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TitleInvestigation of the mechanism of fenfluramine-induced pulmonary phospholipidosis in the rat lung model
AuthorHassan, Mogamat Shafick
SubjectScanning Electron Microscopy (SEM)
SubjectTransmission Electron Microscopy (TEM)
AbstractMagister Pharmaceuticae - MPharm
AbstractThe aim of this study was to investigate the mechanism of fenfluramine-induced pulmonary phospholipidosis, by comparing the profile and levels of induced phospholipids in the rat and the mode of phospholipase inactivation, both relative to that produced by chlorphentermine. Wistar and BD9 rats were injected with fenfluramine (FF) and chlorphentermine (CP) intra-peritoneally daily over a six week period to induce phospholipidosis. The lungs isolated from such treated and untreated animals, were grouped into unlavaged lungs and lungs to be lavaged and from the latter group the alveolar macrophages were isolated. Small sections of the unlavaged lungs were microscopically examined to verify the induction of phospholipidosis. Further the levels of phosphatidyl choline (PC), spingomyelin (SPM), phosphatidyl ethanolamine (PE), phosphatidyl glycerol (PG), phosphatidyl inositol (PI), phosphatidyl serine (PS) and phosphatidic acid (PA) were determined in both groups of lungs using a TLC method. To assess whether the drug-mediated inactivation of the phospholipases (PL) occurred via direct inhibition of the enzymes or via the drug-phospholipid complex, the hydrolysis of the above phospholipids by PL-A or PL-C were monitored using colorimetric methods. The feasibility of the phospholipid-drug complex-mediated mechanism was further explored, by assessing the effect the two drugs had on the phase transition temperature of the phospholipids. Electron microscopy revealed the presence of hypertrophied and elevated counts of alveolar macrophages in the treated-Wistar and -BD9 rats. In the FF- and CP treated Wistar and BD9 rats there were, compared to the saline-treated rats, a 200 % and 235 % increase in macrophage counts, respectively, for the FF-treated rats and a 700 % and 965 % increase in macrophage counts, respectively, for the CP treated rats. The levels of all the phospholipids in the unlavaged lungs of both rat strains were elevated, except that for PG, PS and PA. In both rat strains following the treatment with both drugs the PG levels were not elevated and the PS levels were not elevated following CP treatment. Following the treatment for both drugs, the PA levels were also not elevated in the BD9 rats. Relative to the levels found in the unlavaged lungs of the control rats, the increases ranged from a minimum of 9 to a maximum of 216 %. In general, Wistar rats appeared to be more susceptible to both FF and CP treatment. In both rat strains, lavaging of the lungs considerably reduced the levels of phospholipids remaining in the lung and the differences between the treated and untreated animals became less striking. The addition of FF or CP, whether directly to the enzyme, or in the form of the drug phospholipid complex, resulted in significant decreases in the PL-A-mediated or PL-C-mediated hydrolysis of virtualy all the test phospholipids. The average decrease ranged from 0.811 to 4.04 ,.,.FFAbbb ,.,.1-1sample min-I, for the PL-A activity and 0.023 to 0.827 ,.,.gIp"CC100 ,.,.1-1 sample min-I, for the PL-C activity. In the case of FF, the inhibition of PL-A activity could not be ascribed exclusively to either direct inhibition of the enzyme or reduced susceptibility of the phospholipid substrate-drug complex. The PL-C activity appeared to be inhibited to a greater extent via the phospholipid substrate-drug complex rather than by direct inhibition. On the other hand, CP induced a small, but significantly greater degree of inhibition of PL-A activity, more via direct inhibition, rather than by the phospholipid substrate-drug complex. The PL-C activity appeared to be inhibited to a greater extent via phospholipid substrate-drug complexation than by direct inhibition. From the above data, considered collectively, it was not possible to declare either of the two possible mechanisms as the more likely one for FF or CP-induced inhibition of the phospholipases. The feasibility of the indirect mode was further explored, by determining the phase transition temperatures for the phospholipid-drug complexes of each drug. The addition of each drug caused a depression of the phase transition temperature of all the phospholipids with a .1T"dd ranging from 0.52 to 15.73 °C. This appears to support the notion that both drugs bind to the phospholipids and the differences in the extent of the phase transition temperature depression of the individual phospholipids may indicate differences in the binding capacities of these drugs. The following major conclusions may be drawn from the results of this investigation. Fenfluramine induces a phospholipidosis syndrome in the lungs of Wistar and BD9 rats that are histologically similar to that induced by CP. It induces the elevation of essentially the same phospholipids as CP, primarily in the alveolar spaces and macrophages, and by implication, most likely via similar mechanisms. For both FF and CP, both direct inhibition and phospholipid-drug complex-mediated inhibition of phospholipases were found to be a viable mechanism for this syndrome. The mechanism for FF-induced pulmonary phospholipidosis thus appears to be similar to that of CP; small quantitative differences in essentially similar mechanisms, may explain the differences in the levels of induced phospholipidosis found in this study.
PublisherUniversity of the Western Cape