INSULIN PHARMACOLOGYDr. D. K. BrahmaAssociate ProfessorDepartment of PharmacologyNEIGRIHMS, Shillong

Background

Discovered by Banting and Best - 1921Fredrick G. Banting & Charles H. Best - Canadian Scientist – Extracted Insulin from Dog pancreas

2 chain Polypeptide – 51 amino acids & MW 6000Chain-A has 21 and Chain-B has 30 amino acids – connected by two disulfide bondsSource: Porcine, Bovine and Human (Pork = human)Synthesized in β cells of islets of pancreas – single chain 110 amino acids (Preproinsulin)Proinsulin – 86 amino acids Connecting “C” peptide (35 amino acids) removed by proteolysis in Golgi apparatus – Insulin

Source: https://www.researchgate.net/figure/268872215_fig3_Figure-3-Structure-of-pro-insulin-showing-C-peptide-and-the-A-and-B-chains-of-insulin

Diabetes Mellitus

• Hyperglycaemia, glycosuria, hyperlipidaemia, negative nitrogen balance and ketonaemia

• Pathological changes: thickening of capillary basement membrane, increase in blood vessel wall matrix, and cellular proliferation

• Consequences: Lumen narrowing, atherosclerosis, sclerosis of glomerular capillaries, retinopathy, neuropathy and peripheral vascular insuffficency

• Causes of pathological changes: Enhanced non-enzymatic glycosylation of tissue proteins and accumulation of large amounts of sorbitol

• Glycosylated haemoglobin (HbA1c ) – Index of protein glycosylation

Diabetes Mellitus - Types

• Type I: Insulin dependent DM (IDDM) / Juvenile onset diabetes – low circulating insulin level – prone to ketoacidocis

– destruction of β cells in pancreatic islets– Type IA – antibodies destroying β cells– Type IB: Idiopathic, no β cell antibody detectable– Low degree of genetic predisposition

• Type II Noninsulin-dependent DM (NIDDM): no loss of moderate loss of β cell, low/normal/high insulin in circulation, no anti- β-cell antibody – genetic predisposition

• Causes: (1) Abnormality in gluco-receptor in β cell – needs higher conc. of glucose; (2) Relative β cell deficiency

– Down regulation of insulin receptors in peripheral tissues– Many Hypertensives are hyperinsulinaemic – normoglycaemic but dyslipidaemia,

hyperuricaemia, abdominal obesity (metabolic syndrome) – Insulin Resistance– Excess of hyperglycamic hormones

• Other Types: Type III (!) – LADA and MODY; Type IV – Pancreactomy and gestational diabetes mellitus (GDM)

Regulation of Secretion

Basal condition – 1 U per hour – more after meals

Regulated by – Chemical, hormonal and neural mechanisms

3. Neural: Sypmpathetic and vagal Nerve influennce in islets – (i) alpha-2 stimulation decreases insulin release (predominant) (ii) beta-2 cell stimulation increases Insulin release; (3) Vagal stimulation increases Insulin relese – IP3DAG

1. Chemical (glucosensor) 2. Hormonal

Glucokinase

Glucosensor

Source: Essentials of Medical pharmacology by KD Tripathi – 7th Edition, JAYPEE, 2013

Source: https://courses.washington.edu/conj/bess/humoral/humoralregulation.htm

Actions of Insulin

Meal derived glucose, amino acids, fatty acids and fuel storage Major anabolic hormone – synthesis of glycogen, lipids and proteins1. Facilitates glucose transport across cell membrane – skeletal muscle and fats - Liver, brain,

RBC, WBC and renal medullary cells are independent

2. Intracellular utilization of glucose – phosphorylation to form Glucose-6-PO4 – increased production of glucokinase – also glycogen synthase

3. Inhibits gluconeogenesis from protein, FFA and glycerol (diverted to liver) – by decreasing synthesis of (gene mediated) phosphoenol pyruvate carboxykinase.

4. Inhibits lipolysis in adipose tissues – favours triglyceride synthesis – in diabetes, increased FFA and glycerol (Acetyl-CoA) – ketone bodies

5.Facilitates amino acid entry and their synthesis to protein – also inhibits protein breakdown in muscles and other cells – in its absence excess pyruvate, glucose and urea (negative nitrogen balance)

Insulin - mechanism of action

T – Tyrosine residue; GLUT4 – Glucose transporter, IRS – Insulin receptor substrate protein, T-PrK – Tyrosine protein kinase, Ras – Regulator of cell division and differentiation

Source: Essentials of Medical pharmacology by KD Tripathi – 7th Edition, JAYPEE, 2013

Fate of Insulin

Distributed only extracellularly – given orally gets degraded in GIT

Secreted and injected Insulin – metabolized in liver, kidneys and muscles

First pass metabolism - 50% of Insulin passing through portal vein

Degradation after receptor mediated internalization

Biotransformation – sulfide bonds are reduced – chain A and B are separated – broken down to amino acids

Source - https://www.slideshare.net/aishahadalicia/insulin-and-its-mechanism-of-action-31298888

Preparations of Insulin

Classically – produced from beef and pork pancreas

Contains 1% (10, 000 ppm) other proteins – proinsulin, polypeptides, pancreatic proteins etc.) – potentially antigenic

Replaced with highly purified pork/beef insulin/recombinant human insulin/insulin analogues

Single peak and Monocomponent insulin (MC) – proinsulin <10 ppm – stable, less resistance and lipodystrophy

Unitage/Assay: I U reduces fasting rabbit blood sugar by 45 mg/dl or potency to induce hypoglycaemic convulsion in mice

I mg of International Standard of Insulin = 28 units Radioimmunoassay or enzyme immunoassay

Types of preparations – Regular (Soluble) Insulin

Buffered neutral pH solution unmodified insulin stabilized by small amount of zinc

Forms hexamers around zinc ions – released slowly and gradually by dilution on SC administration

Peak onset 2- 3 hours and lasts for 6-8 hours Drawbacks:

Before meals – early postprandial hyperglycaemia and late post prandial hypoglycaemia – injected ½ to 1 hour before

Do not provide basal level of action – interdigestive period Slow onset of action is not applicable for IV injection

Long acting – modified or retard preparations

Insulin preparations - Purified

Rendered insoluble - complexed with protamine or excess zinc

Lente (Insulin-zinc suspension): 2 types Ultrelente: Large particle size, crystalline and insoluble in water – long acting Semilente: Small particle

size, amorphous – short acting; Lente: 7:3 ratio mixture

Isophane (Neutral Protamine Hagedorn or NPH) insulin: Protamine added just sufficient to complex all insulin molecules

Neither are in free form – neutral pH On injection: dissociate slowly intermediate action Used in combination with regular insulin in 70:30 ratio or 50:50 Injected twice daily before breakfast and dinner (split-regimen)

Available preparations: Highly purified MC pork regular insulin, highly purified MC pork lente, Highly purified MC NPH, highly purified regular insulin and Isophane (30:70 ratio)

Human Insulin

Same amino acid sequence as human insulin - produced by recombinant DNA technology

In Es che richia c o li – proinsulin recombinant bacteria (prb) and in yeast – precursor yeast recombinant (pyr) or by enzymatic modification of porcine insulin

Human actrapid (regular insulin) – 40 U/ml

Human monotard (lente), human insulatard (NPH), Human mixtard (30:50), Insuman (50:50)

Advantages: More water soluble and hydrophobic, more rapid absorption than porcine or bovine, more defined peak, shorter duration of action

Insulin analogues

Recombinant DNA technology, modified pharmacokinetic – greater stability and consistency

Insulin lispro: Reversing Proline and lysine at B 28 and B 29 position – quick acting, just before meals

Insulin aspart: B 28 is replaced by aspartic acid – mimics physiological insulin

Insulin glulisine: Replacing aspartic acid at B 23 by lysine and glutamic acid replacing lysine at B 29 – continuous SC insulin infusion (CSII)

Insulin glargine: Long- acting – precipitates at neutral pH on SC injection – depot created – slow dissociation – 24 hours low blood level – usually at bed time

Reactions and Drug Interactions (DIs)

HYPOGLYCAEMIA: Labile diabetics Causes: Injection of large doses, missing a meal after injection, vigorous exercise Symptoms: Sweating, anxiety, palpitation, tremor – counter regulatory; dizziness,

headache, behavioural changes, visual disturbances, hunger, fatigue, weakness, muscular incoordination etc. - due to deprivation - Below < 40 mg/dl – seizure and coma

Treatment: Glucose orally and IV – Glucagon – 0.5 to 1 mg IV

Local reactions (swelling), lipodystrophy, Allergy, Oedema

Drug Interactions: Beta blockers (beta-2; prolong hypoglycaemia), Thiazides, diuretics, steroids, OCPs (raises blood sugar), acute alcohol ingestion (hypoglycaemia – glycogen depletion), Lithium and aspirin (hypoglycaemia – enhance insulin secretion)

Uses of Insulin

Purpose: Restore metabolism to normal, avoid symptoms due to hyperglycaemia and glycosuria and prevent complications

Indications: Type 1 DM, Post pancreatectomy diabetes and gestational diabetes; Type 2 DM: Not conrolled by diet and exercise, failure of oral hypoglycaemics, under wight, tide over crisis and complications (ketoacidosis)

Treatment: According to requirement and convenience of each patient – by testing urine and blood glucose level

Type 1: usually 0.4 to 0.8 U/kg/day (severity and obesity) Target: obtain basal control – no single daily dose of long/intermediate/short acting ones can fulfill Multiple (2 – 4) injections daily of long and short acting or Long acting with Oral hypoglycaemics (meal

time) Conventionally, s p lit-m ix e d re g im e : mixture of regular with lente/isophane (30:70 or 50;50) – before

breakfast and before dinner

Uses of Insulin – contd.

Basal bolus regime: 3 - 4 daily injections - a long acting (glargine) insulin before breakfast or before bed time with 2-3 meal time injections of short rapidly acting (lispro or aspart)

Other uses: Diabetic Ketoacidosis (Coma), Hyperosmolar (non-ketotic hyperglycaemic) coma

Insulin resistance: Type 2 DM, Age, large body fats, pregnancy, OCPs – acromegally, Cushing`s syndrome, phaeochromocytoma etc.

Acute Insulin resistance: Infection, trauma, surgery, stress etc.

Newer Insulin Delivery devices: Insulin syringe, Pen devices, inhalled insulin, Insulin pumps (CSII) etc.

Summary of Insulin preparations

Short acting: Regular soluble insulin – clear appearance; 6-8 hours – can be mixed with others except glargine

Intermediate acting: Lente, NPH or Isophane – cloudy; 20-24 hours - Regular

Long acting: Glargine and detemir – clear; 24 hours – cannot be mixed with others (can be combined)

Rapid acting: lispro, aspart, glulisine – clear; 3-5 hours – can be mixed with Regular and NPH

Thank youInsulin prefilled syringe

Insulin pump