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Biopharmaceutics > Insulin > Flashcards

Insulin Flashcards

1
Q

How is Insulin synthesized?

A
  1. Pre-proinsulin is synthesized in the cytosol and transported to the ER
  2. cleavage #1 in the ER of the signal peptide (leader sequence), protein folding, and -S-S- formation (Proinsulin)-> Transport to the Golgi
  3. In the Golgi: Packed into vesicles –> cleavage #2 of the C-peptide, the formation of Insulin -> Exocytose of the vesicles with Insulin
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2
Q

Where is the signal peptide and the C-peptide cleaved away?

A

Signal peptide: in the ER Pre-proinsulin to Proinsulin

C-peptide: Golgi Proninsuil to Insulin

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3
Q

How is Glucose uptake into the cell initiated?

A

-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

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4
Q

What else is being synthesized besides GLUT4 transporters, with Insulin binding to Insulin receptors?

A

Proteins and enzymes are required to form Glucose into Glycogen and proteins for cell growth

-in the skeletal muscle, liver, and adipose tissue

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5
Q

Why are E.coli and yeast used as an expression system for Insulin production instead of mammalian cells?

A

-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

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6
Q

What are the two ways of using an expression system for Insulin production?

A

-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

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7
Q

What are the downstream steps of Insulin production?

A

-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

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8
Q

What is the net charge of Insulin at the isoelectric point and at physiological pH?

A

-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

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9
Q

Which type of complex does Insulin form in its active form
and which type of interaction promotes the structure?

A

Dimer form -> through hydrophilic interactions

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10
Q

Which type of complex does Insulin form when it is stored in the pancreas (Islet cells)?

A

Hexamers including bivalent Zinc ions

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11
Q

Which preservative can be used to increase the stability of Insulin?

A

Phenol or m-cresol (Phenolic preservatives)
-> building T6 complexes

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12
Q

Which preservative can be used to increase the stability of Insulin?

A

Phenol or m-cresol (Phenolic preservatives)
-> building R6 complex

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13
Q

Why is the state of aggregation (dimer, hexamer) important?

A

-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

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14
Q

Which property is used to formulate different Insulin forms (rapid or long-lasting)?

A

-Monomer-Mulitmer equilibrium

-determines if Insulin is absorbed directly through the vascular system (rapidly) or lymphatic system (slowly)

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15
Q

How is the predominant form of Insulin absorbed in vivo?

A

In the form of monomers -> 80% vascular -rapidly
(only 20% lymphatic - slow)

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16
Q

What are the two types of Insulin secretion throughout the day in the human body?

A

-tonic secretion: low level, constantly secreted (15 mU/ml)
-phasic (pulsatile) secretion: when taking a meal (60 mU/ml)

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17
Q

What were the chemical stability issues with the first produced Insulin Humulin R?

A

It was acidic and prone to deamidation (Asn to Asp or iso-Asp)

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18
Q

What prevented deamidation and made Insulin more stable?

A
  1. Addition of 2 Zn++ to build T6 hexamer
  2. Addition of 6 Phenolic molecules to build R6 hexamer
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19
Q

Why is the Tmax (time needed to reach peak concentration) of 2-3h for Insulin Humulin R problematic?

A

Because it is hard for the patient to match the time when Insulin concentration reaches the peak, with the time they take their meal

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20
Q

What causes a long Tmax of 2-3h for Humulin R

A

The slow dissociation of the hexamer to monomers

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21
Q

How can the dissociation time of hexamer in Insulin be reduced?

A

By changing the primary structure (swapping amino acids)
-f.e. Pro with Lys –> Lispro (Admelog)
-it reduces the strength of the dimer/hexamer

22
Q

How does the amino acid change in Lispro affect Tmax?

A

Tmax was shorter (max concentration reached in a shorter time)
(quicker onset)

23
Q

How can Lispro and Humulog (Biosimilars) be structurally identical, even if Biosimilars usually are not identical?

A

Because Biosimilars differentiate in PTMs, in the case of Insulin there are no PTMs in the structure

24
Q

Which Insulin form doesn’t contain Zn++?

A

Insulin Glulisine (Apidra)

25
Q

How do surfactants (Tween 20) help increase Tmax and the onset of Insulin Glulisine?

A

Reduces the hydrophobicity and self-association of Insulin monomers –> dissociation is faster

26
Q

Which ingredients cause a fast onset in FIASP Insulin?

A

Vitamin B3 and L-Arginie

27
Q

What causes a rapid onset in AFREZZA (pulmonary)?

A

-Large surface area of the alveoli
-Alveoli are rich in blood vessel

28
Q

Why is AFREZZA Insulin (pulmonary) only used for meal-time control?

A

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

29
Q

Why is Insulin absorption by inhalation not so efficient?

A

Because most of the drugs get stuck in the bronchial tubes or are exhaled due to small particles

30
Q

Why is the drug Insulin NPH cloudy?

A

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

31
Q

How can spike-free and constant levels of Insulin be maintained by NPH Insulin?

A

-Because of the crystalization of basic Protamin (NPH) and acidic Insulin
-the dissolution of the crystal is the rate-limiting step

32
Q

How can a natural flat tonic insulin secretion be achieved with NPH and regular Insulin?

A

combine 70% NPH and 30% regular
-But there is still a peak in the PK curve -> a flat line is the GOAL

33
Q

What makes Glargine a long-lasting Insulin?

A

-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

34
Q

How soluble or unsoluble would the drug with an ISE of 6.9 be in the vial at a pH of 4?

A

Very soluble and transparent solution, because of the difference 6.9 and 4

35
Q

What does the PK profile of Insulin Glargine look like?

A

No peak and it stays at a low level constantly for 8h
-mimics the natural tonic level of Insulin

36
Q

How does the long-acting Insulin detemir (Levemir) work?

A

-Insulin is conjugated with a fatty acid (myristic acid)
-myristic acid binds to Albumin and is released slowly

37
Q

How does the long-acting Insulin degludec (Tresiba) work?

A
  • Conjugated to hexadecanoic acid (fatty acid 16C atoms)
    -forms multi hexamers
    -binds to Albumin is released slowly
38
Q

What is a unique feature of degludec?

A

It forms a multi hexamer

39
Q

Match the Insulin with the shown PK profiles

A

Lecture 19&20; Slide 32

40
Q

Which Insulin is considered as intermediate acting?

A

NPH

41
Q

What does the Insulin regimen usually look like?

A

-Basal Insulin: long-acting Insulin 1x per day
-Bolus Insulin: rapid-acting Insulin 3x per day at mealtime

42
Q

What is the purpose of Phenol or m-cresol in an Insulin formulation?

A

Preservative/stabilizing agent

43
Q

Purpose of glycerol or NaCl

A

To adjust tonicity, reduce pain on injection site

44
Q

Purpose of Sodium Phosphate (NaPO4) or TRIS

Purpose of polysorbate or Tween 20

A

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)

45
Q

Which chemical modification is likely to happen at low pH, give an example

A

-Deamidation for example in Insulin Glargine (pH 4 in the vial)

-not a concern in neutral formulations or suspensions

46
Q

What is the consequence of glycerol or impurities in glycerol in Insulin formulation?

A

Covalently linked Insulin dimers -> not functional anymore
-a severe form of aggregation: Insulin fibrils

47
Q

What causes aggregate formation?

A

-Applying shear (shaking)
-air/liquid interface -> Aggregation
-breaking the cold chain (temperature)

48
Q

Storage of Insulin

A

-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

49
Q

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?

A

-Stimulate Insulin secretion and inhibits Glucagon
-Because there is no Insulin secretion in Type 1 diabetes patients

50
Q

What is a closed-loop Insulin system?

A

-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

51
Q

What type of Insulin is used in closed-loop Insulin systems?

A

Short acting and regular Insulin

Biopharmaceutics (17 decks)

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