EFFECTS OF FORMULATION VARIABLES ON NASAL EPITHELIAL-CELL INTEGRITY -BIOCHEMICAL EVALUATIONS

The effects of pH, osmolarity, type and concentration of buffers on th e nasal mucosal epithelium have been investigated in rats using an in situ nasal perfusion technique. Traditionally, histological approaches which are qualitative and not predicitive of nasal mucosal sensitivit y, have been used to assess the damage to the nasal mucosa. A biochemi cal approach has been used in this report to assess irritation to the nasal mucosa which may provide a priori indication of nasal sensitivit y to chronic use of nasal formulations. The nasal mucosal irritation m ay be predicted by determining the amount of total protein and two enz ymes, 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 ma rkers whereas solutions of pH above 10 caused significant membrane and intracellular enzyme release. Acetate buffers (pH 4.75) at three diff erent concentrations, 0.07, 0.14 and 0.21 M, were used to study the ef fect of buffer concentration on the nasal mucosal integrity. The resul ts indicate that the alteration to the nasal mucosal cells by buffers is concentration dependent. To study the effect of buffer type, four d ifferent buffers, i.e., acetate, adipate, citrate, and phosphate (0.07 M, pH 4.75) were studied. The acetate buffer was found to have the mo st irritation potential when compared to adipate, citrate, and phospha te 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 i sotonic sodium chloride solutions caused minimal mucosal cell aberrati ons while hypotonic solutions caused extensive leakage of LDH. These r esults along with other results from our laboratory may help in design ing well tolerated nasal formulations for chronic use.