Formulation of insulin

Question 1

what are Biomacromolecules/Biologics?

Answer 1

any macromolecule (such as protein, nucleic acid or polysaccharide) of biological origin.
-e.g. insulin

Question 2

how does the delivery and manufacture of Small molecules differ from that of Biologicals

Answer 2

1) Small molecules are chemically synthesised and can be formulated into tablets to be ingested
2) Biologicals are made for example in eggs ( flu vaccine) and administered by injection to reach the site of action

Question 3

Biologicals can be classified into 4 categories. state these 4 categories

Answer 3

1) gene therapy
2) proteins and peptides
3) monoclonal antibodies
4) vaccines

Question 4

name some protein and peptide drugs

Answer 4

1) Insulin
2) Growth hormone
3) Clotting/blood factors

Question 5

outline the three challenges for all biologics

Answer 5

1) Complexity: of biologics higher than typical drugs
2) Instability: of biomacromolecules inherent due to biological origin
3) Availability: often limited by large molecular size

4) also Immunogenicity immune response to drug (hypersensitivity) can lead to loss of efficacy and more serious adverse effects

Question 6

the complexity of biologics often higher than typical small molecule drugs. state what problems are associated with this complexity.

Answer 6

1) More to go wrong, more expensive to manufacture

2) Molecular complexity, Functional complexity, Complex composition

Question 7

Why are Biologics typically so unstable?

Answer 7

1) Instability of biomacromolecules due to biological origin
2) Conditions must be compatible with biological molecules
3) but this means they are good food for microbes
4) many reactions of biological molecules are thermodynamically favourable in these conditions
5) almost all Biologics contain proteins- and proteins are relatively unstable

Question 8

discuss why the delivery of biologics is a problem

Answer 8

1) Availability often limited by large molecular size
2) poor permeability through epithelia
3) Access to tissues from blood vessels ( cant go through blood vessels)
4) cant go through cell membranes (although drug target usually not within cells)

Question 9

Biomacromolecules won’t pass through epithelia or membranes and they cant be formulated orally. explain why biomacromolecules cant be formulated orally and state the route by which they should be administered.

Answer 9

1) Digested by gastric and intestinal components. Oral biomacromolecules = food!
2) Typically can’t use oral delivery => “PARENTERAL”

Question 10

Insulin is key example of a protein drug. outline the 3 ways we formulate insulin

Answer 10

1) by changing the Protein sequence (& source)
2) Physical state of protein can change (phase, crystallinity)
3) change the protein structure e.g. complex with zinc

Question 11

state and explain the two ways by which pharmaceutical proteins are produced/manufactured?

Answer 11

1) Biotechnology: Produced in microbial or animal cell cultures and isolated via chromatographic and filtration steps
- e.g: insulin, erythropoietin, monoclonal antibodies
2) Isolate from blood (or animal tissue)
- Protein isolation focused on reduction of viral contamination
- e.g: albumin and blood clotting factors

Question 12

state the problems with isolating proteins from blood and using biotechnology to manufacture proteins

Answer 12

1) Biotechnology:
- Produce in microbes: non-mammalian protein
- Produce in animal cells: high cost
2) Isolate from blood (or animal tissue)
- Purify from animals- non-human sequence
- Purify from humans- viral infection, BSE prion infection

Question 13

explain why insulin is the most intensely researched therapeutic macromolecule

Answer 13

1) Large number of diabetics
2) Long term (lifetime) treatment
3) Precise, continuous delivery needed for dose control
4) Dire consequence of failure to deliver. too much or too little insulin can kill you

Question 14

outline the source of different types of insulin

Answer 14

1) Purified animal insulin
- Still available: porcine (pig) and bovine (cow; rare use)
2) Technological breakthrough for recombinant DNA technology: “Recombinant” human insulin

Question 15

Variants of human insulin have now been genetically engineered (altered amino acid sequence) with changes to speed of onset and duration of activity. give examples of Rapid-acting Insulins

Answer 15

Onset of action of 15 minutes; Duration of 2-5 hours

1) Insulin lispro (Humalog®),
2) insulin aspart (NovoRapid®),
3) insulin glulysine (Apidra®)

Question 16

outline how Insulin aspart modification works

Answer 16

Insulin aspart – position B28 proline replaced with negatively charged aspartic acid = faster dissociation of hexamer
(an example of formulating by changing amino acid sequence)

Question 17

Long-acting Insulins provide longer effects than intermediate-acting insulins; Normally used once daily to maintain a constant level of insulin (steady state achieved after 2-4 days). give examples of long-acting insulins

Answer 17

1) Insulin glargine (Lantus®)
2) Insulin detemir (Levemir®)
3) Insulin degludec (Tresiba®)

Question 18

explain how Insulin glargine modification works

Answer 18

1) Asparginine residue substituted with glargine at position 21 of A chain = stabilise hexamer interactions
2) Add 2 arginine residues on C-terminus of B chain = less soluble at pH of subcutaneous tissue

Question 19

There are different physical forms of insulin e.g. aggregates/crystals/complexed. explain how we can use this to our advantage in formulation.

Answer 19

1) Modification of aggregation, quaternary structure and crystallinity alters solubility and availability: allows tuning of kinetics of insulin release
2) when injected subcutaneously, a ‘depot’ is formed
Lowest molecular weight monomeric insulin is most rapidly released. Dissociation of aggregates /crystals /complexed insulin results in slow release of active therapeutic

Question 20

as stated earlier another way of formulating insulin is by a change in the structure of protein. explain how complexing to zinc helps to control the release of insulin.

Answer 20

1) Insulin protein can have different quaternary structures
2) 6 single insulin protein molecules forms complex with zinc. 6-mer is ‘stored form’ – also has slower release after injection
3) Single molecule is ‘active form’ – but is released faster by complexing to zinc the release is slower.

Question 21

Insulin regimes are individualised for each patient. outline the following three insulin regimes:

1) One, two or three insulin injections
2) Multiple daily injections
3) Continuous sub-cutaneous insulin infusion

Answer 21

1) One, two or three insulin injections per day of a short-acting or rapid-acting insulin analogue with an intermediate-acting insulin
2) Multiple daily injections. A short-acting or rapid-acting insulin analogue is injected before meals, together with one or more separate daily injections of an intermediate-acting insulin or a long-acting insulin.
3) Continuous sub-cutaneous insulin infusion. A programmable pump delivers a regular or continuous amount of insulin (usually short-acting or rapid-acting insulin analogue) via a SC needle or cannula.

Question 22

summarise the devices used for insulin administration

Answer 22

1) Pre-filled injectors
2) solution for injection plus:
- syringe and needle,
- Injectors, autoinjectors
- Needle-free injectors
- Pumps
3) injectors/pumps

Question 23

how do injectors and pumps work?

Answer 23

1) injector: self-administered subcutaneous injection

2) Pumps: computer controlled infusion through subcutaneous canulla

Question 24

outline the future of insulin delivery and discuss the advantages and disadvantages of each route

Answer 24

1) Needle-free injectors:
- no more needles, many happy users
- supply, complex to use, bruising
2) Inhaled
3) Transdermal
- Challenge:Low bioavailability through skin - high molecular weight, hydrophilic
- precise dosing impossible with cream
4) oral: Market pull drives experimental attempts:
e. g. microencapsulation to protect from gastric degradation
- mechanism of absorption of microencapsulated protein?