Formulations for Parenteral Administration of Insulin, Glulisine Case Study

Insulin Case Study

Insulin Discovery and Structure

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SINGLE USE ONLY
NIP20
Formulations for
Parenteral Administration
Dr Marta Roldo
marta.roldo@port.ac.uk
Insulin - case studyInsulin
· Discovered in 1921 (100 years old !! )
. One of the first resolved crystal
structures
· Protein made of two polypeptides
· A: 21 aa
· B: 30 aa
1
5
10
15
20
H2N- Gly
Val
Glu
Ser
Cys
Ser
Leu
Leu
Asn
Tyr
Asn
-COOH
H2N- Phe Val
Asn
Gin
Leu
Gly
Ser
His
Leu
Val ( Glu
Ala
Leu
Tyr
Leu
Val
Cys
Gly
Glu
10
15
20
Arg
N
C
HOOC- Thr Lys
Pro Thr
Tyr Phe
Phe
C
30
25
N
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(N)
(N)
(C)
monomer
(C)
(N)
(N)
(C)
(N)
(C)V
(C) (N)
dimer
Il℮
Gin
Cys
Cys
Thr
Ile
Tyr
Gin
Glu
Cys
His
Cys
1
5
Gly
hexamer

Insulin Plasma Concentration and Types

Insulin
0.08 -
Insulin ( U/L)
0.04 -
4
0
breakfast snack lunch
dinner snack sleep
wake
Pattern of insulin plasma concentration in
healthy individuals following meals

• rapid-acting
  starts to work
  5-10 minutes
  peaks
  30-90 minutes

types of insulin

• regular-/short-acting
  starts to work
  30 mins - 1 hour
  peaks
  2-5 hours
• intermediate-acting
  intensity
  starts to work
  1-2 hours
  peaks
  4-12 hours
• long-acting
  starts to work
  1-2 hours
  does not peak
  2
  6
  10
  14
  18
  22
  duration (hours)
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Insulin Absorption and Self-Association

-Insulin absorption
subcutaneous
injection
dermal layer
self-association equilibria
multi-hexamer
capillaries
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Long-Acting Insulin Formulations

Insulin: Long-acting

Onset of insulin action	Brand and type	Generic name	Type of insulin
> 60 min	Lantus® Solostar	Insulin Glargine	Onout: 1-2 hrs Podkci no pack Duration: 24 hm # Lantus 0,8 Untokg # NPH 0.3 Units/kg Tiene Ihr ater irpotion
> 60 min	Levemir® FlexPen	Insulin Detemir	Onout: 1-2 fra Pask 3-14 hrs Duration: 24 hr 0 2 4 6 8 10 12 14 10 10 20 22 24 0 20 TOMIÑO 10 0 0 6 12 24 30
> 60 min	Toujeo® SoloStar 300 units/ml	Insulin Glargine	Onout: 6 hra Posic no pock Duration: > 24 hrs

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Detemir (Levemir®) Mode of Action

Insulin: Long-acting - DETEMIR (LEVEMIR®)
detemir
O
A1
A21
ENYCN
B1
FUN CHLCGSHLV
G
RGF
YOPK
B
detemir R6-hexamer
detemir - mode of action
Albumin
Albumin
phenol
R6
T6
Albumin
subcutaneous layer
dihexamers albumin binding
circulation
(SQ depot)

• Myristic acid insulin
  derivative
• Formation of a Zn2+ and
  phenol stabilised hexamer
• Effect:
  · Increased stability of
  subcutaneous depot
  · Increased binding to plasma
  protein
  · Slower clearance from
  insulin receptor
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Glargine (Lantus® Toujeo®) Characteristics

Insulin: Long-acting - Glargine (LANTUS® TUJEO®)
glargine dihexamers
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glargine hexamers
in a crystal lattice

• Insulin modified with 2 arginine
  residues
• Formulated at pH 4 + Zn2+, no
  buffer
• Effect:
  · At pH 7 after injection it precipitates
  as microcrystals
  · Arg-Arg are cleaved by exopeptidases
  · Crystals break and release the
  monomers that are absorbed
  · Tujeo® has higher concentration,
  prologued absorption and no peak

Poll: Strategies to Prolong Insulin Action

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POLL OPEN
Which of the following strategies can be used
to prolong the action of insulin?
1. Destabilise the crystal structure of insulin
2. Enhance the affinity of the insulin analogue for plasma proteins
3. Develop a pH sensitive formula that triggers insulin dissolution at physiological pH
4. Inject insulin intravenously
5. Decrease the concentration of insulin in the formulation

Summary of Insulin Action Prolongation

Insulin: Log-acting summary
To prolong insulin action we can:

• Slow down the dissolution of insulin from the subcutaneous depot
• Increase plasma protein binding thus circulation time
• Increase affinity for the insulin receptor thus slowing down clearance
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Intermediate-Acting Insulin Formulations

Insulin: Intermediate-acting

Onset of insulin action	Brand and type	Generic name	Type of insulin
60 min	Insulatard" Insulatard®	NPH Insulin	Onoet: 1.6 hrs Pool 4-12 hrs Duration: 24 hrs 0 2 4 6 8 10 12 14 16 18 20 22 24 (7)

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NPH (Insulatard®) Mechanism

Insulin: Intermediate-acting - NPH (INSULATARD®)
insulin-protamine complex
(dihexamer)

• INSULIN PROTAMINE COMPLEXES:
  · Complexation with the basic protein
  protamine, a DNA precipitating
  agent, in the presence of Zn2+
  · Further addition of phenol or meta-
  cresol forming Neutral Protamine
  Hagedorn (NPH)
  · Self -conversion of amorphous
  precipitates to microcrystals
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Short-Acting Insulin Formulations

Insulin: Short-acting

Onset of insulin action	Brand and type	Generic name	Type of insulin
15 min	Apidra® Solostar	Insulin Glulisine	Onsat: 6-16 mnd Pask; 1-2 hrl Duration: 3-4 hrs ..... Tine Fri afler ijpoton
15 min	Humalog® KwikPen	Insulin Lispro	Oncet: 0-15 mm Duration: 3.5-4.5 fra
15 min	NovoRapid® FlexPen	Insulin Aspart	Oncet: 10-30 mnc Podkc 1-4 hrs Duration: 8-6 TvG 0 2 4 6 8 10 12 14 15 18 20 22 24 (7)
30 min	Actrapid" Actrapid®	Regular Insulin	OnoRe: 0.5 TYS Pook: 1-8 hrs Duration: 8 fira 0 2 4 0 8 10 12 14 10 18 20 22 24 (r)

Insulin Self-Assembly and Absorption Challenges

Catch 22:
Self assembly of insulin into hexamers:
1. Reduces chemical and physical
degradation
2. Delays absorption from the
subcutaneous depot
How can we obtain fast action to avoid:
1. Immediate post-prandial
hyperglycemia
2. Late post-prandial hypoglycemia
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Lispro (Humalog®) Mechanism

Insulin: Short-acting - Lispro (Humalog®)
Insulin lispro hexamers in pharmaceutical formulation
Site of injection
sonning
Insulin lispro hexamers after injection
Insulin lispro monomers diffuse into capillaries
Insulin lispro monomers
Phenol and zinc diffuse out

• the name lispro indicates the aa
  mutation in this analogue (lysine-
  proline)
• Unstable analogue
• Stabilised by addition of phenols
• Zn2+ hexamers only form in presence of
  phenols
• As phenols diffuse out quickly
  (milliseconds) the hexamers destabilise
  quickly
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Glulisine (Apidra®) Characteristics

Insulin: Short-acting - Glulisine (Apidra®)
Modified Human Insulin
Glulisine:
Replacement of asparagine B3 with lysine
and lysine B29 with glutamic acid
A chain
Gly
S
1
S
Ala
Cys
Glu
20
Thr
(Lys)
Pro
30
1
Asn
S
10
(His
S
Phe
B chain
25
5
GIV
His
10
15
Substitutions favor
rapid dissociation
after SC injection

• the name glulisine indicates the
  insertion of a glutamine and a lysine
  . Net charge at neutral pH is decreased
  by 1
• Stabilised by change of pl (isoelectric
  point)
• No need for hexamers formation to
  stabilise
• This is a Zn2+ free formula
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  S
  Ile
  Gin
  S
  Lys
  5
  Phe
  15
  20
  Gin

Poll: False Statement on Insulin Analogues

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POLL OPEN
Which of the following statements is FALSE?
1. Fast on-set insulin analogues tend to be more prone to degradation
2. Insulin analogues can be stabilised by aminoacid substitutions that change their isoelectric point
3. Amorphous insulin dissolves faster than crystalline insuline
4. Zn2+ free insulin formulations have a slower on-set of action
5. Insulin hexamers are not easily absorbed into circulation

Biphasic Insulin Formulations

Insulin - Biphasic

Onset of insulin action	Brand and type	Generic name	Type of insulin
15 min	NovoMix®30 FlexPen	Biphasic Insulin Aspart	Onset: 10-20 mins Peak: 1-4 hra Duration: 24 hra 0 2 4 6 8 10 12 14 16 18 20 22 24 (hr)
15 min	Humalog® Mix 75/25 KwikPen	Biphasic Insulin Lispro 25%/ Protamine 75%	Onset: 0.5 hra Peak: 2-4 hrs Duration: 12 hra serum IRI 10. 10 -8 Time (hr) after dosing
15 min	Humalog® Mix 50/50 Kwikpen	Biphasic Insulin Lispro 50%/ Protamine 50%	Onset: 0.5 hrs Peak: 2-3 hrs Duration: 12 hrs Humalog Mix:50 - Humalog Basal 12 18 20 24 Time (hr)
15 min	Mixtard® 30	Biphasic 30% Soluble Human Insulin +70% NPH Insulin	10 m Mixtard" 30 Onset: 0.5 hrs Peak: 2-8 hra Duration: 24 hra 0 2 4 6 8 10 12 14 16 18 20 22 24 (hr) UNIVERSITYOF PORTSMOUTH NovoMix" 30 FlexPen" Humalog Mix75/25 ---- Humulin 70/30

Apidra and Admelog Formulations

Insulin
Apidra 100 Units/ml solution for injection in a vial
100 Units insulin glulisine
Active
Metacresol
Stabiliser for hexamers
and preservative
Sodium chloride
Isotonicity
Trometamol (TRIS)
Buffer
Polysorbate 20
Surfactant for stability
Hydrochloric acid,
concentrated
pH adjuster
Sodium hydroxide
pH adjuster
Water for injections
vehicle
Admelog 100 units/ml solution for injection in
cartridge
100 units (equivalent to
3.5 mg) insulin lispro
active
Metacresol
Stabiliser for hexamers
and preservative
Disodium hydrogen
phosphate heptahydrate
buffer
Zinc oxide
Stabiliser for hexamers
Water for injections
vehicle
Hydrochloric acid
pH adjuster
Sodium hydroxide
pH adjuster
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Insulatard Formulation Components

Insulin
Insulatard 40 international units/ml suspension for
injection in vial.
1 ml suspension contains
40 international units
isophane (NPH) insulin
human
active
Zinc chloride
Stabiliser for hexamers
Glycerol
Viscosity enhancer
Metacresol
Stabiliser for hexamers
and preservative
Phenol
Stabiliser for hexamers
and preservative
Disodium phosphate
dihydrate
buffer
Sodium hydroxide
pH adjuster
Hydrochloric acid
pH adjuster
Protamine sulfate
Stabiliser for hexamers
Water for injections
vehicle
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Sustanon Active Ingredients and Absorption

Sustanon

Active/excipient	Function
30 mg Testosterone propionate	Active
60 mg Testosterone phenylpropionate	Active
60 mg Testosterone isocaproate	Active
100 mg Testosterone decanoate	Active
Arachis Oil	Vehicle
Benzyl Alcohol	Preservative

Absorption
A single dose of Sustanon 250 leads to an increase of total plasma testosterone with peak levels of approximately 70nmol/l (Cmax), which are reached
approximately 24-48 h (tmax) after administration. Plasma testosterone levels return to the lower limit of the normal range in males in approximately
21 days.
In female-to-male transsexuals, a single dose of Sustanon 250 repeated every two weeks resulted in mean trough testosterone levels towards the upper
end of the normal male range at 2, 4 and 12 months.
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https://www.medicines.org.uk/emc/product/5373#EXCIPIENTS