Plasma Protein Binding

5.14.1.1 Overview

The role of plasma protein binding (PPB) in the discovery process and the impact this key parameter has on the discovery and clinical process is now becoming fully realized. The pharmacokinetic and pharmacodynamic properties of drugs are greatly influenced by the reversible binding to plasma proteins with the unbound fraction of the drug being responsible for the biological activity. Methods are now available for the rapid determination of free levels in plasma using a multitude of techniques from the traditional dialysis methods to the more recent surface plasmon resonance methodologies.

5.14.1.2 Biological Aspects

Human plasma is known to contain over 60 different proteins, the major component being albumin, which comprises approximately 60% of the total plasma protein.1 The next most abundant and well-characterized protein is a^acid glycoprotein. Human serum albumin has a molecular weight of 66kDa and contains 585 amino acid residues. At least 18 different mutations of human serum albumin have been identified and are primarily due to a single amino acid mutation, accounting for distinct protein-ligand binding.2 The concentration of albumin in a normal healthy adult male is typically 43gL_ with a range of 35-53gL_ 1 Females have approximately a 9% lower concentration (38gL"1)1'3 and this is argued to account for the gender difference in binding of chlorodiazepoxide and warfarin.

In diseased patients the albumin concentration can be significantly different. For patients with nephrotic syndrome, burns, or cirrhosis, the albumin concentration can be less than 10 gL_ 1, i.e., 20-30% of the normal concentration.4 In contrast, the normal plasma concentration (human) of a1-acid glycoprotein is 0.4-1.0gL_ 1 (10-30mM) (MW = 44kDa), and in patients with inflammatory diseases, it can be elevated by up to 4-5-fold.4 Taking into account the significant variation in protein content and concentration in human plasma (and other species) it is useful to determine PPB using large plasma pools containing a statistically reasonable number of donors.2

Plasma contains many other globulins (the name of a family of proteins precipitated from plasma or serum by addition of ammonium sulfate). These can be separated into many subgroups, the main ones being alpha-, beta-, and gamma-globulins which differ with respect to the associated lipid or carbohydrate. Immunoglobulins (antibodies) are in the a and b fractions, lipoproteins are in the c and d fractions. Other substances in the globulin fractions include macroglobulin, plasminogen, prothrombin, euglobulin, antihemomorphic globulin, fibrinogen, and cryoglobulin. The lipoproteins in plasma, of which a1-acid glycoprotein is one, can be further classified into very high-density (VHDL), high-density (HDL), low-density (LDL), and very low-density (VLDL). The higher the density the lower the lipid content. Lipoproteins are macromolecular complexes displaying characteristic sizes, densities, and compositions. All lipoproteins contain protein components, called apoproteins, and polar lipids (phospholipids) in a surface film surrounding a neutral core (free and esterified cholesterol, triglycerides). The plasma lipoproteins vary in composition with respect to the lipid component, because their principal physiological function is to transport lipids in a water-soluble form, but also vary with respect to the polypeptide chain composition. Lipoprotein plasma concentration may vary 5-10-fold. Gamma-globulins generally only marginally account for the plasma binding of drugs.5 Often it is only when a drug is present at very high concentrations that binding to components other than albumin or a1-acid glycoprotein occurs.6 Considerable intersubject variability in the PPB of some compounds (4-5-fold variation in/u,p is not uncommon) and in the concentration of proteins exists even within healthy human volunteers.3 Genetically determined variations in amino acid sequences of human serum albumin can also contribute to variability in binding, and cause higher variability in patients with highly bound drugs. The binding affinities of warfarin, salicylate, and diazepam to five known variants of human serum albumin have been studied.7 The association constants for all three drugs to albumin decreased by a factor of 4-10-fold for the mutations relative to each other.

5.14.1.3 Effect of Plasma Protein Binding on Drug Disposition

The extent of plasma protein binding is extremely important in its influence on many pharmacokinetic parameters. The relationship between the in vitro intrinsic clearance, CLint and the in vivo clearance, CL, may be understood by the use of the well-stirred model, which is given in its most simple form by eqn [1]8:

where QH is the hepatic blood flow, with units of mLmin _ 1 kg _ 1, and/ u b the fraction of compound unbound in blood as defined in Section 5.14.2.1.

For drugs with low hepatic clearance, compared to liver blood flow, the in vivo clearance can be approximated by :

where CLint is the intrinsic clearance of the drug and/u p is the fraction of compound unbound in plasma. Under these conditions clearance is directly proportional to/u p. An example of this behavior is given by the clearance of warfarin in male Sprague-Dawley rats which is proportional to the free fraction of the drug within each individual rat, as shown in Figure 1a. For drugs with high hepatic clearance, the clearance is largely controlled by the liver blood flow, QH, and is independent of the extent of plasma binding9:

CLdQh

This type of behavior is illustrated by propranolol where the clearance in humans does not depend on the human free fraction of drug in plasma12 (Figure 1b).

The degree of plasma protein binding cannot only influence metabolic clearance, but also renal clearance.13 The renal clearance (CLR) of a drug consists of four different processes; glomerular filtration (CLf), active secretion (CLrs),

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