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Vatsala Sadasivan, Rebecca Carrier, David Budil, Albert Saccco, Jr. The United States drug delivery market is expected to grow from $19 billion in 2001 to $41 billion by 2007 [1]. As a result of this, we have various drug delivery methods today including oral, transdermal, nasal, pulmonary, ocular, intravenous, etc. Within this billion dollar industry, oral formulations still control over 60% of the market [1]. The accelerated pace of oral drug delivery system development has been fostered by the need to deliver medications to patients more efficiently and with fewer side effects. A crucial question in the development of any new drug is whether it will be bioavailable (the ability of a drug to be absorbed and used by the body) after oral administration. Unfortunately, many drugs have low oral bioavailability as well as insufficient targeting efficacy. Intestinal mucus is believed to be the main barrier to drug absorption. The mucus mainly affords protection for the underlying epithelial cells. It consists of a slimy, viscous membrane through which a drug must first diffuse before it can be absorbed into the blood stream. There is as yet no clear understanding of the molecular interactions between drug molecules and the mucus membrane. Previous work has focused on developing a macroscopic model for oral drug absorption. In addition to experimental work which includes the cultivation of epithelial cells, diffusion and absorption studies, molecular modeling will be used to gain an understanding of the intestinal mucus membrane as well as to study drug-mucus binding to characterize the extent and nature of these interactions. The mucin glycoprotein, a major component of the mucus membrane is a large and complex molecule with a MW of > 1,000,000 that has been studied persistently since the 1970’s. It is the principal determinant of the viscoselastic properties of the mucus which are believed to inhibit drug diffusion. In spite of this, its structure still remains mostly unknown with its role and relative importance as a drug barrier being the subject of a contentious debate. However, with the breakthrough of DNA sequencing through the human genome project, the major intestinal mucin gene, MUC2 has been sequenced. MUC2 has also been identified as the major gene responsible for cystic fibrosis. From the MUC2 protein sequence, the key tandem repeating sequence has been identified. With the development of sophisticated software and computational capabilities in the past 5-10 years, it is now possible to use computational analyses such as molecular modeling to predict secondary structures and modeling of unknown protein sequences that are intractable to study by other techniques.
Figure 1: Schematic of Intestinal Secretory Mucin (MUC2) [2]. The tools and methods of modern molecular modeling, which has great potential towards the understanding and engineering of oral drug delivery, will be used in our research. Applications of molecular modeling will include homology modeling as a possible approach to determine the molecular structure of mucin. The aim here is to eventually model the mucus membrane to better understand the interactions between drug delivery agents (primarily lipids) and the membrane. Molecular dynamics simulations will be performed to determine diffusion coefficients of various drug molecules and drug delivery agents through the mucus membrane. Molecular dynamics is also a possible conformational search method, which will be utilized in our study. In addition, molecular docking is a potential method that will be used to study interactions between drug molecules (or drug delivery agents) and binding sites. Ultimately, it is hypothesized that molecular modeling can be used as a tool to better understand oral drug delivery.
Figure 2: Example of drug-protein docking. Drug molecule (ligand) shown by space filling. Protein surface colored by its polarity. Mauve (purple-red) areas and blue areas are hydrophilic while the cream area is hydrophobic. Mauve indicates hydrogen acceptors (e.g. C=O) and blue indicates areas of hydrogen donors (e.g. -NH or more generally -XH). As expected since proteins tend to fold so that hydrophobic residues are on the interior and hydrophilic groups are on the exterior, hydrophilic regions dominate the outside of the protein.
Figure 3: Superposition of the backbone N-C-C trace of homology model and 1HCN-B. Pink molecule - homology model of the CTCK (carbon-terminal cysteine knot-like) region of MUC2 and the yellow molecule – 1HCN-B. The balls and cylinders represent the N-C-C trace of the 6 cysteine residues involved in the cysteine knot formation; grey – carbon atoms and blue-nitrogen atoms.
Figure 4: Superposition of the cysteine knots. The background dim rods represent the two molecules; pink rods – homology model of CTCK region of MUC2 and blue rods – 1HCN-B. Cysteine residues shown in ball and cylinder representation.
Figure 5: Cysteine-knot of the Homology Model of the CTCK region of MUC2. Ball and cylinder representation; blue – nitrogen, red - oxygen, grey- carbon and yellow – sulfur.
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