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Extent of distribution

We can now consider factors which alter the extent of drug distribution

Plasma protein binding

Extensive plasma protein binding will cause more drug to stay in the central blood compartment. Therefore drugs which bind strongly to plasma protein tend to have lower volumes of distribution.
Proteins involved
Although drugs are bound to many macromolecules, binding to plasma protein is the most common. Of these plasma proteins, albumin, which comprises 50 % of the total proteins binds the widest range of drugs. Acidic drugs commonly bind to albumin, while basic drugs often bind to alpha1-acid glycoproteins and lipoproteins. Many endogenous substances, steroids, vitamins, and metal ions are bound to globulins.

Table XVIII-4 Proteins with Potential Binding Sites for Various Drugs[3]

Drugs Binding Sites for Acidic Agents
Bilirubin, Bile acids, Fatty Acids,Vitamin C, Salicylates, Sulfonamides,Barbiturates, Phenylbutazone,Penicillins, Tetracyclines, ProbenecidAlbumins
  Binding Sites for Basic Agents
Adenisine, Quinacrine, Quinine,Streptomycin, Chloramphenicol,Digitoxin, Ouabain, CoumarinGlobulins, alpha1, alpha2, beta1, beta2, gamma

Forces involved
Groups on the protein molecules that are responsible for electrostatic interactions with drugs include:

the of lysine and N- terminal amino acids,

the of histidine, the - S- of cysteine, and

the - COO- of aspartic and glutamic acid residues.

In order to achieve reasonably stable complexes, however, it is likely that in most cases the initial electrostatic attraction is reinforced at close range by van der Waal's forces (dipole-dipole; dipole-induced dipole; induced dipole-induced dipole) and hydrogen bonding.

This is suggested by the frequently crucial role of protein configuration in the binding phenomenon. Agents which denature protein may cause the release of bound drug.

Often there may be competition between drugs, in which agents that are bound very tightly, such as coumarin anticoagulants, are able to displace less tightly bound compounds from their binding sites.

Table XVIII-5 Percent Unbound for Selected Drugs[4,5]

DrugPercent Unbound (100 * fu)

Slight changes in the binding of highly bound drugs can result in significant changes in clinical response or cause a toxic response. Since it is the free drug in plasma which equilibrates with the site of pharmacological or toxic response, a slight change in the extent of binding, such as 99 to 98 % bound, which can result in an almost 100 % change in free concentration, can cause very significant alteration in response. For a large number of drugs, including warfarin and phenytoin, drug response will be dependent on free drug concentration. Alteration of free concentration by drug interaction or disease state can alter the intensity of action of these drugs. Examples include phenylbutazone and salicylates displacing tolbutamide to give an increased effect, hypoglycemia.

As you can see from Table XVIII-5, the extent of protein binding can vary considerably from one drug to another.

Protein binding determination
Spectral changes
Most drugs have distinct UV spectra because of the conjugated chromophores in the molecule. When a drug interacts with a protein the UV or visible spectrum may be changed because of alterations in the electronic configuration. These alterations can be quantitated and used to determine the extent of binding. Changes in fluorescence spectra can be used in the same way. Spectra for warfarin[6].

Gel filtration
This involves the use of porous gels that are molecular sieves. They separate components on the basis of size. Low molecular weight drugs are held on the gel whereas bound drug and protein are washed through.

Equilibrium dialysis

Diagram XVIII-3 Equilibrium Across a Semi-permeable Membrane

The protein solution (e.g. plasma) containing drug and a buffer solution are placed on opposite sides of a dialysis membrane.

After a sufficient time (maybe 12- 24 hours), free drug concentration will be the same on either side of the membrane. Protein binding can be determined by measuring the concentration of drug on either side of the membrane. On left the concentration will involve free and bound drug, whereas on the right there is no binding and the concentration will equal to the free drug concentration.


Diagram XVIII-4 Ultrafiltration as a Method of Measuring Protein Binding

A quicker method of separating free and bound drug is the ultrafiltration method. Drug and protein solution are placed in a filter membrane and liquid containing free drug is forced through the membrane by centrifugation.

Protein binding equilibria
With one type of binding site, protein binding can be described mathematically by the equation:

With [D] free drug concentration, [P] total protein concentration with 'n' binding sites per molecule, thus [nP] is the total concentration of protein binding sites and [rP] = [DP] is the concentration of bound drug or bound protein with r drug molecules bound per protein molecule. Typically there may be 1 - 4 binding sites per protein molecule.

Ka = association constant


Figure XVIII-1 Plot of r/[D] Versus r

This can be rearranged to give

thus plotting r/[D] versus r should give a straight line. This is called a Scatchard plot.

Figure XVIII-2, Plot of 1/r Versus 1/[D]

Alternate rearrangement gives

thus a plot of 1/r versus 1/[D] should also give a straight line. This the double reciprocal plot.

With one type of binding site these plots produce straight lines which can be used to determine Ka and n values. With more than one type of binding site, these plots are curved.

Tissue localization of drugs

In addition to plasma protein binding, drugs may bind to intracellular molecules. Certain of these may be actual drug receptors, and the interaction that occurs may represent the molecular basis of the pharmacological action.

The affinity of a tissue for a drug may be for any of several reasons, including binding to tissue proteins (such as albumin) or to nucleic acids, or in the case of adipose tissue, dissolution in the lipid material.

The concentration of chloroquine in the liver is due to the binding of the drug to DNA. Barbiturates distribute extensively into adipose tissue, primarily because of their high lipid solubility. Tetracyclines bind to bone thus should be avoided in young children or discoloration of permanent teeth may occur.

Unlike plasma binding, tissue binding of a drug cannot be measured directly as handling of the tissue results in disruption of the binding. This doesn't mean that tissue binding and changes in tissue binding are not important.

This page was last modified: 12 February 2001

Copyright 2001 David W.A. Bourne

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