Protein binding of drugs and screening of drugs by physicochemical properties

1. Protein Binding of Drugs
Dr. Bhaswat S. Chakraborty

2. Drug-Protein Binding
• Drug protein binding is usually a reversible interaction of
drugs with proteins in plasma
• Also includes the reversible interaction of drugs with red
blood cell and tissue membranes and other blood
constituents
• Binding plasma proteins are:
– Albumin
– α1-acid glycoprotein
– Lipoproteins
• PB can be measured in vitro by ultrafiltration &
equilibrium dialysis

3. D-R Effect
D + P D-P
Ka
Kd
+
R
K2 K1
Central
Compartment
Peripheral
Compartment

4. Higher the Kd, the more the unbound drug; lower the K2 the
more effect of the drug

5. Why is PB Important?
• Important from Biological (PK/PD/Toxicology) point of view not from
Clinical point of view
• PK
– High PB will lead to more drug present in the central blood compartment and
therefore a lower Vd
• Exception: high degree of binding to tissue proteins will also show high Vd
– Low PB: more drug is free to move into tissue and therefore result in a high Vd
– Knowledge of the unbound fraction in blood is essential to the correlation of total
plasma concentration with activity
• Toxicity/PD
– High PB of a compound can show drug toxicity
– A small change (from drug interactions or disease state) in the extent of binding of a
very high PB → large change in drug concentration in plasma
• e.g., phenytoin is 90% PB, can be displaced by co prescribed drugs, e.g., valproic
acid, in particular can increase phenytoin Fu by 30% - 100% → lead to toxicity

6. Different Binding Sites
• Albumin and α1-acid glycoprotein have structurally selective binding sites
for drugs
– Each albumin molecule has at least 6 distinct binding sites for drugs and
endogenous compounds
– 2 of these very tightly and specifically bind long chain fatty acids
– Another site selectively binds bilirubin
– Two major sites (site I and site II) mainly bind acidic drugs
• Site I binds drugs e.g., warfarin and phenylbutazone, whereas site II binds drugs
e.g., diazepam and ibuprofen
– Drugs which bind at the same site can be predicted to displace each other
competitively when co-administered
• α1-acid glycoprotein is an acute phase reactant which has one binding site
selective for basic drugs such as disopyramide and lignocaine.

7. Unbound Fraction
• Unbound fraction (Fu)of a drug is determined by:
– the affinity of the drug for the protein
– the concentration of the binding protein
– the concentration of drug relative to that of the binding protein
• In most cases, drug concentrations at therapeutic doses are well below
those of the binding protein and the Fu is constant across the
therapeutic range of drug concentration
• However, the concentration of α1-acid glycoprotein is relatively low, and
saturation of the binding sites can occur in the therapeutic range
– E.g., disopyramide; Cu increases linearly with dose, but there is a less than
proportionate increase in Ct as saturation occurs causing fraction unbound to
increase
• Albumin concentrations are high, and saturation rarely occurs with drugs
binding to this protein
– An exception is salicylate which has high therapeutic concentrations.

8. Effect of Diseases on Protein Conc.
• The concentration of albumin is decreased in liver disease and
renal disease resulting in decreased drug binding
• α1-acid glycoprotein is an acute phase reactant and concentrations
increase in rheumatoid arthritis and post-myocardial infarction
resulting in increased drug binding in these situations
• The binding affinity can be changed due to competition from
endogenous compounds such as fatty acids, or from other drugs
competing for the same protein binding sites
• In-vitro situations
– warfarin is about 98% bound and 2% unbound (Fu = 0.02). If a competing drug
reduces the binding from 98% to 96%, the in vitro Fu rises from 0.02 to 0.04, a
twofold increase
Birkett D. (1992) Australian Prescriber, 15: 56-57

9. In Vivo change in Fu
• In vivo, two compensating mechanisms operate
• Vd is dependent on the ratio of fraction unbound in blood and
tissues (Fu/Fut)
– If Fu in blood increases (competitive displacement) without a change
in tissue binding, Vd increases as the displaced drug ‘spreads out’
and is bound in the tissues. This happens quickly within minutes to
hours
– Secondly, Fu is a factor in the clearance of total drug (clearance =
fraction unbound X intrinsic clearance), but clearance of unbound
drug is determined only by intrinsic clearance and does not depend
on protein binding
– Therefore, when Fu increases due to displacement, drug is
eliminated more rapidly until the unbound (active) steady state
concentration returns to the starting point
– Resulting in an increase in Fu, a decrease in total drug concentration,
but no change in the steady state Cu

10. In Vivo change in Fu
• Ct at steady state is reduced in proportion to the increase in Fu (this takes
3-5 half-lives to occur)
• In general, the only situation where unbound drug concentration at
steady state is dependent on the degree of protein binding is that of
high hepatic clearance drugs given intravenously (e.g. lignocaine)
• In cases like phenytoin, binding to albumin is reduced in renal failure,
liver disease or in the presence of competing drugs
– In such cases, total concentration measurements are misleading and control
of therapy needs to be based on measurement of unbound phenytoin
concentration
– Such measurements are available in specialized centres

15. binding- 15

16. Clinical Implications
• In nearly all cases where a clinically important protein
binding interaction has been postulated, other mechanisms,
such as concurrent inhibition of drug metabolism, have been
shown to be the in vivo cause of the increase in drug effect
• This is why it is so important in studies of drug interactions to
measure both total and unbound drug in determining the
mechanism(s)
• Except in very rare circumstances, protein binding
displacement in vivo does not result in increased drug effect

17. Screening of Drugs by
Physicochemical Properties

18. The Design Approaches

19. Simplifying

20. Lipophilicity: logP
logP needs to be optimised

21. Lipophilicity
• Lipophilicity is perhaps the most important physical property
of a drug in relation to its absorption, distribution, potency,
and elimination
• Lipophilicity is often an important factor in all of the
following, which include both biological and physicochemical
properties:

22. Good Absorption and Permeation
• There are various guidelines to help, the most well-known of
which is the Lipinski Rule of Five
 molecular weight < 500
 logP < 5
 < 5 H-bond donors (sum of NH and OH)
 < 10 H-bond acceptors (sum of N and O)
• An additional rule was proposed by Veber
 < 10 rotatable bonds
• Otherwise absorption and bioavailability are likely to be poor.
NB This is for oral drugs only.

23. Thank You