In vitro In vivo Correlation

1. Dr. Rajan Swami

2. IN VITRO DISSOLUTION

3. DISSOLUTION
Dissolution is defined as the process by which solid
substances enters in solvent to yield a solution. Stated
simply, dissolution is the process by which a solid
substance dissolves.
Processes involved in the dissolution of solid dosage forms :
 Initial mechanical lag
 Wetting of the dosage form
 Penetration of the dissolution medium into the dosage form
 Disintegration
 Deaggregation of the dosage form and dislodgement of the
granules
 Dissolution
 Occlusion of some particles of the drug

5. Intrinsic Dissolution
The rate of dissolution of a pure pharmaceutical active ingredient when
conditions such as surface area, temperature, agitation or stirring
speed, pH, ionic strength of the dissolution medium is kept constant is
known as intrinsic dissolution rate.
Mathematically, dissolution process can be simply described as
follows:
dM
dT
= KA(Cs - C)
where, M is the mass of the substance remaining to be dissolved,
A is the surface area exposed to the dissolution medium,
Cs is the saturation concentration referred to as solubility in the
dissolution medium,
C is the amount dissolved or the concentration of the drug in solution
at time t,
K is the intrinsic dissolution rate constant or simply the dissolution
rate constant.

6. When C is small, C < 0.15Cs, then dM/dT is proportional to Cs, since
(Cs - C) is large. If this applies, then to a good approximation we may
write
𝒅𝑴
𝒅𝑻
= KACs
 This equation is commonly referred to as a sink-condition equation,
 Under sink conditions, a stagnant film of liquid (dissolution medium)
is adsorbed onto the solid, the thickness of this film being l cm.
 The liquid in the film that is in direct contact with the solid is saturated
with drug in solution.
 The concentration of the drug in solution then drops as the distance
from the dissolving solid surface increases.
 At the end of the film, l cm from the surface, the concentration in the
film is the same as that in the bulk solution, Cb.
 The driving force behind the movement of solute molecules through
the stagnant film is the concentration gradient that exists between the
saturation concentration of the solute, Cs, in the stagnant layer at the
surface of the solid and its concentration on the farthest side of the
stagnant film, Cb.

8. Compendial methods :
When selecting apparatus for
dissolution testing, routine quality
control, new drug development, or
complying with regulatory
requirements, the analyst must
follow the latest issue of
compendia, including revisions.

9. CLASSIFICATION OF DISSOLUTION APPARATUS IN
DIFFERENT PHARMACOPOEIAS

10. USP TABLET CALIBRATORS
 USP Prednisone Tablets RS(Dissolution Calibrator; Disintegrating)
 USP Salicylic Acid Tablets RS(Dissolution Calibrator; Nondisintegrating)
 USP Chlorpheniramine Maleate Extended-Release Tablets RS (Drug Release Calibrator;
Single Unit)

11. USP/NF Method 1 (Rotating Basket Method) :
 The USP/NF rotating basket method of dissolution testing
essentially consists of a 1-in diameter 13/ 8-in-high stainless-steel
40-mesh wire basket rotated at a constant speed ranging between
25 and 150 rpm.
 It is immersed in 900 ml of dissolution medium in a vessel of
1000 ml capacity.
 The medium in the vessel is maintained at a constant temperature
of 37 ±0.5°C by means of a suitable water bath.
 The dosage unit is placed in a dry basket at the beginning of each
test.
 Distance between inside bottom of the vessel and the basket is
maintained at 25±2 mm during the test.
 In case of non-disintegrating dosage forms this apparatus is
superior to Apparatus 2 since it constrains the dosage form in
steady state fluid flow.

12. 12

13. USP/NF Method 2 (Rotating Paddle Method):
 For all practical purposes the compendial specifications
outlined for this method are identical to method 1 except
that the paddle is substituted for the rotating basket.
The metallic or suitably inert, rigid blade and shaft
comprise a single entity. The paddle and blade shaft may
be coated with suitable inert coating.
The dosage form is allowed to sink to the bottom of the
vessel before rotation of the blade is started.
This apparatus is frequently used for both disintegrating
and non-disintegrating dosage form at 50 rpm. Other
agitation speeds are acceptable with proper justification

14. 14

15. USP/ NF Method 3 (Reciprocating Cylinder) :
 The assembly consists of a set of cylindrical, flat bottomed
glass vessels; a set of glass reciprocating cylinders; stainless
steel fittings (type 316 or equivalent) and screens and a motor
and drive assembly to reciprocate the cylinders vertically
inside the vessels and, if desired, index the reciprocating
cylinders horizontally to a different row of vessels.
The vessels are immersed in suitable water bath of any size
that permits holding the temperature at 37 ±0.5°C during the
test.
One advantage of reciprocating cylinder is that
gastrointestinal tract conditions can be easily simulated, as it
is easy to make time dependent pH changes. This apparatus is
most suitable for nondisintegrating (extended release) or
delayed-release dosage (enteric coated) dosage forms.
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16. 16

17. USP Apparatus 4 (Flow-Through Cell) :
The assembly consists of a reservoir and a pump for dissolution medium;
a flow-through cell; a water bath that maintains dissolution medium at 37
±0.5°C.
The pump forces the dissolution medium upwards through the flow-
through cell.
 The pump has a delivery range between 240 and 960 ml/ hr, with the
standard flow rates of 4, 8, and 16 ml/min.
the flow profile is sinusoidal with a pulsation of 120±10 pulses per
minute.
The advantages of flow through cell apparatus most often cited are the
ability to test drugs of very low aqueous solubility in the open loop mode
and the ability to change the pH conveniently during the test.
The disadvantage associated with it might be the operational difficulties
of preparing large volumes of medium for operation in the open loop
mode and the added time in the system set up and cleaning.

18. 18

19. USP Apparatus 5 (Paddle Over Disk) :
The Apparatus 2 is used, with the addition of a stainless steel
disk assembly designed for holding the transdermal system at
the bottom of the vessel.
Temperature is maintained at 32 ± 0.5°C.
A distance of 25 ± 2 mm between the paddle and blade and
the surface of the disk assembly is maintained during the test.
The vessel may be covered during the test to minimize
evaporation.
Disk assembly for holding the transdermal system is designed
to minimize any ‘dead’ volume between the disk assembly and
the bottom of the vessel. Disk assembly holds the system flat
and is positioned such that the release surface is parallel with
the bottom of the paddle blade.

20. 20

21. USP Apparatus 6 (Cylinder) :
 The vessel assembly used is same as Apparatus 1,
except the basket and the shaft is replaced with a
stainless steel cylinder stirring element and to
maintain the temperature at 32 ± 0.5°C during the test.
The shaft and cylinder components of the stirring
element are fabricated of stainless steel to the
specifications .
The dosage units are placed on the cylinder at the
beginning of each test. The distance between the inside
of the vessel and the cylinder is maintained at 25 ± 2
mm during the test.

22. 22

23. USP Apparatus 7 (Reciprocating holder) :
The assembly consists of a set of volumetrically
calibrated or tared solution containers made of glass or
other suitable inert material, a motor and drive
assembly to reciprocate the system vertically and to
index the system horizontally to a different row of
vessels automatically if desired, and a set of suitable
sample holders.
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25. ALTERNATIVE METHODS OF DISSOLUTION
TESTING

26. ROTATING BOTTLE METHOD
ROTATING BOTTLE METHOD
 Mainly used for controlled release beads.
Equipment consist of a rotating rack that
holds the sample drug products in bottles.
The bottles are capped tightly and rotated in
a 37⁰ C temperature bath.
At various times samples are removed from
the bottle, decanted through a 40 mesh
screen and the residues are assayed.
Equal volume of fresh medium is added to
the remaining drug residues within the
bottles and dissolution test is continued.
Disadvantage- manual and tedious.

27. PERISTALSIS METHOD
To stimulate hydrodynamic condition of
GIT tract in an in-vitro dissolution
device.
It consists of rigid plastic cylindrical
tubing fitted with septum and rubber
stopper at both ends.
Dissolution chamber consists of a space
between septum and lower stopper.
The apparatus is placed in beaker
containing the dissolution medium.
Dissolution medium is pumped with
peristaltic action through the dosage
form

28. FRANZ DIFFUSION CELL
Static or flow through diffusion cells are used to characterize invitro
drug release and drug permeation kinetics from a topical drug product
eg: Ointment, cream or transdermal drug product.
The Franz diffusion cell is static diffusion system used to characterize
drug permeation through skin model.
The skin is mounted on the Franz diffusion cell and the drug product is
placed on the skin surface.
The drug permeates across the skin into a receptor fluid compartment
that may be sampled at various times.
 This system is used for selection of appropriate formulation that has
optimum drug delivery.

29. FRANZ DIFFUSION CELL

30. Various equipments and operating variables are associated
with dissolution testing
The variables may or may not exert a pronounced effect on
the rate of dissolution of drug or drug product
Some of these variables are:
The centering and alignment of paddles is critical in
paddle method
Turbulence can create increased agitation, resulting in
higher dissolution rate.
Wobbling and tilting due to worn equipment should be
avoided
PROBLEMS OF
VARIABLE
CONTROL IN
DISSOLUTION
TESTING

31. PROBLEMS OF VARIABLE CONTROL
IN DISSOLUTION TESTING
The basket method is more sensitive to clogging due to
gummy material small pieces can even clog the basket screen
and create a non-sink condition
Dissolved gases in media may form air bubbles on the
surface of dosage form
Composition of product formulation also affects the
dissolution
For eg. Dissolution with paddle method is faster with paddle
method then that of basket method for a tablet of 4-kg
hardness at 50 rpm but for a tablet of 6.8-kg hardness same
dissolution results are obtained at 125 rpm

32. PROBLEMS
OF
VARIABLE
CONTROL
IN
DISSOLUTI
ON
TESTING

33. DISSOLUTION TEST FOR EVALUATION
OF TABLETS
This test is done to show the release of drug to as close as
100% and uniform from batch to batch
Interpretation of results :
Stage 1 : 6 tablets tested and accepted if all the tablets are not
less than the monograph tolerance limit (Q) plus 5%
Stage 2 : additional 6 tablets tested and accepted of the
average of 12 is greater than of equal to Q and no unit less
than Q-15%. If fail then next stage.
Stage 3 : Additional 12 tablets tested and accepted if average
of 24 is greater than of equal to Q and nmt 2 tablets are less
than Q-15%

41.  An in vitro in vivo correlation (IVIVC) is a predictive
mathematical model that describes the relationship between
an in vitro property of a dosage form (primarily dissolution or
drug release) and a relevant in vivo response (primarily a drug’s
plasma concentration or the amount of drug absorbed).
In other terms, IVIVC expresses the relationship between drug
release in a dissolution apparatus and how that translates to the
amount of drug that enters the bloodstream following
administration.

42.  An IVIVC model is recommended by regulatory
authorities for most modified release dosage forms.
 Once a validated IVIVC model has been established,
it can be used to predict (BA/BE) based on in
vitro data that are already available.

43.  The main advantage of IVIVC is that it provides a
mechanism for evaluating the change in in
vivo absorption based on in vitro dissolution changes
when there are small changes in a formulation.
 Another advantage of IVIVC is that it conveys a better
understanding of the drug product itself.

44.  Establishing an IVIVC model can be even more helpful after
the product has been approved by determining the impact of
post-approval manufacturing changes, changes in the site of
manufacture, and issues with individual lots of manufactured
products all without having to repeat costly in vivo BE studies.
CONTD..

45.  The FDA Guidance, “Extended Release Oral Dosage Forms”
is more than 20 years old. At the time of its release, the ability
to precisely predict expected BA characteristics for a product
from its dissolution profile had been a goal.
 The guidance outlines IVIVC development and how to
evaluate predictability, use an IVIVC to establish
specifications for dissolution, and apply an IVIVC as a
surrogate for in vivo BE studies.

46. IVIVC Model
Development
IVIVC Model
Validation
IVIVC
Methodology

47. IVIVC METHODOLOGY
1. Finding in vitro product parameters
2. Developing and validating suitable dissolution method to
predict in vitro drug product performance to establish best
IVIVC sensitive enough to detect subtle changes in in
vivo performance due to changes in one or more of:
• Formulation
• Process parameters
• Drug release patterns
• Fluctuations in environmental conditions

48. 3. Establishing robustness of dissolution method
4. Identifying various factors affecting in vivo drug
release
5. Using the acquired information to develop better prototype
formulations
6. Optimizing the best prototype formulation using validated
dissolution methods and establishing
IVIVC during:
• Post-approval use
• Post scale-up
• Post-approval change(s) in formulation
CONTD..

49. There are five different types of correlation accepted in
as per the FDA guidance:
 Level A,
 Level B
 Level C,
 Multiple level C correlation
 Level D, (a rank order correlation is not Federal acceptable,
therefore have limited significance).

50. It is highest level; point to point relationship between in-vitro dissolution
rate and in-vivo rate of the drug from the dosage form.
Figure1: Correlation between percent
theophylline dissolved in vitro and
percent theophylline absorbed after
administration of extended release
product

54. Using ACAT modelling

55. Transporter role, fraction of drug absorbed, fraction of drug permeated etc.

57. Mean in vitro dissolution time (MDT vitro) of the product is compared to
mean in vivo residence time (MRT).
Least used as MDT and MRT varies.
It utilizes principle of Statistical moment analysis
Drawback: Does not reflect actual in vivo plasma level curves.
Figure 2: Correlation of mean
in vitro dissolution time
(MDT) and mean in vivo
absorption time (MAT)

58.  Level C correlation represents a single point correlation.
 One dissolution time point (t50%, t90%, etc.) is compared to one
mean pharmacokinetic parameter such as AUC, tmax or Cmax
Figure 3: Correlation between
percent drug dissolved in 45
minutes and AUC of plasma
drug-time curve

59. LIMITATION
 Does not reflect entire plasma drug
concentration curve.
 It is the weakest level as partial relationship
between absorption and dissolution is
established.
 So,limited in predicting in vivo drug
performance in early stages of formulation
development when pilot formulations are being
selected.

60.  Relates one or several pharmacokinetic parameters of interest
(Cmax, AUC etc.) to the amount of drug dissolved at several time
points of the dissolution profile.
 It should be based on at least 3 dissolution time points covering
early, middle and late stages of dissolution profile.
 Used to justify bio-waivers, provided that the correlation has been
established over the entire dissolution profile and one or more
pharmacokinetic parameters.

61.  Level D correlation is a rank order and qualitative analysis and
is not considered useful for regulatory purposes.
 It is not a formal correlation but serves as an aid in the
development of a formulation or processing procedure.

62.  Reduces the costs associated with expensive
bioavailability/ bioequivalence studies in human
subjects.
 Serve as a surrogate for more number of human studies
 Speeds up the product development process with
meaningful

63.  Understanding of the product behaviour under in vitro and in
Vivo conditions
 Demonstrates bioequivalence when certain pre-approval
changes are made in formulation, equipment, manufacturing
process or in manufacturing site.
 Improves product quality using more meaningful dissolution
specifications.
CONTD..

66.  When a class II drug is formulated as an ER product, where
solubility and permeability of the drug is site-independent, a good
level A IVIVC is observed.
 However, once the permeability is site-dependent, little or no
IVIVC is expected.

67.  As drug permeation is rate controlling, limited or
no IVIVC is expected.
 Class III drugs, such as proteins and peptides;
require the technologies that address to fundamental
limitations of permeability.

68.  Class IV drugs exhibit significant problems for effective oral
delivery and no IVIVC is expected in this class.
 This class of drugs presents a major challenge for
development of DDS and the route of choice for administering
such drugs is parenteral with the formulation containing
solubility enhancers

69.  The objective of IVIVC is to successfully predict the
outcome(in vivo profile) using a given model and test
condition (in vitro profile).
 The focus is on predictive performance of the model and
therefore, the prediction error is evaluated. Depending on
the intended application of an IVIVC and the therapeutic
index (TI) of the drug, evaluation of internal and/or external
predictability may be appropriate.

70.  Evaluation of internal predictability is based upon the
initial data used to define the IVIVC model.
 Internal predictability is applied to IVIVC established
using formulations with three or more release rates for
wide therapeutic index drug exhibiting conclusive
prediction error.
 Average percent prediction error (%PE) of 10% or
less, with none greater than 15% is acceptable.
 If criteria are not met, proceed to evaluation of external
predictability.

71.  Evaluation of external predictability is based on additional
test data sets. The formulations with different release
rates provide the optimal test of an IVIVC’s predictability.
 Average percent prediction error (%PE) of 10% or
less, with none greater than 20% is acceptable.

72. REFERENCE
 WHO
 http://www.who.int/medicines/areas/quality_safety/quality_a
ssurance/BE-invivo-studies-guidance-QAS15-
622_21052015.pdf?ua=1
 From Biostudies to Biowaivers using IVIVC:A Favorable but
Fastidious Sojourn.
 Essentials of Pharmaceutics; Remington;Dissolution;21st
Edition;Volume 1; Page 63-80.