Biotin binding proteins: Concepts and role in targeted drug delivery

Drug targeting using biotin binding proteins may hold the answer for drugs which have not been able to show their effects because they cannot gather in effective concentrations at the target, say Aditya Pattani and Manan Desai

The conventional drug delivery systems lack control over two parameters determining drug action — where the drug is released (i.e. site) and when the drug is released (i.e. rate). A lot of work has been done on both the aspects of controlling the site and the rate of drug release, which not only provides increased cost-benefit ratio but also aids in reducing the administration of high dosage of drugs.

This reduces adverse effects and increases patient compliance. One of the modern methods of targeting the drug to a particular site is the use of ligand binding proteins. In this article various aspects of ligand binding proteins and its application for drug targeting are briefly reviewed. The emphasis being on the class of biotin binding proteins, which have been widely studied.

Chemistry of ligand binding proteins

The function of all proteins is dependent upon the binding of other molecules or ligands to these proteins. For example many proteins bind to ligands in order to regulate gene expression or enzymatic activity or to transport molecules around. This binding is highly specific and is dependent on the geometry of the binding site usually referred to as ligand binding sites.

Ligand binding sites are usually depressions on the surface of the protein and the size and shape of it determines the nature of the ligand bound. The binding is due to the complimentarity between the ligand and the binding site, it may be either steric complimentarity and/or physico chemical complimentarity.

Ligand binding proteins and drug delivery

The ligand binding proteins offer excellent systems that have a great potential for the controlled and targeted delivery of small molecules. They naturally function to bind small molecules drugs and peptides usually with high specificity. Many of them possess binding properties which are susceptible to useful environmental stimuli and controlling parameters, allowing the design of sophisticated mechanisms of controlled release and uptake of ligands.

Interactions between a macromolecule and its ligand are usually tight and non-covalent. The complex between the two can be dissociated by a change in the environmental conditions. For example based on this concept if a ligand is attached to a given protein, which releases the drug only at a given site, due to specific environment there, then the drug can be targeted to that particular site. Many classes of ligand binding proteins exist such as biotin binding proteins, lipid binding proteins, periplasmic binding proteins, lectins etc. Further discussion here will focus only on the biotin binding proteins.

Biotin binding proteins

Of all the ligand binding proteins, Avidin and Streptavidin are perhaps the most studied. They have an extremely high affinity for biotin.(Kd = 6 x 10-16M for avidin & 4x 10-14 M for streptavidin)

Avidin is a basic tetrameric glycoprotein obtained from egg white. Each of the four monomers is capable of binding one molecule of biotin, thus the entire 62.4 Kda protein can bind four Biotin molecules. Probably due to its cationic nature and due to glycosylation, free avidin has a very short plasma half-life (approximately 1 min) and is rapidly taken up by the kidney and liver. Removal of the glycosyl group of avidin and chemical modification may result in a prolonged half-life.

Streptavidin is secreted by streptomyces avidnii, unlike avidin it is a non-glycoprotein. It exists as a tetramer approximately 53 Kda.

The primary structures of both the proteins are similar, the proteins are constructed of eight anti parallel strands forming a classic barrel.The biotin binding site contains a number of aromatic and polar amino acids involved in the ligand binding being positioned to provide a precise fit for biotin. The amino acids provide a hydrophobic box surrounding the biotin molecule. The immunogenic nature of streptavidin/Avidin is a point of concern. However, because most people have been exposed to egg avidin and because oral antigens are known to be tolerogenic, a certain degree of tolerance to avidin can be expected.

Role of biotin binding proteins in drug targeting

The basic concept behind the use of biotin binding proteins for targeted drug delivery is the affinity of avidin and streptavidin have for biotin. Thus wherever biotin will be circulating in the blood, it would be localised at the site at which the biotin binding proteins are present and vice-versa. If a drug moiety is attached with the biotin it will also reach the site at which the biotin binding proteins are present. Thus, the system for targeting consists of (1) the biotin binding protein which is either chemically modified or has an attached antibody so as to guide the biotin binding protein to the specified site and (2) The drug which is biotinylated.

Attachment of the drug directly to the antibody or a site directed protein may be troublesome in many cases as the large size of the protein/antibody may hamper the activity of the drug. Biotin is quite a small molecule and usually does not hamper the activity of the therapeutic agent. Also, biotinylation reactions for many substances have been worked out, making this system feasible to use for targeted drug delivery. This theory has been used, applied in practice and has been found useful for drug targeting. Some examples have been given below to give a better understanding of the theory.

Targeting 5-flouro uridine to the liver:

It was observed that substitution of trinitrophenyl groups on strepavidin lead to high and prolonged accumulation in the mouse liver, following intravenous administration. The drug 5-Fluoro Uridine was attached to a high molecular weight carrier — carboxymethyl dextran and this conjugate was charged with 2-4 biotinyl groups for complexing with the trinitrophenyl modification of Streptavidin. Studies show that specific liver accumulation of this conjugate took place and that the metabolic processes required to generate the active metabolite also took place. The liver targeting of 5-fluoro uridine was thus demonstrated.

Targeting of vasoactive intestinal peptide analogue to brain:

T.Yoshikawa et al have used the fact that OX-26 mouse monoclonal antibody undergoes rapid transcytosis through the brain capillary endothelial wall which makes up the blood brain barrier in vivo, owing to the high concentration of transferrin receptor on the blood brain barrier. Biotin can be delivered to the brain as a covalent conjugate with of avidin and the monoclonal antibody for the transferrin receptor.Thus drugs bound to biotin may be targeted to the brain. Pharmacological effects in the brain caused by the systemic administration of neuropeptides are prevented by poor transport of the peptide through the brain vascular endothelium.

A chimeric peptide was formed by the linkage potent Vasoactive intestinal peptide analogue, which had been monobiotinylated and linked to a drug transport vector. The vector consisted of a covalent conjugate of Avidin and OX- 26 monoclonal antibody. Thus the vasoactive intestinal peptide could be successfully delivered across the blood brain barrier to exhibit a pharmacological effect.

Targeting of tumour necrosis factor A to cancer cells:

The clinical use of tumour necrosis factor a (TNF) as an anticancer drug is limited to local or loco regional administration due to the dose limiting systemic toxicity. TNF can exert anti-tumour effect against human cancer if the systemic toxicity is kept under control. The therapeutic index of systemically administered human or murine TNF can be increased by tumour pre targeting strategies based on the biotin avidin system.

Pretargeting the s.c. mouse WEHI- 164 fibro carcinoma and RMA lymphoma, genetically engineered to express Thy 1.1 antigen on the cell membrane was achieved by the use of biotinylated anti Thy 1.1 antibody and avidin. Initially the bitinylated antibody was given followed by avidin followed by TNF-Biotin. Thus the TNF could be specifically directed to the cancer cells.

Conclusion

Today, cancer patients are suffering more due to the side effects of chemotherapy than the disease. The culprit is the fact that the chemotherapeutic agent is a poison for both the cancer as well as the rapidly dividing cells. Many peptide drugs are hydrophilic, waiting for delivery to the brain. Some drugs are not able to show their effects because they cannot gather in effective concentrations at the site at which they are expected to show their activity. Drug targeting using biotin binding proteins may hold the answer.

Aditya Pattani is with MUICT, Mumbai. E-mail: Pharmadi04@yahoo.co.in, Manan Desai is with Institute of Science, Mumbai