The effect of the traditional medicine phela on p-glycoprotein and multidrug resistance-associated protein 2 drug transporters in the gastrointestinal tract of a rat model
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Date
2015-11
Authors
Binyane, Moleboheng Emily
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
English: Phela is the herbal preparation of four African traditional medicinal plants, and is under the development by the Medical Research Council (MRC) as an immune stimulant for immune compromised individuals. Patients might use Phela with other medicines; therefore, the herb-drug interactions profiling of Phela is important. Membrane drug-transporters such as P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) are considered important factors in determining the pharmacokinetic parameters of drugs such as paclitaxel (PTX) and methotrexate (MTX), respectively. Inhibition or induction of transport might result in drug interactions with other drugs transported by these respective transporters. Moreover, significant herb-drug interactions involving P-gp and MRP2 have been described. Therefore, the effect of Phela on P-gp and MRP2 in the gastrointestinal tract of a rat model was investigated here.
First, a high performance liquid chromatography (HPLC) method for determination of PTX in plasma was developed. It involved liquid-liquid extraction of 100 μl plasma, spiked with PTX, extracted with diethyl ether: dichloromethane (2:1), followed by centrifugation. The supernatant was evaporated to dryness under a stream of nitrogen, reconstituted, and 100 μl was injected into the HPLC. The sample was eluted with a mobile phase of sodium phosphate buffer (pH 2): acetonitrile (60:40, v/v) over a C8 (1) (4.6 X 250 mm) 5 μ analytic column at 1 ml/min. PTX was detected by UV at 230 nm. Docetaxel (DTX) was used as the internal standard. Under these conditions, DTX and PTX eluted at retention times of 6.595 and 6.038 minutes, respectively. The average calibration curve (0-15 μg/ml) was linear with a regression equation of y = 0.1931x + 0.0705, and correlation coefficient (r) of 0.9973. The method was used successfully in animal experiments to measure PTX in the plasma of treated rats.
Thereafter, a preliminary experiment was conducted in vitro to establish whether Phela has a direct/ physical effect on PTX, using a direct drug interaction testing experiment in buffer, as well as Slide-A-Lyzer® dialysis. During the direct drug interaction experiment, buffer was spiked with 10 μg/ml of PTX with or without 3.85 mg/ml Phela, and PTX concentrations were determined by HPLC. Then, using a Slide-A-Lyzer® dialysis cassette, the time of equilibrium of PTX was determined by monitoring the changes in PTX concentrations over 12 hours, in plasma containing 230 μg/ml PTX and buffer. Thereafter, the potential of an interaction was tested by adding 88.55 mg/ml Phela to the same experiment after 8 hours of incubation, and monitoring PTX concentrations after 10 and 12 hours by HPLC. In the first experiment, Phela had no direct effect on PTX concentrations, while in the second experiment the time of equilibrium of PTX was estimated at 8 hours. After Phela was added, PTX concentrations and its free fraction (fu) remained unchanged. Therefore, it was concluded that there is no interaction between Phela and PTX in vitro.
This final part of the study was undertaken to investigate the effect of Phela on P-gp and MRP2 transporters. PTX and cyclosporin A (CyA) were used as the respective substrate and inhibitor of P-gp, while MTX and probenecid (PRO) were those of MRP2. Ethical approval was obtained and male Sprague-Dawley (SD) rats (200-250 g) were used. The animal experiment was divided into two parts. In Part I, three groups of 40 rats each received a one-off oral dose of PTX-only (10 mg/kg); PTX & CyA (10 mg/kg); or PTX & Phela (15.4 mg/kg), while in Part II, three groups of 40 rats each received a one-off oral dose of MTX-only (10 mg/kg); MTX & PRO (20 mg/kg); or MTX & Phela (15.4 mg/kg). For each group, 5 rats were sacrificed after 0.5, 1, 2, 4, 6, 8, 10, and 12 hours. Blood was analysed for full blood count, liver function, and PTX and MTX concentrations. CyA and PRO increased the area under the plasma concentration-time curve (AUC) of PTX and MTX, respectively, whereas Phela had no effect on the AUC of PTX or MTX.
Overall, no direct interaction between PTX and Phela was observed both in vitro and in vivo, and there were also no interactions between MTX and Phela in vivo. Phela did not inhibit P-gp or MRP2. This implies that Phela will most probably not be involved in herb- drug interactions of membrane transporter origin. Therefore, the doses of drugs that are transported by P-gp and MRP2 need not be adjusted when co-administered with Phela.
Afrikaans: Membraan gekoppelde middel transporters soos P-glikoproteïen (P-gp) en multimiddel weerstand verwante proteïen 2 (MRP2) word beskou as belangrike faktore in die bepaling van die farmakokinetiese parameters van middels soos paclitaxel (PTX) en metotreksaat (MTX), onderskeidelik. Inhibisie of induksie van vervoer kan lei tot interaksies met ander middels wat ook deur hierdie onderskeie transporters vervoer word. Verder is beduidende plant- geneesmiddelinteraksies met P-gp en MRP2 reeds beskryf. Daarom word die effek van Phela op P-gp en MRP2 in die spysverteringskanaal van 'n rot-model hier ondersoek. Eerstens, is 'n hoëdrukvloeistof-chromatografie (HPLC) metode vir die bepaling van PTX in plasma ontwikkel. Dit het behels vloeistof-ekstraksie van 100 μl plasma, waartoe die PTX gevoeg is met diëtieleter: dichlorometaan (2:1), gevolg deur sentrifugering. Die supernatant is met ‘n stikstofstroom verdamp tot droog, hersaamgestel, en 100 μl is in die HPLC ingespuit. Die monster is geëlueer met 'n mobiele fase van natrium-fosfaat- buffer (pH 2): asetonitriel (60:40, v/v) oor 'n C8 (1) (4.6 X 250 mm) 5 μ analitiese kolom teen 1 ml/min. PTX is bepaal deur UV by 230 nm. Docetaxel (DTX) is gebruik as die interne standaard. Onder hierdie toestande het DTX en PTX onderskeidelik geëlueer teen retensietye van 6,595 en 6,038 minute. Die gemiddelde kalibrasie-kurwe (0-15 μg/ml) was liniêr met 'n regressievergelyking van y = 0.1931x + 0.0705 en korrelasie- koëffisiënt (r) van 0.9973. Die metode is suksesvol in diere-eksperimente gebruik om PTX te meet in die plasma van behandelde rotte. Hierna is 'n voorlopige in vitro eksperiment uitgevoer om te bepaal of Phela 'n direkte / fisiese effek het op PTX. 'n direkte middelinteraksie-eksperiment sowel as Slide-A-Lyzer® dialise is uitgevoer en PTX konsentrasies is gemeet met HPLC. Phela het geen direkte effek op PTX konsentrasies. Die tyd om ekwilibrium van PTX te bereik is geskat op 8 ure. Nadat Phela bygevoeg is, het PTX konsentrasies en die vry-fraksie (fu) onveranderd gebly. Daarom word die gevolgtrekking gemaak dat daar geen interaksie tussen Phela en PTX in vitro is nie. Die finale deel van die studie is onderneem om die effek van Phela op P-gp en MRP2 transporters te ondersoek. PTX en siklosporien A (CYA) is onderskeidelik gebruik as die substraat en die inhibeerder van P-gp, terwyl MTX en probenesied (PRO) soortgelyk gebruik was vir MRP2. Etiese goedkeuring is verkry en manlike Sprague-Dawley (SD) rotte (200-250 g) is gebruik. Die proefdier-eksperiment is in twee dele verdeel. In Deel I het drie groepe van 40 rotte elk 'n eenmalige dosis gekry van PTX-alleen (10 mg/kg); PTX & CYA (10 mg/kg); of PTX & Phela (15.4 mg/kg), terwyl in Deel II, drie groepe van 40 rotte elk 'n eenmalige dosis gekry het van MTX-alleen (10 mg/kg); of MTX & PRO (20 mg/kg); of MTX & Phela (15.4 mg/kg). Uit elke groep is 5 rotte geslag na 0.5, 1, 2, 4, 6, 8, 10, en 12 uur. Bloed is ontleed vir volbloedtelling, lewerfunksie, en PTX en MTX konsentrasies. CYA en PRO het die area onder die plasma-konsentrasie tyd-kurwe (AUC) van PTX en MTX, onderskeidelik verhoog, terwyl Phela geen effek op die AUC van PTX of MTX getoon het nie. Algeheel, is daar geen direkte interaksie tussen PTX en Phela waargeneem beide in vitro en in vivo nie, en daar was ook geen interaksie tussen MTX en Phela in vivo nie. Phela het nie P-gp of MRP2 inhibeer nie. Dit impliseer dat Phela waarskynlik nie betrokke sal wees in plant-geneesmiddel-interaksies van membraan-vervoerder- oorsprong nie. Daarom hoef die dosisse van middels wat deur P-gp en MRP2 vervoer word, nie aangepas te word wanneer dit saam met Phela toegedien word nie.
Afrikaans: Membraan gekoppelde middel transporters soos P-glikoproteïen (P-gp) en multimiddel weerstand verwante proteïen 2 (MRP2) word beskou as belangrike faktore in die bepaling van die farmakokinetiese parameters van middels soos paclitaxel (PTX) en metotreksaat (MTX), onderskeidelik. Inhibisie of induksie van vervoer kan lei tot interaksies met ander middels wat ook deur hierdie onderskeie transporters vervoer word. Verder is beduidende plant- geneesmiddelinteraksies met P-gp en MRP2 reeds beskryf. Daarom word die effek van Phela op P-gp en MRP2 in die spysverteringskanaal van 'n rot-model hier ondersoek. Eerstens, is 'n hoëdrukvloeistof-chromatografie (HPLC) metode vir die bepaling van PTX in plasma ontwikkel. Dit het behels vloeistof-ekstraksie van 100 μl plasma, waartoe die PTX gevoeg is met diëtieleter: dichlorometaan (2:1), gevolg deur sentrifugering. Die supernatant is met ‘n stikstofstroom verdamp tot droog, hersaamgestel, en 100 μl is in die HPLC ingespuit. Die monster is geëlueer met 'n mobiele fase van natrium-fosfaat- buffer (pH 2): asetonitriel (60:40, v/v) oor 'n C8 (1) (4.6 X 250 mm) 5 μ analitiese kolom teen 1 ml/min. PTX is bepaal deur UV by 230 nm. Docetaxel (DTX) is gebruik as die interne standaard. Onder hierdie toestande het DTX en PTX onderskeidelik geëlueer teen retensietye van 6,595 en 6,038 minute. Die gemiddelde kalibrasie-kurwe (0-15 μg/ml) was liniêr met 'n regressievergelyking van y = 0.1931x + 0.0705 en korrelasie- koëffisiënt (r) van 0.9973. Die metode is suksesvol in diere-eksperimente gebruik om PTX te meet in die plasma van behandelde rotte. Hierna is 'n voorlopige in vitro eksperiment uitgevoer om te bepaal of Phela 'n direkte / fisiese effek het op PTX. 'n direkte middelinteraksie-eksperiment sowel as Slide-A-Lyzer® dialise is uitgevoer en PTX konsentrasies is gemeet met HPLC. Phela het geen direkte effek op PTX konsentrasies. Die tyd om ekwilibrium van PTX te bereik is geskat op 8 ure. Nadat Phela bygevoeg is, het PTX konsentrasies en die vry-fraksie (fu) onveranderd gebly. Daarom word die gevolgtrekking gemaak dat daar geen interaksie tussen Phela en PTX in vitro is nie. Die finale deel van die studie is onderneem om die effek van Phela op P-gp en MRP2 transporters te ondersoek. PTX en siklosporien A (CYA) is onderskeidelik gebruik as die substraat en die inhibeerder van P-gp, terwyl MTX en probenesied (PRO) soortgelyk gebruik was vir MRP2. Etiese goedkeuring is verkry en manlike Sprague-Dawley (SD) rotte (200-250 g) is gebruik. Die proefdier-eksperiment is in twee dele verdeel. In Deel I het drie groepe van 40 rotte elk 'n eenmalige dosis gekry van PTX-alleen (10 mg/kg); PTX & CYA (10 mg/kg); of PTX & Phela (15.4 mg/kg), terwyl in Deel II, drie groepe van 40 rotte elk 'n eenmalige dosis gekry het van MTX-alleen (10 mg/kg); of MTX & PRO (20 mg/kg); of MTX & Phela (15.4 mg/kg). Uit elke groep is 5 rotte geslag na 0.5, 1, 2, 4, 6, 8, 10, en 12 uur. Bloed is ontleed vir volbloedtelling, lewerfunksie, en PTX en MTX konsentrasies. CYA en PRO het die area onder die plasma-konsentrasie tyd-kurwe (AUC) van PTX en MTX, onderskeidelik verhoog, terwyl Phela geen effek op die AUC van PTX of MTX getoon het nie. Algeheel, is daar geen direkte interaksie tussen PTX en Phela waargeneem beide in vitro en in vivo nie, en daar was ook geen interaksie tussen MTX en Phela in vivo nie. Phela het nie P-gp of MRP2 inhibeer nie. Dit impliseer dat Phela waarskynlik nie betrokke sal wees in plant-geneesmiddel-interaksies van membraan-vervoerder- oorsprong nie. Daarom hoef die dosisse van middels wat deur P-gp en MRP2 vervoer word, nie aangepas te word wanneer dit saam met Phela toegedien word nie.
Description
Keywords
Membrane drug-transporters, P-glycoprotein, Multidrug resistance-associated protein 2, Pharmacokinetic parameters, Paclitaxel, Methotrexate, Herb-drug interactions, Phela, High performance liquid chromatography, The area under the plasma concentration-time curve, Drugs -- Testing, Pharmacology, Experimental, Traditional medicine, Rats as laboratory animals, Gastrointestinal system, Multidrug resistance, P-glycoprotein, Dissertation (M.Med.Sc. (Pharmacology))--University of the Free State, 2015