Recombinant Insulin

Year of Introduction: 1982, marking the production of human insulin recombinantly in E. coli for therapeutic use.
1990s Onwards: Development of multiple variants of insulin produced recombinantly.

First Method:
- A and B chains of insulin are expressed separately in E. coli.
- In-vitro mixing occurs under oxidizing conditions to form di-sulphide bonds, resulting in a product known as human insulin crb.
Alternate Method (Eli Lilly Development):
- Involves the overexpression of pre-proinsulin in E. coli.
- This method includes in-vitro proteolytic cleavage of the C-peptide, leading to human insulin prb.

Chromatographic Purification Techniques:
1. Ion Exchange: Separation based on charge.
2. Size Exclusion: Separation based on size.
3. Hydrophobic Interaction Chromatography (often reverse-phase HPLC): Based on relative hydrophobicity.
- These methods yield a high purity insulin product (~99% purity), significantly reducing immunogenic response and contaminants associated with E. coli.

pH: Recombinant human insulin and its analogues are usually formulated at neutral pH, with the exception of insulin glargine (addressed in future slides).
Additives:
- Zinc: Serves as a stabilizer aiding in hexamer formation.
- Antimicrobial Agents: Such as phenol and m-cresol, which also assist in hexamer formation.
- Isotonic Agents: Electron solutions such as chloride, acetate, and glycerol to minimize tissue damage and injection pain.
- Buffer: Such as sodium phosphate, to prevent pH drift.

Achieved via site-directed mutagenesis or post-translational modification, the goals include:
- Creating slower or faster-acting variants of insulin.
- Developing variants with higher affinity for insulin receptors, thus requiring less insulin per dose.
Targeted Amino Acids: Mutations often occur at amino acid positions: A1, A5, A19, A21, B10, B16, B23-25.
- Example mutation: His to Glu at position B10 increases activity in-vitro by 5-fold but doesn't necessarily reflect in-vivo activity, indicating a focus on mutations impacting oligomer formation.

Mechanisms: Recombinant human insulin generally forms hexamers when injected at therapeutic concentrations. Upon entering blood and combining with zinc, hexamer formation occurs at contact points.
Monomerization: In-vivo must first reconfigure the insulin into monomers, slowing activity.
Engineered Variants: Insulins that do not revert to multimeric structures are faster acting. Suggested contact points for oligomerization include B8, B9, B12-13, B16, and B23–28, facilitating mutations for improved speed.

Insertion of Charged or Bulky Amino Acids: Added at B-chain oligomerization points to promote charge repulsion or steric hindrance, facilitating faster absorption from injection sites.
Possible practical deployment of these engineered insulins includes injections at mealtime rather than pre-meal.
Example Product: Insulin lispro (Humalog) as the first approved short-acting insulin variant.

Total of 150 insulin products approved, as per HPRA website.
Types of Insulin Available:
- Native recombinant human insulin
- Long-Acting: Glargine, Detemir, Degludec
- Fast-Acting: Glulisine, Aspart, Lispro
- Mixed formulations such as insulin degludec with Liraglutide (GLP-1 receptor agonist), and mixes of both long and fast-acting insulins for mealtime glucose control.

Insulin Glulisine: First produced by Sanofi-Aventis, approved in early 2004. Engineered to disrupt monomer-monomer interactions.
- B29 Lys to Gln substitution - disrupts monomer - monomer interaction
- B3 Asn to Lys substitution - disrupt Zn stabilisation of hexamer
Insulin Aspart: Developed by Novo Nordisk, available since 1999, it incorporates Aspartic acid substitution at B28 to enhance charge repulsion and suppress hexamer formation - faster acting monomer.
Insulin Lispro: Notable for altering the B28-B29 proline-lysine sequence. The change diminishes critical hydrophobic interactions, substantially lowering the dimerization constant compared to native insulin.

Production: Expressed in E. coli. Modifications include the addition of 2 Arg at the C-terminal of the B chain and substituting asparagine at A21 for glycine.
Characteristics: Lowers pI from 5.4 to 6.7, fully soluble at pH 4.0. PH increase post-subcutaneous injection causes precipitation and allows slow release over 24 hours, necessitating once-daily dosing.

Developed by Novo Nordisk, approved in 2004. Characterized by the deletion of Thr B30 and addition of a C14 fatty acid at Lys B29. This alteration facilitates reversible binding to albumin, which elongates absorption duration, allowing for consistent blood levels of insulin.

Ultra-long-acting insulin, first approved in 2014. Characterized by a similar B29 modification with a 16 fatty acid di-chain, it exhibits slow monomerization and prolonged action duration exceeding 42 hours.

Insulin Type	Onset (h)	Peak Activity (h)	Duration (h)
Regular	~1	2-3	5-8
Lispro	Within 15 min	~1	3-5
Aspart	Within 15 min	1-3	3-5
Glulisine	0.25 - 0.5	0.5 - 1	4
Glargine	1.5	Flat	24
Detemir	3-4	6-8	Up to 20-24
Degludec	1	9	42

Hypoglycemia: Leading to excessively low blood glucose levels.
Weight Gain: Often arises from hypoglycemia, necessitating high-calorie intake and increased fatty acid uptake.
Local Reactions: Allergic responses to insulin or additives; lipoatrophy has been minimized with recombinant insulins.
Cancer Risks: Retrospective studies indicate a potential correlation between insulin dosages and cancer due to the mitogenic properties of insulin, though limitations exist.

The predominant method is parenteral subcutaneous injection. Additional methods include:
- Continuous Subcutaneous Insulin Infusion: Utilized with rapid-acting insulin formulations.
- Pulmonary Delivery: Using aerosol devices, though largely withdrawn due to poor patient acceptance.
- Oral Delivery: Tablet forms are in consideration.

Long-Acting Insulin Innovations: Novo Nordisk is working on a once-weekly long-acting insulin (LAI287 - Icodec), now in phase 3 trials for both Type 1 and Type 2 Diabetes.
Oral Insulin Therapies: Various companies are developing oral insulin options to enhance convenience and effectiveness.
Stem Cell Therapy: Advancements in potential therapies for Type 1 diabetes, focusing on regenerative and improved management solutions.

Topics covered include: Insulin structure and function, diabetes, insulin therapy, recombinant human insulins, modified analogues (fast and long-acting), purification and formulation processes, adverse effects, delivery modalities, and future trends in insulin therapies.