When a drug enters the bloodstream, it can exist in two forms: freely dissolved or temporarily bound to plasma proteins. This interaction, known as plasma protein binding, significantly influences how a drug behaves within the body. Understanding the extent of this binding is an important step in drug discovery and development, as it helps determine how a drug will be distributed, processed, and ultimately eliminated.
The Role of Plasma Proteins
Plasma contains several proteins that act as carriers for natural body molecules and drugs. Albumin is the most abundant protein in human plasma, and many small-molecule drugs, fatty acids, and peptides can bind to it. Another significant plasma protein involved in drug binding is alpha-1 acid glycoprotein (AAG). When a drug binds to these proteins, it is a reversible interaction, creating a balance between the bound and unbound forms of the drug in the bloodstream.
Why Plasma Protein Binding Matters for Drugs
The extent to which a drug binds to plasma proteins has a direct impact on its effectiveness and how the body handles it. Only the unbound, or “free,” fraction of a drug is able to leave the bloodstream and reach its intended target tissues to exert a therapeutic effect. This unbound drug is also the portion that can be metabolized by the liver or excreted by the kidneys.
High plasma protein binding means a larger portion of the drug remains in the bloodstream, temporarily “sequestered” by the proteins. This can limit the amount of free drug available to distribute into tissues, potentially affecting the drug’s therapeutic impact. Extensive binding can also slow down the rate at which the drug is broken down by the body (metabolism) and removed (excretion), which might prolong its presence in the system. Therefore, accurately measuring the unbound fraction is important for determining appropriate drug dosages and predicting potential drug interactions where one drug might displace another from its protein binding sites.
Measuring Plasma Protein Binding
Several laboratory techniques are used to determine the extent of plasma protein binding. These methods aim to separate the unbound drug from the protein-bound drug to quantify the free concentration.
Equilibrium Dialysis
Equilibrium dialysis is a widely accepted method. In this technique, a semipermeable membrane separates a compartment containing drug-spiked plasma from a compartment with a protein-free buffer. The system is allowed to equilibrate, allowing the unbound drug to pass through the membrane while proteins and bound drug remain in their respective compartments.
Ultrafiltration
Ultrafiltration is another common method where a drug-spiked plasma sample is placed in a well with a filtration membrane at the bottom. Centrifugation then forces the unbound drug through the filter, leaving the larger protein-bound drug behind.
Ultracentrifugation
Ultracentrifugation is a third technique that uses high centrifugal forces to separate bound and unbound drug. After these separation steps, the concentration of the unbound drug in the protein-free compartment is measured using analytical techniques like liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Factors Influencing Binding
The degree of plasma protein binding can be influenced by a variety of factors, both related to the patient and the drug itself.
Patient Factors
Patient-specific factors include physiological conditions such as age, as well as the presence of kidney or liver diseases, which can alter the levels or binding capacity of plasma proteins. Severe liver disease might reduce albumin production, potentially leading to a higher unbound fraction of drugs that primarily bind to albumin.
Drug Factors
Drug-specific factors also play a role. The concentration of the drug in the plasma can affect binding. The presence of other drugs can lead to competition for the same binding sites on plasma proteins, which might displace a bound drug and increase its unbound concentration. The physicochemical properties of the drug, such as its lipophilicity (fat-solubility) and charge, also determine its affinity for specific plasma proteins.