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dc.contributor.advisorWalubo, A.
dc.contributor.authorBekker, Zanelle
dc.date.accessioned2018-07-31T12:43:48Z
dc.date.available2018-07-31T12:43:48Z
dc.date.issued2010
dc.identifier.urihttp://hdl.handle.net/11660/9051
dc.description.abstractEnglish: Nevirapine is associated with hypersensitivity reactions such as skin rash and hepatotoxicity, hampering its use for HIV prophylaxis. The mechanism of nevirapine induced hepatotoxicity remains unknown and was postulated to be immune mediated. As recently reported, several drugs have shown to induce toxicity by immune activation. Therefore, the aim of this study was to determine the role of the immune system in nevirapine induced hepatotoxicity. A high performance liquid chromatography method for the determination of nevirapine in rat plasma was developed. It involved protein precipitation with perchloric acid, followed by solid phase extraction on C18 cartridges. The mobile phase was tetraethylammoniumphosphate (TEAP) buffer and acetonitrile (60:40, v/v) and was run over a Luna C18 (4.60x150 mm) 5μ analytical column at 1 ml/min. The eluent was detected by UV at 210 nm. Nevirapine and chlorzoxazone eluted at 2.6 and 5.2 minutes, respectively. The average 5 day calibration curve (0 – 10 μg/ml) was linear with a regression equation of y=0.012x+0.051, and the correlation coefficient (r2) was 0.9985. The method was used to measure nevirapine concentrations in rat plasma. The animal experiment consisted of an acute and chronic phase. During the acute phase male Sprague-Dawley rats were divided into 3 groups of 10 rats each. Groups were dosed as follows: S+NVP, LPS+S and LPS+NVP. From each group, 5 rats were sacrificed at 6 and 24 hours after dosing. During the chronic phase, animals were divided into 2 groups of 15 rats each, to which nevirapine was administered daily. From each group, 5 rats were administered with LPS or saline, just before the daily nevirapine dose on days 7, 14 and 21. The animals were sacrificed 24 hours later. Blood was tested for ALT, full blood count, IL-2, IFN-γ, TNF-α and nevirapine concentrations. Liver sections were sent for histopathology testing. In the acute phase, the S+NVP group exhibited increased ALT at 6 and 24 hours. Liver injury was characterised by mild cloudy swelling (6 hours), and hepatocellular vacuolar degeneration (24 hours). Although LPS+S led to increased ALT at 6 hours, which normalised by 24 hours, abnormal liver histology was observed on both occasions as swollen cytoplasm and narrow sinusoids. The LPS+NVP group showed increased ALT at 6 hours, which returned to normal by 24 hours. Interestingly, liver histology was normal on both occasions, indicating that LPS+NVP prevented hepatotoxic effects of either drug. Whereas all three groups caused increased IL-2, it was higher in the LPS+NVP group, implying that LPS was responsible for the increase during the acute phase. Nevirapine concentrations at 6 hours were higher in the S+NVP group, but by 24 hours they were higher the LPS+NVP group. During the chronic phase, S+NVP caused liver injury on days 7 and 14 as demonstrated by increased ALT with centrilobular hepatocellular degeneration on day 7. However, by day 14 and 21 the ALT had normalised and liver histology was unaffected. Whereas the LPS+NVP group exhibited increased ALT on days 7 and 14, the corresponding liver histology was normal on all three occasions, illustrating further that LPS attenuates nevirapine induced hepatotoxicity. By day 21, S+NVP caused profound lymphocytosis, while LPS+NVP caused neutrophilia. S+NVP and LPS+NVP exhibited increased IL-2 and IFN-γ, but both cytokines were higher in the S+NVP group. TNF-α was elevated in both groups on day 7, and thereafter fell, remaining higher in the LPS+NVP group. Nevirapine concentrations were higher in the LPS+NVP group than in the S+NVP group. In conclusion, it was demonstrated that nevirapine is a slow onset immune stimulant, which caused nevirapine induced hepatotoxicity, and LPS prevented the hepatotoxicity. These observations suggest that immune manipulation may help to prevent nevirapine induced hepatotoxicity.en_ZA
dc.description.abstractAfrikaans: Nevirapien word geassosiëer met hipersensitiwiteitsreaksies soos veluitslae en hepatotoksisiteit wat die gebruik daarvan vir MIV profilakse belemmer. Die meganisme van nevirapien-geïnduseerde hepatotoksisiteit is onbekend en word gepostuleer om immuun-bemiddeld te wees. Soos onlangs aangemeld, veroorsaak verskeie middels hepatotoksistieit deur immuun-aktivering. Die doel van hierdie studie was dus om die rol van die immuunsisteem in nevirapien-geïnduseerde hepatotoksisiteit te bepaal. ‘n Hoëdrukvloeistof-chromatografiemetode vir die bepaling van nevirapien in rotplasma is ontwikkel. Dit behels proteïen presipitasie met perchloraatsuur, gevolg deur vastefaseëkstraksie met C18 kolomme. Die mobiele fase het bestaan uit tetraetielammoniumfosfaat (TEAP) buffer en asetonitriel (60:40, v/v) en was gechromatografeer deur ‘n Luna C18 (4.60x150 mm) 5μ analitiese kolom teen 1 ml/min. Die eluant is bepaal deur UV teen 210 nm. Nevirapien en chlorzoksasoon het onderskeidelik teen 2.6 en 5.2 minute gëelueer. Die gemiddelde 5 dag kalibrasiekromme (0 – 10 μg/ml) was liniêr met ‘n regressievergelyking van y=0.012x+0.051, en die korrelasie-koëffisiënt (r2) was 0.9985. Die metode is suksesvol gebruik om nevirapienkonsentrasies in rotplasma te bepaal. Die diereëksperiment het bestaan uit `n akute- en chroniese fase. Gedurende die akute fase is manlike Sprague-Dawley rotte opgedeel in 3 groepe van 10 rotte elk. Die groepe is as volg behandel: saline+nevirapien, lipopoliesakkaried+saline en lipopoliesakkaried+nevirapien. Uit elke groep is 5 rotte geslag teen 6 en 24 uur na middeltoediening. Gedurende die chroniese fase is die diere opgedeel in 2 groepe van 15 rotte elk, waaraan nevirapien daagliks toegedien is. Uit elke groep is 5 rotte behandel met LPS of saline, net voor die daaglikse nevirapiendosis, op dae 7, 14 en 21. Die diere is 24 uur later geslag. Bloed is getoets vir ALT, volbloedtelling, IFN-γ, IL-2, TNF-α en nevirapienkonsentrasies. Lewermonsters is gestuur vir histopatologiese ondersoek. In die akute fase het die S+NVP groep verhoogde ALT gedemonstreer teen 6 en 24 uur. Lewerskade was gekarakteriseer deur ligte swelling (6 uur) en hepatosellulêre vakuolêre degenerasie (24 uur). Alhoewel LPS+S gelei het tot verhoogde ALT teen 6 uur, wat herstel het teen 24 uur, was abnormale lewerhistologie waargeneem as geswolle sitoplasma en vernoude sinusoïedes tydens beide intervalle. Die LPS+NVP groep vertoon verhoogde ALT teen 6 uur, wat genormaliseer het teen 24 uur. Dit was merkwaardig dat die lewerhistologie normaal was tydens beide gevalle, wat aandui dat LPS+NVP die hepatotoksiese effekte van beide middels voorkom het. Al drie groepe het verhoogde IL-2 veroorsaak, met die hoogste vlakke in die LPS+NVP groep, wat aandui dat LPS verantwoordelik was vir die verhoging tydens die akute fase. Nevirapien konsentrasies was hoër in die S+NVP groep teen 6 uur, maar teen 24 uur was dit hoër in die LPS+NVP groep. Gedurende die chroniese fase het S+NVP lewerskade veroorsaak teen 7 en 14 dae, soos aangetoon deur verhoogde ALT met sentrilobulêre hepatosellulêre degenerasie teen dag 7. Teen dae 14 en 21 het die ALT genormaliseer en die lewerhistologie was normaal. Alhoewel die LPS+NVP groep verhoogde ALT getoon het teen dae 7 en 14, was die lewerhistologie normaal tydens al 3 intervalle, wat weereens aandui dat LPS nevirapiengeïnduseerde hepatotoksisiteit geatenueer het. Teen dag 21 het S+NVP merkwaardige limfositose veroorsaak, terwyl LPS+NVP gelei het tot neutrofilie. Beide S+NVP en LPS+NVP het gelei tot verhoogde IL-2 en IFN-γ, maar beide sitokiene was hoër in die S+NVP groep. TNF-α was verhoog in beide groepe teen dag 7, waarna dit gedaal het, maar was steeds die hoogste in die LPS+NVP groep. Nevirapienkonsentrasies was hoër in die LPS+NVP groep as in die S+NVP groep. Gevolglik is daar gedemonstreer dat nevirapien ‘n stadige imuunstimulant is en nevirapien-geïnduseerde hepatotoksisiteit veroorsaak het, en dat LPS nevirapien-geïnduseerde hepatotoksisiteit voorkom het. Die waarnemings stel voor dat immuunmanipulasie mag help om nevirapien-geïnduseerde hepatotoksisiteit te voorkom.en_ZA
dc.description.sponsorshipDepartment of Pharmacology, University of the Free Stateen_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectNevirapineen_ZA
dc.subjectHepatotoxicityen_ZA
dc.subjectImmune systemen_ZA
dc.subjectHigh performanceen_ZA
dc.subjectLiquid chromatographyen_ZA
dc.subjectLipopolysaccharideen_ZA
dc.subjectCytokinesen_ZA
dc.subjectDrugs -- Metabolismen_ZA
dc.subjectAIDS (Disease) -- Treatmenten_ZA
dc.subjectLiver -- Diseasesen_ZA
dc.subjectDissertation (M.Med.Sc. (Pharmacology))--University of the Free State, 2010en_ZA
dc.titleThe role of the immune system in nevirapine induced hepatotoxicity in a rat modelen_ZA
dc.typeDissertationen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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