adenosine triphosphate - binding cassette transporters , such as p - glycoprotein ( pgp ) , breast cancer resistance protein and multidrug resistance protein 4 have a pivotal role in controlling brain distribution of drugs . these transporters are expressed in the luminal membrane of brain capillary endothelial cells that form the blood  brain barrier ( bbb ) and are capable of effluxing a multitude of chemically diverse compounds , often in cooperation with transporters expressed at the abluminal membrane , from brain into blood . transporter - mediated drug  drug interactions at the bbb are of great concern as they may lead to altered brain distribution of drugs and hence to severe central nervous system side effects . studies in mice have shown that genetic knockout of pgp can result in up to 2,600% increases in brain - to - plasma ratios of drugs , which are selective pgp substrates ( e.g. , ivermectin ) . however , for drugs that are in addition to pgp recognized by other efflux transporters at the bbb much smaller increases in brain distribution will be achieved when only pgp is inhibited , as the other non - inhibited transporter(s ) may compensate the function of pgp . to better understand whether results from rodent studies translate to humans it is important to assess the effect of transporter inhibition on drug brain distribution in humans . positron emission tomography ( pet ) imaging with radiolabelled pgp substrates , such as [ c]verapamil or [ c]n - desmethyl - loperamide , in combination with administration of pgp inhibitors has now made it possible to directly study pgp - mediated drug  drug interactions at the human bbb . two pgp inhibitors have so far been used in clinical pet studies , the immunosuppressant cyclosporine a and the non - marketed third - generation pgp inhibitor tariquidar . whereas cyclosporine a caused only small increases in brain uptake of [ c]verapamil and the use of higher doses is limited by safety concerns , tariquidar is relatively well tolerated at doses required to inhibit pgp at the bbb . administration of tariquidar at a dose of 6  mg / kg body weight was shown to result in 129% and 223% increases in brain distribution of ( r)-[c]verapamil and [ c]n - desmethyl - loperamide , respectively , in humans . however , as these effects were smaller than those observed in rodent studies it was not clear whether complete inhibition of pgp at the human bbb had been achieved . knowing the maximum possible effect of pgp inhibition on brain distribution of a pgp substrate in humans is important to better understand the safety risks associated with pgp - mediated drug  drug interactions at the human bbb and to obtain much needed scaling factors for extrapolation of data from rodents to humans . in previous studies , pet scans with ( r)-[c]verapamil or [ c]n - desmethyl - loperamide were performed at approximately 1  hour after completion of an intravenous tariquidar infusion . as tariquidar plasma concentrations rapidly decline after end of infusion , we investigated in the present study the effect of continuous infusion of tariquidar for the duration of the pet scan on ( r)-[c]verapamil brain distribution . the study was registered with eudract ( number 2012 - 005796 - 14 ) , approved by the ethics committee of the medical university of vienna , and conducted in accordance with the declaration of helsinki and its amendments . from all subjects written informed consent was obtained before enrollment into the study . five healthy men ( mean age : 273 years , mean weight : 7914  kg ) were included into the study . subjects were required to be free of any medication for at least 14 days before start of the study . subjects underwent two consecutive 60-minute dynamic ( r)-[c]verapamil pet scans ( mean injected activity : 40016  mbq ) on a ge advance scanner ( general electric medical systems , milwaukee , wi , usa ) and serial arterial blood sampling via the radial artery as described previously . one hour before start of the second pet scan an intravenous infusion of tariquidar was started at an infusion rate of 375  ml / h and maintained until the end of the scan ( mean infusion time 1233  minutes ) . for formulation of the infusion solution , a stock solution ( 70  ml ) of 7.5  mg / ml of tariquidar free base in 20% ethanol/80% propylene glycol ( azatrius pharmaceuticals pvt ltd , london , uk ) was diluted with aqueous dextrose solution ( 5% , w / v , 805  ml ) to a final concentration of 0.6  mg tariquidar free base per ml . three venous blood samples ( 5  ml ) were collected at the beginning , in the middle and at the end of the second pet scan to measure plasma concentrations of tariquidar by a previously described liquid chromatography tandem mass spectrometry assay . an arterial input function was constructed by correcting total activity counts in arterial plasma for polar [ c]metabolites of ( r)-[c]verapamil as described previously . plasma protein binding of ( r)-[c]verapamil in blood samples obtained immediately before each pet scan was determined as described before . all subjects underwent t1-weighted 3 tesla magnetic resonance imaging on an achieva 3.0  t scanner ( philips medical systems , best , the netherlands ) . the pet and magnetic resonance data were processed with analyze 8.0 ( biomedical imaging resource , mayo foundation ) and spm5 ( wellcome department of imaging neuroscience , ucl ) software and whole - brain gray matter time - activity curves were extracted using the hammersmith n30r83 three - dimensional maximum probability atlas of the human brain as described previously . in addition , the pituitary gland was manually delineated on axial slices of coregistered pet and magnetic resonance data sets with pmod 3.6 software ( pmod technologies ltd , zrich , switzerland ) . a standard 2-tissue 4-rate - constant ( 2t4k ) compartment model was fitted to the time - activity curves from 0 to 60  minutes after radiotracer injection as described previously . differences in outcome parameters of scan 1 and 2 were tested using the wilcoxon matched pairs test ( statistica 6.1 , statsoft inc . , concentration - effect relationship was analyzed by nonlinear regression based on a sigmoidal model with variable slope using the prism 5.0 software ( graphpad software , la jolla , ca , usa ) . in a previous study in which we administered tariquidar as a short infusion over 30  minutes during an ( r)-[c]verapamil pet scan we observed that ( r)-[c]verapamil brain concentrations were rising in immediate response to tariquidar infusion and were rapidly falling after the end of infusion in parallel with declining tariquidar plasma concentrations ( supplementary figure i ) . this suggested that the pgp - inhibitory effect of tariquidar at the bbb is transient . this is in good agreement with another study , which demonstrated that higher increases in brain uptake of [ c]n - desmethyl - loperamide can be obtained in humans when the pet scan is performed during rather than after tariquidar administration . the transient inhibition of pgp at the bbb by tariquidar prompted us to perform in this study two consecutive ( r)-[c]verapamil pet scans , a first baseline scan and a second scan during continuous infusion of tariquidar , in analogy to a previously described pet protocol using cyclosporine a as pgp inhibitor . we started the tariquidar infusion at 60  minutes before start of the second pet scan and maintained it throughout the pet scan , which resulted in the administration of a mean tariquidar dose of 5.91.0  mg / kg body weight . in four out of five subjects previously known , mild adverse reactions that were most likely related to tariquidar occurred ( metallic taste in the mouth : n=3 and dizziness : n=1 ) . mean tariquidar plasma concentration during the pet scan was 2,873517  nmol / l ( supplementary table i ) , which was 23 times higher than in our previous study . tariquidar administration had no significant effect on the fraction of non - protein bound ( r)-[c]verapamil in plasma ( scan 1 : 0.0840.016 , scan 2 : 0.1030.016 ; p=0.11 ) , which was in good agreement with the previous results . brain time - activity curves were approximately four times higher in scan 2 as compared with scan 1 ( figure 1a ) . the pet data were analyzed with kinetic modelling using a 2t4k model , which was shown in previous studies to be the model of choice for modelling of [ c]verapamil pet data after pgp inhibition , to obtain vt and the transfer rate constant from plasma into brain k1 as outcome parameters of interest ( table 1 ) . vt and k1 values were significantly increased by 27378% and 25974% , respectively , relative to baseline scans ( p=0.043 ) . mean vt and k1 values in scan 2 were 1.7 and 1.6 times higher than mean vt and k1 values in the highest tariquidar dose group ( 8  mg / kg ) of our previous study . interestingly , the increases in brain uptake of ( r)-[c]verapamil observed in the present study were of similar magnitude as the increases observed for [ c]verapamil in another study in non - human primates during administration of a high dose of cyclosporine a , which presumably led to complete pgp inhibition at the bbb ( mean 372% increase in [ c]verapamil k1 ) . it has been hypothesized that uptake of [ c]verapamil in the posterior pituitary gland , which has fenestrated capillaries and is therefore not protected by the bbb , may provide an estimate of brain uptake of [ c]verapamil in the absence of pgp function . we therefore analyzed distribution of ( r)-[c]verapamil to the pituitary gland ( figures 1a and 1b ) . while vt in the pituitary gland was 5.31.7 times higher than in whole - brain gray matter in scan 1 ( p=0.043 ) , vts in whole - brain gray matter and pituitary gland were comparable and not significantly different in scan 2 , which supported the assumption that we were approaching complete inhibition of pgp at the bbb ( table 1 ) . moreover , vt in the pituitary gland was similar in scans 1 and 2 , which suggested that distribution of ( r)-[c]verapamil to this region is not restricted by pgp ( table 1 ) . it should be noted , however , that the pituitary gland lies in proximity of two arteries and is smaller ( mean volume of interest : 0.110.02  cm ) than the spatial resolution of our pet scanner ( 6  mm ) so that quantification of radioactivity in the pituitary gland may be affected by partial volume and/or spill - over effects . we used the data obtained in this study and data from a previous study to extend our previously published concentration - effect curve of tariquidar for enhancement of brain uptake of ( r)-[c]verapamil . a sigmoidal curve provided good fits of the combined data ( figure 1c ) giving an estimated half - maximum effect concentration ( ec50 ) of 2,248 nmol / l , which was higher than the previously estimated ec50 of tariquidar in humans ( 868  nmol / l ) . the estimated maximum vt in brain ( 3.5 ) was comparable to vt in the pituitary gland in scan 2 ( 3.060.72 ) . the ratio of nonprotein bound plasma concentration of a transporter inhibitor to its in vitro inhibitory constant ( ki ) has been proposed to predict the propensity of an inhibitor to achieve significant transporter inhibition in vivo . in our study , the fraction of unbound tariquidar in human plasma has been determined before as 0.00630.0021 , corresponding to unbound tariquidar plasma concentrations in our study ranging from 11 to 26 the ki of tariquidar for inhibition of transport of [ h]verapamil by human pgp is 8.5  nmol / l ( unpublished data ) giving a ratio of the unbound tariquidar plasma concentration to ki ranging from 1.3 to 3.0 . since brain distribution of lipophilic pgp substrates , such as ( r)-[c]verapamil or [ c]n - desmethyl - loperamide , may be dependent on cerebral blood flow ( cbf ) , it has been suggested that extraction fraction ( e ) given as k1 divided by cbf may be a more suitable parameter of pgp function than k1 or vt . however , since we did not measure cbf in the present study , we were only able to estimate e based on cbf values available from the literature ( 0.5  ml / g per minute for whole - brain gray matter ) . normalization of k1 values in scan 2 to cbf provided e values of 0.300.04 and 0.440.12 in whole - brain gray matter and pituitary gland , respectively , which indicated that for ( r)-[c]verapamil extraction may not reach 100% even in the absence of pgp activity . it should be noted , however , that cbf in the pituitary gland may differ from cbf in whole - brain gray matter so that our e estimate for the pituitary gland may not be accurate . in a previous study in rats a 932% increase in ( r)-[c]verapamil brain vt over baseline scans was observed after complete inhibition of pgp at the bbb by tariquidar . according to our concentration - effect curve ( figure 1c ) , the estimated maximum vt in human brain is 3.5 , which allows predicting a maximum vt increase in humans of 367% . this is in good agreement with the prediction made in another study for [ c]verapamil ( + 400% ) . this indicates that for ( r)-[c]verapamil the scaling factor between rats and humans for vt increase after complete pgp inhibition is 2.5 . it is , however , not known whether this scaling factor will also apply to other substrate - inhibitor combinations than ( r)-[c]verapamil and tariquidar . in conclusion , our results show that substantial inhibition of pgp can be safely achieved at the human bbb using an experimental ( nonmarketed ) pgp inhibitor , which results in pronounced increases in brain delivery of a radiolabelled pgp substrate . this approach may be of interest to improve brain penetration of therapeutic drugs , which are cleared from brain by pgp . moreover , we experimentally verified the hypothesis that pet analysis of baseline uptake ( i.e. , without administration of a pgp inhibitor ) of a radiolabelled pgp substrate in the pituitary gland gives an estimate of brain uptake when pgp is fully inhibited , thus providing the possibility to predict the maximum possible effect of pgp - mediated drug  drug interactions at the bbb . finally , our data provide much needed scaling factors for extrapolation of rodent data to humans in pharmacokinetic modelling .