kinetics of protein binding

1. KINETICS OF PROTEIN
BINDING, BIOEQUIVALANCE,
NON COMPARTMENTAL
ANALYSIS
Presented by
Grandhi Sandeep Ganesh

2. PROTEIN BINDING
The macromolecules such as protein, DNA or adipose are
particularly responsible for complex formation with drug. Such
interaction of drug with protein is called protein binding of drug.
The proteins commonly involved in binding with drugs are albumin,
lipoproteins and al-acid-glycoprotein (AGP).
The bound drug is kept in the blood stream while the unbound
component may be metabolized or excreted.
The unbound drug shows the pharmacological action while the
bound drug is neither metabolized nor excreted due to its
pharmacokinetic and pharmacodynamic inertness.
protein + drug = protien-drug complex

3. KINETICS OF PROTEIN BINDING
If P represents proteins and D the drug ,applying law of mass action.
P+D PD
At equilibrium, Ka=
[PD]= Ka [P][D] …………(1)
where,
[P]=concentration of free protein
[D]=concentration of free drug
[PD]=concentration of protein-drug complex
Ka = association rate constant
Kd =dissociation constant

4. Ka > Kd indicates forward reaction i.e. protein-drug binding is
favored. If Pt is the total concentration of protein present , bound and
unbound, then :
Pt =[PD]+[P]………….(2)
If r is the number of moles of drug bound to total moles of protein,
then,
r= ……….(3)
Substituting the value of [PD] from equation (1) in equation (3)
r = = ………(4)
Equation (4) holds when there is only one binding site on the protein
drug complex is 1:1 complex

6. if N is number of binding sites available per mole of protein , then:
r = …….(5)
the value of association constant ka and number of binding sites then
N can be obtained by plotting equation four in different ways.
1. Direct plot
2. Scatchard plot
3. Klotz plot/lineweaver-burke plot (double reciprocal plot)
4. Hitchcock plot

7. DIRECT PLOT
It is made by plotting r vs [D]. Note
that all binding sites are occupied by
the drug. The protein is saturated and
plateau is reached
r=N where r=N/2 , [D]=1/ka

8. SCATCHARD PLOT
It is made by transforming equation (5)
into linear form
r=
r+r ka [D]
r=N ka [D]-r ka [D]
therefore,
=N ka -r ka
A plot of r/[D] vs r yields a straight line
slope line=- ka y-intercept=N ka and x-
intercept=N

9. KLOTZ PLOT/LINEWEAVER-
BURKE PLOT(DOUBLE
RECIPROCAL PLOT
The reciprocal of equation
=
A plot of 1/r vs 1/[D] yields a straight line
with slope 1/N ka and y-intercept 1/N

10. HITCHCOCK PLOT
It is made by re-writing equation (5) as
=1+ ka [D]
Dividing both sides with N ka gives
=

11. BIOEQUIVALENCE STUDIES
 Equivalence:
It is a relative term that compares drug products with respect to a
specific characteristic or function or to a defined set of standards. There are
several types of equivalences.
 Chemical Equivalence:-
It indicates that two or more drug products contain the same
labeled chemical substance as an active ingredient in the same amount.
 Pharmaceutical Equivalence:-
This term implies that two or more drug products are identical in
strength, quality, purity, content uniformity and disintegration and dissolution
characteristics. They may, however, differ in containing different excipients.

12. Bioequivalence:-
It is a relative term which denotes that the drug substance
in two or more identical dosage forms, reaches the systemic
circulation at the same relative rate and to the same relative extent
i.e. their plasma concentration-time profiles will be identical without
significant statistical differences.
When statistically significant differences are observed in the
bioavailability of two or more drug products, bioinequivalence is
indicated.
Therapeutic Equivalence:-
This term indicates that two or more drug products
that contain the same therapeutically active ingredient elicit identical
pharmacological effects and can control the disease to the same
extent.

13. TYPES OF BIOEQUIVALENCE STUDIES
Bioequivalence can be demonstrated either-
In vivo, or
In vitro
OBJECTIVE
If a new product is intended to be a substitute for an approved medicinal
product as a pharmaceutical equivalent or alternative, the equivalence with this product
should be shown or justified. In order to ensure clinical performance of such drug
products, bioequivalence studies should be performed

14. In vivo Bioequivalence Studies:-
The following sequence of criteria is useful in assessing the need for invivo studies
1.Oral immediate-release products with systemic action-
• Indicated for serious conditions requiring assured response.
• Narrow therapeutic margin.
• Pharmacokinetics complicated by absorption <70o/o or absorption window,
nonlinear kinetics, presystemic limination >70%.
• Unfavorable physiochemical properties, e.g. low solubility, metastable
modification, Instability, etc.
• Documented evidence for bioavailability problems.
• No relevant data available, unless justification by applicant that invivo study is
not necessary.
• Non-oral immediate-release products.
• Modified-release products with systemic action

15. INVITRO BIOEQUIVALENCE STUDIES
1. The drug product differs only in strength of the active substance It contains,
provided all the following conditions hold-
Pharmacokinetics are linear
The qualitative composition is the same.
The ratio between active substance and the excipients is the same, or (in the case of
small strengths) the ratio between the excipients is the same.
Both products are produced by the same manufacturer at the same production site.
A bioavailabilty or bioequivalence study has been performed with a original
product.
Under the same test conditions, the in vitro
Dissolution rate is the same.

16. 2.The drug product has been slightly reformulated or the manufacturing method has
been slightly modified by the original manufacturer in ways that can convincingly
be argued to be irrelevant for the bioavailability.
3.The drug product meets all of the following requirements -
The product is in the form of solution or solubilised form (elixir, syrup, tincture,
etc).
The product contains active ingredient in the same concentration as the approved
drug product.
The product contains no excipients known to significantly affect absorption
of the active ingredient.
4.An acceptable IVIVC and the invitro dissolution rate of the new product is
equivalent with that of the already approved medicinal product, Moreover,
The product is intended for topical administration (crean1,ointment,gel,etc.)
for local effect.

17. BIOEQUIVALENCE STUDY DESIGNS
A. STANDARD BIOEQUIVALENCE STUDY DESIGNS (NON-
REPLICATE):
This is the most common design where in two formulations are compared
in a randomized two-period, to-sequence single dose crossover design.
The order of treatment administration in a crossover experiment is called
a sequence and the time of treatment administration is called a period.
The treatment periods should be separated by a wash out period sufficient
to ensure that drug concentration are below the lower limit of
bioanalytical quantification in all subjects at the beginning of the second
period normally 5 elimination half lives are necessary to achieve this.

18. 2. ALTERNATIVE BIOEQUIVALENCE STUDY DESIGNS
1. Parallel design:
It is used for drug substances with very long half-life,
e.g.Fingolimod.
2. Replicate designs:
For substances with highly variable pharmacokinetic
characteristics,e.g.lansoprazole. The advantages of replicate study
designs may be that they allow comparisons of within-subject variances
for the test and reference products.
a) Partial replicate: where in one treatment (test or reference) is
administered to the same subject on to separate occasions.
b) Full replicate : where in both treatments of test and reference product
are administered to the same subject on to separate occasions.

19. BIOEQUIVALENCE PROTOCOL
1. Title
a) Principle investigator
b) Project number and date
2. Study objective
3. Study design
a) Design
b) Drug products
I. Test products
II. Reference product
c) Dosage regimen
d) Sample collection schedule
e) Housing
4. Study population
a) Subjects
b) Subject selection
5. Clinical procedures
a) Dosage and drug administration
b) Biological sampling schedule
6. Ethical considerations
7. Facilities
8. Data analysis
9. Drug accountability
10. appendix

20. NON COMPARTMENTALANALYSIS
The non compartment analysis, also called as model-independent
method, does not require the assumption of specific compartment
model.
This method is however, based on the assumption that the drugs or
metabolites follow linear kinetics, and on this basis, this technique
can be applied to any compartment model.
The compartmental approach, based on the statistical moments
theory, involves collection of experimental data following a single
dose of drug. If one considers the time course of drug concentration
in plasma as a statistical distribution curve, then

21. MRT=
where,
MRT = mean residence time
AUMC= area under the first-moment curve
AUC = area under the zero-moment curve
AUMC is obtained from a plot of product of plasma drug concentration and
time t from zero to infinity.
mathematically it is expressed by equation;
AUMC =

22. AUC AND AUMC
PLOTS

23. AUC is obtained from a plot of plasma drug concentration versus time from zero
to infinity.
mathematically it is expressed by equation:-
AUC =
Practically, the AUMC and AUC can be calculated from the respective graphs by
the trapezoidal rule ( the method involves dividing the curve by a series of
vertical lines into a number of trapezoids, calculating separately the area of each
trapezoid and adding them together).
MRT is defined as the average amount of time spent by the drug in the body
before being eliminated. In this sense, it is the statistical moment analogy of half-
life t1/2 .
In effect MRT represents bolus dose to be eliminated. The values will always be
greater when the drug is administered in a fashion other than I.v bolus

24. APPLICATIONS
It is widely used to estimate the important pharmacokinetic
parameters like bioavailability, clearance and apparent volume of
distribution.
The method is also useful in determining half-life, rate of absorption
and first-order absorption rate constant of the drug

25. ADVANTAGES
Ease of derivation of pharmacokinetic parameters by simple
algebraic equations.
The same mathematical treatment can be applied to almost any drug
or metabolite provided they follow first-order kinetics.
DISADVANTAGES
It provided limited information regarding the plasma drug
concentration-time profile more often, it deals with averages.
The method does not adequately treat non-linear cases.

26. REFERENCES
1. Biopharmaceutics and pharmacokinetics by-treatise by
D.M.Brahmankar, Sunil B.jaiswal
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2880414/
3. https://www.sciencedirect.com/topics/neuroscience/drug-binding