The pharmacokinetic interactions between valproic acid and acyclovir assessed in vitro and in a rabbit model

Abstract

English: Valproic acid is an antiepileptic drug that is widely used for treatment of epilepsy, while acyclovir is an antiviral drug indicated for treatment of infections caused by herpes simplex type I & II and varicella-zoster viruses. Given the high prevalence of people with conditions for which chronic use of valproic acid is indicated, and the notion that valproic acid increases the antiviral activity of acyclovir, it is not uncommon for the two drugs to be used concomitantly. As such, recent reports on the interaction between valproic acid and acyclovir with break through convulsions were a cause for concern. Since understanding the mechanism of this interaction is vital to the establishment of concrete guidelines on the use of the two drugs in patients, the aim of this study was to investigate the possible pharmacokinetic interaction between acyclovir and valproic acid. First, a high performance liquid chromatography (HPlC) method for analysis of acyclovir in plasma was developed. It involved simple protein precipitation of 200 IJl of plasma with perchloric acid, followed by centrifugation after which 20 IJl of the supernatant was injected in the HPlC. The sample was eluted with acetonitrile: octanesulfonic acid: ammonium acetate-citrate (vol.lvol.; 5%:11.88%:83.12%) at 1.5 ml/min over a luna C18 (4.60 x 150 mm) 51Janalytical column. Gancyclovir was used as the internal standard. Under these conditions, gancyclovir eluted at 3.4 min and acyclovir at 4.5 min. Over the calibration range of 10 - 100 IJg/ml, linearity was demonstrated by a linear regression equation of y = 0.03196 - 3.207x with a regression coefficient r² = 0.995, and accuracy by a percentage coefficient of variation (CV%) of less than 15%. The method was successfully used to analyze acyclovir in a rabbit treated with acyclovir single dose. Thereafter, the possibility of a direct interaction between acyclovir and valproic acid in vitro was investigated by monitoring the concentrations of valproic acid and acyclovir at different pH (pH 7.4 or pH 3 or pH 10) and temperatures (25°C and 37°C) when mixed in a 1:1 molar ratio or prepared separately in phosphate buffer. The samples were incubated at 25°C for 2 hours and a further 1 hour at 370C, and aliquots were drawn at 10 min., 2 and 3 hours to measure the concentration of valproic acid and acyclovir (n=3). The average concentrations of valproic acid and acyclovir from the samples containing the single drug were not different (P > 0.05) from those in the mixture of both drugs at the different temperatures and pH. However, when the temperature and pH were evaluated separately, there was a trend whereby, at high temperature (37°C), the concentrations of acyclovir (percentage detected) tended to be higher in the mixture (87%) than when it was alone (84%), while those of valproic acid tended to be lower in the mixture (89%) than when it was alone (92%). This same trend was observed at acid or alkaline pH. In conclusion, although temperature and pH did not induce significant effects on the concentrations of both acyclovir and valproic acid, increased concentrations of acyclovir were associated with reduced concentration of valproic acid when the two drugs were mixed under constrained conditions. These observations suggested a possible direct interaction between the two drugs This final part of the study was undertaken to investigate the effect of coadministration of valproic acid and acyclovir on the pharmacokinetic parameters of each other in a rabbit model. Fifteen white New Zealand rabbits were divided into 3 groups A, Band C whereby group A received acyclovir only, group B received valproic acid only, and group C received a combination of acyclovir and valproic acid. In a cross-over design, the intravenous route was studied first, followed by the oral route after a two-week wash out period. Blood samples were drawn over a 10 hr period and the pharmacokinetic parameters were derived from the concentrations. After intravenous administration, the area under the plasma concentration time curve (AUC) and plasma concentrations of acyciovir in group C were higher than in group A, while the volume of distribution (Vd) and plasma clearance (CLp) of acyciovir in group C were only 12.8% and 10.36% of those of group A, respectively. A similar trend was observed after oral administration. However, the bioavailability (F) of acyclovir was 8.4% in group A versus 1.5% in group C. Of note, the concentrations and kinetic parameters of valproic acid between the two groups after oral and intravenous administration were not different. In conclusion, co-administration of single doses of acyclovir and valproic acid led to reduced oral bioavailability of acyclovir, but increased concentrations of acyclovir due to reduced volume of distribution and clearance and this was most probably due to inhibition of the membrane transport proteins for acyclovir by valproic acid. Overall, a simple and accurate HPLC method for analysis of acyclovir in plasma was successfully developed, and a possibility of direct interaction between the two drugs was observed both in vitro and in vivo. These observations call for a cautious approach to the concomitant use of the two drugs until human studies are done.

Afrikaans: Valproaatsuur is 'n breë spektrum anti-epileptiese geneesmiddel wat algemeen aangewend word in die behandeling van epilepsie. Hierteenoor is asiklovir 'n antivirale geneesmiddel wat ge-indikeerd is in die behandeling van herpes simpleks tipe I en II asook varisella-zoster virale infeksies. Vanwee die hoë voorkoms van pasiënte met toestande waarvoor chroniese valproaatsuur gebruik aangedui is, tesame met die wete dat valproaatsuur die antivirale aktiwiteit van asiklovir verhoog, is dit nie ongewoon om 'n kombinasie van dié geneesmiddels aan pasiënte voor te skryf nie. Om hierdie rede is die onlangse gevallestudie van 'n interaksie tussen valproaatsuur en asiklovir, wat aanleiding gegee het tot deurbraakkonvulsies, 'n rede tot kommer. Dit is noodsaaklik om die meganisme van dié interaksie te verstaan vir die daarstelling van konkrete riglyne aangaande die gebruik van die middels in pasiënte en daarom was die doel van hierdie studie dus 'n ondersoek na die moontlike interaksie tussen asiklovir en valproaatsuur. Eerstens is 'n hoë druk vloeistof chromatografie (HPLC) metode ontwikkel waarmee die plasmakonsentrasie van asiklovir bepaal kan word. Die metode het die volgende behels: proteïen presipitering van 200 IJl plasma met perchloor suur, gevolg deur sentrifugering en inspuiting van 20 IJl van die bodrywende vloeistof in die HPLC. Die monster is oor 'n Luna C18 (4.60 x 150 mm) 51J analitiese kolom gedra deur asetonitriel: oktanesulfoniese suur: ammonium asetaat-sitraat (vol./vol.; 5%: 11.88%:83.12%) teen 1.5 ml/min en gansiklovir is as interne standaard gebruik. Onder hierdie toestande het die piek van gansiklovir teen 3.4 min. en die van asiklovir teen 4.5 min. uitgekom. Oor 'n kallibrasle reikwydte van 10 - 100 !-lg/ml is lineariteit gedemonstreer deur die liniëre regressie vergelyking y = 0.03196 - 3.207x met 'n regressie koëffisiënt r² = 0.995, en akkuraatheid deur 'n persentasie koëffisiënt van variëring (CV%) van minder as 15% . Die metode is suksesvol gebruik om die plasmakonsentrasie van asiklovir te bepaal in die plasma van 'n konyn na toediening van 'n enkeldosering asiklovir Hierna is die moontikheid van 'n direkte in vitro interaksie tussen asiklovir en valproaatsuur ondersoek deur die konsentrasies van asiklovir en valproaatsuur te monitor by verskillende pH (pH 7.4 of pH 3 of pH 10) en temperature (250C en 370C) gemeng in 'n 1:1 molêre verhouding, of afsonderlik, in fosfaatbuffer voorberei. Die monsters is geïnkubeer teen 250C vir 2 ure en daarna vir nog 1 uur teen 37oC, deelvolumes is geneem teen 10 min., 2 en 3 ure om die konsentrasie van asiklovir en valproaatsuur te bepaal (n=3). Die gemiddelde konsentrasies van asiklovir en valproaatsuur van die monsters wat die afsonderlike geneesmiddels bevat het, het nie verskil (P > 0.05) van die wat die kombinasie geneesmiddel bevat het by die verskillende temperatuur en pH nie. Nietemin, toe die uitwerking van temperatuur en pH op die konsentrasie van die geneesmiddels apart van mekaar geëvalueer is, is 'n neiging geïdentifiseer waar, teen hoër temperatuur (370C), die konsentrasie van asiklovir (persentasie gemeet) hoër was in die monsters wat beide middels bevat het (87%) vergelyk met die monsters wat In enkel geneesmiddel bevat het (84%); in teenstelling hiermee was die konsentrasie van valproaatsuur (persentasie gemeet) laer in die monsters wat beide middels bevat het (89%) vergelyk met die monsters wat In enkel geneesmiddel bevat het (92%). Dieselfde neiging is geobserveer met suur en alkaliese pH. Samevattend dus: alhoewel temperatuur en pH nie 'n statisties betekenisvolle effek op die konsentrasies van asiklovir en valproaat suur gehad het nie, is verhoogde konsentrasies van asiklovir geassosiëer met verlaagde konsentrasies van valproaat suur wanneer die twee geneesmiddels gemeng is onder uiterste fisiologiese toestande. Hierdie observering suggereer die moontlikheid van In direkte interaksie tussen die twee geneesmiddels. Die finale deel van hierdie studie is ondemeem om die effek van kombinasietoediening van asiklovir en valproaatsuur op die farmakokinetiese parameters van elke geneesmiddel in In konynmodel te ondersoek. Vyftien wit New Zealand konyne is verdeel in 3 groepe A, B en C. Groep A het slegs asiklovir ontvang, groep B het slegs valproaatsuur ontvang en groep C het 'n kombinasie van asiklovir en valproaatsuur ontvang. In In oorkruis studie-ontwerp is die intraveneuse roete eerste bestudeer, gevolg deur In twee weke uitwasperiode waarna die middels oral toegedien in. Bloedmonsters is getrek oor In 10 h periode en die farmakokinetiese parameters is afsonderlik verkry vanaf die konsentrasie versus tydprofiel van elke dier. Na intraveneuse toediening was die area onder die plasma konsentrasie versus tydkurwe (AUC) en die plasma konsentrasies van asiklovir in groep C hoër as dié in groep A, terwyl die volume van distribusie (Vd) en plasma opruiming (CLp) van asiklovir in groep C respektief slegs 12.8% en 10.36% van die waarde van groep A was. In Soortgelyke neiging is gevind na orale toediening. Nietemin was die biobeskikbaarheid (F) van asiklovir 8.4% in groep A versus 1.5% in groep C. Interessant is dat gedurende hierdie studie geen statisties betekenisvolle verskil in die plasma konsentrasies sowel as kinetiese parameters van valproaatsuur tussen groep B en groep C gevind na oraal en intraveneuse toediening nie. Samevattend dus, kombinasie toediening van In enkel dosering asiklovir en valproaatsuur het gelei tot verminderde biobeskikbaarheid van asiklovir maar verhoogde plasma konsentrasies van asiklovir as gevolg van 'n verlaging in volume van distribusie en opruiming en dit was mees waarskynlik as gevolg van inhibisie van membraan transport proteïene van asiklovir deur valproaatsuur. Ten slotte dus, 'n eenvoudig dog akkurate HPLC metode is suksesvolontwikkel waarmee die konsentrasie van asiklovir in plasma bepaal kan word en die moontlikheid van In direkte interaksie tussen asiklovir en valproaatsuur is waargeneem beide in vitro en in vivo. Hierdie waarneming maan tot versigtige gebruik van hierdie geneesmiddels in kombinasie in pasiënte totdat verdere menslike studies gedoen is.