Protein Binding

One of the underlying principles of clinical pharmacology is that only the unbound, free drug is pharmacologically active and that only in this form can a drug cross a biological membrane or interact with a receptor. Interaction with the receptor results in a biochemical change leading to a physiological response.

Figure 2.7. Concentrations of guanethidine in tissues following intravenous administration21

Figure 2.7. Concentrations of guanethidine in tissues following intravenous administration21

Drugs bind to a number of plasma proteins including albumin, lipoproteins, and gamma globulins, and the more extensively the drug is bound, the lower will be the drug activity available to exert a pharmacological effect. Since the concentrations of these proteins change in disease, and as a function of nutrition, the effect of the drug may be significantly and unpredictably modulated. For example, most antimicrobials bind primarily to albumin, while basic drugs including erythromycin, clindamycin, and trimethoprim bind to the "acute-phase" serum protein ([[alpha]] 1-acid glycoprotein). Moreover, binding is not limited to proteins in the serum. Because albumin is the principal protein in interstitial fluid, substantial binding to this protein and to other tissue constituents, takes place. Differential concentrations of these proteins, and cell debris, leads to marked alterations in peripheral drug concentrations around an inflamed area such as a wound or abscess.


The ventricles of the brain lie within the ventricular compartment and are surrounded by approximately 150 ml of cerebrospinal fluid (CSF) which is secreted by the choroid plexi and circulates around the brain before being absorbed into the venous circulation. The entire fluid volume surrounding the brain is replaced every five hours in normal subjects. As a result it is rarely practical to deliver drugs by injection of a bolus into the ventricular space, since they are efficiently cleared before they can diffuse into the brain tissue. As a result the only practical means of delivering a drug effectively to the whole brain volume is via the systemic circulation. A significant feature of the blood flow in the brain is that the capillaries are very close together (of the order of 40 micrometres), so that any substance passing into the brain from the circulation will equilibrate within seconds through the whole of the brain tissue.

Unfortunately, delivery of drugs to the brain from the systemic circulation is difficult due to the presence of the so called blood brain barrier. This is not so much a physical structure as the absence of the usual mechanisms which allow drugs to be transferred across the capillary wall in other tissues. The junctions separating the capillary endothelial cells are extremely tight, eliminating the possibility of paracellular transport, and there is an almost complete absence of pinocytosis across the endothelial membrane. It also appears that there is a specific transport molecule, P-glycoprotein, which actively back-transports a wide range of drugs out of the apical cells, preventing their passage into the brain tissues22. A number of workers have attempted to open channels through the endothelium by infusing osmotic agents such as mannitol. Although this does lead to an increase in permeability, extensive side effects are observed, including seizures in experimental animals, and the practicality of this method seems limited. It is also possible to increase the permeability of the endothelium pharmacologically using bradykinin23. However, the most widely studied pathways for drug absorption are those which are part of the normal endothelial function, i.e. simple diffusion in the lipid membrane, and receptor-mediated active transport.

Uptake by diffusion

In order for a molecule to cross the endothelial capillary by diffusion, it must have two properties; it must be highly lipid soluble, and of relatively low molecular weight. A number of studies have examined the dependence of uptake on molecular weight, and an upper limit between 400 and 700 Daltons is generally accepted24 25. The absorption of hydrophilic molecules can be increased by administering them as hydrophobic prodrugs. A good example of this is provided by the absorption of morphine, codeine (methylmorphine) and heroin (diacetylmorphine) which have LogP (calculated) of 0.24, 1.14 and 1.86 respectively. Codeine has a blood-brain permeability of approximately 10 times that of morphine, while that of heroin is 10 times greater still.

Receptor-mediated transport

The brain capillary endothelium possesses a number of receptors which allow the transport of specific materials. These include small molecule nutrient receptors, such as those for hexose, amino acids, amines, and a number of peptide and protein receptors, including those for insulin, transferrin and IGF-II26. It is possible to take advantage of these pathways for the delivery of mimetic drugs; for example, dopamine is poorly transported through the blood brain barrier, and its administration is of no direct therapeutic value, but the amino-acid analogue L-DOPA is transported through the phenylalanine receptor and can thus exert a useful pharmacological effect27.

A number of reserchers have attempted to make use of these active pathways to develop blood brain barrier vectors which can improve drug uptake. A typical example is the OX26 monoclonal antibody to the endothelial transferrin receptor28. The antibody binds efficiently to the receptor, and is actively transported across the endothelium. The drug can be attached to the antibody using conventional avidin-biotin methods which do not degrade the activity of the drug. This technique has been used to deliver vasoactive intestinal peptide (VIP) attached to OX26 to the brain, resulting in a significant increase in hemispheric brain blood flow; the peptide alone was not significantly absorbed and showed no pharmacological effect29. It has also been used in an attempt to deliver antisense gene therapy agents to the brain30. Antisense oligonucleotide-OX26 conjugates were not delivered efficiently since they bound to plasma proteins, but peptide nucleic acid—OX26 conjugates did not suffer from this problem and were efficiently transported into the brain. More recently the antibody has been grafted to the surface of liposomes containing daunomycin and successfully used to produce elevated drug levels within the brain tissue.

The most significant difficulty with these vector methods is that the receptors being used in the target tissue occur in a wide range of organs; for example transferrin receptors are present on most cells since they are required for the active uptake of iron. It is thus important to distinguish between brain uptake and brain targeting; vector based conjugates may improve brain uptake but will not produce targeting, with the drug being absorbed in a wide range of organs. Many published studies are particularly vague on this point and one is led to suspect that it is a significant problem.

A number of polyionic macromolecules are also actively transported into the brain capillary endothelium by absorptive transcytosis. This mechanism is distinct from the receptor-mediated pathways discussed above since it involves the physical adsorption of the macromolecule (usually a polycation) to the negatively charged membrane by charge interactions, after which internalization occurs. Molecules transported in this way include lectins such as wheat germ agglutinin and polycationic proteins such as cationized albumin31. This approach has been used to deliver radiolabelled antibody diagnostics; for example a monoclonal antibody to the amyloid protein which is characteristic of Alzheimers disease. The antibody itself was not significantly taken up by brain tissue, but localized in the brain in dogs after it had been converted into a polycationic protein by reaction with hexamethylene diamine32.

Colloidal delivery

We have already mentioned the use of liposomes coated with the OX26 transferrin antibody which successfully increased brain levels of daunomycin. In this case, it was unclear whether or not the liposomes were internalized in the endothelial cells or simply adhered to the surface, thus providing a greater concentration gradient to assist passive diffusion. It has also been found that cyanoacrylate microspheres coated with polysorbates can be used to deliver a number of hydrophilic analgesic peptides33 34. The mechanisms of this effect are also uncertain but may be due to a surface receptor recognizing the sorbate block of the surfactant.

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