Protein therapeutics insulin

List the four insulin formulations

Regular insulin - contains zinc and creaol duration for 5-8 hours

NPH insulin - cresol zinc protonamine and phenol longer acting - the protonamine sticks to insulin so insulin slowly gets released

Lente insulin - zinc and methyl paraben longer acting

Ultralente - cresol zinc phenol. Protamine long acting

Charge wise what does insulin contain

-2 charge due to presence of lysine, histidine arginine

And contains N terminus which is positively charged nh2

And C terminus carboxylic acid which is negatively charged

Acidic groups include all the negatively charged amino acids like glutamic acid and aspartic acid since can lose proton and positively charged include histidine lysine arginine which accept proteins and are basic groups

Briefly how do you produce recombinant insulin

Get human insulin producing gene and get plasmid from bacteria and cut plasmid through restriction enzymes

Then put together to form recombinant dna

Then put Recombinant dna in recombinant bacteria allow it to multiply in fermenter and then extract purify the human insulin to produce lots of human insulin in large quantities

What do insulin properties depend on

Depend on ph temperature and concentration ionic strength

Describe different ph from monomeric form to hexameric to crystallisation

Monomeric insulin - ph 2 and low conc

Then ph 4- dimers form as ph increases

Then above ph 4 complex forms is made and this forms precipitates as amorphous aggregates at ph 4-7

Then amorphous precipitate crystallise at ph 5.6-6

How can you make insulin work faster

Monomer is biologically active and it acts on receptors

Can weaken propensity of insulin self associating - prevent insulin associating in to dimer

By charge repulsion - try to reduce electrostatic attraction between positive and negative charges

How does insulin lispro work different to normal insulin

Insulin lispro works faster since swap positions of proline a cyclic amino acid with lysine which is positively charged

Proline exchanges b28 and b29 causes lysine and glutamic acid to move further away so less electrostatic interaction so less likely to form dimers

Pushes equilibrium away from forming hexamers and instead allows dimers to form quicker then in to monomers and bind to receptor to have effect faster - less likely to make stable hexamers - they're less stable so form dimer quickly into monomer to bind to receptor and have effect

What's the role of zinc in insulin formulation

3 dimers and 2 zinc sites form hexamers

Zinc prevents formation of insulin aggregates

More zinc increases duration - more zinc means more hexamers formed which take longer to dissociate

Name the three types of fasting acting insulins

Lispro - b28 and b29 swap

Aspart novolog- aspartate gets substituted with lysine - so euqaliribrium shifts away forming hexamers means faster rate of action and faster absorption

Glulisine apidra - glu glu interaction is unfavourable means less tendency of dimer and zinc forming hexamers and more likely to have faster rate of action and no clogging

How does the concentration of faster acting insulins compare to normal insulin

Higher concentrations of monomers released in fast acting insulin compared to normal

How does lantus glargine work

It's at ph 4 and it's clear , you inject it subcutaneously in to tissues - body tissue are at high ph neutral 7.4, ph difference causes lantus glargine to go through structural modification forming micropreciptates

Then they slowly release insulin steadily

And take it once a day

How does detemir levimer work

B chain thr(30) gets deleted

Then b chain lys(29) and e amino group gets myrisolated (c14)

Then lipid moiety slows down absorption

Then binds to albumin in circulation

Takes longer to absorb through endothelium

See less weight gain in type two diabetes

What does basal insulin do

Constant peak of insulin throughout the day minimal peaks in activity

What ways have been introduced to increase duration of insulin

Binding to insulin - in determir - once in circulation can bind to albumin

Can have high zinc formulations like zente insulin - more zinc means more hexamers made take longer to dissociate so longer Duration of action

Poorly soluble insulin protoamine like in nph insulin - protoamine binds to insulin allows insulin to be slowly released over time

Ph dependent precipitation - eg lantus glargine - low ph 4 and then inject subcutaneously into tissue and forms higher neutral ph causes structure to be modified into micrprecipitatea which bind to albumin and releases slowly

Can link hexamers together, multimeric insulin - longer duration of action

What are the different conformational states of insulin hexamers

Consists of 6 insulin molecules - can adopt conformational states such as ph temperature presence of zinc ions

Have tense state - more zinc ions at neutral ph hexamers have limited flexibility - t6

Them relaxed state - lower ph, low ph and less zinc ions - hexamers less tightly packed

Then T3r3 - adding zinc ions stabilises tense state then decrease in ph can favour relaxed state

how does degludec work?

Inject subcutaneously causes multi hexamers then pk/pd half life is 25 hours

Then it's in the presence of phenol and zinc form stable soluble dihexamers

Then after injection - the phenol diffuses away then that reorganises to form multi hexamers chains - lasts long time

Then zinc slowly diffuses to release insulin monomers

Then terminal carboxylic acid and fatty acid chains are essential

And they coordinate with zinc atom of the neighbouring hexamer

What stages are degludec in

When inject formulation its in T3R3 state

And when in multihexamer its T6 state - tense state more zinc ions more stable

How do ultrafast insulins act

Form monomer really quickly and exert effect

What do all the insulin analogues contain

Lispro, aspart, glulisine, detimer all contain cresol and phenol

Lispro and aspart contain m cresol and phenol

What excipients does lispro contain and what do they do

Zinc - allow for hexameric formation

Cresol - preservative prevents microbial contamination , maintains ph

Phenol - prevents microbial growth prevents insulin from oxidative degradation