Effect of protein binding on PK/PD

Clinical pharmacology weekly meeting

24 Feb. 2015
Sean Oosterholt
Protein Binding

• At therapeutic concentrations in
plasma, many drugs exist mainly
in bound form.
• The most important plasma protein in relation to
drug binding is albumin. Albumin binds many
acidic drugs and a smaller number of basic drugs
– Other plasma proteins include ß-globulin, a-acid
glycoprotein and Lipoproteins
Function of Protein Binding proteins

• Serum albumins are important in regulating blood volume

by maintaining the osmotic pressure of the blood
compartment
– They also serve as carriers for molecules of low water solubility this
way isolating their hydrophobic nature.

• ß-globulins are a subgroup of globulin proteins produced

by the liver or immune system
– Mostly involved with transport.

• α-acid glycoprotein is an alpha-globulin and act as a

carrier of basic and neutrally charged lipophilic compounds
• The amount of a drug that is bound to protein depends on three factors:
– the concentration of the drug
– its affinity for the binding sites
– the concentration of protein.

• As an approximation, the binding reaction can be regarded as a simple association of

the drug molecules with a finite population of binding sites, analogous to drug–receptor
binding:

𝐷 + 𝑆 ⇌ 𝐷𝑆
free Binding complex
drug site

• The usual concentration of albumin in plasma is about 0.6 mmol/l (4 g/100 mL).
With two sites per albumin molecule, the drug-binding capacity of plasma
albumin would therefore be about 1.2 mmol/L.
– For most drugs, the total plasma concentration required for a clinical effect is much less than 1.2
mmol/l, so with usual therapeutic doses the binding sites are far from saturated, and the concentration
bound [DS] varies nearly in direct proportion to the free concentration [D].
– Under these conditions the fraction bound: [DS]/([D] + [DS]) is independent
of the drug concentration.
Some drugs, such as tolbutamide
work at plasma concentrations at
which the binding to protein is
approaching saturation
This means that adding more drug
to the plasma increases its free
concentration disproportionately.

• Doubling the dose of such a

drug can therefore more than
double the free
(pharmacologically active)
concentration.
• It is the unbound drug that is pharmacologically
active
– Both in metabolism in the liver or any pharmacological
effect.

• The fraction of drug that is free in

aqueous solution can be less than
1%,
– small differences in protein binding
(e.g. 99.5 versus 99.0%) can have
large effects on free drug concentration
and drug effect.
– Differences are common between
human plasma and plasma from
species used in preclinical drug testing
Conditions in which the plasma
concentration of major plasma proteins are
altered
Conditions Change in concentration
Albumin hepatic cirrhosis +
burns +
nephritic syndrome +
pregnancy +
a-glycoprotein myocardial infarcts -
surgery -
trauma -
rheumatoid arthritis -
Drug-drug interactions involving protein
binding

Before After % increase in

Displacement Displacement unbound fraction

Drug A
% bound 95 90
% unbound 5 10 +100
Drug B
% bound 50 45
% unbound 50 55 +10
Influence of drug binding on
pharmacokinetic parameters

Volume of Distribution:
• When concentrations are measured as “total plasma concentration” the
volume of distribution can become very small due to protein binding.

Example: Warfarin binds for 99% to plasma Albumin

• Plasma Concentrations after a 10mg Warfarin dose
– Total = 1 mg/L
– Bound = 0.99 mg/L
– Unbound = 0.01 mg/L
• Apparent Volume
– Total = 10 mg/1 mg/L = 10 L
– Unbound = 10 mg/0.01 mg/L = 1000 L
Volume of distribution

• Small volumes of distribution

– Warfarin: 10L
– Gentamicin: 18L

• Low volume of distribution does not necessarily mean plasma

protein binding.
• Both Warfarin and Gentamicin have low volumes of distribution.
– Gentamicin is highly ionized and does not cross membranes very well.
– The volume of distribution of Gentamicin is close to the physical
volume of extracellular fluid.
Clearance

• Clearance is depends on the free drug

concentration and not on protein binding.
• However, only the free fraction of drug can be
cleared
𝑄𝑜𝑟𝑔𝑎𝑛 ∙ 𝑓𝑢 ∙ 𝐶𝑙𝑖𝑛𝑡
𝐶𝑙𝑜𝑟𝑔𝑎𝑛 =
𝑄𝑜𝑟𝑔𝑎𝑛 + 𝑓𝑢 ∙ 𝐶𝑙𝑖𝑛𝑡
Importance of protein binding

In most cases Clinically not very important

– Generally less than one third of the drug in the body in bound to plasma proteins even in
the most extreme cases.
• A change in unbound fraction from 1% to 10% releases less than 5% of the total amount of drug
in the body, and produces at most a %5 increase in pharmacologically active unbound drug at the
site of action.
– Displacement of drugs from the binding site does not happen very often
• Drugs that are “displacers” are few and rarely used therapeutically

Exception:
• IV bolus dose can cause rapid displacement of substances bound to plasma
proteins before redistribution happens
• Translating between species or from in vitro to in vivo
– Protein binding can have big differences between species
in vitro to in vivo

minimum inhibitory
concentration (MIC) is
the lowest concentration of
an antimicrobial that will
inhibit the visible growth of
a microorganism

Dashed line: Control growth

Filled squares: Drug without albumin
Filled circles: Drug with albumin
White squares: drug without albumin in a concentration equivalent to in vivo unbound
drug concentrations
Discussion

Plasma protein binding does not automatically result in

clearance restrictions.
• Example: Ciclesonide: bound for 99% to proteins but
clearance is close to hepatic blood flow
– Intrahepatic re-equilibration between bound and free drug
can occur so fast that the vast majority of drug can be
metabolized as the blood crosses the liver.
• Same could be said about the pharmacodynamic
effect, if association/dissociation is quick
Guidelines

• EMA:
– “The degree of protein binding of the investigational drug
should be determined before phase I. If the investigational
drug is extensively protein bound to a specific binding site
and present at concentrations saturating the binding sites,
the risk of displacement of other drugs known to be
subject to clinically relevant displacement interactions
could be evaluated in vitro at a time point relevant for the
clinical development program. If a clinically relevant
interaction is predicted based on in vitro data, an in vivo
study measuring unbound concentrations could be
considered.”