Protein binding of drugs

Dr. M.S. Muthu

Associate Professor
Department of Pharmaceutical Engg. & Technology
IIT, BHU, Varanasi
Contents
1. Protein binding introduction

2. Significance of protein binding in pharmacokinetics of drugs

3. Kinetics of protein binding, Scatchard plot

4. Experimental methods of determining protein binding

4.1 Equilibrium dialysis method/ Inferences

4.2 Dynamic dialysis method

5. Significance of protein Binding

1. Protein binding
Protein binding refers to complex formation between small molecules (drug molecules) and blood

protein such as albumins and globulins.

The principal binding force for complexation is hydrophobic interactions.

The level of albumin is remarkably high in blood and plays a crucial role in the drug protein

binding.

Based on the health condition the albumin concentration may vary from 3.5 to 4.5 g/100 ml.

Hydrophobic compounds such as long chain fatty acids are found to be avidly bound to HSA.

The extent of order of binding of drugs to various plasma proteins is -

albumin > α1 acid glycoprotein > lipoprotein > globulins.

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1. Protein binding/ Examples
2. Significance of protein-binding in pharmacokinetics of drugs

Decrease the distribution of drugs by reducing concentration of free form of the drug.

Inactivate a drug by reducing the concentration of free form of the drug needed for action.

Retard the excretion of a drug since only the free form of the drug is excreted.

Alter the duration of action of a drug by slowly releasing the free form of the drug.
3. Kinetics of protein binding

Where K = Association constant

[P] = concentration of unbound
protein
[PD] = Concentration of protein drug
complex
[Pt] = total protein concentration,

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 3. Kinetics of protein binding
❑ [PD]/ Pt = r represent the

average no. of drug

[PD] = [ K [D] [ Pt]] – K [D] [PD] molecules bound per mole of

Or [PD] + K [D] [PD] = K [D] [ Pt] proteins.

[PD](1+K[D]) = K [D][Pt]
❑ Where v = is the no. of sites
Or [PD]/ Pt = K [D] / 1+ K [D]
in class of proteins.
r = K [D]/1 + K [D]

If “v” is no. of independent/ equivalent site, then equation can be written-

r = v K [D]/1 + K [D]

Continue….
 3. Kinetics of protein binding (Scatchard plot)
❑ The Scatchard plot yields straight

line when only one class binding

This equation can be modified to obtain a plot and is called Scatchard plot
sites is present.

r = v K [D]/1 +K [D] ❑ For n classes binding sites, a plot

r (1 + K [D]) = v K [D] of r/[D] against r shows a
r + r K [D] = v K [D]
curvature suggesting more than
r = v K [D] – r K [D]
one class of binding sites.
= [D] (v K- r K)
Or r/ [D] = v K- r K

Continue….
 3. Kinetics of protein binding (Scatchard plot/ Example)
Scatchard plot shown for the binding of bishydroxycoumarin to

human serum albumin at 20° and 40 ° C in Fig.

The curvatures in both the plots at 20° and 40 °C indicate the

presence of more than one type of binding sites.

Scatchard plot
4. Experimental methods of determining protein binding
A number of methods such as equilibrium dialysis, ultrafiltration and electrophoresis are available.

The most commonly used technique is the equilibrium dialysis.

4.1 Equilibrium dialysis method

In this method, a protein solution is enclosed within a membrane (semi-permeable membrane

such as celophane) which is permeable to small drug molecules and not to macromolecule.

The protein solution within the membrane is contacted with a solution of drug.

The solution is agitated slightly until equilibrium has been achieved.

Samples from both sides of the membrane are withdrawn and analysed.

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4. Experimental methods of determining protein binding.
4.1 Equilibrium dialysis method/ Inferences

If the concentration of drug with in the membrane = conc. Of drug

outside the membrane, it indicates no protein binding

If the concentration of drug with in the membrane > conc. Of drug

outside the membrane, it indicates the protein binding.

The concentration is increased because both bound and unbound

drug are present

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 4.2 Dynamic dialysis method
This method is based on the rate of disappearance of drug from a

dialysis cell.

This rate of disappearance is proportional to the concentration of the

unbound drug. This experimental can be used -

a) To know the solubility of a drug in the presence and absence of

protein. This may be used to access the degree of nitration between

various species, solvent, drug and proteins.

b) To study the influence of protein on the partitioning behaviour of

drug molecules between an aqueous an organic solvent that are

immiscible.
5. Significance of protein Binding

Plasma protein bound drugs are unable to penetrate biological membranes. Hence they cannot be

excreted or biotransformed. Only free form of the drug is pharmacologically active.

Plasma protein-drugs act as a reservoir which releases the drug in the free form resulting

pharmacological responce. This effect prolongs the duration of action of the drugs. E.g.,

sulphamethoxydiazine and hydroxycobalamine.

The pharmacological response to a drug is due to attainment of particular concentration of the drug

at the site of action and to its interaction with the receptor site.

Protein binding may lead to increase in solubility of certain drugs. Example discoumarol.

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5. Significance of protein Binding
Drugs may exhibit competitive binding due to differences in the affinities of the drugs for the binding sites

in the protein molecule such competitive binding may be advantageous or disadvantageous. For example,

displacement of antibiotics by other drugs provides better effect due to the release of free antibiotic for

action. But displacement of protein bound an anticoagulants may cause excessive bleeding.

The concentration of a drug in the plasma is often a measure of drug distribution in the body since the

drug distributed in various organs of the body show a dynamic equilibrium. There are exceptions.

Chlorpromazine binds to lipoproteins of brain tissue fifty times more than the binding with blood proteins.

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5. Significance of protein Binding
If a drug protein binding is very high, the concentration of free form of the drug may not be

sufficient to elicit pharmacological activity. This can be remedied by giving sufficiently more

amount of drug to provide the required concentration for activity.

Drugs not only bind to plasma proteins in the blood but also to the constituents of the tissues

perfused by the blood supply. If drug binding is more to plasma proteins than to tissues, more drug

will be retained in the blood. This affects distribution of the drug and there for the apparent volume

of distribution will be low. If drug binding is more to the tissues than to the plasma proteins,

retention of drug in the tissues is more and this affects the distribution of drug. In this case the

apparent volume of distribution will be high.

Dose = C x Vd
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5. Significance of protein Binding
Protein binding drug affects clearance also. If the excretion ratio for a tissue is high, protein binding

has little effect on the clearance. On the other hand, if the excretion ratio for the issue is small, the

clearance depends upon protein binding. In the latter case, only the free drug is cleared Volume

distribution and elimination rate constant become dose dependent if protein is saturated by the drug. In

such a situation, high loading dose is recommended to saturate the proteins followed by lower

maintenance doses.
Thank You