British Journal of Pharmacology (2003) 138, 1367–1375
`
`& 2003 Nature Publishing Group All rights reserved 0007–1188/03 $25.00
`www.nature.com/bjp
`
`Nonlinear accumulation in the brain of the new taxoid TXD258
`following saturation of P-glycoprotein at the blood–brain barrier in
`mice and rats
`
`*,1Salvatore Cisternino, 1Fanchon Bourasset, 2Yves Archimbaud, 2Dorothe´ e Se´ miond, 2Ge´ rard
`Sanderink & 1Jean-Michel Scherrmann
`
`1INSERM U26, Hoˆ pital Fernand Widal, 200 Rue du Faubourg Saint-Denis, 75475 Paris cedex 10, France and 2Aventis Pharma,
`Drug Metabolism and Pharmacokinetics, 13 Quai Jules Guesde, B.P.14, 94403 Vitry sur Seine, France
`
`1 TXD258, a new taxoid antitumor agent, is a poor substrate for the P-glycoprotein (P-gp) in Caco-2
`cells. In this study, we investigated the amount of drug accumulating in the brains of rats and mice
`under a variety of conditions (dose and infusion time, species and plasma concentration) using
`conventional in vivo pharmacokinetic techniques and in situ brain perfusion.
`2 Mice were infused with radiolabeled TXD258 at 15, 30, 45 and 90 mg m2 for 45 s or 1 h and rats
`were infused with 15 and 60 mg m2 over 2.3 min. The radioactivity in the plasma and brains was
`measured. The brain concentrations of TXD258 in mice and rats were maximal from 2 min to 1 h
`postinfusion and radioactivity was still detectable at 168 h. While the plasma concentration of
`TXD258 increased linearly in mice with the infused dose, the brain content increased more than
`proportionally with the dose between 15 and 90 mg m2. This nonlinear uptake of TXD258 also
`occurred in the plasma and brain of the rat.
`3 These findings suggest that the protein-mediated efflux across the blood–brain barrier (BBB)
`becomes saturated. In situ brain perfusion studies confirmed that TXD258 is a P-gp substrate at the
`BBB of mice and rats. The P-gp of both species was saturated at the half-inhibitory concentration
`(B13 mm) produced by i.v. infusion.
`4 Thus, the observed nonlinear accumulation of TXD258 in the brain seems to occur by saturation
`of the P-gp at the rodent BBB. This saturation could have several advantages, such as overcoming a P-
`gp-mediated efflux, but the nonlinear pharmacokinetics could increase the risk of toxicity.
`British Journal of Pharmacology (2003) 138, 1367–1375. doi:10.1038/sj.bjp.0705150
`Keywords: Blood–brain barrier; in situ brain perfusion; multidrug resistance; P-glycoprotein; taxanes
`
`Abbreviations: AUC, area under the curve; BBB, blood–brain barrier; CNS, central nervous system; Kp, brain-to-plasma area
`under the curve ratio; MDR, multidrug resistance; P-gp, P-glycoprotein; PS80, polysorbate 80
`
`Introduction
`
`TXD258 is a new taxoid that has an in vivo spectrum of
`antitumor action similar to that of docetaxel;
`it stabilizes
`microtubules against cold-induced depolymerization (Bissery
`et al., 2000). TXD258 also inhibits the growth of tumor cells
`expressing the mdr1 gene in vitro. The pharmacological
`treatment of brain diseases is often complicated by the
`inability of potent drugs to pass across the blood–brain barrier
`(BBB), which is formed by the tight endothelial cell junctions
`of
`the brain capillaries.
`In vivo,
`intravenous TXD258
`suppresses
`implanted B16/TXT-resistant melanomas and
`intracerebral glioblastoma, suggesting that the drug is able to
`cross the BBB and/or the blood–brain tumor barrier (Bissery
`et al., 2000; Dykes et al., 2000). Other pharmacokinetic studies
`indicate that TXD258 is active when given orally to mice and is
`recognized as a marginal substrate of P-glycoprotein (P-gp)
`efflux pump in human Caco-2 colon carcinoma cells, largely
`used to predict in vivo oral absorption (Bissery et al., 2000).
`These properties have prompted the development of TXD258
`
`*Author for correspondence;
`E-mail: Salvatore.Cisternino@fwidal.inserm.fr
`
`for clinical use, with potential to treat brain metastases.
`Therefore, more detailed pharmacokinetic studies are needed
`to investigate the amount of drug given systemically that
`reaches the brain, as this can be influenced by the dose and
`duration of the drug infusion. Our pharmacokinetic studies
`were performed in mice and rats, using two methods. In the
`first conventional method, we measured the drug concentra-
`tions in the plasma and brain over time. TXD258 was given
`intravenously at different doses and over various infusion
`times. In the second method, we used in situ brain perfusion to
`determine the parameters of TXD258 uptake across the BBB
`in rats and mdr1a-deficient or control mice. We showed that
`the transport of TXD258 across the BBB is mediated by P-gp.
`This transport could be saturated by vascular concentrations
`of TXD258 greater than 13 mm. These data explain why uptake
`by the brain was nonlinear in the conventional pharmacoki-
`netic dose range and emphasize how a delivery rate above or
`below the plasma TXD258 concentration that saturates P-gp
`at BBB could be critical for controlling the amount of TXD258
`in the brain parenchyma and consequently available to treat
`brain tumors. This new taxoid shows that the P-gp at the BBB
`
`AVENTIS EXHIBIT 2052
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`
`

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`1368
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`S. Cisternino et al
`
`TXD258 transport by P-glycoprotein at the BBB
`
`can be saturated and that this saturation is responsible for the
`difference between the concentration of a drug in the brain and
`its systemic concentration.
`
`Methods
`
`Chemicals and reagents
`
`TXD258 was manufactured by Aventis (Vitry, France). [14C]-
`TXD258 was provided by NENt Life Science Products
`(Boston, MA, U.S.A.). Its molecular weight of 835.95 g mol1
`and its structure is shown in Figure 1.
`The tracer had a specific activity of 1.99 GBq mmol1, and a
`radiochemical purity of 97.7% determined by h.p.l.c. [3H]-
`sucrose (377.4 GBq mmol1) was obtained from NENt Life
`Science Products (Paris, France). Polysorbate 80 (PS80) and
`ethanol were purchased from Prolabo (Paris, France). Glucose
`5%, (7)-verapamil hydrochloride and dimethyl sulfoxide were
`purchased from Meram (Melun, France), Sigma (St. Quentin
`Fallavier, France) and Merck Eurolab (Strasbourg, France),
`respectively. Liquid scintillation cocktails were purchased from
`Packard (Rungis, France). All other chemicals were commer-
`cial products of analytical grade.
`
`Animals
`
`The studies were performed on female CD2F1/Crl BR mice
`(18–23 g, 6–8 weeks old) and female Crl:CDs-(SD)BR rats
`(180–220 g, 6–8 weeks old), provided by Charles River
`Laboratories
`(Elbeuf, France). Adult
`female CF-1 mice
`(mdr1a(+/+) and (/), 30–40 g, 6–8 weeks old) were bred
`in-house from progenitors genotyped for mdr1a P-gp that were
`initially obtained from Charles River Laboratories (Wilming-
`ton, MA, U.S.A.). Tap water and diet were provided ad
`libitum. The animals used in these studies were handled and
`maintained in accordance with the requirements of the E.E.C.
`Guideline (1986) and U.S. Federal Guidelines (1985). Com-
`pliance with the above legislation was ensured by adhering to
`the standards set forth in the Guide for the Care and Use of
`Laboratory Animals, DHHS Publication No. (NIH) 86–23,
`revised 1985.
`
`Pharmacokinetic studies
`
`Formulation, dose and administration The poor TXD258
`solubility prompted us to use a similar administration protocol
`used for preclinical taxoid drug docetaxel studies that involved
`PS80, ethanol and relative high amount of glucose 5% (Bissery
`[14C]-TXD258 was diluted in PS80/ethanol/
`et al., 1995).
`glucose 5% and was administered via the femoral vein using an
`
`Figure 1 Chemical structure of [14C]-TXD258.
`
`British Journal of Pharmacology vol 138 (7)
`
`infusion pump (Harvard PHD 2000, Harvard apparatus,
`Holliston, MA, U.S.A.), at 25 ml kg1 for female mice to give
`doses of 15, 30, 45 or 90 mg m2 and at 10 ml kg1 for female
`rats to give doses of 15 and 60 mg m2. The proportion of each
`vehicle PS80/ethanol/glucose 5% for TXD258 administration
`at 15 mg m2 was 0.3/1.0/98.7% and 0.4/1.4/98.2% for mice
`and rats, respectively. Each dose was perfused at a constant
`rate of 1 ml min1 and the duration of the perfusion was
`approximately 45 s for mice and 2.3 min for rats. TXD258 was
`also given to female mice at doses of 30 and 90 mg m2 using a
`perfusion rate of 8.3 ml min1 for approximately 1 h.
`
`Sampling Mice and rats were anesthetized with isoflurane
`and exsanguinated via the abdominal aorta at times optimized
`for the metabolites profile: 0.03, 0.25, 1, 6, 24, 72 and 168 h
`after dosing. Blood and brain samples were collected from four
`mice and two rats at each time point. Blood samples were
`collected into heparinized syringes. The plasma was separated
`by centrifugation at 3000 g for 15 min. The plasma samples
`obtained at each time point were pooled to obtain enough
`material for quantifying metabolites. Thus, the resulting data
`are averages. Brains were lyophilized and kept frozen at –801C
`until analyzed.
`
`Total radioactivity analysis
`The total radioactivity in
`plasma and brain samples was determined by liquid spectro-
`metry using a Beckman LS 6000 SC spectrometer equipped
`with an external standard system (number H). A quench curve
`was generated using [14C] quenched standards supplied by
`Beckman. Samples were counted for up to 10 min (0.5%
`precision). D.p.m. values of less than twice the background
`were considered to be insignificant. Plasma samples (0.5 – 1 ml)
`were added directly to the liquid scintillation cocktail Hionic-
`Fluor (Beckman). Duplicate samples of freeze-dried homoge-
`nized brain were processed in a sample oxidizer (Packard model
`307) and the 14CO2 formed was trapped in 9 ml Carbosorb
`(Packard). The carbosorb was mixed with 12 ml of Permafluor
`E+ (Packard) liquid scintillation cocktail for counting.
`
`Metabolite analysis The parent drug and metabolites in
`plasma and brain extracts were measured at 1 and 6 h after the
`end of infusion by h.p.l.c. with on-line radiochemical detector.
`Aliquots of plasma were subject to solid phase extraction with
`Oasis HLB cartridges (Waters) equilibrated with methanol and
`demineralized water. The eluates were concentrated and
`aliquots (50 ml) were analyzed by h.p.l.c. A lyophilized sample
`of brain was weighed and sonicated for 15 min with 20.5 ml
`ethyl acetate, water/formic acid (0.1%), (100/2.5, v v1). The
`homogenate was stirred and centrifuged at 13,000 r.p.m. for
`10 min. The resulting supernatant was evaporated to dryness,
`taken up in 0.5 ml of a mixture containing dimethyl sulfoxide,
`methanol/acetone (15%), (50/50, v v1), vortexed for 2 min and
`clarified by centrifugation at 15,000 r.p.m. for 10 min. The
`extraction procedure was repeated once, the supernatants were
`combined and a 50 ml aliquot analyzed by h.p.l.c. The Mercks
`h.p.l.c. analytical system consisted of a L6200A gradient
`elution system pump with autosampler model AS4000, a diode
`array detector (L7450) operating at 230 nm and a Berthold
`model LB507B on-line radioactivity detector equipped with a
`500 ml flow cell. Samples were separated on a Symmetry C8
`column (250 4.6 mm, 5 mm) connected to a Symmetry C8
`guard column (Waters). Elution was performed under gradient
`
`

`
`S. Cisternino et al
`
`TXD258 transport by P-glycoprotein at the BBB
`
`1369
`
`conditions using succession of six steps from 0 to 170 min with
`a mobile phase of water/trifluoroacetic acid 0.01% and
`acetonitrile, at a flow rate of 0.6 ml min1 and at room
`temperature. Peaks of radioactivity were quantified on the
`radioactivity detector by integrating the area under each peak.
`The calculated intra- and interday coefficients of variation
`were below 15%.
`
`Pharmacokinetic analysis The pharmacokinetic analysis
`was carried out using a noncompartmental model with
`WinNonlins software (Version 1.0, Scientific Consulting
`Inc., U.S.A.). The following brain and plasma parameters
`were determined, Cmax, Tmax, and AUC(0–t). The area under the
`radioactivity concentration decay curves between 0 and t (h)
`were computed from the experimental points by the trapezoi-
`dal method. The partition coefficient Kp was calculated as the
`ratio of brain AUC0 – 168 h over plasma AUC0 – 168 h.
`
`In situ brain perfusion studies
`
`Surgical procedure and perfusion technique The transport
`of [14C]-TXD258 into the brains of rats and mice was measured
`using the in situ brain perfusion method (Takasato et al., 1984;
`Smith, 1996; Dagenais et al., 2000). Animals were anesthetized
`by i.p. injection of a mixture of xylazine (Bayer, Puteaux,
`France) and ketamine (Panpharma, Fouge` res, France), at 8/
`140 mg kg1 for mice and 4/70 mg kg1 for rats.
`Briefly, the right common carotid artery was catheterized
`with heparin-filled polyethylene tubing (0.30 mm i.d. 
`0.70 mm o.d. for mice; 0.76 mm i.d.  1.22 mm o.d. for rats,
`Biotrol Diagnostic, Chennevie` res-les–Louvre, France). The
`common carotid artery was ligated on the heart side before
`inserting the catheter. In mice, the external carotid was ligated
`rostral to the occipital artery at the bifurcation of the common
`carotid artery. In rats, the external carotid and occipital
`arteries were ligated. Body temperature was maintained at 37 –
`381C during surgery using a rectal thermistor connected to a
`temperature monitor. The syringe containing the perfusion
`fluid was placed in an infusion pump (Harvard pump PHD
`2000, Harvard Apparatus) and connected to the catheter.
`Before perfusion, the thorax of the animal was opened, the
`heart was cut and perfusion immediately started with a flow
`rate of 2.5 ml min1 for mice and 10 ml min1 for rats. The
`perfusion fluid consisted of bicarbonate-buffered physiological
`saline (mm): 128 NaCl, 24 NaHCO3, 4.2 KCl, 2.4 NaH2PO4,
`1.5 CaCl2, 0.9 MgCl2 and 9 d-glucose. The solution was gassed
`with 95% O2 and 5% CO2 to obtain a pH of 7.4 and warmed
`to 371C in a water bath. Compounds were added to the
`perfusate at the appropriate concentration. Ethanol and PS80
`did not exceed 0.08 and 0.02% in the perfusate, respectively.
`Each animal was perfused with [14C]-TXD258 plus [3H]-
`sucrose (11.1 kBq ml1) to check the physical integrity of the
`BBB. Perfusion was terminated after 60 s by decapitating the
`animal. The brain was removed from the skull and dissected
`free on ice. The right cerebral hemisphere was placed in a tared
`vial and weighed. Aliquots of the perfusion fluid were also
`collected and weighed to determine tracer concentrations in
`the perfusate. Samples were digested in 2 ml of Solvable
`(Packard) at 501C and mixed with 9 ml of Ultima gold XR
`scintillation cocktail (Packard). Both labels were counted
`simultaneously in a Packard Tri-Carb model 1900 TR
`(Packard).
`
`Transport studies The transport of [14C]-TXD258 into the
`brain was first measured in mice perfused with 4.5, 10, 12.5, 15,
`20, 25 and 30 mg ml1 drug and in rats perfused with 2.5, 4.5,
`10, 12.5, 15, 25 and 30 mg ml 1 drug. These were the concen-
`trations measured in previous pharmacokinetic and toxicoki-
`netic studies. We then measured the influence of P-gp on the
`uptake of [14C]-TXD258 by the brain using a drug concentra-
`tion of 4.5 mg ml1 (B5.4 mm) and 150 mm (7)-verapamil, in the
`perfusion fluid. These studies were conducted on rats, wild-
`type mice and P-gp deficient mdr1a(/) mice.
`A ‘wash-out’ procedure was also used to study the trans-
`efflux zero and trans-inhibition of the compound (Stein, 1986).
`One syringe (syringe A) of a dual-syringe infusion pump
`(Harvard Apparatus) contained the bicarbonate-buffered
`(11.1 kBq ml1;
`physiological
`saline plus
`the
`radiotracer
`B5.4 mm) and the other (syringe B) contained saline, no
`tracer, but with or without (7)-verapamil (150 mm). The
`carotid catheter was connected to a four-way valve (Hamilton,
`Bonnaduz, Switzerland). After the carotid cannulation was
`completed and the appropriate connections were made, syringe
`A was discharged at 2.5 ml min1 (mice) or 10 ml min1 (rats)
`for 60 s. Syringe A was switched off and syringe B was
`switched on simultaneously to wash-out the capillary space for
`30 s. The animal was decapitated and the brain removed to
`measure its radioactivity.
`
`Distribution in brain microvascular and parenchymal
`compartments To distinguish molecules that are trapped
`in endothelial cells from those that reach the brain parenchyma
`by transcytosis, distribution of [14C]-TXD258 in the brain
`microvascular and parenchymal compartments was assessed in
`rats using the capillary depletion method of Triguero et al.
`(1990) as modified by Rousselle et al. (2000). We used the
`wash-out procedure described above with syringe B containing
`compound-free bicarbonate-buffered saline in order to remove
`nonspecific adsorption and free circulating labeled compounds
`from the vascular space (Triguero et al., 1990). At the end of
`the wash-out,
`the right cerebral hemisphere was rapidly
`removed, weighed and homogenized (15 pestel strokes) in
`3.5 ml buffer (10 mm HEPES, 141 mm NaCl, 4 mm KCl, 1 mm
`NaH2PO4, 2.8 mm CaCl2, 1 mm MgSO4 and 10 mm d-glucose,
`pH 7.4) on ice. Chilled 37% dextran solution (4 ml) was added
`to obtain a final dextran concentration of 18.5%. All
`homogenizations were performed rapidly at 41C. An aliquot
`of homogenate was removed and the remainder was centri-
`fuged at 5400 g for 15 min at 41C in a swinging-bucket rotor.
`The pellet and supernatant were carefully separated and
`counted in the liquid scintillation counter. The pellet was
`composed mainly of brain capillaries and the supernatant
`reflected the parenchyma.
`
`Calculation of BBB transport parameters All calculations
`were made as described by Smith (1996). Brain vascular
`volume (Vvasc; ml g1) was estimated from the tissue distribu-
`tion of [3H]-sucrose, which diffuses very slowly across the
`BBB, using the following equation:
`
`V ¼ X
`C
`where X* (d.p.m. g1) is the amount of sucrose measured in the
`right brain hemisphere and C*perf (d.p.m. ml1) is the concentra-
`tion of labeled sucrose in the perfusion fluid. Transport across
`
`perf
`
`ð1Þ
`
`British Journal of Pharmacology vol 138 (7)
`
`

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`
`TXD258 transport by P-glycoprotein at the BBB
`
`the BBB was expressed in terms of the apparent distribution
`volume (Vbrain) and the transport coefficient
`(Kin). The
`apparent distribution volume was calculated from the radio-
`activity in the right brain hemisphere using the following
`equation:
`
`ð2Þ
`
`Vbrain ¼ Xbrain
`Cperf
`where Xbrain (d.p.m. g1) is the calculated amount of [14C]-
`TXD258
`in the
`right
`cerebral hemisphere
`and Cperf
`(d.p.m. ml1) is the labeled TXD258 tracer concentration in
`the perfusion fluid. Brain tissue radioactivity was corrected for
`vascular contamination with the following equation:
`ð3Þ
`Xbrain ¼ Xtot VvascCperf
`where Xtot (d.p.m. g1) is the total quantity of tracer measured
`in the tissue sample (vascular+extravascular).
`Brain uptake, expressed as Kin (ml s1 g1), was calculated
`from
`
`Kin ¼ Vbrain
`T
`where T is the perfusion time (s).
`The perfusion time used in single-time uptake studies was
`long enough to ensure that at
`least 40% of
`the total
`radioactivity in the tissue was outside the vascular space
`(XbrainX0.4Xtot; Takasato et al., 1984).
`
`ð4Þ
`
`BBB transport modeling As the relation between Kin values
`and the TXD258 perfusate concentration was sigmoidal, an
`empirical Hill
`function was derived for assessing kinetic
`transport parameters. Kinetic analyses were carried out using
`nonlinear regression with the least-squares method of Systat
`5.01 (Systat Inc., IL, U.S.A.) to fit the equation:
`
`Kin ¼ Kin;min þ ðKin;max Kin;minÞCn
`50 þ Cn
`ICn
`where C is the TXD258 concentration in the perfusate, Kin,min
`is the minimal and Kin,max the maximal brain coefficient
`transport (Kin) value measured for [14C]-TXD258, n is the Hill
`coefficient, and IC50 is the concentration at which brain
`transport was half-maximally inhibited.
`
`ð5Þ
`
`Statistical analysis Data are presented as means 7s.d. for
`four to eight animals, unless specified otherwise. Student’s
`unpaired t-test was used to identify significant differences
`between groups when appropriate. All the tests were two-tailed
`and statistical significance was set at Po0.05. The error values
`associated with the kinetic parameters (IC50, Kin,min, Kin,max) are
`asymptotic standard errors returned by a nonlinear regression
`routine and are a measure of the certainty of the best-fit value.
`
`Results
`
`Pharmacokinetics in plasma and brain
`
`The plasma and brain total radioactivity kinetics in rats and
`mice following a single short intravenous administration of
`[14C]-TXD258 at 15 mg m2 are shown in Figure 2. The other
`doses tested gave similar plasma and brain dispositions (data
`not shown). The maximal plasma concentration of TXD258
`radioactivity was reached in the first sample, that is, at the end
`
`British Journal of Pharmacology vol 138 (7)
`
`Figure 2 Average changes in the plasma and brain concentrations
`of TXD258 over time. [14C]-TXD258 (15 mg m2) was infused into
`mice for 45 s and into rats for 2.3 min. Samples were taken from four
`mice and two rats at each time and pooled to obtain an average
`analytical determination.
`
`infusion, after both short and long infusions. The
`of
`concentration then rapidly decreased up to 6 h, and slowly
`thereafter up to 168 h, for all doses in both species. The
`TXD258-related radioactivity rapidly penetrated into the brain
`and the concentration was maximal in brain 2 min after the
`end of a short infusion at doses lower than 90 mg m2 in both
`mice and rats, and 15 min after the end of short infusion of
`90 mg m2 in mice. The maximal brain concentration in mice
`occurred 15 min after the end of the 1 h infusion of TXD258 at
`30 mg m2 and 60 min after an infusion of 90 mg m2 (Table 1).
`The brain radioactivity decreased slowly thereafter, but was
`still detectable for up to 168 h (Figure 2).
`Radio-h.p.l.c. analysis of plasma showed that unchanged
`drug was the major compound, accounting for at least 60% of
`the radioactivity at 1 h and 100% at 6 h after infusion in mice,
`and about 84% at 1 h and 76% at 6 h in rats, at all the
`perfusion times and doses studied. One of the five metabolites,
`docetaxel, accounted for 2–11% of the total plasma radio-
`activity 1 h after the end of both short or long infusions for all
`the doses studied in both species. Only TXD258 was detected
`in the brains of mice after infusion of the lowest dose.
`Unchanged drug represented about 90% of the total radio-
`activity in the brain at the end of the short infusion of TXD258
`at 45 and 90 mg m2, and about 72% of the brain radioactivity
`after a long infusion at 90 mg m2. Docetaxel was not detected
`in the brains of either rats or mice at any of the doses of
`TXD258 studied.
`
`Dose ranging effect on plasma and brain Cmax and AUC
`
`The values of Cmax and AUC0 – 168 h determined from the total
`plasma radioactivity increased proportionally with the dose in
`mice between 15 and 90 mg m2, but greater than proportion-
`ally with the dose in rats between 15 and 60 mg m2 (Table 1
`and Figure 3). The brain AUC0 – 168 h increased eight-fold
`greater than proportionally with the dose in mice and 2.5-fold
`greater in rats, at the highest dose investigated compared to the
`lowest dose. However, higher doses were not investigated in
`rats because of
`the toxicity of TXD258, which possibly
`prevented a clear demonstration of the nonlinear brain uptake
`of TXD258. The brain-to-plasma AUC0–168 h
`ratios also
`
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`S. Cisternino et al
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`TXD258 transport by P-glycoprotein at the BBB
`
`1371
`
`infusion. Doses of 90 mg m2 infused for 45 s or 1 h resulted in
`dramatically higher Kp values of 2.3 and 1.7, respectively
`(Table 1). When Kp values are compared between 30 and
`90 mg m2, the values are increased 6.1- and 2.6-fold for 45 s
`and 1 h infusion, respectively. This increase in Kp could be
`attributed to the saturation of an active efflux transport at the
`BBB. The lower Kp values obtained following the 1 h infusions
`could, in part, be explained by a lower maximal peak plasma
`after the long infusion; it was 7 mg ml1 after the long infusion
`and 32 mg ml1 after the short infusion of 90 mg m2.
`
`Effect of the concentration of TXD258 on transport into
`the brain of wild-type mice and rats
`
`The effect of the TXD258 concentration on drug transport
`into the brain was investigated by in situ brain perfusion. More
`[14C]-TXD258 was taken up by the brain when the perfusate
`concentrations were over 10 mg ml1 (B11 mm) of TXD258 in
`both rats and mice, suggesting the saturation of an active
`efflux process at the BBB (Figure 4). The variation in the
`blood – brain transport coefficient Kin with the TXD258
`concentration fitted a sigmoid equation with a Hill coefficient
`of 23 and TXD258 IC50 values of 12.970.4 mm in rats and
`13.070.4 mm in mice. The Kin values at lower concentrations
`(Kin,min) were 0.6370.01 and 0.8370.01 ml s1 g1 and Kin
`values at higher concentrations (Kin,max) were 1.9770.01 and
`2.7670.01 ml s1 g1 in mice and rats, respectively (Figure 4).
`The differences between the Kin in mice and rats were
`statistically significant (Po0.001), whereas the IC50 values in
`the two species were not different. The Kin ratios between the
`high and low concentrations were about 3. The amount of
`drug in the brain after infusions of concentrations from 2.5 to
`30 mg ml1 showed that the BBB was not damaged by TXD258
`as the [3H]-sucrose vascular volumes remained within the
`normal range (data not shown).
`
`Involvement of P-glycoprotein in the transport of [14C]-
`TXD258 across the BBB
`
`We evaluated the importance of P-gp for the transport of
`TXD258 across the BBB using two approaches. First, brain
`trans-influx zero experiments were performed in rats and mice
`with 4.5 mg ml1 [14C]-TXD258, as this concentration does not
`saturate P-gp. The Kin in mdr1a(/) mice was about three-
`fold higher than in the wild-type mice (Figure 5). Perfusion of
`rats and wild-type mice with [14C]-TXD258 (4.5 mg ml1) plus
`the P-gp modulator (7)-verapamil (150 mm) increased the
`
`Figure 3 (a) Relations between the average areas under the curves
`(AUC0 – 168 h) or maximal concentration (Cmax; inset) in the brains
`and plasma of mice infused intravenously with [14C]-TXD258 at 15,
`30, 45 and 90 mg m2 for 45 s. Values in parentheses represent the
`increases in the AUC obtained at 30, 45 or 90 mg m2 compared to
`the value measured at 15 mg m2.
`(b) Rat brain and plasma
`AUC0 – 168 h or Cmax (inset) values. The rats were given an intra-
`venous infusion of [14C]-TXD258 at 15 and 60 mg m2 lasting 2.3 min.
`
`increased more than proportionally as the doses increased
`(Table 1).
`
`Effect of doses and infusion times on [14C]-TXD258 in
`the brain and plasma of mice
`
`These plasma concentrations produced AUC brain-to-plasma
`ratios of B1.1 for the short infusion and 0.7 for the long
`
`Table 1 Brain and plasma pharmacokinetic parameters obtained after the intravenous infusion of mice and rats with [14C]-TXD258
`
`Infusion time
`
`Dose (mg m2)
`Cmax plasma (mg ml1)
`Cmax brain (mg ml1)
`Tmax brain (min)
`AUC0–168 h plasma (mg h ml1)
`AUC0–168 h brain (mg h ml1)
`Kp
`
`15
`6.4
`0.31
`2
`9.7
`6.8
`0.70
`
`Mice
`
`45 s
`
`1 h
`
`Rats
`2.3 min
`
`30
`8.4
`0.39
`2
`15.4
`16.5
`1.07
`
`45
`13.6
`0.92
`2
`24.7
`57
`2.31
`
`90
`32.2
`6.1
`15
`52.1
`339
`6.51
`
`30
`6
`0.39
`15
`16
`10.6
`0.66
`
`90
`6.98
`0.45
`60
`26.9
`45.4
`1.69
`
`15
`0.55
`0.09
`2
`1.6
`3.9
`2.44
`
`60
`4.31
`0.49
`2
`11
`39.1
`3.55
`
`The parameters (Cmax, Tmax, AUC0–168 h) were calculated by a noncompartmental model using WinNonlin software. The partition
`coefficient (Kp) was calculated as the ratio of the brain AUC0–168 h to the plasma AUC0–168 h.
`
`British Journal of Pharmacology vol 138 (7)
`
`

`
`1372
`
`S. Cisternino et al
`
`TXD258 transport by P-glycoprotein at the BBB
`
`[14C]-
`Figure 4 Concentration-dependent brain transport of
`TXD258 (expressed as a brain transport coefficient Kin) measured
`by in situ brain perfusion in rats and mice. Animals were perfused
`with TXD258 via the common carotid artery for 60 s. Dotted (rats)
`and solid (mice) curves represent data fitted with the Hill equation
`and an apparent TXD258 IC50 value of B13 mm. Data are presented
`as means7s.d. of n¼ 4 – 8 animals per point.
`
`(Kin; ml s1 g1) was
`Figure 5 The brain transport coefficient
`measured by in situ brain perfusion in P-gp-proficient mice (a) and
`rats (b) with or without the chemical P-gp modulator (7)-verapamil
`(150 mm) and in P-gp-deficient mdr1a(/) mice. Each group of
`animals was perfused with [14C]-TXD258 at a noninhibiting
`concentration (5.4 mm). P-gp chemical modulation or disruption
`produced an approximately three-fold increase in the brain TXD258
`transport in mice and a 4.7-fold increase in rats. Data are presented
`as means 7s.d. of n¼ 5 – 8 animals per group. ***Po0.001
`compared to the control group.
`
`brain uptake 2.9-fold in mice and 4.7-fold in rats over the
`values for control animals perfused with TXD258 alone (no
`[14C]-TXD258 Kin at a
`verapamil)
`(Figure 5). Moreover,
`saturating dose (30 mg ml1) in mdr1a(/) mice was not
`
`British Journal of Pharmacology vol 138 (7)
`
`Figure 6 The apparent distribution volume (Vd; ml g1) of [14C]-
`TXD258 was measured in controls after [14C]-TXD258 had been
`accumulated for 60 s. The animals in the two other groups,
`underwent
`a
`30 s
`tracer-free
`‘wash-out’ with or without
`(7)-verapamil (150 mm) after the 60 s of brain accumulation of
`[14C]-TXD258. Both mice and rats were perfused with [14C]-TXD258
`at 5.4 mm. Data are presented as means 7s.d. of n¼ 5 – 6 animals
`per group. *Po0.05, ***Po0.001, compared to their respective
`controls.
`
`different as compared to the [14C]-TXD258 Kin observed with a
`nonsaturating concentration (4.5 mg ml1) in mdr1a(/) mice
`(data not shown). The trans-efflux zero process was also
`investigated in wild-type mice and rats after 60 s of accumula-
`tion of [14C]-TXD258 followed by a 30 s wash-out with a drug-
`free and tracer-free bicarbonate-buffered saline. The brain
`distribution volume was 1.2-fold lower in mice and 2.5-fold
`lower in rats (Figure 6) than in control animals not given the
`wash-out. Finally, a trans-inhibition study was conducted on
`(150 mm)
`mice and rats by adding (7)-verapamil
`to the
`bicarbonate-buffered saline during the 30 s wash-out. The
`brain distribution volumes measured after this treatment were
`not different from the control values observed in rat and
`mouse not given the wash-out
`(Figure 6). We therefore
`conclude that P-gp at
`the BBB controls the uptake of
`TXD258 across the BBB.
`
`Distribution in the brain microvascular and parenchymal
`compartments of rats
`
`These brain perfusion experiments were performed to deter-
`mine the percentage of TXD258 in the brain parenchyma. The
`distributions of [14C]-TXD258 in brain capillaries and par-
`enchyma were measured in rats subjected to perfusion and a
`30 s wash-out using the capillary depletion method of Triguero
`et al. (1990). This procedure distinguishes between the fraction
`of TXD258 remaining in the endothelial cells and the drug that
`has crossed the abluminal endothelial membrane to enter the
`
`

`
`S. Cisternino et al
`
`TXD258 transport by P-glycoprotein at the BBB
`
`1373
`
`brain parenchyma. The washing procedure removed radio-
`active compounds from the vessel lumen space. About 6% of
`the [14C]-TXD258 was associated with the brain capillaries,
`and about 94% of [14C]-TXD258 was in the brain parenchyma.
`
`Discussion
`
`The taxanes are a new family of antineoplastic drugs. The
`family includes the natural compound paclitaxel (Taxols) and
`the more recently prepared hemisynthetic docetaxel (Taxo-
`teres), which are remarkably potent against various cancers
`(for review, see Lin & Ojima, 2000). Despite their clinical
`success and the fact that docetaxel
`is more effective than
`paclitaxel in inhibiting the growth of tumoral cells (Clarke &
`Rivory, 1999; Ferlini et al., 2000), some tumors such as brain
`malignancies respond poorly to these drugs. Moreover, despite
`its lipophilicity at a log P(octanol/water) of 3.5, paclitaxel does
`not readily cross the intact BBB of either rodents (Eiseman
`et al., 1994; Klecker et al., 1994) or humans (Heimans et al.,
`1994). The physiologically abundant P-gp at the luminal
`surface of the brain endothelial cells that form the BBB might
`be the main reason why paclitaxel and docetaxel are poorly
`diffusing into the brain. P-gp is involved in the active efflux of
`a broad spectrum of substrates from the brain to the blood
`vessel lumen (Ambudkar et al., 1999; for review, see Tsuji &
`Tamai, 1997; Schinkel, 1999).
`TXD258 is a new semisynthetic compound that has
`interesting pharmacological properties. It is active against
`many tumors in vivo,
`including human glioblastoma cells
`implanted in the brain of mice (Dykes et al., 2000; Vrignaud
`et al., 2000) and interacts less strongly with P-gp than the older
`taxanes. We therefore determined the brain and plasma
`pharmacokinetics of TXD258 in rodents. TXD258 was
`prepared in the same excipients as the commercially available
`form of Taxoteres, which is
`free of cremophor EL.
`Pharmacokinetic studies in rodents indicate that TXD258
`enters the