in a number of clinical situations , concomitant use of two or more drugs is imperative . in particular in chronic disease treatment course , patients must take more than one drug for an extended period of time .
in this course , many drug interactions have received a great deal of attention to avoid any side effects to the patients .
the greater the number of drugs prescribed to a patient , the greater the risk of drug interactions . in an epidemiological study , conducted at a community hospital to investigate the clinical relevance of drug - drug interactions ( ddis ) ,
it was reported that incidence of interactions raises up to 7% in those patients taking 610 drugs and 40% in those taking 1620 drugs a day . beside the off target effects
, ddis can lead to termination of drug development in early stages , refusal of approval , very strong prescribing restrictions , and withdrawal of drugs from market . furthermore , frequent and severe side effects due to ddis in elderly patients because of the impaired physiological functions are reportedly increasing , necessitating the requirement of more information regarding the ddis , while giving any combination therapy .
many of the major pharmacokinetic ( pk ) interactions between drugs are due to cytochrome 450 ( cyp450 ) enzymes , whose genetic expressions are being affected ( induction or inhibition ) by previously or simultaneously administered drugs . in addition , such interactions may exist at the level of absorption , distribution , clearance , and transporters .
such interactions are investigated in terms of the pk parameter changes of the concerned drug .
these substantial changes in pk parameters may be used to indicate clinical importance of ddis , when substrate and interacting drugs are likely to be given simultaneously either for short periods or chronically for an extended periods of time during the combinational therapy . the present study aimed to evaluate the influences of coadministration of antiepileptic drugs ( aeds ) on an antimalarial candidate 99/411 pharmacokinetic ( pk ) profile .
99/411 is a novel 1 , 2 , 4 trioxane derivative , antimalarial drug candidate with peroxide scaffold essential for its pharmacological activity [ 4 , 5 ] .
this molecule has currently entered into phase i clinical trial . during the drug developmental stages , in vivo drug interaction studies
are usually carried in experimental animals because of the difficulties to conduct these studies in humans at this stage [ 6 , 7 ] .
these studies are further helpful in predicting the drug interactions in human beings , although results obtained in the experimental animals do not ensures the existence of the same in humans , provided extrapolation of these findings is carefully done by considering physiochemical variables in higher species as an important function of the body weight across the species . in this study ,
our investigational drug 99/411 was considered as a substrate and the interacting agents chosen were fda approved antiepileptic drugs ( aeds ) including phenytoin ( pht ) , carbamazepine ( cbz ) , and gabapentin ( gb ) .
the rationale behind selecting aeds as interacting drugs is being strengthened by the fact that the seizures ( characteristics of epilepsy ) are one among the common feature of malaria regardless of its severity .
seizures are found to occur in about 85% of the cases diagnosed of cerebral malaria [ 10 , 11 ] .
thus the coexistence of these two diseases leads to a combinational therapy involving simultaneous administration of aeds and antimalarials , making the importance of investigating ddis .
  pure ( > 99% ) reference standards of 99/411 and 97/63 ( used as is ) ( figures 1(a ) and 1(b ) ) were obtained from medicinal & process chemistry division , csir - central drug research institute ( cdri ) , lucknow , india .
acetonitrile , hplc grade , was purchased from thomas baker ( chemicals ) limited ( mumbai , india ) .
analytical grade ammonium acetate for buffer preparation was obtained from sd fine - chemicals limited ( mumbai , india ) .
drug free rat plasma was collected from healthy male and female sprague - dawley ( sd ) rats provided by national laboratory animal centre ( nlac ) , cdri .
healthy male and female sd rats obtained from nlac of cdri , india , were weighed 200250  g on the day of dosing .
prior to the studies , animals were acclimatized for 4 days in proper ventilated polypropylene cases in standard laboratory conditions with regular 12  h light - dark cycle , temperature ( 22  2c ) , and relative humidity ( 55  5% ) .
guidelines approved by the  animal experimentation ethics committee  and good laboratory practice ( glp ) were followed throughout the animal experimentation .
the studies were single dose oral pk , designed to estimate the effect of concurrent administration of aeds on pk profile of 99/411 .
each study was carried out in two groups of experimental animals , every group comprising three animals ( n = 3 ) .
studies were conducted separately on male and female sd rats to assess the intersex differences in the drug interaction pattern .
subjects were given 12  mg / kg of 99/411 ( control ) and 99/411 followed by coadministration of aeds ( 42  mg / kg each ) in separate sets of experimental animals .
blood samples for pk analysis were collected before dosing and at the following time points 0.083 , 0.33 , 0.75 , 1 , 1.5 , 2 , 4 , 6 , 8 , 12 , 24 , 36 , 48 , 60 , 72 , and 96 hours after dose .
fresh oral formulations of 99/411 and each aeds were prepared in neutralized arachis oil ( as a suspension ) at 12 and 42  mg / kg dose , respectively .
both the formulations were subjected to quality control ( qc ) , stability , and homogeneity test to ensure the strength before dosing .
blood samples collected for quantitative estimation of 99/411 were processed by centrifugation within 30  min of sample collection to obtain plasma and were stored at 70c till analysis .
10  l of is was spiked into each study samples followed by a double step liquid - liquid extraction procedure using in n - hexane to extract the drugs and is .
plasma concentration was determined by using a fully validated lc - ms / ms method .
mass spectrometric detection was performed on an api 4000 lc - ms / ms mass spectrometer ( applied biosystems , mds sciex , usa ) with analyst 1.4 software .
product ion transitions ( ammonium adducts of analyte and is ) , at m / z 412.3 to 185.1 and 418.2 to 119.1 were used for quantification of 99/411 and is respectively .
the assay was linear over the range 1.56200  ng / ml with loq 1.56  ng / ml . coefficients of determination ( r ) were > 0.99 for standard curves generated .
precision and accuracy of the method was determined by analyzing qcs at 1.50 , 80 and 160  ng / ml . all accuracies were within 15% of the nominal concentrations with standard deviation of < 8% .
the primary endpoints for these studies were area under the curve ( auc0 ) , maximum plasma concentration ( cmax ) , time to attain cmax ( tmax ) , elimination half - life ( t1/2 ) , mean residence time ( mrt ) , and relative bioavailability ( rb ) , when 99/411 was given alone and coadministered with aeds in male and female sd rats .
these pk parameters were calculated by noncompartmental analysis using winnonlin software ( version 1.5 , pharsight corporation ) .
the estimated pk parameters for 99/411 alone and coadministered with aeds were statistically compared using two - tailed student 's t - test . in all the tests , a probability level of significance was kept at  = 0.05 .
to evaluate the coadministered influences of aeds on pk profile of 99/411 , its baseline pk profile was generated at 12  mg / kg in healthy male and female rats .
after oral dose administration , auc0 values were found to be higher in female rats in comparison to male rats , but with no statistical significant difference ( table 1 ) .
 statistically no significant difference was observed in t1/2 , tmax , cmax , auc0 , and mrt in terms of p value ( p > 0.05 ) .
the plasma concentration - time profiles ( mean  sd , n = 3 ) for 99/411 in male and female rats have been shown in figure 2 . in male rats ,
mean values of t1/2 , tmax , cmax , auc0 , and mrt for 99/411 upon coadministration of 99/411 with pht were 2.10  0.30  h , 7.00  1.00  h , 489.00  166.4  ng / ml , 2521.93  619.89  ngh / ml , and 17.58  2.85  h ,
while for female rats values were 4.65  0.67  h , 4.00  1.18  h , 493.85  73.14  ng / ml , 2160.70  488.58  ngh / ml , and 12.04  1.00  h , respectively .
pk profiles after oral coadministration of 99/411 with pht have been given in table 1 .
intersex statistical analysis of pk parameters for 99/411 upon coadministration with pht in male and female rats revealed that no significant difference existed between male and female pk profiles in terms of p values which were found > 0.05 for respective parameter .
however , rb was found to be enhanced in male rats , while a decrease was seen in female rats ( table 1 ) .
the plasma concentration - time profiles ( mean  sd , n = 3 ) for 99/411 upon pht coadministration in male and female rats have been shown in figure 3 .
mean values of t1/2 , tmax , cmax , auc0 , and mrt for 99/411 upon coadministration of 99/411 with cbz were 3.16  0.49  h , 6.50  0.50  h , 495.25  58.42  ng / ml , 2534.84  299.69  ngh / ml , and 13.84  1.56  h in male rats , while 3.49  1.09  h , 4.00  1.18  h , 484.74  60.50  ng / ml , 2095.20  298.73  ngh / ml , and 12.17  0.98  h for female rats , respectively ( table 1 ) .
pk profiles after oral coadministration of 99/411 with cbz have been given in table 1 .
intersex statistical analysis of pk parameters for 99/411 upon coadministration with cbz in male and female rats revealed that no significant difference existed between male and female pk profiles in terms of p values .
however , like pht coadministration , rb was found to be enhanced in male rats , while a decrease was seen in female rats ( table 1 ) .
the plasma concentration - time profiles ( mean  sd , n = 3 ) for 99/411 upon cbz coadministration in male and female rats have been shown in figure 4 .
mean values of t1/2 , tmax , cmax , auc0 , and mrt for 99/411 upon coadministration of 99/411 with gb were 3.16  0.49  h , 6.50  0.50  h , 495.25  58.42  ng / ml , 2534.84  299.69  ngh / ml , and 13.84  1.56  h in male rats , while 5.02  1.09  h , 2.00  1.11  h , 449.64  77.95  ng / ml , 4560.86  1396.05  ngh / ml , and 14.05  1.56  h for female rats , respectively .
pk profiles after oral coadministration of 99/411 with gb have been given in table 1 .
intersex statistical analysis of pk parameters for 99/411 upon coadministration of 99/411 with gb in male and female rats revealed that no significant difference existed between male and female pk profiles in terms of p values .
however , rb was found to be decreased in male rats , while an increase was seen in female rats ( table 1 ) . the plasma concentration - time profiles ( mean  sd ,
n = 3 ) for 99/411 upon gb coadministration in male and female rats have been shown in figure 5 .
this study was conducted to evaluate the influence of concurrent administration of 99/411 with fda approved aeds .
keeping in view , the effect of species and sex differences on the pk profile of concerned drugs , study was conducted in healthy male and female sd rats [ 12 , 13 ] .
first of all , the baseline pk profile was generated in male and female rats .
due to fluctuating plasma concentration - time profile ( figure 2 ) the use of noncompartmental analysis was preferred .
therefore , pk parameters of 99/411 were obtained by noncompartmental analysis and are listed in table 1 .
pht , one of the frequently used aed , is well known to lower the plasma levels of a number of drugs , which reduces therapeutic efficacy of concurrently administered drugs .
statistical comparison of pk parameters of 99/411 have shown no significance difference ( in terms of p values > 0.05 ) from baseline pk profile , indicating that pht coadministration has little influence on pk parameters of 99/411 .
rb of 99/411 was observed to be enhanced by about 20% in male rats , while a decrease of about 13% was found in female rats . when pk profiles of male and female rats were statistically compared to evaluate intersex effect ,
it was observed that significance difference existed for t1/2 values , while for other pk parameters no significance difference existed ( table 1 ) .
although individual differences in the expression of drug transporters along git and intersex hormonal differences may be responsible for the differences in the rb , but in context to this study , the alteration in rb values is due to individual variations rather than any realistic differences among male and female rats .
however , such differences in pharmacokinetics and pharmacodynamics due to gender differences are well reported in animals and humans .
gender is one among the influential variable that contributes significantly to differences in pharmacokinetics including absorption , distribution , metabolism , and excretion .
its widespread and long term use with other medications enhance the possibilities of drug interactions .
study was carried out to evaluate the influence of cbz on pk profile of 99/411 .
it was found that rb of 99/411 was enhanced by about 20% in male rats , while a decrease of about 15% was found in female rats , similar to that of 99/411 coadministration with pht , but statistically nonsignificant .
no significant differences were observed too for other pk parameters of 99/411 in both the sexes . like pht and cbz ,
it does not bind to plasma proteins nor is metabolized and does not induce / inhibit liver enzymes , reducing the likelihood of drug interaction with other drugs .
although this drug is rapidly absorbed in small intestine by carrier mediated transport system , which is saturable .
this leads to the possibility of ddi in the absorption phase of 99/411 . in an in situ single
pass intestinal perfusion study , intestinal transporters have been reported to play a role in 99/411 absorption .
study revealed that there was slight decrease in the systemic exposure ( auc0 ) of 99/411 in male rats , while an increased auc0 was found for female rats . in terms of rb , rb of 99/411
was significantly increased by about 80% in female rats , while no effect was observed in male rats .
thus , a significant difference was observed intersexually in the interaction pattern influencing the rbs .
the rb was almost unaltered in male rats , while significantly enhanced in female rats .
in male rats , there was a shift in tmax from 2  0.00 to 6.50  0.50  h , indicating that gb coadministration interacted at absorption level , which might have reduced absorption rate of 99/411 ; however , systemic exposure was unaltered .
conversely , in female rats , tmax was shifted from 4.50  1.53 to 2.00  1.11 indicating fast absorption of 99/411 upon gb coadministration which might have resulted into enhanced systemic exposure of 99/411 .
overall , these studies explicitly indicated that coadministration of these aeds does not exert any significant effect on pk of 99/411 , except for gb coadministration in female rats . a similarity was observed in the interaction pattern of pht and cbz , where an increase in the rb of 99/411 was reported in male rats , while a decrease in female rats , which was nonsignificant though .
in contrast , opposite pattern was found for gb coadministration , where a significant increase of about 80% was reported in female rats , while no effect was reported in male rats .
however , these studies were conducted on a randomized selection basis of animal group ( n = 3 ) ; results represented herein do not negate the variations in the interaction pattern .
these findings could not be directly utilized to estimate and predict existence of similar interactions in humans , unless predictions are not rationalized through allometric scaling approaches , keeping in view all the anatomical , physical , and chemical variations across species .
 
 objective . the study aimed to evaluate the influences of coadministration of antiepileptic drugs ( aeds ) on an antimalarial candidate 99/411 pharmacokinetic ( pk ) profile 
 . method . for this 
 , single oral dose pk drug interaction studies were conducted between 99/411 and fda approved aeds , namely , phenytoin ( pht ) , carbamazepine ( cbz ) , and gabapentin ( gb ) in both male and female sd rats , to assess the coadministered and intersexual influences on 99/411 pk profile . results . 
 studies revealed that there were no significant alterations in the pk profile of 99/411 upon pht and cbz coadministration in both male and female rats , while systemic exposure of 99/411 was significantly increased by about 80% in female rats upon gb coadministration . in terms of auc 
 , there was an increase from 2471  586 to 4560  1396  ngh / ml . 
 overall , it was concluded that simultaneous administration of aeds with 99/411 excludes the requirements for dose adjustment , additional therapeutic monitoring , contraindication to concomitant use , and/or other measures to mitigate risk , except for gb coadministration in females . 
 these findings are further helpful to predict such interactions in humans , when potentially applied through proper allometric scaling to extrapolate the data . 
1. Introduction
2. Material and Method
3. Results
4. Discussion
5. Conclusion