Plasma Protein Binding

Dr. Muhammad Usman

Dr. Muhammad Usman
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Drug Distribution
• After a drug is absorbed systemically from the site of
administration, the drug molecules are distributed throughout
the body by the systemic circulation.
• The location and extent of distribution depends on;
1. Physicochemical properties of drugs

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2. Patients’ characterstics (Organ perfusion and blood flow)
• The drug molecules are distributed to;
1. Target sites (Intra-cellular or extra-cellular)
2. Eliminating organs (Liver, Kidney etc.)
3. Non-eliminationg tissues (Brain, skin muscles etc.)
4. Cross placenta
5. May bound to plasma proteins 2
 Dr. Muhammad Usman
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Protein Binding of Drugs
• Many drugs interact with plasma or tissue proteins or with
other macromolecules (such as melanin and DNA) to form a
drug–macromolecule complex.
• The formation of a drug–protein complex is often named
drug–protein binding which may be a reversible or an

Dr. Muhammad Usman

irreversible process.

Reversible

Irreversible
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 Protein Binding of Drugs
• Irreversible drug–protein binding is usually a result of
chemical activation of the drug, which then attaches
strongly to the protein or macromolecule by covalent
chemical bonding.

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• It accounts for certain types of drug toxicity that may
occur;
1. Over a long period of time e.g Carcinogenesis
2. Within relatively short period of time e.g Hepatotoxicity
of high doses of acetaminophen is due to the formation
of reactive metabolite intermediates that interact with
liver proteins 5
However, most drugs bind to proteins by reversible process
Protein Binding of Drugs
• Reversible drug–protein binding implies that the drug
binds the protein with weaker chemical bonds, such as
o Hydrogen bonds
o Van der Waals forces.

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• The amino acids that compose the protein chain have
hydroxyl, carboxyl, or other sites available for reversible
drug interactions.

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Protein Binding of Drugs
• Reversible protein binding is of major concern for
pharmacokinetics as the drug-protein complex can not
transverse the capillary walls and therefore has a restricted
distribution.
• Moreover, protein bound drug is usually pharmacologically

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inactive.
• In contrast, the free drug crosses cell membrane and is
therapeutically active

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Drug Binding Proteins
• Drugs may bind to various macromolecular components
in the blood, including
1. Albumin,
2. a1-acid glycoprotein,

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3. Lipoproteins,
4. Immunoglobulins (IgG), and
5. Erythrocytes (RBC).

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Drug Binding Proteins
1. Albumin
❑ Molecular weight = 65,000 to 69,000 Da
❑ Synthesized in liver and is major component of plasma
proteins that are responsible for reversible drug binding.

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❑ Distributed in plasma and extracellular fluids of skin,
muscles and various other tissues.
❑ Interstitial fluid Alb concentration is about 60% of that in
plasma.
❑ Normal Alb concentration is 3.5% to 5.5% W/V (4.5 mg/dL).
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Albumin (Cont.)
Functions
❑ Alb is responsible for maintaining the osmotic pressure
of blood. Oncotic pressure or Colloidal Osmotic Pressure
(COP)

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❑ Transmission of endogenous substances such as Free
fatty acids, bilirubin, hormones, divalent cations e.g
Calcium, Zinc etc.
❑ Binding and transportation of exogenous substances
(drugs etc) in plasma
❑ Weak acidic drugs such as salicylates, phenylbutazone,
penicillins etc. bind to albumin 10
Drug Binding Proteins
2. Alpha-1-acid glycoprotein (AAG)
❑ Also known as orosomucoid
❑ Molecular weight = 44,000 Da
❑ Plasma concentration of AAG is 0.4% to 1%

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❑ Binds primarily to basic drugs
o Propranolol,
o Imipramine
o Lidocain
o Saquinavir
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 Drug Binding Proteins
Alpha-1-acid glycoprotein (AAG)
❑ It has only one drug binding site in each molecule.
Therefore, binding to α-1-acid glycoprotein is saturable and
displaceable
❑ AAG concentration significantly increases in various

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diseases (Cancer, inflammatory diseases) or following
trauma (Burns, surgery).
❑ Change in concentration of AAG can alter the
pharmacokinetics disposition and pharmacological actions
by altering the free drug fraction in plasma or at the site of
action.
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 Drug Binding Proteins
3. Globulins (α, β and ɤ-globulins)
❑ Produced in liver as well as by immune system.
❑ Normal concentration in blood is 2.6 to 3.5 g/dL
❑ Play important role in liver function.
❑ Globulins exert oncotic pressure

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❑ May be responsible for the plasma transport of certain
endogenous substances such as corticosteroids.
❑ The globulins have a low capacity but high affinity for the
binding of these endogenous substances.
❑ The deficiency of globulins may lead to oedema and
increased susceptibility to infections due to decreased
immunogloblins 13
 Drug Binding Proteins
4. Erythrocytes
❑ The major cell body in blood is erythrocyte. Approx 95% of
total cellular components of blood
❑ Major protein in erythrocytes is Hemoglobin which is 7-8
time more than albumin

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❑ Molecular weight of Hb is 64,500 Da which is comparable
to Albumin.
❑ Different drugs bind to blood cells
❑ Acetazolamide
❑ Diphenyldanton
❑ Pentazocin
❑ Tetrahydrocnnabinol
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Factors affecting Protein
Binding
1. Drug related factors
• Physicochemical properties of the drug
• Total concentration of the drug in the body
2. Protein related factors
• Quantity of protein available for drug–protein binding

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• Quality or physicochemical nature of the protein synthesized
3. Affinity between drug and protein
4. Drug Interactions
• Competition for the drug by other substances at a protein-binding site.
• Alteration of the protein by a substance that modifies the affinity of
the drug for the protein; for example, aspirin acetylates lysine residues
of albumin
5. Pathophysiological conditions 15
• Uremic and hepatic patients
Effect of drug Displacement on
PK and PD

Intensive
Drug Concentration
Pharmacological action

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Drug
Displacement

Volume of Distribution Elimination

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Relationship of plasma drug-Protein binding
to the Distribution and Elimination
In General

Protein Binding Clearance

• Hepatic elimination:
Protein binding leads to reduced hepatic elimination due to;

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1. Reduced entry of drug to hepatocytes
2. Bound drug may not be available as substrate for enzyme
action
• Renal elimination:
Protein-bound drugs act as larger molecules. The elimination
half-lives of some drugs such as the cephalosporins, are
generally increased when the percent of drug bound to plasma
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proteins increases
 70% appears
in bile

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Some cephalosporins are excreted by both renal and biliary secretion.
The half-lives of drugs that are significantly excreted in the bile do
not correlate well with the extent of plasma protein binding. 18
 Dr. Muhammad Usman
A drug that is both extensively bound and actively secreted by the
kidneys, such as penicillin, has a short elimination half-life, because
active secretion takes preference in removing or stripping the drug
from the proteins as the blood flows through the kidney.
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Relationship between Vd and
elimination half life
𝑪𝑳 = 𝒌 × 𝑽𝒅

𝟎. 𝟔𝟗𝟑
𝑪𝑳 = × 𝐕𝐝
𝒕𝟏/𝟐

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𝑽𝒅
𝒕𝟏/𝟐 = 𝟎. 𝟔𝟗𝟑
𝑪𝑳
Mechanistically, a relatively low plasma drug
concentration from a given dose may be resulted from
(1) Extensive distribution into tissues
(2) Extensive distribution into tissues due to protein binding in
peripheral tissues, and/or
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(3) lack of drug plasma protein binding
Clinical Examples
1. Dirithromycin
Vd = 800 L (504 L-1041 L)
t1/2 = 44 h
CL = 13.6 to 62.4 L/h

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2. Tenoxicam
Vd = 9.6 L (7.5 L to 11.5 L)
t1/2 = 67 h
CL = 0.106 L/h

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Restrictive vs Non-restrictive
elimination
• Restrictive Elimination:
When a drug is tightly bound to a protein, only the unbound
drug is assumed to be metabolized; drugs belonging to this
category are described as restrictively eliminated.

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• Nonrestrictive elimination:
Some drugs may be eliminated even when they are protein
bound; drugs in this category are described as nonrestrictively
eliminated.
Nonrestrictively cleared drugs are normally rapidly eliminated
since protein binding does not impede the elimination process.
Examples: morphine, metoprolol, and propranolol.
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Restrictive vs Non-restrictive
elimination
• Restrictive Elimination:
Affected with change in drug or protein concentration however,
free drug concentration remains same
• Nonrestrictive elimination:

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Change in drug or protein concentration has no effect on
elimination but affects the free drug concentration

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Methods for studying drug
Protein binding
1. Equilibrium Dialysis
2. Dynamic dialysis
3. Diafiltration

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4. Ultrafiltration
5. Gel Chromatography
6. Spectrophotometry
7. Electrophoresis
8. Optical rotary dispersion and circulatory
dichroism 24
Equilibrium Dialysis
Semi-
Drug Molecule Protein permeable
membrane

Dr. Muhammad Usman

1 2

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CD(Total)
Equilibrium Dialysis
Semi-
Drug Molecule Protein permeable
membrane

Dr. Muhammad Usman

1 2

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CD(f) CD(f) CD(B)

CD(Total)
 𝑪𝑫𝑻𝒐𝒕𝒂𝒍 = 𝑪𝑫𝒇 + 𝑪𝑫𝒇 + 𝑪𝑫𝑩𝒐𝒖𝒏𝒅
At equilibrium
𝑪𝑫𝑻𝒐𝒕𝒂𝒍 = 𝟐 𝑪𝑫𝒇 + 𝑪𝑫𝑩𝒐𝒖𝒏𝒅

𝑪𝑫𝑩𝒐𝒖𝒏𝒅 = 𝑪𝑫𝑻𝒐𝒕𝒂𝒍 − 𝟐 𝑪𝑫𝒇

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Free fraction of drug
𝑪𝑫𝒇
𝒇=
𝑪𝑫𝑻𝒐𝒕𝒂𝒍
Fraction Bound
𝒃=𝟏−𝒇
Percentage Bound = b X 100 27
Case study
Calculate the plasma protein binding of
drug “X”. The total initial concentration of
drug in apparatus is 120 mg/L, while the

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drug concentration in chamber 1 at
equilibrium is 38 mg/L.

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 𝑪𝑫𝑻𝒐𝒕𝒂𝒍 = 𝟏𝟐𝟎 𝒎𝒈/𝑳
𝑪𝑫𝒇 = 𝟑𝟖 𝒎𝒈/𝑳
𝟐 𝑪𝑫𝒇 = 𝟑𝟖 × 𝟐 = 𝟕𝟔 𝒎𝒈/𝑳
𝑪𝑫𝑩𝒐𝒖𝒏𝒅 = 𝑪𝑫𝑻𝒐𝒕𝒂𝒍 − 𝟐 𝑪𝑫𝒇
𝑪𝑫𝑩𝒐𝒖𝒏𝒅 = 𝟏𝟐𝟎 − 𝟕𝟔 = 𝟒𝟒 𝐦𝐠/𝐋
Free fraction of drug

Dr. Muhammad Usman

𝑪𝑫𝒇 𝟕𝟔
𝒇= = = 𝟎. 𝟔𝟑𝟑
𝑪𝑫𝑻𝒐𝒕𝒂𝒍 𝟏𝟐𝟎
Fraction Bound
𝒃 = 𝟏 − 𝒇 = 𝟏 − 𝟎. 𝟔𝟑𝟑 = 𝟎. 𝟑𝟔𝟕
Percentage Bound = 0.367 X 100 = 36.7 %
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Dynamic Dialysis

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Buffer
Solution
Dynamic Dialysis

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CD(f) CD(B) CD(f) Buffer

Solution
 𝑪𝑫𝑻𝒐𝒕𝒂𝒍 = 𝑪𝑫𝒇 + 𝑪𝑫𝒇 + 𝑪𝑫𝑩𝒐𝒖𝒏𝒅
At equilibrium
𝑪𝑫𝑻𝒐𝒕𝒂𝒍 = 𝟐 𝑪𝑫𝒇 + 𝑪𝑫𝑩𝒐𝒖𝒏𝒅

𝑪𝑫𝑩𝒐𝒖𝒏𝒅 = 𝑪𝑫𝑻𝒐𝒕𝒂𝒍 − 𝟐 𝑪𝑫𝒇

Dr. Muhammad Usman

Free fraction of drug
𝑪𝑫𝒇
𝒇=
𝑪𝑫𝑻𝒐𝒕𝒂𝒍
Fraction Bound
𝒃=𝟏−𝒇
Percentage Bound = b X 100 32
Considerations in the study of
drug protein binding
1. Equilibrium between free and bound drug must be
maintained
2. The method must be valid over a wide range of drug and
protein concentration
3. Drug adsorption onto the apparatus walls, membrane or

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other components must be avoided
4. Protein denaturation must be avoided
5. The method must be capable of detecting both reversible
and irreversible protein binding
6. The method should NOT introduce interfering substances
such as organic solvents
7. The results of in-vitro method should allow extrapolation to
in-vivo situations 33
 Drug Interactions at Protein
binding
Acidic Acidic
Alpha-1 Acid Basic Basic
Albumin
Glycoprotein

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Acidic Drugs Basic Drugs
Generally form salt
Generally ending with “ine”
with cations
For Example:
For Example
Atropine
Diclofenac Na+, K+
Morphine
Montelukast Na+
Amphetamine
Aspirin
Tubocurarine etc
Form salts with anions (Cl-, Br- , Sulphate etc)
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 Distribution Decreased
Implications of Protein

Displacement Interaction
Binding

Duration of action Increased

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Nephrotic
Disease
Syndrome

Hypoalbuminemia
Dialysis
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Dose adjustment required for
acidic drugs
Dialysis and drugs
• Requirements
1. Less Protein Binding
2. Less Volume of distribution

Amphetamines

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Verapamil
A spirin
O pioids M ethanol
I mipramine (TCAs) L ithium
Digoxin, Diazepam (BZP)
Dialysis 37
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