1020

Because of the technical difficulties with our clamp protocol in the changeover from

intravenous to subcutaneous insulin we also analysed the glucose metabolism data in these

type 2 subjects from time 60 minutes. When calculated from 60 min the area over the curve

of suppression of Ra was significantly greater for HD (984 ± 135 mg/kg) compared to NPH

(535 ± 79 mg/kg)(p<0.04). The area under the glucose Rd curve was 672 ± 255 mg/kg for

HD and 797 ± 149 mg/kg for NPH (p=NS).

101

Figure 40. Graph of Type 2 Subjects Mean Glucose Ra and Rd Expressed as a change from

T=60 minutes.

Type 2 Subjects Glucose Ra and Rd Expressed as Change from T=60mins

2.5

.5e 1.5

I 1I 0.5

§ 0

% -0.5

I:40 300 360 420 480 540 600 660 720 780 840 900 960

-1.5

Time mins

- NPH Ra change NPH Rd change HD Ra change HD Rd change

Figure 41. Graph of Type 2 Subjects Mean Plasma Glucose Tracer:Tracee Ratio.

T ype 2 Subjects M ean Plasm a G lucose T racerT racee R atio

0 .16 n

I I T t t I I t T t I I t I T T T I I I - I ---------------

1-------- 1--------ÔtT 4 -

■ M ean N P H

■ M ean L D

M ean H D

-180 -1 2 0 -60 0 60 120 180 240 300 360 420 48 0 540 60 0 660 720 78 0 840 90 0 960

Tim e m ins

102

Table 15. Type 2 Subjects Glucose Metabolism.

LD NPH HD

Baseline Ra mg/kg/min 1.64 ±1.99 1.66 ± 0.15 1.67 ± 0.26

Maximum change from

baseline Ra mg/kg/min

1.58 ±0.13 ** 2.07 ±0.19 2.01 ± 0.28

AOC Ra mg/kg 1015.57 ±102.91 1164.99 ±71.87 1242.29 ±200.76

Baseline Rd mg/kg/min 1.83 ±0.17 1.90 ±0.16 1.83 ±0.24

Maximum change from

baseline Rd mg/kg/min

0.33 ±0.17 1.32 ±0.49 1.12 ±0.53

AUC Rd mg/kg -70.9 ±153.94 270.3 ±219.65 463.63 ± 355.48

* Indicates a statistical y significant difference between LD and HD.



Glycerol Metabolism

There was no difference in the glycerol Ra at baseline for the three insulins, being 2.26 ±

0.33 pmol/kg/min for LD, 1.64 ± 0.23 pmol/kg/min for HD and 2.14 ± 0.55 pmol/kg/min

for NPH. There was also no difference in the maximum change from baseline between the

three insulins being 0.80 ± 0.26 pmol for LD, 0.72 ± 0.23 pmol for HD and 1.12 ± 0.41

pmol for NPH. There was however significantly lower area under the curve for HD glycerol

Ra than NPH glycerol Ra (p<0.04), but no difference in the areas under the curve between

LD and HD and LD and NPH. The areas under the curve were 321.53 ±271.7 pmol for LD,

329.34 ± 239.21 pmol for HD and 654.5 ± 345.3 pmol for NPH.

103

Table 16. Type 2 Subjects Glycerol Metabolism.

LD NPH HD

Baseline Ra pmol/kg/min 2.26 ± 0.33 2.14 ±0.55 1.64 ±0.23

Maximum change from


0.80 ± 0.26 1.12 ±0.41 0.72 ± 0.23

AOC Ra pmol/kg 321.53 ±271.7 654.5 ±345.3 329.34 ± 239.21***




Figure 42. Graph of Type 2 Subjects Mean Glycerol Rate of Appearance.

Type 2 Subjects Mean Glycerol Rate of Appearance (Ra)

2t5-

285

-60 60 420180 300 540 660 780 900 1020

Mean H • Mean N ■ Mean L

Time mins

NEFA Metabolism

There was no difference between the plasma NEFA concentrations at baseline. Plasma

NEFA concentrations were more suppressed following HD than ED (p<0.05), but there was

104

no difference between HD and NPH or NPH and LD with minimum concentrations for the

three insulins being 0.23 ± 0.04 |Limol/l, 0.13 ± 0.03 |imol/l and 0.13 ± 0.05 imol/1 for LD,

HD and NPH respectively. There was no difference in the change from baseline between

the three insulins nor was there a difference in the areas over the curve.

Table 17. Type 2 Subjects NEFA Results.

LD NPH HD

Baseline NEFA pmol/I 0.55 ±0.12 0.48 ±0.12 0.43 ± 0.06

Maximum change from

baseline NEFA pmol/l

0.31 ±0.15 0.35 ±0.13 0.30 ±0.09

AOC NEFA pmol/l 75.48 ± 142.0 187.15 ±97.43 137.88 ±156.03



Indicates a statistically significant difference between NPH and HD.

Figure 43. Graph of Type 2 Subjects Mean Plasma Non Esterified Fatty Acid

Concentrations.

Type 2 Subjects Mean Plasma Non Esterified Fatty acid (NEFA)Concentrations

180 -60 60 180 300 420 540 660 780 900

Mean LD Mean NPH Mean HD

Time mins

105

IGF-1 Plasma Concentrations

There was no difference in the baseline, maximum or mean concentrations of plasma lGF-1

between the three insulins. However there was a significant rise from baseline found in all

three insulins (p<0.03). The plasma concentrations are shown in the table below.

Table 18. Type 2 Subjects lGF-1 Results.

LD NPH HD

Baseline IGF-1 concentration nmol/1 15.03 ±0.94 13.83 ±0.69 14.44 ±0.97

Mean IGF-1 concentration nmol/1 15.36 ±0.74 14.98 ±0.86 15.59 ±0.92

Maximum IGF-1 concentration nmol/1 18.47 ±1.00 17.82± 1.11 19.92 ± 1.48

* Indicates a statistically significant cifference between LD and HD.


***lndicates a statistically significant difference between NPH and HD.

Figure 44. Graph of Type 2 Subjects Mean Plasma Insulin Like Growth Factor-1

Concentrations

Type 2 Subjcts Mean Plasma Insulin Like Growth Factor-1 (IGF-1) Concentrations

25

MeanNPFI ■ Mean LD Mean HD

'----------------------r - 0 -

-180 -60 60 180 300 420 540 660 780 900 1020

Time mins

106

IGFBP-1 Plasma Concentrations

All three insulins suppressed IGFBP-1 concentrations. There was no significant difference

in the baseline or minimum concentrations, nor was there a difference in the change for

baseline or the areas over the curve. These results are shown in the table below.

Table 19. Type 2 Subjects IGFBP-1 Results.

LD NPH HD

Baseline IGFBP-1

concentration pg/l

13.08 ±7.70 22.08 ±16.19 10.73 ±5.63

Minimum IGFBP-1 Plasma

concentration pg/1

3.8 ±1.75 5.72 ±2.88 2.92 ±1.51

Maximum change from

baseline pg/l

9.28 ± 6.00 16.36 ±13.33 7.82 ±4.14

AOC 4713 ±3106.1 9033.1 ±7870.3 3976.5 ±1981.8

* Indicates a statistically significant difference between LD and F[D.



107

Figure 45. Graph of Type 2 Subjects Mean Plasma Insulin Like Growth Factor Binding

Protein-1 Concentrations.

Type 2 Subjects Mean Plasma Insulin Like Growth Factor-1 (IGFBP-1) Concentrations

2

60-180 -60 180 300 420 540 660 780 900 1020

• Mean LD Mean HD

• Mean NPH

Time mins


Mean AUC (0-960mins) was 1577.6 ± 219.39 nmol, 3131.5 ± 261.92 nmol and 141.8 ±

31.76 nmol for LD, HD and NPH respectively. There was no individual difference in the

time to reach maximum concentration (Tmax) for the three insulins being 638.3 ± 86.98

mins, 625.3 ± 48.49 mins and 565.0 ± 62.57 mins for LD, HD and NPH respectively. The

maximum concentration (C max) was greater for HD (4996.7 ± 189.84 pmolF^) than LD

(2660.0 ± 311.02 pmoir^). C max for NPH was 208.8 ± 108.1 pmoH'\ A doubling in insulin

detemir dose resulted in an approximately two-fold increase in AUC and Cmax.

C-peptide

C-peptide at baseline was similar in each insulin study being 0.46±0.12 nmol/1, 0.49+0.17

nmol/1 and 0.52±0.14 nmol/1 for LD. NPH and HD respectively. C-peptide suppressed

following each insulin and there was no difference in this suppression between the insulins

with minimum c-peptide 0.32±0.08 nmol/1, 0.26+0.07 nmol/1 and 0.27+0.07 nmol/1

following LD, NPH and HD respectively.

108

The plasma concentrations of C-peptide decreased slightly after dosing with insulin detemir

and NPH insulin in subjects with type 2 diabetes.

Figure 46. Graph of type 2 Subjects Mean Plasma C-Peptide Concentrations.

Type 2 Subjects Mean Plasma C-Peptide Concentrations

0.8

0.7

0.6

0 0.51

^ 0.4

0.3

0.2

0.1

0

!

I . .T

r •: " ;; " ::' Î I T T

t i . . \^ N /-----

Mean NPH Mean HD Mean LD

0 120 240 360 480 600 720 840 960

Time Mins

Glucagon



The mean glucagon plasma concentrations were highest in healthy subjects and lowest in

subjects with type 1 diabetes and subjects with type 2 diabetes were between the two other

subject groups.

109

Combined results

Subjects with diabetes are more insulin resistant than those without, and therefore the data

from the three groups of subjects (Healthy, Type 1 diabetes and Type 2 diabetes) were

analysed further by combining the results and comparing the effect of the equipotent doses

of detemir and NPH. The doses of insulin given for healthy subjects (9 nmol/kg insulin

detemir and 0.3 iu/kg NPH ) were half that of the doses given to the subjects with diabetes

(18 nmol/kg insulin detemir and 0.6 iu/kg NPH), in spite of this the doses required similar

amounts of glucose to be infused to maintain euglycaemia throughout all the groups. It may

therefore be justified to pool the data as shown below.

Figure 47. Graph of Mean Glucose Infusion Rates for Each Subject Group.

Mean Glucose Infusion Rates For Each Subject Group

S0Û60 2 s0 1 cÜ

0.5

0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960

Healthy •Type 1 Type 2

Time mins

Plasma Glucose

The plasma glucose thoughout the clamp period (0-960mins) was lower in healthy subjects

than in those with type 1 diabetes (p<0.0001) or type 2 diabetes (p<0.0001) being 4.94 ±

0.06 mmol in the healthy group, 6.63 ±0.15 in subjects with Type 1 diabetes and 6.27 ±

0.22 in subjects with Type 2 diabetes. There was no difference between the plasma glucose

110

in the clamp period between the two groups of subjects with diabetes. With plasma glucose

pooled across the subject groups there was no difference in the plasma glucose for those

subjects treated with NPH (5.90 ± 0.22 mmol) compared with those treated with insulin

detemir (5.88 ± 0.21 mmol)(p=NS).

Glucose Infusion Rate

There were no differences in the glucose infusion rates either between the subject groups

(healthy, type 1, and type 2 subjects), or between the two treatments (detemir and NPH)

when analysed for total glucose infused, mean GIR, maximum GIR and GIR at 960 mins.

Table 20. Combined Glucose and Glucose Infusion Rate Data.

Healthy

subjects

Type 1

subjects

Type 2

Subjects

Detemir NPH

Area under

the curve

GIR, mg/kg

2055.99 ±

256.95

1776.1 ±

246.71

1771.5 ±

196.75

1819.0 ±

191.95

1936.3 ±

195.6

Maximum

GIR

mg/kg/min

3.79 ± 0.49 3.63 ±0.51 3.12 ±0.44 3.16 ±0.35 3.89 ± 0.42

Mean GIR

mg/kg/min

2.12 ±0.29 1.84 ±0.26 1.76 ±0.21 1.83 ±0.20 2.00 ± 0.22

GIR at 960

mins

mg/kg/min

1.64 ±0.15 1.40 ±0.39 1.98 ±0.20 1.83 ±0.23 1.51 ±0.42

I l l

Figure 48. Graph of Mean Glucose Infusion Rates for Each Insulin from the Combined

Data from each Subject Group.

Mean Glucose Infusion Rates for each insulin in all subject Groups

5

0.5

0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960

Mean D • Mean NPH

Time mins

Glucose Metabolism

There were no significant differences found in the effect of the two insulins on glucose Ra

when compared at baseline, minimum Ra, maximum change from baseline, area over the

curve Ra or by ANOVA (p=0.06). Baseline Rd was similar for the two insulins but NPH

had a significantly higher maximum Rd compared with detemir (p<0.04) and also had a

greater change from baseline (p<0.02). There area under the curve for Rd appeared lower

for detemir, but the differences were not statistically significant. Using ANOVA the

differences in the effect on Rd did not quite reach statistical significance p=0.05. Of the

mean glucose lowering effect of insulin detemir over the 960 minutes 1.39 ± 0.08

mg/kg/min glucose was infused to overcome Ra (79%) and 0.37 ± 0.07 mg/kg/min glucose

was infused to overcome Rd (21%), whereas for NPH this was 1.29 ± 0.08 mg/kg/min for

Ra (65%) and 0.68 ± 0.10 mg/kg/min for Rd (35%)(p<0.001).

12

Table 2 1 . Combined Glucose Metabolism Data.

Detemir NPH

Baseline Ra mg/kg/min 1.87±0.11 1.95 ±0.08

Minimum Ra mg/kg/min -0.23 ± 0.09 -0.19 ±0.12

Maximum change from baseline Ra mg/kg/min 2.09 ±0.14 2.14±0.12

AOC Ra mg/kg 1375.3 ± 111.5 1282.2 ±84.1

Baseline Rd mg/kg/min 1.97±0.10 1.97 ±0.08

Maximum Rd mg/kg/min 3.18 ±0.25 3 ^ 8 ± 0 JT

Maximum change from baseline Rd mg/kg/min 1.21 ±0.25 1.90 ±0.29

AUC Rd mg/kg 367.38 ± 159.9 625.4 ± 140.4

Figure 49. Graph of Mean Glucose Ra and Rd for each insulin from the combined data from

each subject group.

Data from the three subject groups combined to show Mean Glucose Ra & Rd

Mean D Rd Mean D Ra Mean NPH Rd Mean NPH Ra

a

-60 0 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960

Time mins

13

Figure 50. Bar charts showing the relative proportion of glucose lowering effect from effect on glucose Ra and glucose Rd of each insulin using data combined from the three subject groups.

Bar chart showing mean effect on Glucose Ra and Rd following injection with Detemir

B Mean D Rd □ Mean D Ra

Time mins

B a r c h a r t s h o w i n g m e a n e f f e c t o n G l u c o s e R a a n d R d f o l l o w i n g i n j e c t i o n w i t h N P H

| o ^

JLJJT r 4 4 l r i n T r r

■ M e a n N P H R d

□ M e a n N P H R a

UO 360 480 720 <%0

T i m e m i n s

114

Figure 51. Pie chart showing the overall mean proportion of effect of each insulin on

glucose Ra and Rd using data combined from the three subject groups.

Pie chart showing overall mean effect of Detemir on Glucose Ra and Rd

21%

79%

□ Ra ■ Rd

Pie chart showing overall mean effect of NPH on glucose Ra and Rd

35%

65%

□ Ra ■ Rd

15

Chapter 4

Discussion

In the 1990s the link between metabolic control and development of microvascular

complications was firmly established by the diabetes control and complications trial research

group (DCCT). The DCCT was a study in 1441 subjects with type 1 diabetes. They were

randomised to either intensive control (with basal bolus regime and strict targets) or

conventional treatment. Those in the intensive group had a mean HbAlc of 7.1% whereas

those in the conventional treatment group had an HbAlc of 9.0%. There was a lower rate of

complications in the intensive group with a reduction in: the risk of retinopathy of 45%,

development of microalbuminuria by 35% and clinical neuropathy by 60%. For every 10%

reduction in HbAlc (ie a reduction in HbAlc from 10% to 9%) there was about 35% risk

reduction of retinopathy, 25-44% risk reduction for nephropathy and 30% risk reduction for

neuropathy. However the risk of hypoglycaemia increased threefold in the intensive

treatment group. The United Kingdom Prospective diabetes study research group (UKPDS)

looked at intensive versus conventional treatment in patients with type 2 diabetes, and

showed risk reduction in microvascular endpoints, myocardial infarction, cataract extraction,

retinopathy and albuminuria.

These studies were large, well-controlled and highly regarded and demonstrated the

importance of good control for reducing the risk of complications. As a result of these

studies the objective now in the treatment of diabetes is not the avoidance of multiple

injections but the imitation of normal physiology.

116

Figure 52. Diagram of the Physiological Plasma Insulin Secretion Profile.

Physiological serum insulin secretion profileB reakfast Lunch Dinner

500-1

g 400 -

I300-

r

§

I* 2 0 0 -

s100

6 a.m. 10 a.m. 2 p.m. 6 p.m. 10 p.m. 2 a.m. 6 a.m.T im e

Polonsky KS et al. N E n g lJ M ed 1996;334:777-83

In people without diabetes insulin is released constantly from the pancreas at a “basal level”

with boluses of insulin released at meal times to cover additional prandial requirements.

Bolus (mealtime) insulin release from the pancreas limits hyperglycaemia after meals by an

immediate sharp rise and a peak at 1 hour (Bell et al., 1996). At first insulin regimes were

felt not to be satisfactory because the meal time bolus was with regular insulin. This insulin

when injected subcutaneously existed in hexameric form and therefore must dissociate into

dimers and monomers before it could be absorbed into the capillary circulation

(Cunningham et al, 1955). Conventional “short acting” or regular insulin had onset of

action about 1 hour after injection and peak action at 2-4 hours (Howey et al, 1994). Thus

patients were advised to inject insulin around 30 minutes before a meal (an instruction that

was rarely adhered to), and to take snacks between meals to compensate for prolonged

hyperinsulinaemia, which can lead to hypoglycaemia (Barnett, 2001). Rapid acting

analogues were designed to mimic the prandial release of insulin. They have fast absorption

times with onset of action after 20 to 30 minutes and peak action at about 1-2 hours, leaving

a shorter duration of action than regular insulin (Howey et al, 1994) and as such more

117

closely mimic physiological insulin release in response to meals. All the pivotal studies

involving several thousand patients with Type 1 and Type 2 diabetes showed significant

reductions in postprandial glucose excursions using the rapid acting insulin analogues

compared with human soluble insulin (Owens et al, 2000, Anderson et al 1997a, Anderson

et al, 1997b and Kang et al, 1991). This was entirely expected based on the more rapid

absorption and higher peak insulin levels demonstrated in earlier phase studies. These

registration studies, however did not show a significant improvement in glycated

haemoglobin, although there was a significant improvement in patient satisfaction (Barnet,

2001). The latter was primarily related to the fact that patients could take their rapid acting

insulin analogue immediately prior to eating a meal, rather than half an hour before.

Furthermore the incidence o f hypoglycaemia was reduced in those using rapid acting insulin

analogues compared to those using regular insulin as their bolus (Brunelle et al, 1998,

Holleman et al 1997).

Because these analogues did not normalise blood glucose control as hoped attention was

turned to basal insulins. It was recognised that little or no improvements in basal insulins

had occurred since the 1930’s when the concept of crystalline insulins as a method of

protraction was conceived. It is accepted that these longer acting insulins do not imitate the

basal insulin production from the pancreas, but rather have peaks of action after around 6-8

hours and had variable absorption, resulting in huge differences in daily basal insulin levels

(Home, 1999). Blood glucose variability correlates with mean glycaemic exposure and risk

of hypoglycaemia (Moberg et al, 1994), and also with mortality (Muggeo et al 2000).

Somogyi (1959) observed that the blood glucose and glycosuria of diabetic patients often

showed wide fluctuations even when both carbohydrate content of the diet and insulin dose

are unchanged.

With conventional crystalline suspensions of insulin some of this can be explained by poor

mixing techniques, with variations in the actual concentration of insulin injected varying in

one study from 5-214% (Bolli and Owens, 2000). Studies have shown that fluctuations in

absorption rate are the main cause for unpredictable insulin activity, accounting for as much

as 80% of the variability in the action of NPH insulin or insulin lente (Guthrie, 2001).

118

Laurizten et al (1979) injected subjects with radiolabelled NPH insulin over a 12 day period,

they found that absorption varied greatly from full to only 19% on some doses. Chen et al

(2003) also observed large variations in absorption of NPH insulin after subcutaneous

injection with 50% of the original dose of insulin found at the injection site for around 12

hours on one day and around 96 hours on another in the same patient. Luzio et al (2003)

also describes large variations in insulin absorption rates with 50% absorption between 7

and 18.2 hours for NPH and 10.2 and 42 hours for glargine. There is also considerable

variation in the absorption of NPH depending on the site of injection (Lauritzen et al, 1982),

and the time of day that that site is used (Chen et al, 2003).

The drive now was to design an insulin to mimic the secretion of basal insulin in healthy

subjects so to suppress glucose production between meals and overnight. Renewed interest

in prolonging the duration of action of insulin has led to developments along different

strategic routes. The expectation that proinsulin would increase basal insulin concentrations

was short lived because of concerns on cardiovascular safety that arose from clinical studies

(Owens et al, 2001). Proinsulin’s metabolite, des (64,65)-human proinsulin had an even

shorter period of action than NPH insulin and was discontinued (Owens et al, 2001).

Surprisingly, the strategy to slow absorption and clearance by strengthening hexameric

interactions in the form of cobalt (Ill)-insulin complex also proved unsuccessful (Burge et

al, 1997). However, increase of the isoelectric point of insulin from 5.4 towards neutrality

thereby causing precipitation or crystallisation at the site of injection, delays absorption, and

prolongs the effect of insulin (Markussenet al, 1987). The first acid soluble extended action

insulin analogue made by this method was Novosol Basal GlyA21, ArgB27, ThrB30-NH2-

insulin (Jorgensen et al, 1989), which was discontinued because of escalating dose

requirements (reduced bioavailability) possibly associated with local inflammatory reactions

at the site of injection. On the basis of the same concept, a di-arginylinsulin analogue

known as insulin glargine was developed that had a much slower and more reproducible rate

of subcutaneous absorption than NPH insulin (Owens et al, 2000), resulting in slower onset

and extended hypoglycaemic effect (Lepore et al, 2000, Heineman et al 2000). An

additional phenol molecule bound to the hexamer of crystalline insulin glargine might

further contribute to its protracted action.

119

Insulin glargine is the first of these long acting insulin analogues to make it to the market,

being launched in 2002. Plasma glucose and insulin concentrations vary less between

patients when insulin glargine is used compared to ultralente insulin (Lepore et al, 2000,

Scholtz et al 1999). Comparison of insulin glargine with NPH insulin in isoglycaemic

clamp studies shows that a bolus subcutaneous injection of NPH insulin (0.3iu/kg) produces

a definite peak effect within 3-6 hours, before returning to baseline at 16 hours. By contrast

insulin glargine plateaus at a concentration 2-3 times lower than NPH within 6-8 hours,

remains essentially unchanged for 24 hours (Heinemann et al, 2000) and is unaffected by the

site of injection (Owens and Barnett, 2000). In studies comparing insulin glargine with

NPH as part of a basal bolus regime, subjects using insulin glargine had a greater reduction

in fasting blood glucose (Pieber et al, 2000, Ratner et al, 2000, Rosenstock et al, 2000,

Raskin et al, 2000), less variability in blood glucose (Rosenstock et al, 2000, Raskin et al,

2000), a lower frequency of hypoglycaemia (predominantly nocturnal) (Pieber et al, 2000,

Ratner et al, 2000) and a fall in glycosylated haemoglobin (Pieber et al, 2000). However

injection site pain which is negligible in the NPH group is experienced in 6.1% with insulin

glargine, presumably because of the acidic pH (4.0) of insulin glargine (Raskin et al, 2000).

Insulin glargine has also been compared to NPH insulin in patients with type 2 diabetes.

Both insulins were given at bedtime in poorly controlled circumstances in which insulin

naïve patients were allowed to continue oral agents (Yki-Jarvinen and Westerbacka, 2000).

Overall glycated haemoglobin improved to the same extent in each group, however there

was a lower frequency of nocturnal hypoglycaemia in the glargine treated group. Similarly

in patients with type 2 diabetes changing from NPH once or twice daily to glargine at

bedtime, there was a lower risk of nocturnal hypoglycaemia and equivalent glycaemic

control (Rosenstock et al, 2001). In these studies no differences in doses were recorded, but

either no weight gain (Rosenstock et al, 2001), or less weight gain with insulin glargine

(Wang et al, 2003), than with NPH.

Insulin detemir is a long acting soluble insulin analogue, with a 14 C fatty acid chain

attached to position B29 and omission of the amino acid residue at position B30 of the

human insulin molecule (Brunner et al, 2000). The fatty acid residue associates with free

120

fatty acid binding sites on albumin in interstitial fluid and plasma (Kurtzhals et al, 1995)

developing an equilibrium between free and albumin bound analogue. Only the free fraction

of insulin detemir permeates the capillary wall entering the blood stream and binding to

albumin again. To reach peripheral target tissues (muscle and adipose) free insulin detemir

must again pass the capillary wall. The principle of protraction is based on slow release

from albumin and strong self-association at the injection site (Havelund et al, 2004, Brunner

et al, 2000). Insulin detemir uniquely among the long acting insulin analogues does not

form a precipitate at the site of injection and this property has been considered to be of key

importance in eliminating a potential source of variability (Lindholm, 2002). It is also likely

that plasma albumin binding may buffer the effect of short duration changes in the depot

absorption rate, thereby limiting the pharmacodynamic variability (Kurtzals, 2004). This

favourable action was found in a study reported by Heise et al (2004) and Bott et al (2003)

compared the pharmacokinetic, pharmacodynamic profiles and within subject variability of

NPH, detemir and glargine in patients with Type 1 diabetes. Subjects received 4 identical

doses of an insulin on four different dosing days, 18 subjects were randomised to each

insulin. In this study reproducibility of the glucose infusion rate curves were analysed and it

was found that the probability of reproducibility was worst for NPH insulin, followed by

glargine and that detemir had the most reproducible curve. The coefficient of variation for

pharmacodynamics as observed by glucose infusion rate and pharmacokinetics as assessed

by insulin concentration area under the curve was significantly lower with detemir than NPH

and glargine. This favourable action may result in lower frequencies of unpredicted hypo or

hyperglycaemia. A study reported by Pieber et al (2002) looking at duration of action,

pharmacodynamic profile and between subject variability of insulin detemir in subjects with

type 1 diabetes, found a linear dose response relationship for pharmacokinetic and

pharmacodynamic properties of detemir. It reported a longer duration of action at a dose of

0.4u/kg (20 hours) than NPH at a dose of 0.3iu/kg (13 hours). They also reported less

between subject variation.

Capillary endothelial cells in adipose and muscle limit the transfer of insulin detemir from

the circulation into the extravascular extracellular space (Havellund et al, 2004), whereas in

the liver the sinusoids are lined by highly fenestrated epithelial cells, between which there

121

are large gaps. The sinusoids also have no basal lamina. Because of the large gaps in the

endothelial cell layer and the absence of a continuous basal lamina, no significant barrier

exists between blood plasma in the sinusoid and the hepatocyte plasma membrane (Ross,

2003). Insulin detemir can therefore pass freely to this “space of Disse” (the perisinusoidal

space) Gray, 1973, Reichen, 1999). Hepatocytes will be freely exposed to insulin detemir

possibly resulting in a greater effect of this insulin analogue on the liver than on peripheral

tissue.

Insulin detemir’s binding properties may provide an alternative theory for insulin detemir’s

apparent hepatoselectivity. The albumin binding properties of insulin detemir have been

extensively studied using an immobilised human serum albumin (HSA) preparation

(Kurtzhals et al, 1995 and 1997). The binding constant of insulin detemir for HSA was

determined to be 2.4 x 10 M'^ at 23°C and 1.0 x 10 M'^ at 37°C in this assay system.

Based on this binding constant, insulin detemir is estimated to be 98.4% bound in plasma

(37°C, 0.6mM albumin), which is consistent with the above determined plasma protein

binding (98.8%) of insulin detemir (Kurtzhals et al, 1995). We can see therefore that

detemir has a higher binding affinity to albumin at colder temperatures. Core body

temperature is higher than that at the periphery (Kim et al, 1998). This may mean that a

higher proportion of detemir is free (that is not bound to albumin) in the core (ie at the liver)

whereas in the periphery (that is in adipose and muscle tissue) detemir will be more tightly

bound to albumin and less will pass through the capillary wall to act on target cells. This

may explain why detemir’s hepatoselective effects in people with type 2 diabetes is more

difficult to demonstrate, as these people tend to have a higher proportion of their adipose

tissue centrally (that is intra-abdominal fat deposits) where temperatures are more likely to

resemble core body temperatures, and thus detemir will be less bound to albumin and more

freely able to pass the capillary wall and act on target adipose cells.

122

Why is it important for an insulin to act more on the liver than in the periphery?

Glucose metabolism:

As discussed earlier the brain almost exclusively utilizes glucose as a source of energy

(Cryer 1997). The brain has high energy requirements (required to maintain the ionic

gradient across nerve membranes, critical for maintenance of brain activity) estimated at

approximately 1 mg/kg/min corresponding to about lOOg glucose per 24 hours, however it

has low energy reserves (enough for only a few minutes), and is unable to synthesize

glucose (Bolli, 1998). Intricate mechanisms to protect the supply of glucose to the brain

have therefore evolved. Only the liver and the kidney are able to release glucose. This

release results from the presence of glucose-6-phosphatase in these organs which liberates

glucose into the bloodstream (Bolli and Fanelli, 1999). It is generally believed that the liver

makes the major quantitative contribution during this state, whereas the kidney has a minor

role, except during prolonged starvation, when it is estimated that the kidney may contribute

almost half of glucose production (Bolli 1998, Cahill et al,1966, and Cahill, 1970).

Blood glucose is sensed in the brain (Frohman and Nagai, 1976, Ritter et al, 1981, Borg et

al, 1995), pancreas(Taborsky et al, 1998) and liver (Connolly et al, 1996), each of these

organs is involved in the counter-regulation response to low blood glucose in healthy

subjects. A fall in blood sugar concentration that may occur in the postabsorptive state leads

initially to a reduction in insulin secretion, by direct action of glucose on p-cells of the islets

of Langerhans (Cryer 1997, Bolli, 1998). This leads to a reduction in the plasma insulin

concentration in the portal circulation, and therefore a reduction in the suppression of

hepatic glucose output (or a rise in hepatic glucose output). If the blood glucose continues

to drop counter-regulatory hormones are stimulated and released.

As discussed earlier in the introduction section on glucose metabolism, all counter

regulatory responses to hypoglycaemia increase hepatic glucose output either directly alone

or in combination with an indirect effect by the stimulation of lipolysis (Bolli, 1998), some

also decrease peripheral glucose uptake. In normal physiology these defence mechanisms

123

are appropriate as insulin is released into the portal circulation and has its first effects at the

liver, and the liver is essential in the production of glucose to support brain metabolism

(Bolli, 1998). In insulin dependent diabetes no insulin is released by the pancreas

exogenous insulin is administered by subcutaneous delivery, it circulates peripherally first,

and is controlled by pharmacokinetics, with peak actions occurring 1 to 8 hours after

injection (Bolli, 1998). The insulin profile does not resemble physiological insulin profiles

of a normal 24 hour day, and is unable to respond to changing insuhn need (Amiel, 1998,

Cryer et al, 2003). In this situation the liver is relatively underinsulinised and peripheral

tissue over insulinised. In the event of hypoglycaemia, the first line of counter regulation

(that is a decrease in insulin release) is not possible, insulin levels remain inappropriately

high suppressing hepatic glucose output, increasing peripheral glucose uptake and inhibiting

lipolysis. Further counter regulation has evolved to exert itself primarily at the liver,

suppression of hepatic glucose output is thus easily overcome since in these circumstances

as the liver is relatively underinsulinsed however the peripheral over insulinisation presents

a more difficult obstacle, and therefore it could be argued that greater counter regulation has

to occur to prevent a further fall in plasma glucose and maintain the brain’s glucose supply.

This may cause later hyperglycaemia as a result of the sustained release of counter

regulatory hormones and their prolonged duration of action may be a factor in the overall

deterioration in glycaemic control seen in patients with frequent hypoglycaemia.

Hypoglycaemia is a common problem encountered in the treatment of diabetes. In one

study on 411 patients with type 1 diabetes (Pramming et a l, 1991) patients had 1.8 episodes

of mild hypoglycaemia per patient per week, and 0.027 episodes of severe hypoglycaemia

per patient per week. Others have suggested people with diabetes have an average of 2

episodes of symptomatic hypoglycaemia a week (Reichard, 1991, Macleod et al, 1993), and

an estimated 2-4% of deaths of people with diabetes are attributable to hypoglycaemia

(Laing et al, 1999). Socially this also causes problems to patients with diabetes with around

three times the percentage of people with type 1 diabetes being refused car insurance than

those without diabetes (Songer et a l, 1991). In the DCCT and UKPDS studies rates of

severe hypoglycaemia were higher for a given HbAlc in conventionally treated patients than

those on intensive treatment regimes, however those on intensive treatment regimes were

124

more likely to have more frequent hypoglycaemia. Hypoglycaemia seems to be the price to

be paid for improving rates of progression to complication.

Duration of diabetes is associated with decreases in the hormonal response to

hypoglycaemia (Bolli et al., 1983). This can lead to a vicious circle of hypoglycaemia

causing impaired physiological responses to hypoglycaemia, causing reduced awareness to

hypoglycaemia, causing increased vulnerability to further episodes and thus more

hypoglycaemia.

Lipid metabolism and weight gain:

The counterregulatory hormone response to insulin acts primarily by increasing glucose Ra.

This is less effective against exogenous insulin, which acts mainly to increase glucose Rd.

Thus an insulin analogue exerting greater effects on the liver than the periphery has the

potential to reduce hypoglycaemic episodes and may reduce weight gain through less

snacking to avoid hypoglycaemia. Peripheral hyperinsulinaemia increases peripheral

glucose uptake and lipogenesis and decreases lipolysis contributing to the weight gain

associated with insulin therapy (Andreani, 1999). Kruszynska et al (1985) investigated the

effects on carbohydrate metabolism of peripheral insulin delivery compared to portal insulin

delivery. They found in rats that hepatic glycogen concentrations are lower in rats receiving

insulin into the systemic circulation compared to those rats receiving insulin via the portal

circulation. Others have shown that sensors in the liver to increased intracellular ATP

concentrations affect eating behaviour via signals transmitted via vagus afferents from the

liver to the brain (Mayer, 1991, Freidman, 1995, Langhans, 1996). Although the control of

eating behaviour remains a highly complex and controversial subject with many factors

having been shown to influence it. Nonetheless recent large-scale clinical trials comparing

detemir and NPH provide support for the theory that by acting in a more physiological way

detemir has advantages over NPH. These trials have shown that nocturnal hypoglycaemic

episodes are reduced and weight gain is lower with insulin detemir when compared to NPH

insulin (Russell-Jones et a l , 2002, De Leeuw et al 2002, Vague et al 2003).

125

IGF-l/GH secretion:

In patients with type 1 diabetes growth hormone levels have been found to be raised

throughout the day with increased frequency and amplitude of pulsatility and exaggerated

responses to a wide variety of stimuli (Tamborlane et al, 1979). Although there is a marked

increase in growth hormone, IGF-1 levels are at the low end of normal or are suppressed

(Tan et al, 1986). IGF-1 levels are not normalised with intensive therapy or by maintainence

of good control (Amiel et al, 1984). Delivery of insulin via the portal system influences

hepatic IGF-1 production. In rats intraperitoneal insulin given to mimic portal insulin

increases IGF-1 mRNA expression and systemic IGF-1 levels (Russell-Jones DL, 1992),

Griffen et al (1987) also provided evidence that insulin delivered via the portal circulation is

more effective than systemic insulin at restoring IGF-I levels in diabetic rats.

Subcutaneously delivered insulin leads to unphysiological low portal insulin and this may

explain why, despite intensive s.c. insulin therapy, circulating IGF-I levels in diabetic

patients remain either low or at best low normal (Rudolf et al 1981). IGF-1 itself has a

negative feedback effect on GH secretion. It seems likely that in IDDM and NIDDM the

low level of IGF-1 is sensed at the hypothalamus/pituitary level, and GH is hypersecreted

(Simpson et al, 1998). This will lead to a distorted GH/IGF-1 axis with raised GH and

inappropriately low IGF-1. This elevated GH contributes to the insulin resistance and

causes the “dawn phenomenon” which is difficult to control (Fowelin, et al, 1991, Periello et

al, 1990). Changes in GH levels have been implicated in the development of microvascular

complications, particularly retinopathy but also nephropathy (Chiarelli et al, 2000). Low

levels of circulating IGF-1 have also been implicated to be associated with neuropathy

(Migdalis et al, 1995). Some authors have suggested that relative underinsulinisation of the

liver may play an important role in the development of microvascular complications

(Sonksen et al, 1993, Wurzberg et al 1995, Janssen et al, 2000). It is possible therefore to

speculate that an insulin that has greater effect at the liver may be associated with reduced

frequency of microvascular complications.

126

Is detemir hepatoselective?

From other studies; A study reported by Russell-Jones et al (2004) compared once daily

nocturnal injection of detemir and NPH in a 6 month multicentre randomised controlled

trial. They found that treatment for 6 months with insulin detemir resulted in slight lowering

of HbAlc, lowering of fasting plasma glucose, less fluctuation in blood glucose levels

especially at night, less nocturnal hypoglycaemia in the last 5 months of treatment and a

relative decrease in weight in comparison to the group treated with NPH. Vague et al (2003)

reported the efficacy and tolerability of detemir compared to NPH in a basal bolus regime

using asp art as the bolus insulin. They also reported that detemir had similar metabolic

control judged by HbAlc but found a lower within subject variation in fasting blood

glucose, a flatter and more stable blood glucose profile during the night, a lower risk of

hypoglycaemia, a relative decrease in weight gain and a good safety profile. De Leeuw et

al (2002) reported a study using NPH or detemir as a twice daily basal regime, and using

aspart as the bolus insulin. Again it was found that detemir had similar glycaemic control

over 12 months, significantly lower risk of hypoglycaemia and a significant and favourable

weight reduction in comparison to NPH. Pieber et al (2004) reported a study using a basal

bolus regime with aspart as the bolus insulin and twice daily basal injections of detemir or

NPH. The NPH was given at breakfast and bedtime, in one detemir arm detemir was given

as the NPH (ie breakfast and bedtime) and in the other it was given at breakfast and at

suppertime. Compared to NPH detemir provided significantly lower fasting plasma glucose

in both arms, significantly lower within subject variation in both arms and less weight gain.

Thus suggesting that detemir may be administered more flexibly than NPH. Home et al

(2004) reported a similar study with type 1 subjects randomised to receive their basal insulin

in a basal bolus regime as either NPH morning and bedtime, detemir morning and bedtime

or detemir 12 hours apart. Again they found detemir produced significantly lower fasting

plasma glucose, lower within subject variability, lower nocturnal hypoglycaemia, less

weight gain and flexible administration. Haak et al (2004) reported a study with type 2

individuals using a basal bolus regime with aspart as the bolus insulin, and twice daily NPH

or detemir. They found lower risk of hypoglycaemia, less weight increase and similar

glycaemic control. Hermansen et al (2004) compared an analogue basal bolus regime of

127

twice daily detemir (morning and bedtime) with aspart as the bolus insulin to a human

insulin regime of twice daily NPH (morning and bedtime) with actrapid as the bolus insulin.

Subjects had type 1 diabetes and were randomised in an open label trial for 18 weeks. They

found the analogue regime lowered HbAlc more than the human insulin regime and caused

less nocturnal hypoglycaemia.

From this study: The equivalence in glucose infusion rate profiles is of key importance

when interpreting the comparative effects of two insulin preparations on glucose Ra and

glucose Rd. Insulin exhibits a different proportion of action at each of these sites at varying

concentrations. In diabetic patients (Brown et al 1978) and in diabetic dogs (Issekutz et al

1974) it has been demonstrated that peripheral infusion of insulin at low rates of

0.5mU/kg/min without euglycaemia reduces hepatic glucose output without increase in

glucose Rd. Thus at low concentrations of insulin the majority of the glucose lowering

effect occurs by suppression of glucose Ra, with only a small effect on glucose Rd. At

higher concentrations the majority of action occurs by glucose Rd once glucose Ra has

effectively been completely suppressed (Sonksen and Sonksen, 2000). Therefore results

from studies that compare the effects of two insulin preparations on glucose Ra and Rd

where the glucose infusion profile is not equivalent must be treated with caution as the

difference in effect may be purely related to dose.

In this study we have demonstrated in healthy subjects that 9nmol/kg insulin detemir is

equipotent to 0.3 iu/kg NPH, with a very similar time action profile, we have also

demonstrated in subjects with Type 1 diabetes that 18nmol/kg insulin detemir is equipotent

to 0.6 iu/kg NPH, with a similar time action profile There were small but significant

differences in the effect of this dose of insulin detemir on glucose Ra and a lesser non

significant effect on glucose Rd compared to the equipotent dose of NPH suggesting a

greater effect on liver than on peripheral tissue. In the group with type 1 diabetes there were

also small but significant differences in the effect of this dose of insulin detemir on glucose

Rd but no measurable differences in the effect on glucose Ra (which at this dose of insulin

is almost completely suppressed) suggesting NPH has a greater effect on the periphery than

an equipotent dose of insulin detemir.

128

The reduced effect of insulin detemir on peripheral tissues was confirmed by the

measurements of NEFA concentration. The suppression of NEFA concentrations by insulin

detemir was less than with an equipotent dose of NPH, both in healthy subjects and to a

lesser extent in those with type 1 diabetes (the effect in subjects with type 1 diabetes was

further clouded by apparent although non statistical differences at baseline). It has been

suggested that insulin’s suppressive effect on peripheral lipolysis with a decrease in NEFA

concentrations is an important signal mediating an indirect effect of insulin on glucose Ra

(Bergman, 2000). The demonstration in this study, that detemir has greater effect on

glucose Ra but less effect on NEFA concentration than an equipotent dose of NPH insulin,

suggests that the action of detemir on glucose Ra is independent of NEFA delivery to the

liver.

In type 2 subjects the results are more difficult to determine. There was great inter

individual variation in the response to the insulins. There were also technical difficulties

with interpreting the clamp results as both the Ra and Rd for each individual fell after the

baseline bloods had been taken and the injection given at time 0 reaching a nadir at around

60 minutes, suggesting that the patients were not at steady state at the start of the clamp.

This is probably a result of the technical difficulties associated with the study design of

controlling the blood glucose using intravenous insulin and converting to subcutaneous

insulin at time 0 even though the delayed absorption of these insulins would mean that

subjects would effectively be without insulin for a short period at the beginning of the study.

It is not clear why the effect of this is more pronounced in the subjects with Type 2 diabetes.

However when analysed using time 60 minutes as baseline there was greater effect on

glucose Ra with insulin detemir than the equipotent dose of NPH, and a small (although not

significant) reduction in the effect on glucose Rd for detemir compared to NPH.

Looking at the effect on lipid metabolism in subjects with type 2 diabetes findings are

similar to those in subjects with type 1 diabetes and healthy subjects, in that there was less

suppression of glycerol Ra with insulin detemir compared with NPH (although this

difference again was clouded by apparent although non statistical differences at baseline)

129

and less effect on NEFA suppression, although the latter did not reach statistical

significance. There were no measurable differences in these insulins effects on IGF-1 or

IGFBP-1 in subjects with type 1 or type 2 diabetes.

When analysed together as a combined set the data from these 19 subjects show the glucose

lowering effect of these insulins are not different. Insulin detemir suppresses hepatic

glucose output equivalently but has less effect on peripheral glucose uptake. This further

supports our hypothesis.

The clinical implications of an insulin that may act in a more hepatoselective way are

exciting. As described above by acting more physiologically detemir may give protection

from profound hypoglycaemia and may reduce the frequency of hypoglycaemic episodes.

Studies so far indeed indicate this reduced hypoglycaemia risk. This is of great importance

to patients in whom the fear of hypoglycaemia is often a barrier to improved control and

thus lowered risk of micro and macrovascular complications. There have been no studies

with long enough follow up to determine the long term benefits of detemir with respect to its

potential effects on reducing microvascular complications as a result of its differential effect

on IGF-1. These advantages may only become apparent with decades of use of this insulin.

Limitations of this work:

This study had many limitations, lack of resources and man-power meant that it was not

feasible to attempt to achieve more than 6 analysable sets of data in each group of patients.

This in itself meant that we performed nearly 1400 hours of euglycaemic clamping. With

low subject numbers it was difficult at times to demonstrate statistically significant

differences in our results although similar trends ran throughout our findings.

Constraints of metabolic study:

Metabolic studies often provide such results with day to day intra-individual variation

making interpretation more difficult. Also the calculations used in these studies rely on

130

assumptions to determine glucose Ra and glucose Rd, and data entered in these calculations

undergoes a smoothing program which may result in important differences being minimised.

As described above it is of utmost importance that the glucose infusion rate profiles be as

equivalent as possible for comparison between two insulins (ie their equipotency must be as

near as possible) for interpretation of differences to be truly due to differences in insulin

action and not merely differences due to dose. We were exceptionally fortunate that in the

healthy subjects the glucose infusion rates were inseparable and thus the small differences

that we found could be truly attributed to effect rather than dose.

These differences appear small but if each dose results in 10% greater action at the liver than

the periphery as demonstrated in the healthy subjects in this study, then the cumulative

effect over the years of administration may be clinically relevant.

The results from the two groups of patients with diabetes has been more difficult to

interpret. The studies were technically much more difficult, in order to avoid “hangover

effect” from any other long acting insulin, patients were asked to omit their long acting

insulin from 24 hours prior to the study. However we know from data presented above that

NPH has highly variable absorption, and therefore it is possible that some of the NPH

previously injected was still acting thus accounting for the greater intra-individual variation

in results in this group of patients. Also lowering blood glucose after presentation on the

study day was not without difficulties, as rapid lowering meant difficulties arose during the

baseline period and looking at the data from both diabetic groups it is clear that subjects

were not at steady state at baseline or for the 30-60 minutes following subcutaneous

injection as the calculations clearly show falls in both Ra and Rd during this time period.

The type 2 patient subject group also shows considerable variation. The very nature of this

group of patients is variable given that they represent a spectrum of diabetes from insulin

resistance to decreased insulin production. Therefore as a group their results are more

variable. This means that the small differences we demonstrated in other groups in this

study were more difficult to demonstrate in this group of patients. Also this group consisted

of some patients with higher abdominal girth (although this was not actually measured) and

131

as previously discussed this may lead to a greater effect of detemir on central deposits of

adipose tissue and thus less relative effect on the liver. Given these considerations it may

have been prudent to increase the numbers of subjects studied in the type 2 group, or

controlled for other variables such as abdominal girth.

In conclusion this study shows that when compared to an equipotent dose of NPH, detemir

has a greater effect on glucose metabolism in the liver than in peripheral tissues. This

reduced effect of insulin detemir on peripheral tissues was confirmed by less of an effect on

NEFA concentrations than NPH. This suggests that this insulin analogue has the potential to

imitate the normal insulin gradient, thus mimicking normal insulin physiology more closely

than any other insulin currently available. This may mean that this insulin has therapeutic

advantages over other currently available insulin preparations.

132

Chapter 5

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