FACTORS AFFECTING PROTEIN-DRUG BINDING
Factors affecting protein-drug binding can be broadly categorized as—
1. Drug related factors
a. Physicochemical characteristics of the drug
b. Concentration of drug in the body
C. Affinity of a drug for a particular binding component
2. Protein/tissue related factors
a. Physicochemical characteristics of the protein or binding agent
b. Concentration of protein or binding component
c. Number of binding sites on the binding agent
3. Drug interactions
a. Competition between drugs for the binding site (displacement interactions)
b. Competition between the drug and normal body constituents
c. Allosteric changes in protein molecule
4. Patient related factors
a. Age
b. Intersubject variations
c. Disease states
1. DRUG RELATED FACTOR
a. Physicochemical Characteristics of the Drug
As mentioned earlier, protein binding is directly related to the lipophilicity of drug. An increase in lipophilicity increases the extent of binding
b. Concentration of Drug in the Body
The extent of protein-drug binding can change with both changes in drug as well as protein concentration. The concentration of drugs that bind to HSA does not have much of an influence, as the therapeutic concentration of any drug is insufficient to saturate it. However, therapeutic concentration of lidocaine can saturate AAG with which it binds as the concentration of AAG is much less in comparison to that of HSA in blood.
c. Drug-Protein/Tissue Affinity
Lidocaine has greater affinity for AAG than for HSA. Digoxin has more affinity for proteins of cardiac muscles than those of skeletal muscles or plasma. Iophenoxic acid, a radio-opaque medium, has so great an affinity for plasma proteins that it has a half-life of 2½ years.
2. PROTEIN/TISSUE RELATED FACTORS
A. Physicochemical Properties of Protein/Binding Component
Lipoproteins and adipose tissue tend to bind lipophilic drugs by dissolving them in their lipid core. The physiologic pH determines the presence of active anionic and cationic groups on the albumin molecules to bind a variety of drugs.
B. Concentration of Protein/Binding Component
Among the plasma proteins, binding predominantly occurs with albumin, as it is present in a higher concentration in comparison to other plasma proteins. The amount of several proteins and tissue components available for binding, changes during disease states. This effect will be discussed in the subsequent sections.
C. Number of Binding Sites on the Protein
Albumin has a large number of binding sites as compared to other proteins and is a high capacity binding component. Several drugs are capable of binding at more than one site on albumin, e.g. fluocloxacillin, flurbiprofen, ketoprofen, tamoxifen and dicoumarol bind to both primary and secondary sites on albumin. Indomethacin is known to bind to 3 different sites. AAG is a protein with limited binding capacity because of its low concentration and low molecular size. Though pure AAG has only one binding site for lidocaine, in presence of HSA, two binding sites have been reported which was suggested to be due to direct interaction between HSA and AAG.
3. DRUG INTERACTIONS
A. Competition Between Drugs for the Binding Sites (Displacement Interactions)
When two or more drugs can bind to the same site, competition between them for interaction with the binding site results. If one of the drugs (drug A) is bound to such a site, then administration of another drug (drug B) having affinity for the same site results in displacement of drug A from its binding site. Such a drug-drug interaction for the common binding site is called as displacement interaction. The drug A here is called as the displaced drug and drug B as the displacer. Warfarin and phenylbutazone have same degree of affinity for HSA. Administration of phenylbutazone to a patient on warfarin therapy results in displacement of latter from its binding site. The free warfarin may cause adverse hemorrhagic reactions which may be lethal. Phenylbutazone is also known to displace sulphonamides from their HSA binding sites. Displacement interactions can result in unexpected rise in free concentration of the displaced drug which may enhance clinical response or toxicity. Even a drug metabolite can affect displacement interaction.
Clinically significant interactions will result when:
I. The displaced drug (e.g. warfarin) —
1. Is more than 95% bound.
2. Has a small volume of distribution (less than 0.15 L/Kg).
3. Shows a rapid onset of therapeutic or adverse effects.
4. Has a narrow therapeutic index.
II. The displacer drug (e.g. phenylbutazone) —
1. Has a high degree of affinity as the drug to be displaced.
2. Competes for the same binding sites.
3. The drug/protein concentration ratio is high (above 0.10).
4. Shows a rapid and large increase in plasma drug concentration.
It will be worthwhile to mention here that, both the concentration of the displacer drug and its affinity for the binding site with respect to that of the drug to be displaced, will determine the extent to which displacement will occur.
For a drug that is 95% bound, a displacement of just 5% of the bound drug results in a 100% rise in free drug concentration. If the displaced drug has a small volume of distribution, it remains confined to the blood compartment and shows serious toxic responses. On the contrary, if such a drug has a large Vd, it redistributes into a large volume of body fluids and clinical effects may be negligible or insignificant. The increase in free drug concentration following displacement also makes it more available for elimination by the liver and the kidneys (Fig. 4.3). If the drug is easily metabolisable or excretable, its displacement results in significant reduction in elimination half-life.
Fig. 4.3. Fate of a drug after displacement interaction
Displacement also becomes insignificant with the use of more selective, potent, low dose drugs.
Besides the direct displacement interaction discussed above, indirect interactions are also possible. For example, the use of heparin as an anticoagulant activates lipoprotein lipase, an enzyme which metabolises triglycerides to free fatty acids. Heparin co-administration with drugs has also been shown to result in decreased protein binding of propranolol, quinidine, etc. via its effects on fatty acid levels.
B. Competition Between Drugs and Normal Body Constituents
Among the various normal body constituents, the free fatty acids are known to interact with a number of drugs that bind primarily to HSA. The free fatty acid level is increased in several physiologic (fasting), pathologic (diabetes, myocardial infarction, alcohol abstinence) and pharmacologically induced conditions (after heparin and caffeine administration). The fatty acids, which also bind to albumin, influence binding of several benzodiazepines and propranolol (decreased binding) and warfarin (increased binding). Bilirubin binding to HSA can be impaired by certain drugs and is of great concern in neonates whose BBB and bilirubin metabolising capacity are not very efficient. Acidic drugs such as sodium salicylate, sodium benzoate and sulphonamides displace bilirubin from its albumin-binding site. The free bilirubin is not conjugated by the liver of the neonates and thus crosses the BBB and precipitates the condition called as kernicterus (characterized by degeneration of brain and mental retardation).
C. Allosteric Changes in Protein Molecule
This is yet another mechanism by which drugs can affect protein-binding interactions. The process involves alteration of the protein structure by the drug or its metabolite thereby modifying its binding capacity. The agent that produces such an effect is called as allosteric effector, e.g. aspirin acetylates the lysine fraction of albumin thereby modifying its capacity to bind NSAIDs like phenylbutazone (increased affinity) and flufenamic acid (decreased affinity).
4. PATIENT RELATED FACTORS
A. Age
Modification in protein-drug binding as influenced by age of the patient is mainly due to differences in the protein content in various age groups.
i. Neonates: Albumin content is low in newborn; as a result, the unbound concentration of drug that primarily binds to albumin, for example phenytoin and diazepam, is increased.
ii. Young infants: An interesting example of differences in protein-drug binding in infants is that of digoxin. Infants suffering from congestive cardiac failure are given a digitalizing dose 4 to 6 times the adult dose on body weight basis. This is contrary to one's belief that infants should be given low doses considering their poorly developed drug eliminating system. The reason attributed for use of a large digoxin dose is greater binding of the drug in infants (the other reason is abnormally large renal clearance of digoxin in infants).
iii. Elderly: In old age, the albumin content is lowered and free concentration of drugs that bind primarily to it is increased. Old age is also characterized by an increase in the levels of AAG and thus decreased free concentration is observed for drugs that bind to it. The situation is complex and difficult to generalize for drugs that bind to both HSA and AAG, e.g. lidocaine and propranolol.
B. Intersubject Variations
Intersubject variability in drug binding as studied with few drugs showed that the difference is small and no more than two fold. These differences have been attributed to genetic and environmental factors.
c. Disease States
Several pathologic conditions are associated with alteration in protein content. Since albumin is the major drug binding protein, hypoalbuminaemia can severely impair protein-drug binding. Hypoalbuminaemia is caused by several conditions like aging, CCF, trauma, burns, inflammatory states, renal and hepatic disorders, pregnancy, surgery, cancer, etc. Almost every serious chronic illness is characterized by decreased albumin content. Some of the diseases that modify protein-drug binding are depicted in Table 4.3. Hyperlipoproteinaemia, caused by hypothyroidism, obstructive liver disease, alcoholism, etc., affects binding of lipophilic drugs.
Influence of Disease States on Protein-Drug Binding
Putting in a nutshell, all factors, especially drug interactions and patient related factors that affect protein or tissue binding of drugs, influence:
1. Pharmacokinetics of drugs: A decrease in plasma protein—drug binding i.e. an increase in unbound drug concentration, favours tissue redistribution and/or clearance of drugs from the body (enhanced biotransformation and excretion).
2. Pharmacodynamics of drugs: An increase in concentration of free or unbound drug results in increased intensity of action (therapeutic/toxic).
