Photo-control of iNOS inhibition in RAW 264.7 cell culture by near infrared multiphoton photolysis of a caged iNOS selective inhibitor (Bhc -1400W)

• Main Author: Perdicakis, Basil S
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2004
• Subjects: Chemical engineering
• ISBN: 9780612920248; 0612920240

Motivation. It is well established that the inability of current drug delivery protocols to selectively target diseased tissue regions is a major problem associated with systemic drug administration. This Ph.D. research was part of a multi-disciplinary project aimed at the creation of a novel class of photolabile drugs that may be selectively activated in specific tissue regions. The benefits of this drug delivery approach include reduced drug toxicity and real time control of active drug concentration. This novel class of drug has the potential to be applied against a variety of illnesses including stroke and cancer. Background. Nitric Oxide (NO) is a gaseous biological messenger that is critically involved in a number of physiological processes and is implicated in a diverse number of diseases. The control of NO production in the body is of considerable importance for the prevention and treatment of several physical ailments and diseases including stroke, heart disease, diabetes; and arthritis. NO is produced in mammalian tissues by three distinct isoforms of the enzyme nitric oxide synthase (NOS). The tissue damage that occurs during inflammatory processes such as stroke may be significantly reduced by inhibiting one of the three NOS isoforms known as inducible NOS (iNOS). A compound known as 1400W selectively inhibits iNOS over the other two NOS isoforms. Ideally, the inhibition of iNOS should be confined to the vicinity of the inflammation so as not to interfere with essential NOS activity in other regions of the body. Multiphoton (MP) absorption is a relatively new technique in which molecules are excited to higher energy levels by absorbing two infrared photons in a short time period. The net effect is that when a specialized laser, a mode-locked near infrared laser, is focused through an objective lens a restricted volume of MP absorption is produced about the focal point of the objective lens. A caging molecule known as Bhc is efficient at absorbing MP radiation and using this energy to break the covalent bond between itself and another molecule. Hypothesis. It was hypothesized that photo-control of iNOS inhibition in cell culture could be achieved by near infrared multiphoton photolysis of a caged iNOS selective inhibitor (Bhc-1400W). Results. A compound known as Bhc-1400W was synthesised, and a method of quantifying MP uncaging of Bhc-1400W using a NOS enzyme assay was developed. The techniques developed during this phase of research were used to screen trans 3,4-cyclopropyl L-arginine analogues as isoform selective inhibitors of NOS, and to develop a method for the calculation of dissociation constants of noncompetitive tight-binding activators. Techniques to improve the data obtained from enzyme kinetic experiments performed in multi-well plate format were also developed. These advances will be valuable in the screening of new caged inhibitors. Bhc-1400W was proven to be a weak iNOS inhibitor in comparison to 1400W. Following exposure of Bhc-1400W to MP irradiation, MP uncaged Bhc-1400W was produced and was shown to recover the full inhibitory potency of 1400W. Exposure of cells to the 1400W, Bhc-1400W, MP uncaged Bhc-1400W, and MP radiation doses utilized was not cytotoxic. The efficacy of the proposed drug delivery strategy was demonstrated in cell culture. Mathematical models were developed that supported experimental results. Due to the spatially confined nature of MP absorption, our approach has the potential to effect spatial and temporal control of drug delivery. The research hypothesis was proven, and development of this technology will continue as critical measures of success were met. The successful development of this novel technique may be potentially utilised to combat not only stroke but also a variety of other ailments.