PLASMA PROTEIN BINDING and DISTRIBUTION

Here we get to listen terms like blood plasma, blood serum, protein binding etc. 

Why is it so important to know these things? First let us understand these terms.

Blood Plasma: Fresh blood containing an anticoagulant when centrifuged, blood cells settle at the bottom; the supernatant liquid from this centrifugation is called blood plasma (or simply plasma).  Blood plasma without clotting factors is called Blood Serum.  Blood contains several proteins. Interestingly, drugs can bind to these proteins.

As mentioned earlier (1) blood carries the drug through-out the body (2) blood contains several proteins which might have affinity (high or low depending on the chemical nature of the drug) towards the drugs -  Therefore, drug in the blood can exist as protein-bound-drug or unbound drug (free drug).  Not surprisingly, it is the unbound (free) drug which exhibits the pharmacologic effects.  However, it should also be remembered that the same free drug may also be metabolized or excreted.  Plasma protein binding by the drug is reversible process.  During the course of travel if a more affinity protein or receptor is met, then the plasma protein bound drug will move (dissipate from the plasma protein) towards such protein or receptor. The bound portion may act as a reservoir from which the drug is slowly released as the unbound form. Since the unbound form is being metabolized and/or excreted from the body, the bound fraction will be released in order to maintain equilibrium.

What percentage of compound is plasma protein bound? To which component of plasma protein did the compound bind? What is the free fraction available to get to the target?

In many cases, drug efficacy is determined by the concentration of free drug (unbound), rather than the total concentration in plasma.

If the drug is highly bound to plasma proteins, the amount of drug available to reach the target is reduced. Subsequently, the efficacy of that compound may be significantly reduced.  Therefore, information on the free drug fraction is essential for drug development and may be helpful in correlating with in vivo efficacy.

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Rapid equilibrium dialysis (RED) is an accurate and reliable method for determining the degree to which a compound binds to plasma proteins.  Human or specific species of interest plasma spiked with test compound at 100x dilution of stock solution (typically 10 mM in DMSO) is added to the center chamber of a commercial plate based RED device. Blank, isotonic sodium phosphate buffer is added to the peripheral chamber of the RED device and the plate is incubated at 37°C for 4 hours. Equilibrium of free compound is achieved by the diffusion of the unbound compound across the dialysis membrane. Aliquots from each chamber (plasma and PBS) are collected and the concentrations of compound in each sample are determined by LC/MS/MS. Adjustments are made for non-specific binding.

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Distribution is the movement of drugs throughout the body. Determined by the blood flow to the tissues, it is ability of the drug to enter the vasculature system and the ability of the drug to enter the cell if required.

The protein bound drug is in equilibrium with the free drug. That means that once the free drug enters the target tissue then the protein bound drug will be released to maintain equilibrium.

After absorption most drugs are distributed in the blood to the body tissue where they have their effect. The degree to which the drug is likely to accumulate in the tissue is dependent on the lipophilicity and local blood flow to the tissue. Highly perfused organs receive most of the drugs.

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Synthetic Organic Chemists can simply apply organic chemistry rules to understand these phenomena.  In this case, plasma protein can be viewed as a protecting group for the drug.  Therefore, unbound drug can be viewed as unprotected version.  In synthetic organic chemistry we regularly observe that unprotected functional groups may be prone to undesired reactions - similarly, protein unbound drug is prone to metabolism as it is exposed to reactive enzymes in the body. But not surprisingly, it is the same unbound (free) drug which exhibits the pharmacologic effects. 

Now we know that free drug exhibits pharmacologic effect but is also prone to metabolize.  Therefore, it is important to get the plasma protein binding data in the initial stages of drug discovery projects itself.

Guy's having experience in medicinal chemistry projects hopefully agree with me that predicting Plasma Protein Binding is very difficult. Therefore, rather than concentrating on modifying the compound in hand for altering plasma protein binding property, one should focus on increasing the drug affinity to the target and reducing its clearance problems.