Equilibrium dialysis method, a classic method for determining plasma protein binding rate

 Drugs are bound to plasma proteins to varying degrees in plasma, and the degree of binding can affect the drug's absorption, distribution, metabolism, and excretion in vivo, which in turn can affect the pharmacodynamic behavior of the drug. In general, after absorption into the bloodstream, only free drug reaches the site of action and produce pharmacological activity. 

Equilibrium dialysis is one of the most commonly used method to determine the free concentration of medicines based on the equilibrium principle of drug binding. It is the classical method to study drug plasma protein binding rate.

It has been found that the critical physiological and pharmacological functions of proteins require the participation of small drug molecules, which can promote or inhibit the protein functions through their interaction with proteins. The binding of drugs to plasma proteins can affect the biological activity of drugs. Therefore, determining the drug plasma protein binding rate is more important for drug development.

1. Equilibrium dialysis method

Plasma protein binding is one of the most critical pharmacological parameters in animals, affecting the drug's free concentration in vivo and, thus, the drug disposal process. Plasma protein binding studies of drugs in higher animals have been developed over time. 

Drug plasma protein binding can be measured by equilibrium dialysis, which uses a dialysis membrane to separate the protein solution from the buffer, establishing a state of equilibrium between the two in which only small molecular weight drug small molecules can pass. The driving force of dialysis is diffusion pressure, which is created by the concentration gradient across both sides of the membrane. 

The rate of dialysis is related to the thickness of the membrane, the concentration gradient of the dialyzed small molecule solute across both sides of the membrane, and the dialysis temperature. Equilibrium dialysis can directly measure the number of small drug molecules bound to the protein, which is the key to analyzing the binding of protein to small molecule substances, thus enabling the number of binding sites and binding constants to be derived.

2. The Effect of Plasma Protein Binding on Drugs

After various drugs bind to plasma proteins at a specific rate, there will be both bound and free types in the plasma, and free pills have drug activity. The free drug has drug activity. The drug bound to plasma protein becomes a bound drug, temporarily loses its pharmacological activity, and is stored in the blood, acting as a drug reservoir. It is of great importance for the drug action and its maintenance time. Generally, drugs with high protein binding rates have slow elimination in vivo, long duration of action, and smooth effect. Drugs with low binding rates are eliminated quickly in vivo, while the time of action is short, and there are large fluctuations in efficacy.

When two or more drugs are combined, they can compete for the plasma protein binding site. The drug with strong binding capacity can replace the drug with weak binding capacity from the protein binding site, increasing the number of free forms of the latter and leading to enhanced efficacy and toxic reactions. The extent of the effect can vary depending on the volume of distribution of the latest in the body. Only drugs with high plasma protein binding, a small volume of distribution, slow elimination, and low therapeutic index are clinically relevant for such interactions. 

The determination of plasma protein binding rate belongs to the content of in vitro pharmacokinetic research. Medicilon has rich experience in vitro research on pharmacokinetics, including metabolic stability, P450 induction and inhibition, metabolic pathway, and metabolite identification, with animals involved, including rats, mice, rabbits, dogs, and monkeys.

3. Application of equilibrium dialysis method to determine plasma protein binding rate

For example, one researcher used the equilibrium dialysis method to determine the plasma protein binding rate of nine components in Inula cappa extract. The researchers used equilibrium dialysis to determine the extract's plasma protein critical rate of nine elements. The biological samples were processed by methanolic precipitation of protein. Then the researchers used the plasma and buffered solutions to determine the concentration of chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, scopolamine, etc.

The nine components showed good linearity (r≥0.999) in the concentration range studied. The accuracy, precision, extraction recovery, and stability followed the requirements for determining biological samples. There was no interference from endogenous impurities. The average protein binding rates of the nine components in human and rat plasma ranged from (41.07±0.046)% to (94.95±0.008)% and (37.66±0.043)% to (97.46±0.013)%, respectively. The results showed differences in the plasma protein binding rates of nine components of Inula cappa extract in humans and rats.

Other researchers used equilibrium dialysis to simulate the binding process of LS-177 to plasma proteins in vivo and used UPLC-MS/MS to determine the content of LS-177 in dialysis external and internal fluids and to calculate the binding rate of LS-177 to plasma proteins in rats, beagle dogs, and humans. The results showed that the established method for determining the LS-177 protein critical rate was simple, stable, and reliable, and the plasma protein binding rate of LS-177 did not have significant concentration dependence and species differences.

The equilibrium dialysis method has the advantages of simple operation, easy temperature control, adjustable pH value, and low equipment cost in determining drug plasma protein binding rate. However, disadvantages such as long equilibration time, change of solution volume, and degradation of the measured substance caused by heating or metabolism may be caused by too long a dialysis time. Therefore, when using equilibrium dialysis to determine the plasma protein binding of drugs, drug-protein interactions need to be combined with other methods to obtain more accurate information about the action.

[1] Equilibrium dialysis method to determine the plasma protein binding of nine components of Inula cappa extract [J].