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Oral co-administration of elacridar and
ritonavir enhances plasma levels of oral
paclitaxel and docetaxel without affecting
relative brain accumulation
J J M A Hendrikx*
,1,2
, J S Lagas
1
, E Wagenaar
2
, H Rosing
1
, J H M Schellens
3,4
, J H Beijnen
1,4
and A H Schinkel
2
1
Department of Pharmacy and Pharmacology, Slotervaart Hospital, PO 90440, 1006 BK Amsterdam, The Netherlands;
2
Division of
Molecular Oncology, The Netherlands Cancer Institute, PO 90203, 1006 BE Amsterdam, The Netherlands;
3
Department of Clinical
Pharmacology, The Netherlands Cancer Institute, PO 90203, 1006 BE Amsterdam, The Netherlands and
4
Department of
Pharmaceutical Sciences, Utrecht University, PO 80082, 3508 TB Utrecht, The Netherlands
Background: The intestinal uptake of the taxanes paclitaxel and docetaxel is seriously hampered by drug efflux through
P-glycoprotein (P-gp) and drug metabolism via cytochrome P450 (CYP) 3A. The resulting low oral bioavailability can be boosted by
co-administration of P-gp or CYP3A4 inhibitors.
Methods: Paclitaxel or docetaxel (10 mg/kg) was administered to CYP3A4-humanised mice after administration of the P-gp
inhibitor elacridar (25 mg kg
1
) and the CYP3A inhibitor ritonavir (12.5 mg kg
1
). Plasma and brain concentrations of the taxanes
were measured.
Results: Oral co-administration of the taxanes with elacridar increased plasma concentrations of paclitaxel (10.7-fold, Po0.001)
and docetaxel (four-fold, Po0.001). Co-administration with ritonavir resulted in 2.5-fold (paclitaxel, Po0.001) and 7.3-fold
(docetaxel, Po0.001) increases in plasma concentrations. Co-administration with both inhibitors simultaneously resulted in further
increased plasma concentrations of paclitaxel (31.9-fold, Po0.001) and docetaxel (37.4-fold, Po0.001). Although boosting of orally
applied taxanes with elacridar and ritonavir potentially increases brain accumulation of taxanes, we found that only brain
concentrations, but not brain-to-plasma ratios, were increased after co-administration with both inhibitors.
Conclusions: The oral availability of taxanes can be enhanced by co-administration with oral elacridar and ritonavir, without
increasing the brain penetration of the taxanes.
The taxane anticancer agents paclitaxel (Taxol) and docetaxel
(Taxotere) share the baccatin core ring structure (Vaishampayan
et al, 1999). Currently, paclitaxel and docetaxel are routinely
applied intravenously (i.v.) to treat several types of cancer, such as
non-small cell lung cancer, ovarian, breast, gastric, prostate and
head-and-neck cancer (Gligorov and Lotz, 2004; Koolen et al, 2010a).
The development of oral formulations of paclitaxel and
docetaxel is the focus of preclinical and clinical research in our
groups because oral administration has many advantages over i.v.
administration (Schellens et al, 2000; Koolen et al, 2010a). Oral
administration is more practical and convenient for patients as oral
medication can be taken by the patient at home, whereas
i.v. administration requires hospitalisation during infusion. Oral
administration in the home situation also reduces treatment cost.
Moreover, oral administration enables other dosing schedules such
as metronomic therapy (e.g., continuous or frequent treatment
*Correspondence: Dr JJMA Hendrikx; E-mail: Jeroen.Hendrikx@slz.nl
Revised 19 March 2014; accepted 27 March 2014; published online 29 April 2014
&2014 Cancer Research UK. All rights reserved 0007 – 0920/14
FULL PAPER
Keywords: P-glycoprotein (P-gp/MDR1); cytochrome P450 3A (CYP3A4); paclitaxel; docetaxel; oral bioavailability
British Journal of Cancer (2014) 110, 2669–2676 | doi: 10.1038/bjc.2014.222
www.bjcancer.com | DOI:10.1038/bjc.2014.222 2669
with low doses of anticancer drugs), which can increase efficacy of
taxane treatment and reduce adverse effects caused by high plasma
concentrations of docetaxel or paclitaxel (Jiang et al, 2010; Wu
et al, 2011).
A major limitation in the concept of oral administration of
taxanes is, however, the low oral availability of paclitaxel and
docetaxel (Schellens et al, 2000; Koolen et al, 2010a). Paclitaxel and
docetaxel have poor aqueous solubility and upon oral administra-
tion, intestinal uptake is seriously hampered by drug efflux through
P-glycoprotein (P-gp/MDR1/ABCB1), and systemic exposure is
further limited by drug metabolism via cytochrome P450 (CYP) 3A
(Sparreboom et al, 1997; van Asperen et al, 1997; Meerum Terwogt
et al, 1998; van Asperen et al, 1998; Bardelmeijer et al, 2004; Lagas
et al, 2006; van Waterschoot et al, 2009; Hendrikx et al, 2013).
P-glycoprotein is a member of the ATP-binding cassette efflux
transporter family and is expressed in multiple tissues such as
intestine, liver and kidney, but also at the blood–brain barrier
(BBB) (Gottesman and Ambudkar, 2001). P-glycoprotein-
mediated transport limits drug absorption across intestinal cells
and brain penetration across the BBB. In enterocytes, P-gp pumps
back absorbed taxanes into the intestinal lumen, whereas at the
BBB, taxanes are pumped back into the systemic circulation. In
liver and kidney, P-gp increases drug excretion by active efflux
transport into the bile and urine (Glaeser and Fromm, 2008).
CYP3A is a member of the CYP superfamily and CYP enzymes
are responsible for most phase-I drug metabolism (Thelen and
Dressman, 2009). CYP enzymes are mainly expressed in the liver,
but some CYP members are also expressed in enterocytes. CYP3A
is the most abundant CYP enzyme in liver and intestine,
representing 40% and 80% of the total CYP enzymes expressed
in each tissue, respectively (Paine et al, 2006). Docetaxel is
primarily metabolised by enzymes of the CYP3A subfamily,
whereas paclitaxel is metabolised by both CYP3A4 and CYP2C8
(Vaishampayan et al, 1999). In contrast to CYP3A, CYP2C8 is only
expressed in liver cells (Paine et al, 2006).
Although docetaxel is a good P-gp substrate, transport of
paclitaxel by P-gp is even more efficient. In addition, paclitaxel
metabolism is not solely CYP3A dependent. Therefore, it was
assumed that oral bioavailability of paclitaxel was primarily limited
by P-gp, and that of docetaxel primarily by CYP3A. However, in
mice, complete vs single knockout of Mdr1a/b and/or Cyp3a genes
resulted in further increased plasma exposure of paclitaxel and
docetaxel alike after oral administration, suggesting that both
systems are important for oral availability of both taxanes
(van Waterschoot et al, 2009; Hendrikx et al, 2013). The importance
of CYP3A4 for paclitaxel metabolism was further supported by our
finding that human CYP3A4 metabolises paclitaxel far more
efficiently than the mouse Cyp3a enzymes (Hendrikx et al, 2013).
Therefore, a promising strategy to boost the oral availability of
these taxanes is combining oral formulations of paclitaxel or
docetaxel with inhibitors of both P-gp and CYP3A4. In (pre)
clinical studies, it has already been demonstrated that the area
under the plasma concentration–time curves (AUC) after oral
administration of paclitaxel and docetaxel can be strongly
enhanced in both mice and humans by co-administration of the
potent CYP3A4 inhibitor ritonavir (Bardelmeijer et al, 2002;
Oostendorp et al, 2009; Koolen, 2011; Hendrikx et al, 2013). In
addition, co-administration of the potent P-gp inhibitor elacridar
results in increased oral plasma AUC of paclitaxel in mice and
humans (Bardelmeijer et al, 2000; Malingre et al, 2001).
There are potential risks involved when the oral bioavailability
of paclitaxel and docetaxel is increased by inhibition of metabolising
enzymes and drug transporters. For instance, co-administration of
oral elacridar in mice resulted in increased brain penetration of i.v.
administered paclitaxel by inhibition of P-gp at the BBB (Kemper
et al, 2003). Therefore, boosting oral uptake of taxanes using an
oral P-gp inhibitor might increase the relative risk of CNS toxicity.
Furthermore, oral administration of docetaxel or paclitaxel to mice
lacking both P-gp and Cyp3a resulted in substantially higher
plasma levels than administration of the taxanes to mice lacking
either P-gp or Cyp3a alone. Simultaneous inhibition of P-gp and
CYP3A by drugs that are co-administered with orally administered
taxanes may lead to further increased plasma levels of the taxanes
and therefore an increased risk of side effects and toxicity in
clinical practice.
In the present preclinical study, we examined whether we could
substantially increase the oral availability of taxanes by simulta-
neous inhibition of P-gp and CYP3A using oral co-administration
of elacridar and ritonavir, and to what extent this would affect P-gp
transport at the BBB.
MATERIALS AND METHODS
Drugs and chemicals. Paclitaxel, docetaxel, elacridar HCl and
ritonavir were purchased from Sequoia Research Products (Oxford,
UK). Drug-free lithium-heparinised human plasma was obtained
from Bioreclamation LLC (New York, NY, USA). All other
chemicals were of analytical grade and obtained from commercial
sources.
Animals. In compliance with Dutch legislation, mice were housed
and handled according to institutional guidelines, and approval of
the local (NKI) animal care and use committee was obtained before
the start of experiments. Mice were kept in a temperature-
controlled environment with a 12-h light/12-h dark cycle and
received a standard diet (AM-II, Hope Farms, Woerden, The
Netherlands) and acidified water ad libitum. In this study, Cyp3a
knockout mice with specific expression of human CYP3A4 in liver
and intestine (Cyp3a
/
Tg-3A4
Hep/Int
) were used (van Herwaarden
et al, 2007). The strain had a 499% FVB genetic background.
Cyp3a
/
Tg-3A4
Hep/Int
mice were used as there is a species
difference for paclitaxel in CYP3A substrate specificity or enzyme
activity between endogenous murine Cyp3a and human CYP3A4
(Hendrikx et al, 2013). Experiments comparing paclitaxel PK in
wild-type mice may therefore underestimate the impact of human
CYP3A on paclitaxel pharmacokinetics in patients. The difference
between species is minimised using Cyp3a
/
Tg-3A4
Hep/Int
mice.
A basic difference in docetaxel metabolite formation was not observed
between human, wild-type mice and Cyp3a
/
Tg-3A4
Hep/Int
mice
(Hendrikx et al, 2013). In all experiments, male mice of 9–14 weeks
of age were used.
In vivo analysis of plasma pharmacokinetics. Before the experi-
ments, stock solutions containing 6 mg ml
1
paclitaxel, 6 mg ml
1
docetaxel, 15 mg ml
1
elacridar HCl, 7.5 mg ml
1
ritonavir or
15 mg ml
1
elacridar HCl and 7.5 mg ml
1
ritonavir in ethanol:-
polysorbate 80 (1 : 1, v/v) were made and stored at 20 1C. On the
day of the experiments, stock solutions were diluted with water
(1 : 5, v/v) to obtain solutions for administration. Animals were
fasted 2 h before oral drug administration to minimise variation in
absorption. Paclitaxel and docetaxel were administered orally at a
dose of 10 mg kg
1
of bodyweight, ritonavir was administered
orally at a dose of 12.5 mg kg
1
of bodyweight, and elacridar was
administered orally at a dose of 25 mg kg
1
of bodyweight. Oral
administration was performed by gavage into the stomach using a
blunt-ended needle. In case of co-administration with ritonavir,
elacridar or ritonavir and elacridar, the booster(s) were orally
administered 15 min before oral taxane administration.
Sample collection. For determining plasma pharmacokinetics,
multiple blood samples (B50 ml) were collected from the tail vein
at 15 min and at 1, 2, 4, 8 and 24h using heparinised capillary tubes
(Oxford Labware, St Louis, MO, USA). All time point samples were
derived from the same mouse. At the last time point of sequential
BRITISH JOURNAL OF CANCER Elacridar and ritonavir boost oral taxanes
2670 www.bjcancer.com | DOI:10.1038/bjc.2014.222
sampling (48 h), blood was taken by cardiac puncture. Blood
samples were centrifuged at ambient temperature at 8000 gfor
5 min and subsequently plasma was collected. All samples were
stored at 20 1C until analysis.
For brain accumulation studies, blood samples at 2 h were taken
by cardiac puncture and brain tissue was isolated. Blood samples
were centrifuged at ambient temperature at 8000 gfor 5 min and
subsequently plasma was collected. Brain tissue was homogenised
in 1% bovine serum albumin. All samples were stored at 20 1C
until analysis. Brain-to-plasma ratios at t¼2 h were calculated per
mouse by dividing the brain concentration by the corresponding
plasma concentration.
Bioanalytical analysis. Previously developed liquid chromato-
graphy assays coupled with tandem mass spectrometry detection
(LC-MS/MS) were used to quantify paclitaxel (Stokvis et al, 2004)
and docetaxel (Kuppens et al, 2005). Labelled structure analogues
were used as internal standards. In summary, mouse plasma
samples of 20 ml were diluted with 180 ml of human plasma.
Human plasma was used for dilution of the samples as the
concentrations in the undiluted mouse plasma were outside the
calibration range, and also to mimic the calibration standards that
were in human plasma. Brain samples were not diluted as
concentrations were too low to quantify after dilution in some
samples. To 200 ml of diluted plasma sample or homogenised brain
sample, 25 ml of internal standard working solution was added.
Subsequently, the samples were mixed briefly, tertiary-butyl methyl
ether was added and the samples were shaken for 10 min at 1250
r.p.m. The samples were centrifuged at 23 000 g, snap-frozen and
the organic layer was collected. After evaporation of the organic
layer, the samples were reconstituted with reconstitution solvent
and an aliquot was injected into the LC-MS/MS system.
Calibration standards in human plasma in a range of 0.25–
1000 ng ml
1
or 0.25–500 ng ml
1
were used for quantification of
paclitaxel and docetaxel, respectively.
Pharmacokinetic calculations and statistical analysis. Pharmaco-
kinetic parameters, including the AUCs, were calculated using
the software package PK Solutions 2.0.2 (SUMMIT, Research
Services, Ashland, OH, USA). The AUC
0–last time point
was
calculated by trapezoid calculation using observed data points.
The total AUC extrapolated to infinity (AUC
0–inf
) was computed
by combining AUC
0–last time point
with an extrapolated value. One-
way ANOVA was used when multiple groups were compared and
the Bonferroni post hoc correction was used to accommodate
multiple testing. The two-sided unpaired Student’s t-test was used
when treatments or differences between two groups were
compared. Data that did not show normal distribution were log-
transformed to normalise the distribution of the data sets and
enable statistical comparison. The Kolmogorov–Smirnov test was
used to test for normal distribution. During all statistical analyses,
differences in group sizes were considered in the calculations.
Differences were considered statistically significant when Po0.05.
All data are presented as geometric mean±s.d.
Addition of previously reported data. Previously, we published
AUCs of paclitaxel after oral administration of 10 mg kg
1
paclitaxel with and without 12.5 mg kg
1
ritonavir to Cyp3a
/
Tg-3A4
Hep/Int
mice (five and seven animals were used, respectively)
(Hendrikx et al, 2013). These data were compared with
plasma concentrations after oral administration of 10 mg kg
1
paclitaxel with and without 12.5 mg kg
1
ritonavir obtained in
this study (six and four animals were used, respectively).
Previously obtained results were not statistically different from
the results in the present study (data not shown). Therefore,
these results were also used to decrease the number of animals
neededforthisstudy.
Comparison with previously reported data in knockout mice.
To estimate the extent of P-gp inhibition by elacridar and Cyp3a
inhibition by ritonavir, plasma exposure after chemical inhibition
was compared with plasma exposure after complete knockout of
P-gp or Cyp3A. Previously reported plasma AUCs
0–inf
after oral
administration of 10 mg kg
1
paclitaxel (Hendrikx et al, 2013) or
10 mg kg
1
docetaxel (van Waterschoot et al, 2009) to mice
lacking P-gp, Cyp3a or both were compared with AUCs
0–inf
after
chemical inhibition as obtained in this study. All plasma AUCs
were normalised for their matching control group, and these
relative plasma AUCs were used for comparison.
RESULTS
Paclitaxel exposure after oral co-administration with elacridar
and/or ritonavir. To study the effect of the P-gp inhibitor
elacridar and the CYP3A inhibitor ritonavir on oral bioavailability
of paclitaxel, we orally administered 10 mg kg
1
paclitaxel to the
CYP3A4-humanised Cyp3a
/
Tg-3A4
Hep/Int
mice and combined
paclitaxel administration with 25 mg kg
1
elacridar and/or
12.5 mg kg
1
ritonavir.
Oral co-administration of paclitaxel and elacridar or paclitaxel
and ritonavir resulted in increased plasma concentrations of
paclitaxel (Figure 1). The area under the plasma concentration–
time curve from 0 extrapolated to infinity (AUC
0–inf
) was 10.7-fold
higher after co-administration with elacridar than after single
paclitaxel administration (Po0.001). These results in humanised
mice are in line with the previously observed 6.6-fold increase in
paclitaxel AUC after oral co-administration of paclitaxel and
elacridar at the same dose to wild-type mice (Bardelmeijer et al,
2000). Co-administration of paclitaxel and ritonavir resulted in an
increase in the AUC
0–inf
of 2.5-fold (Po0.001). However, this
boosting effect with ritonavir was clearly less than that of elacridar
co-administration. Co-administration of paclitaxel with both
elacridar and ritonavir together resulted in further increased
plasma concentrations of paclitaxel (31.9-fold higher than single
paclitaxel administration; Po0.001). The increases in oral
AUC
0–inf
of paclitaxel after chemical inhibition with elacridar or
ritonavir, alone or in combination, were comparable to the
increases in oral AUC
0–inf
after complete genetic knockout of
P-gp or Cyp3a, alone or in combination (Table 1). These data
suggest that virtually complete inhibition of both P-gp and
CYP3A4 (intestinal and hepatic) was achieved with the combina-
tion elacridar and ritonavir.
Docetaxel exposure after oral co-administration with elacridar
and/or ritonavir. Parallel to the paclitaxel experiments, we
studied the effect of elacridar and/or ritonavir co-administration
on the oral bioavailability of docetaxel. In the CYP3A4-humanised
mouse model, we observed a 7.3-fold increase in AUC
0–inf
after
oral administration of docetaxel and ritonavir when compared with
AUC
0–inf
after single docetaxel administration (Po0.001;
Figure 2). Oral co-administration of docetaxel and elacridar
resulted in a four-fold increase compared with single docetaxel
administration (Po0.001). The AUC
0–inf
of docetaxel after
boosting with elacridar was not significantly different from the
AUC
0–inf
after boosting with ritonavir (P40.05). As observed for
paclitaxel, co-administration of docetaxel with both elacridar and
ritonavir resulted in a further increase in AUC
0–inf
(37.4-fold
higher than single docetaxel administration; Po0.001). The
increase in oral AUC
0–inf
of docetaxel after chemical inhibition
with elacridar was comparable to the increase in oral AUC
0–inf
after complete genetic knockout of P-gp. However, the increase
after chemical inhibition with ritonavir was modestly, but
significantly (Po0.01), lower than after complete genetic knockout
of Cyp3a, and the same was true for combined CYP3A4 and P-gp
Elacridar and ritonavir boost oral taxanes BRITISH JOURNAL OF CANCER
www.bjcancer.com | DOI:10.1038/bjc.2014.222 2671
inhibition compared with full Cyp3a and P-gp knockout
(Po0.001; Table 1). These data suggest that for docetaxel in the
transgenic mice, the inhibition of intestinal and hepatic CYP3A4
by ritonavir was not entirely complete.
Brain concentrations of taxanes after oral co-administration
with elacridar and/or ritonavir. As brain accumulation could
potentially be increased after boosting of oral taxanes with a P-gp
inhibitor, we measured brain concentrations 2 h after oral
administration of paclitaxel or docetaxel, that is, roughly around
the plasma t
max
. Two effects might occur: first, increased taxane
brain concentrations simply as a consequence of the higher plasma
levels of the taxanes; and second, a further, disproportionate
increase in brain concentration relative to the plasma levels
because of inhibition of P-gp at the BBB, and/or possibly saturation
of P-gp activity at the BBB because of the much higher plasma
taxane levels. The second effects could result in poorly predictable
alterations in CNS toxicity of the taxanes. As these effects are most
likely to occur when plasma levels of both taxanes and inhibitors
are high, we chose the 2-h time point for sampling. Maximum
plasma concentrations of docetaxel and paclitaxel are reached at
2–4 h after oral administration. We did not measure plasma
concentrations of the inhibitors in this experiment, but maximum
plasma concentrations are reached in wild-type mice around 2 h
after oral administration of elacridar (Bardelmeijer et al, 2000) or
ritonavir (Supplementary Figure 1).
Brain concentrations of paclitaxel were significantly increased
after co-administration with elacridar (Po0.01 vs single paclitaxel
administration), but not after co-administration with ritonavir
(P40.05; Figure 3B). Co-administration of paclitaxel with both
Table 1. Area under the plasma concentration–time curve of paclitaxel and docetaxel after oral administration of 10 mgkg
1
paclitaxel or 10 mg kg
1
docetaxel in Cyp3a
/
Tg-3A4
Hep/Int
mice
Control group
a
P-gp inhib./KO CYP3A inhib./KO CYP3A and P-gp inhib./KO
Oral paclitaxel
AUC
0–inf
(ng hml
1
) after inhibition 314±74 3373±725 780±412 10002±2652
Fold vs control 1 10.7 2.5 31.9
Number of animals 11 8 11 7
AUC
0–inf
(ng hml
1
) in KO mice 320±224 3954±825 471±174 8830±1999
Fold vs control 1 12.4 1.5 27.6
Number of animals 10 5 9 5
Oral docetaxel
AUC
0–inf
(ng hml
1
) after inhibition 157±67 626±182 1146±281 5869±2520
Fold vs control 1 4.0 7.3 37.4
Number of animals 5 5 5 5
AUC
0–inf
(ng hml
1
) in KO mice 228±130 645±272 2627±1011 16466±2020
Fold vs control 1 2.8 11.5 72.2
Number of animals 6 6 6 7
Abbreviations: AUC
0–inf
¼area under the plasma concentration–time curve from 0 extrapolated to infinity; CYP3A ¼cytochrome P450 3A; Cyp3a
/
Tg-3A4
Hep/Int
¼Cyp3a KO mice with specific
expression of human CYP3A4 in liver and intestine; inhib.¼inhibition; KO¼knockout; P-gp¼P-glycoprotein (MDR1;ABCB1). Values represent the mean±s.d. Animals (5–11) per group were
used. Both drugs were administered as a single dose or co-administered with an oral dose of the CYP3A4 inhibitor ritonavir (12.5 mgkg
1
), the P-gp inhibitor elacridar (25mg kg
1
) or both.
Data are compared with previously reported data after oral administration of paclitaxel (Hendrikx et al, 2013) or docetaxel (van Waterschoot et al, 2009) to wild-type mice, P-gp KO
(Mdr1a/b
/
), Cyp3a KO (Cyp3a
/
) and combined P-gp and Cyp3a KO mice (Cyp3a/Mdr1a/b
/
).
a
When murine P-gp and human CYP3A are inhibited, the control group reflects single drug administration in Cyp3a
/
Tg-3A4
Hep/Int
mice. When murine P-gp and murine Cyp3a are knocked
out, the control group reflects single drug administration in wild-type mice.
10 000
AB
15 000
10 000
5000
AUC0–inf (ng×h ml–1)
0
PAC
PAC+ELC
PAC+RTV
PAC+ELC+RTV
PAC
PAC+ELC
PAC+RTV
PAC+ELC+RTV
1000
Paclitaxel (ng ml–1)
100
10
1
0 10203040
Time (h)
Figure 1. Panel Ashows plasma concentration–time curves in Cyp3a
/
mice expressing human CYP3A4 in liver and intestine (Cyp3a
/
Tg-3A4
Hep/Int
) after oral administration of 10 mg kg
1
paclitaxel. Paclitaxel was administered alone or co-administered with 25mgkg
1
oral
elacridar, 12.5 mg kg
1
oral ritonavir or both elacridar and ritonavir. Panel Bshows the area under the plasma concentration–time curves from 0
extrapolated to infinity (AUC
0–inf
). Data are presented as individual data points and lines represent the mean. Differences in AUC
0–inf
between all
groups were statistically significantly different (Po0.001), unless stated otherwise (NS, P40.05). Values represent the means±s.d. In all, 8–11
animals per group were used. Abbreviations: ELC ¼elacridar; PAC¼paclitaxel; RTV ¼ritonavir.
BRITISH JOURNAL OF CANCER Elacridar and ritonavir boost oral taxanes
2672 www.bjcancer.com | DOI:10.1038/bjc.2014.222
elacridar and ritonavir resulted in a similar increase in brain
concentrations as after co-administration of paclitaxel and
elacridar (P40.05 vs paclitaxel and elacridar administration;
Po0.001 vs single paclitaxel administration). However, correcting
for the increased plasma levels after boosting (Figure 3A), brain-
to-plasma ratios were not statistically different between the groups
(P40.05 for all comparisons; Figure 3C). These data suggest that
the relative brain accumulation of paclitaxel was not altered by
elacridar and ritonavir co-administration, despite the substantially
increased plasma levels of paclitaxel and the circulating elacridar
levels.
Co-administration with elacridar also increased docetaxel brain
concentrations (Po0.01 vs single docetaxel administration;
Figure 4B). In contrast to paclitaxel brain concentrations, docetaxel
brain concentrations were substantially increased after co-admin-
istration with ritonavir to comparable levels as seen after co-
administration with elacridar (P40.05 vs docetaxel and elacridar
administration; Po0.001 vs single docetaxel administration), thus
more or less following the pattern of effects of the inhibitors on
docetaxel plasma concentrations. Brain concentrations of docetaxel
were further increased after co-administration with both ritonavir
and elacridar (Po0.001 vs single docetaxel administration).
However, the increase in docetaxel brain concentrations was
primarily caused by the increased plasma concentrations after
boosting (Figure 4A), as brain-to-plasma ratios were not
statistically significantly different between any of the treatment
groups (P40.05 for all comparisons; Figure 4C).
DISCUSSION
Our data with CYP3A4-humanised mice show that it is possible to
markedly enhance the plasma AUC of oral paclitaxel and docetaxel
(30- to 40-fold) by orally co-administering elacridar and ritonavir.
Each inhibitor contributed substantially to the overall AUC
increase, although the contribution of elacridar was stronger for
paclitaxel and that of ritonavir for docetaxel. Yet, at the same time,
the relative brain accumulation of the taxanes (corrected for the
increased plasma levels) was not increased. This indicates that
neither the circulating elacridar levels, nor the increased plasma
taxane levels were sufficient to substantially inhibit or saturate the
1500
ABC
80 0.4
0.3
0.2
0.1
0.0
Brain/plasma ratio
NS
**
NS
NS NS
NS
60
40
20
0
Paclitaxel plasma
concentration (ng ml–1)
Paclitaxel brain
concentration (ng g–1)
1000
500
0
PAC
PAC+ELC
PAC+RTV
PAC+ELC+RTV
PAC
PAC+ELC
PAC + R T V
PAC +ELC+RTV
PAC
PAC+ELC
PAC + R T V
PAC +ELC+RTV
Figure 3. Plasma and brain concentrations of paclitaxel in Cyp3a
/
mice expressing human CYP3A4 in liver and intestine 2 h after oral
administration of 10 mg kg
1
paclitaxel. Paclitaxel was administered alone or co-administered with 25 mg kg
1
oral elacridar, 12.5 mg kg
1
oral
ritonavir or both elacridar and ritonavir. Panels reflect plasma concentrations (panel A), brain concentrations (panel B) or brain-to-plasma ratios
(panel C). Data are presented as individual data points and lines represent the mean. Differences in plasma or brain concentrations between
groups were statistically significantly different (Po0.001), unless stated otherwise (NS: not significant, P40.05 or **Po0.01). Differences in brain-
to-plasma ratios between all groups were not statistically significant. Values represent the means±s.d. Five animals per group were used.
Abbreviations: ELC ¼elacridar; PAC¼paclitaxel; RTV ¼ritonavir.
10 000
AB
10 000
8000
AUC0–inf (ng×h ml–1)
6000
4000
2000
0
DOC
DOC+ELC
DOC+RTV
DOC+ELC+RTV
DOC
DOC+ELC
DOC+RTV
DOC+ELC+RTV
1000
100
Docetaxel (ng ml–1)
10
1
0102030
Time (h)
40
Figure 2. Panel Ashows plasma concentration–time curves in Cyp3a
/
mice expressing human CYP3A4 in liver and intestine (Cyp3a
/
Tg-3A4
Hep/Int
) after oral administration of 10 mg kg
1
docetaxel. Docetaxel was administered alone or co-administered with 25mg kg
1
oral
elacridar, 12.5 mg kg
1
oral ritonavir or both elacridar and ritonavir. Panel Bshows the area under the plasma concentration–time curves from 0
extrapolated to infinity (AUC
0–inf
). Data are presented as individual data points and lines represent the mean. Differences in AUC
0–inf
between all
groups were statistically significantly different (Po0.001), unless stated otherwise (NS, P40.05). Values represent the means±s.d. Five animals per
group were used. Abbreviations: ELC ¼elacridar; PAC ¼paclitaxel; RTV ¼ritonavir.
Elacridar and ritonavir boost oral taxanes BRITISH JOURNAL OF CANCER
www.bjcancer.com | DOI:10.1038/bjc.2014.222 2673
taxane export activity at the BBB. These data suggest that it may be
possible to greatly enhance the oral availability of taxanes in
patients by co-administration with oral elacridar and ritonavir,
without invoking the risk of increased CNS toxicity of the
taxanes.
To estimate the extent of P-gp inhibition by elacridar and
CYP3A4 inhibition by ritonavir, plasma exposures after chemical
inhibition were compared with plasma exposures after complete
knockout of P-gp and/or Cyp3a (Table 1). Plasma AUCs
0–inf
of
paclitaxel and docetaxel were comparable after complete knockout
of P-gp and after chemical inhibition of P-gp by elacridar. This
suggests that the intestinal and hepatic inhibition of P-gp was
complete at the used dose of elacridar.
The plasma AUCs
0–inf
of paclitaxel were similar after complete
gene knockout of Cyp3a and inhibition of CYP3A4 by ritonavir,
but the plasma AUC
0–inf
of docetaxel was slightly lower after
ritonavir inhibition than upon Cyp3a knockout. The difference is
not substantial (Table 1), but it can probably be attributed to
incomplete inhibition of human CYP3A4 in the CYP3A4-
humanised mice at later time points, as the ritonavir concentra-
tions likely drop considerably after a few hours. Although not
tested in these mice, ritonavir levels in plasma of wild-type mice
receiving 12.5 mg kg
1
oral ritonavir drop substantially after 2 h
(Supplementary Figure 1), which may result in incomplete
CYP3A4 inhibition at later time points. The effect may be more
obvious for docetaxel than for paclitaxel because docetaxel
metabolism is more strongly CYP3A dependent (van Waterschoot
et al, 2009).
Previously reported data showed that complete knockout of
both P-gp and Cyp3a resulted in higher plasma concentrations of
orally applied taxanes than single knockout of P-gp or Cyp3a (van
Waterschoot et al, 2009; Hendrikx et al, 2013). We show here that
chemical inhibition of P-gp and CYP3A4 with both elacridar and
ritonavir likewise further increased plasma concentrations of orally
applied taxanes. In humans, oral formulations of paclitaxel and
docetaxel were thus far tested with only one of these boosters. In
patients, oral availability of paclitaxel is boosted by elacridar (Malingre
et al, 2001) or ritonavir (Koolen, 2011) and oral availability of
docetaxel is boosted by ritonavir (Oostendorp et al, 2009; Marchetti
et al, 2012). Our results suggest that boosting of orally
applied taxanes by both elacridar and ritonavir might
further increase plasma exposure of taxanes in patients.
Moreover, combined inhibition of P-gp and CYP3A4 may
result in decreased interpatient and intrapatient variability in
oral taxane pharmacokinetics. Xenobiotics, similar to other drugs,
herbal derivatives or environmental pollutants can cause clinically
relevant P-gp and CYP3A4 induction via Pregnane X receptor
regulation (Wojnowski, 2004; Xu et al, 2005; di Masi et al, 2009).
Complete inhibition of both P-gp and CYP3A4 in intestine and
liver by elacridar and ritonavir can eliminate the effects of
xenobiotic-related induction of these detoxifying proteins, and
thereby decrease variability in taxane pharmacokinetics after oral
administration. Polymorphisms of genes encoding for P-gp and
CYP3A4 are currently not known to be related to variability in
pharmacokinetics of i.v. administered taxanes (Bosch et al, 2006;
Jabir et al, 2012), but after oral administration, polymorphisms of
these genes can become more important. Interpatient variability in
oral bioavailability of docetaxel is decreased when docetaxel is
co-administered with inhibitors of CYP3A or P-gp (Stuurman et al,
2013). Not only polymorphisms, but also incomplete CYP3A4
inhibition by various other co-administered drugs might
contribute to variable oral taxane plasma exposure (Koolen et al,
2010b; Quinney et al, 2013). Complete inhibition of CYP3A by
ritonavir might eliminate this risk. It should be noted that, as P-gp
and CYP3A4 act at both the intestinal and hepatic level, the risk of
interpatient and intrapatient variability after oral administration of
taxanes is likely higher than after i.v. administration of taxanes, as
two potentially variable barriers need to be passed instead of one.
This underscores the importance of reducing potential sources of
variability by using effective CYP3A and P-gp inhibitors in oral
taxane regimens.
Boosting orally applied taxanes with elacridar and ritonavir
potentially increases the relative brain penetration of taxanes and
would thereby increase the risk of brain toxicity, by either
substantial inhibition of BBB P-gp by the circulating elacridar or
saturation of BBB P-gp activity because of the highly increased
plasma taxane levels, or a combination of both. We found here that
brain concentrations were increased after co-administration of the
taxanes with elacridar and ritonavir, but brain-to-plasma ratios
were not. This indicates that the increased brain concentrations
after oral co-administration of the taxanes with elacridar or
ritonavir were merely a consequence of the increased plasma
concentrations. Kemper et al (2003) showed a three-fold increase
in brain-to-plasma ratios in wild-type mice at 1 h after adminis-
tration of 10 mg kg
1
intravenously administered paclitaxel due to
25 mg kg
1
orally administered elacridar (brain-to-plasma ratios
after administration of paclitaxel with and without elacridar were
0.08 and 0.22, respectively). This three-fold increase was
3000
ABC
2000
NS 100
80
60
40
20
0
NS 0.15
0.10
0.05
0.00
NS
**
** **
**
1000
Docetaxel plasma
concentration (ng ml–1)
Docetaxel brain
concentration (ng g–1)
Brain/plasma ratio
0
DOC
DOC+ELC
DOC+RTV
DOC+ELC+RTV
DOC
DOC+ELC
DOC+RTV
DOC+ELC+RTV
DOC
DOC+ELC
DOC+RTV
DOC+ELC+RTV
Figure 4. Plasma and brain concentrations of docetaxel in Cyp3a
/
mice expressing human CYP3A4 in liver and intestine 2 h after oral
administration of 10 mg kg
1
docetaxel. Docetaxel was administered alone or co-administered with 25 mg kg
1
oral elacridar, 12.5 mg kg
1
oral
ritonavir or both elacridar and ritonavir. Panels reflect plasma concentrations (panel A), brain concentrations (panel B) or brain-to-plasma ratios
(panel C). Data are presented as individual data points and lines represent the mean. Differences in plasma or brain concentrations between
groups were statistically significantly different (Po0.001), unless stated otherwise (NS: not significant, P40.05 or **Po0.01). Differences in brain-
to-plasma ratios between all groups were not statistically significant. Values represent the means±s.d. Five to six animals per group were used.
Abbreviations: DOC ¼docetaxel; ELC ¼elacridar; RTV ¼ritonavir.
BRITISH JOURNAL OF CANCER Elacridar and ritonavir boost oral taxanes
2674 www.bjcancer.com | DOI:10.1038/bjc.2014.222
comparable with the increase in brain-to-plasma ratios as observed
after i.v. administration of paclitaxel to P-gp knockout mice. Brain
concentrations were not further increased when the elacridar
dose was increased to 100 mg kg
1
. Both findings suggest that
25 mg kg
1
oral elacridar can largely, if not completely, inhibit
BBB P-gp activity. However, in our experiments, we observed no
increase in brain-to-plasma ratios after oral co-administration of
the same dose of paclitaxel and elacridar. This can most likely be
explained by the initially far higher plasma levels of paclitaxel after
i.v. administration compared with those after oral administration.
When operating close to saturation, P-gp at the BBB will be more
sensitive to partial inhibition (Kalvass et al, 2013). The absence of
increased brain-to-plasma ratios in the experiments by Kemper
et al (2003) at 4 h after administration of i.v. paclitaxel and oral
elacridar (when plasma concentrations of paclitaxel are much
lower) further supports this interpretation (brain-to-plasma ratios
in wild-type mice after administration of paclitaxel with and
without elacridar were 0.9 and 0.8, respectively, whereas brain-to-
plasma ratios in knockout mice were 2.7 after i.v. administration of
paclitaxel at this time point). Collectively, our data suggest that at
modest plasma concentrations of paclitaxel (and presumably also
docetaxel), P-gp in the BBB has little or no effect on the relative
brain accumulation of taxanes.
CONCLUSIONS
Comparison of the results in our study with previously reported
data obtained from oral administration of taxanes to knockout
mice showed that orally administered elacridar and ritonavir at
comparatively low doses can completely (for paclitaxel), or almost
completely (for docetaxel) inhibit intestinal and hepatic P-gp and
CYP3A4 activity.
We also demonstrated that co-administration of the taxanes
with elacridar and ritonavir simultaneously resulted in a further
increase in plasma levels of the taxanes. In contrast, relative brain
accumulation of the taxanes was not affected after boosting with
oral elacridar. Even at the highly increased plasma concentrations
of taxanes after boosting with both elacridar and ritonavir, relative
brain accumulation was still similar as seen after boosting with
elacridar, or even in otherwise untreated CYP3A4-humanised
animals.
We therefore believe that it will be worthwhile testing
whether simultaneous inhibition of P-gp and CYP3A may
provide a relatively safe strategy to boost plasma exposure of
orally applied taxanes in patients, as relative brain exposure is
unlikely to be higher than that in the currently used i.v.
schedules.
CONFLICT OF INTEREST
The research group of AHS receives revenue from commercial
distribution of some of the mouse strains used in this study. JHMS
and JHB are inventors on patents on the application of oral taxane
formulations. The remaining authors declare no conflict of interest.
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Supplementary Information accompanies this paper on British Journal of Cancer website (http://www.nature.com/bjc)
BRITISH JOURNAL OF CANCER Elacridar and ritonavir boost oral taxanes
2676 www.bjcancer.com | DOI:10.1038/bjc.2014.222
