Insulin Flashcards
How is Insulin synthesized?
- Pre-proinsulin is synthesized in the cytosol and transported to the ER
- cleavage #1 in the ER of the signal peptide (leader sequence), protein folding, and -S-S- formation (Proinsulin)-> Transport to the Golgi
- In the Golgi: Packed into vesicles –> cleavage #2 of the C-peptide, the formation of Insulin -> Exocytose of the vesicles with Insulin
Where is the signal peptide and the C-peptide cleaved away?
Signal peptide: in the ER Pre-proinsulin to Proinsulin
C-peptide: Golgi Proninsuil to Insulin
How is Glucose uptake into the cell initiated?
-Insulin binds to Insulin-receptor a dimer Tyrosin-Kinase
-Tyrosin-Kinase starts signaling pathway (phosphorylation of transcription factors)
-Synthesis of GLUT4 transporters for Insulin uptake
What else is being synthesized besides GLUT4 transporters, with Insulin binding to Insulin receptors?
Proteins and enzymes are required to form Glucose into Glycogen and proteins for cell growth
-in the skeletal muscle, liver, and adipose tissue
Why are E.coli and yeast used as an expression system for Insulin production instead of mammalian cells?
-mainly because the structure of Insulin is less complex
-complex structures require mammalian cells for production
-Insulin doesn’t have as much glycosylation, so Ecoli which is unable to create PTMs can produce it
What are the two ways of using an expression system for Insulin production?
-use 2 different expression systems to produce each chain A and chain B and use enzymes to recombine those through disulfide bridging
-use 1 expression system to create Proinsulin and cleave away the C-peptide through PTM
What are the downstream steps of Insulin production?
-Cell lysis and isolation from inclusion body (if E.coli used)
-Isolation of the protein through filtration
-Folding, cleavage, and PTM with enzymes
-Purification (washing) through several chromatographic steps
-Crystallization -> Lyophilization -> Packaging
What is the net charge of Insulin at the isoelectric point and at physiological pH?
-pH at IPE is 5.7 -> net charge is 0
-it will have low solubility at IPE
-physiological pH is 7.4 -> negative net charge -> more soluble
Which type of complex does Insulin form in its active form
and which type of interaction promotes the structure?
Dimer form -> through hydrophilic interactions
Which type of complex does Insulin form when it is stored in the pancreas (Islet cells)?
Hexamers including bivalent Zinc ions
Which preservative can be used to increase the stability of Insulin?
Phenol or m-cresol (Phenolic preservatives)
-> building T6 complexes
Which preservative can be used to increase the stability of Insulin?
Phenol or m-cresol (Phenolic preservatives)
-> building R6 complex
Why is the state of aggregation (dimer, hexamer) important?
-To predict its onset
-a hexamer has a greater MW -> will be absorbed through the lymphatic system
-> takes longer compared to Insulin monomer molecules that are absorbed directly into the vascular system
Which property is used to formulate different Insulin forms (rapid or long-lasting)?
-Monomer-Mulitmer equilibrium
-determines if Insulin is absorbed directly through the vascular system (rapidly) or lymphatic system (slowly)
How is the predominant form of Insulin absorbed in vivo?
In the form of monomers -> 80% vascular -rapidly
(only 20% lymphatic - slow)
What are the two types of Insulin secretion throughout the day in the human body?
-tonic secretion: low level, constantly secreted (15 mU/ml)
-phasic (pulsatile) secretion: when taking a meal (60 mU/ml)
What were the chemical stability issues with the first produced Insulin Humulin R?
It was acidic and prone to deamidation (Asn to Asp or iso-Asp)
What prevented deamidation and made Insulin more stable?
- Addition of 2 Zn++ to build T6 hexamer
- Addition of 6 Phenolic molecules to build R6 hexamer
Why is the Tmax (time needed to reach peak concentration) of 2-3h for Insulin Humulin R problematic?
Because it is hard for the patient to match the time when Insulin concentration reaches the peak, with the time they take their meal
What causes a long Tmax of 2-3h for Humulin R
The slow dissociation of the hexamer to monomers
How can the dissociation time of hexamer in Insulin be reduced?
By changing the primary structure (swapping amino acids)
-f.e. Pro with Lys –> Lispro (Admelog)
-it reduces the strength of the dimer/hexamer
How does the amino acid change in Lispro affect Tmax?
Tmax was shorter (max concentration reached in a shorter time)
(quicker onset)
How can Lispro and Humulog (Biosimilars) be structurally identical, even if Biosimilars usually are not identical?
Because Biosimilars differentiate in PTMs, in the case of Insulin there are no PTMs in the structure
Which Insulin form doesn’t contain Zn++?
Insulin Glulisine (Apidra)
How do surfactants (Tween 20) help increase Tmax and the onset of Insulin Glulisine?
Reduces the hydrophobicity and self-association of Insulin monomers –> dissociation is faster
Which ingredients cause a fast onset in FIASP Insulin?
Vitamin B3 and L-Arginie
What causes a rapid onset in AFREZZA (pulmonary)?
-Large surface area of the alveoli
-Alveoli are rich in blood vessel
Why is AFREZZA Insulin (pulmonary) only used for meal-time control?
Because it has a quick onset and can be taken right at mealtime
-it is not a daylong Insulin bc it gets metabolized and eliminated quickly
Why is Insulin absorption by inhalation not so efficient?
Because most of the drugs get stuck in the bronchial tubes or are exhaled due to small particles
Why is the drug Insulin NPH cloudy?
Because it is a suspension made of basic protein combined with acidic Insulin forming crystals dissolved in a suspension
-the crystals are big enough to create a cloudy appearance
How can spike-free and constant levels of Insulin be maintained by NPH Insulin?
-Because of the crystalization of basic Protamin (NPH) and acidic Insulin
-the dissolution of the crystal is the rate-limiting step
How can a natural flat tonic insulin secretion be achieved with NPH and regular Insulin?
combine 70% NPH and 30% regular
-But there is still a peak in the PK curve -> a flat line is the GOAL
What makes Glargine a long-lasting Insulin?
-Glycine replaces arginine -> shifts the ISE from 5.7 to 6.9
-6.9 is closer to physiological pH (7.4) -> so in the body, the drug will be unionized and will micro precipitate and delay the dissolution in the subcutaneous space -> slowly going into circulation
How soluble or unsoluble would the drug with an ISE of 6.9 be in the vial at a pH of 4?
Very soluble and transparent solution, because of the difference 6.9 and 4
What does the PK profile of Insulin Glargine look like?
No peak and it stays at a low level constantly for 8h
-mimics the natural tonic level of Insulin
How does the long-acting Insulin detemir (Levemir) work?
-Insulin is conjugated with a fatty acid (myristic acid)
-myristic acid binds to Albumin and is released slowly
How does the long-acting Insulin degludec (Tresiba) work?
- Conjugated to hexadecanoic acid (fatty acid 16C atoms)
-forms multi hexamers
-binds to Albumin is released slowly
What is a unique feature of degludec?
It forms a multi hexamer
Match the Insulin with the shown PK profiles
Lecture 19&20; Slide 32
Which Insulin is considered as intermediate acting?
NPH
What does the Insulin regimen usually look like?
-Basal Insulin: long-acting Insulin 1x per day
-Bolus Insulin: rapid-acting Insulin 3x per day at mealtime
What is the purpose of Phenol or m-cresol in an Insulin formulation?
Preservative/stabilizing agent
Purpose of glycerol or NaCl
To adjust tonicity, reduce pain on injection site
Purpose of Sodium Phosphate (NaPO4) or TRIS
Purpose of polysorbate or Tween 20
Phosphate (NaPO4) or TRIS: Buffering agent
Polysorbate or Tween 20: Surfactants prevent hydrophobic interactions and aggregation to the vial (Albumin can also be used to prevent aggregation to the vial)
Which chemical modification is likely to happen at low pH, give an example
-Deamidation for example in Insulin Glargine (pH 4 in the vial)
-not a concern in neutral formulations or suspensions
What is the consequence of glycerol or impurities in glycerol in Insulin formulation?
Covalently linked Insulin dimers -> not functional anymore
-a severe form of aggregation: Insulin fibrils
What causes aggregate formation?
-Applying shear (shaking)
-air/liquid interface -> Aggregation
-breaking the cold chain (temperature)
Storage of Insulin
-Cool and away from sunlight
-in the fridge (2-6°C), not frozen
-28-56 days
-resuspension of two types of Insulin by gently shaking
-Acidic formulation should not be mixed with neutral pH formulation
How does GLP-1 (Glucagon-like peptide) in Xultophy and Soliqua work?
Why does it only work in Type 2 diabetes and not in Type 1?
-Stimulate Insulin secretion and inhibits Glucagon
-Because there is no Insulin secretion in Type 1 diabetes patients
What is a closed-loop Insulin system?
-Pump with Insulin placed under the skin
-at the same time it measures Blood glucose
-the device secretes Insulin as needed depending on the blood sugar level (might be pulsatile or tonic)
-> Measure blood glucose and then release considered as a LOOP
What type of Insulin is used in closed-loop Insulin systems?
Short acting and regular Insulin
