Effects of formulation variables on nasal epithelial cell integrity: Biochemical evaluations

Abstract

The effects of pH, osmolarity, type and concentration of buffers on the nasal mucosal epithelium have been investigated in rats using an in situ nasal perfusion technique. Traditionally, histological approaches which are qualitative and not predicative of nasal mucosal sensitivity, have been used to assess the damage to the nasal mucosa. A biochemical approach has been used in this report to assess irritation to the nasal mucosa which may provide a priori indication of nasal sensitivity to chronic use of nasal formulations. The nasal mucosal irritation may be predicted by determining the amount of total protein and two enzymes, lactate dehydrogenase (LDH, EC 1.1.1.27), a cytosolic enzyme and 5′-nucleotidase (5′-ND, EC 3.1.3.5), a membrane-bound enzyme released during perfusion. To determine the effect of pH on the nasal mucosa, phosphate buffers ranging in pH from 2 to 12 were utilized. Solutions within a pH range of 3–10 caused minimal release of the biochemical markers whereas solutions of pH above 10 caused significant membrane and intracellular enzyme release. Acetate buffers (pH 4.75) at three different concentrations, 0.07, 0.14 and 0.21 M, were used to study the effect of buffer concentration on the nasal mucosal integrity. The results indicate that the alteration to the nasal mucosal cells by buffers is concentration dependent. To study the effect of buffer type, four different buffers, i.e., acetate, adipate, citrate, and phosphate (0.07 M, pH 4.75) were studied. The acetate buffer was found to have the most irritation potential when compared to adipate, citrate, and phosphate buffers. To determine the effects of unionized and ionized species of a buffer, 0.025 M benzoate buffers at pH 3.2 and 5.2 were studied. The results indicate that the unionized species of benzoic acid causes more cellular perturbation than the ionized species. Hypertonic and isotonic sodium chloride solutions caused minimal mucosal cell aberrations while hypotonic solutions caused extensive leakage of LDH. These results along with other results from our laboratory may help in designing well tolerated nasal formulations for chronic use.