Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis.

Mucositis is a debilitating adverse effect of chemotherapy and radiation treatment. It is important to develop a simple and reliable in vitro model, which can routinely be used to screen new drugs for prevention and treatment of mucositis. Furthermore, identifying cell and molecular stresses especially in the initiation phase of mucositis in this model will help towards this end. We evaluated a three-dimensional (3-D) human oral cell culture that consisted of oral keratinocytes and fibroblasts as a model of oral mucositis. The 3-D cell culture model was irradiated with 12 or 2 Gy. Six hours after the irradiation we evaluated microscopic sections of the cell culture for evidence of morphologic changes including apoptosis. We used microarrays to compare the expression of several genes from the irradiated tissue with identical genes from tissue that was not irradiated. We found that irradiation with 12 Gy induced significant histopathologic effects including cellular apoptosis. Irradiation significantly affected the expression of several genes of the NF-kB pathway and several inflammatory cytokines, such as IL-1B, 1L-8, NF-kB1, and FOS compared to tissue that was not irradiated. We identified significant upregulation of several genes that belong to damage-associated molecular patterns (DAMPs) such as HMB1, S100A13, SA10014, and SA10016 in the 3-D tissues that received 12 Gy but not in tissues that received 2 Gy. In conclusion, this model quantifies radiation damage and this is an important first step towards the development 3-D tissue as a screening tool.

Suitability of caspofungin for aerosol delivery: physicochemical profiling and nebulizer choice.

BACKGROUND: Aerosolized antifungal therapy is a promising route of drug delivery for pulmonary aspergillosis due to attainment of high localized concentrations. Caspofungin, a new antifungal agent with proven efficacy against invasive aspergillosis, has ideal potential for aerosolization. STUDY OBJECTIVE: To examine in vitro the suitability of caspofungin for aerosol administration by characterizing factors that influence efficacy and airway tolerance of aerosol delivery: physicochemical properties, aerodynamics of drug particles, and efficiency of nebulizing systems. DESIGN: Physicochemical characteristics of caspofungin solutions (10 mg/mL and 30 mg/mL) were analyzed: osmolality, pH, viscosity, and surface tension. A time-of-flight aerosol spectrometer API Aerosizer was used to determine aerosol particle size and distribution. Drug output was quantified by high-performance liquid chromatography assay. Nebulizer efficiency was measured by drug output and respirable fraction (percentage of aerosolized particles with a 1 to 5 mum aerodynamic diameter) and compared among three jet nebulizer/compressor systems: device 1, Micromist (Hudson RCI; Temecula, CA)/Pulmo-Aide (model 5650D; DeVilbiss; Somerset, PA); device 2, Sidestream MS 2400/Envoy model IRC 1192 (Invacare; Elyria, OH); and device 3, Pari LC Star/Proneb Ultra (Pari Respiratory Equipment; Midlothian, VA). MEASUREMENTS AND RESULTS: Caspofungin requires 0.9% NaCl rather than sterile water as the diluent and addition of 0.3N NaOH buffer to adjust acidity of solutions (pH 6.17 to 6.26) in order to achieve optimal physicochemical properties for airway tolerability (osmolality, 150 to 550 milliosmol per kilogram; chloride ion, 31 to 300 mmol/L; and pH 7.4). The drug output rate increased with higher concentrations of drug solution: device 1, 4.0 mg/min vs 12.5 mg/min; device 2, 5.4 mg/min vs 14.7 mg/min; and device 3, 2.3 mg/min vs 12 mg/min, respectively. The percentage of particles within the respirable range varies depending on device and concentration of drug solutions (10 mg/mL vs 30 mg/mL): device 1, 85% vs 38%; device 2, 44% vs 57%; and device 3, 83% vs 93%, respectively. CONCLUSION: Caspofungin solution with adjustments appears to have physicochemical and aerodynamic characteristics suitable for aerosolization when used with either the Pari LC Star/Proneb Ultra or Micromist/Pulmo-Aide devices. Further in vivo testing is warranted.

Effects of cyclosporin A on model lipid membranes.

Cyclosporin A (CSA) is a widely used immunosuppressant drug for transplant therapy, however its limitation is its toxicity. The effect of CSA on model membranes such as dimyristoyl phosphatidylcholine (DMPC) bilayers was studied using small-angle X-ray diffraction and differential scanning calorimetry (DSC). CSA abolishes the pretransition and affects the transition of DMPC model membranes in a concentration-related manner as is shown by DSC. CSA induces a second peak at the high temperature side of the main transition, which is interpreted as a phase separation between areas rich and poor in CSA concentration. Small angle X-ray diffraction shows that the repeat distance of the DMPC bilayers in the lamellar Lalpha state increases as a function of concentration up to 10 mol% and remains constant thereafter. Furthermore, CSA affects the fatty acyl chains of the bilayer, especially the part of the chain proximal to the head group. In conclusion, CSA, as both small-angle X-ray diffraction and DSC show, affects in a concentration-wise manner the DMPC model membranes and perturbs the bilayer, in particular the acyl chain region.