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`http://jnci.oxfordjournals.org/
`
` at New York University on February 2, 2015
`
`In Cancer Epidemiology and Prevention
`(Schottenfeld D, Fraumeni JF Jr, eds).
`Philadelphia: Saunders, 1982, pp 855-870
`(23) Sievert RM: A circulating physical depart-
`ment for standardising the roentgen radia-
`tion used in therapy. Acta Radiol 5:457-
`467, 1926
`the system of
`(24) Thoraeus R: Features of
`periodical inspection adopted for roentgen
`therapy installations in Sweden, and some
`experiences of the inspection work. Acta
`Radiol 33:253-280, 1950
`(25) Mattsson B, Rutqvist L-E: Some aspects on
`validity of breast cancer, pancreatic cancer
`and
`lung cancer registration
`in Swedish
`official statistics. Radiother Oncol 4:63—70,
`1985
`the Biological Effects of
`(26) Committee on
`Ionizing Radiations Health Effects of Ex-
`posure to Low Levels of Ionizing Radiation.
`BEIR V. Washington, DC- Natl Acad Press,
`1990
`(27) Boice JD Jr, Harvey EB, Blettner M, et al:
`Cancer
`in
`the contralateral breast after
`radiotherapy for breast cancer. N Engl J
`Med 326:781-785, 1992
`(28) Storm HH, Andersson M, Boice JD Jr, et
`al: Adjuvant
`radiotherapy and
`risk of
`contralateral breast cancer. J Natl Cancer
`Inst 84 1245-1250, 1992
`(29) Hrubec Z, Boice JD Jr, Monson RR, et al
`Breast cancer after multiple chest
`fluo-
`roscopies: second follow-up of Massachu-
`setts women with tuberculosis. Cancer Res
`49:229-234, 1989
`
`Notes
`Affiliations of authors- A. Mattsson, L. E.
`Rutqvist (Oncologic Center), B -I Rud£n (De-
`partment of Hospital Physics), P. Hall, N.
`Wilking
`(Department of General Oncology),
`Radiumhemmet, Karolinska Hospital, Stockholm,
`Sweden.
`to: Anders Mattsson, B.A.,
`Correspondence
`Oncologic Center, Karolinska Hospital, S-104 01
`Stockholm, Sweden.
`Supported by the National Institute of Radia-
`tion Protection (project No. P548/89) and the
`Cancer Society in Stockholm.
`We thank Mrs. Ulla Cassel for expert assist-
`ance in data collection, coding, and entering data
`into the computer; Mr. Lars Jaegerfalk for help
`with the programming; Associate Professor Lars-
`Erik Holm, Professor Timo Hakulinen, and Mr.
`Hemming Johansson for fruitful discussions and
`valuable comments on
`the manuscript; Mrs.
`Elisabeth Bjurstedt for skilful assistance, and all
`the helpful people at the different registries
`Manuscript received February 17, 1993, re-
`vised May 25, 1993; accepted July 19, 1993.
`
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`
`Measurement of Cremophor
`EL Following Taxol:
`Plasma Levels Sufficient to
`Reverse Drug Exclusion
`Mediated by the Multidrug-
`Resistant Phenotype
`
`Lorraine Webster, Martha
`Linsenmeyer, Michael Millward,
`Carmel Morton, James Bishop,
`David Woodcock*
`
`Background: Paclitaxel (Taxol) is
`the first of a new class of cytotoxic
`agents with activity against tumors
`resistant to other drugs. For clinical
`use, paclitaxel is currently formu-
`lated in a vehicle of 50% ethanol and
`50% polyethoxylated surfactant Cre-
`mophor EL (Cremophor). We have
`previously shown
`that Cremophor
`will block the P-glycoprotein drug
`efflux pump
`responsible
`for
`the
`multidrug-resistant phenotype. Over-
`expression of P-glycoprotein is one
`mechanism of in vitro resistance to a
`number of currently used cytotoxic
`agents including paclitaxel. Purpose:
`Our aim was to develop a bioassay to
`measure plasma levels of Cremophor
`and
`to determine whether or not
`plasma levels of Cremophor achieved
`during paclitaxel therapy are suffi-
`cient to inhibit the activity of the
`P-glycoprotein. Methods: All patients
`studied had histologically proven,
`advanced ovarian carcinoma with
`measurable or evaluable disease and
`had
`received at
`least one prior
`platinum-containing
`regimen. The
`bioassay used
`flow cytometry
`to
`measure the increase in equilibrium
`intracellular daunorubicin
`levels in
`multidrug-resistant human T-cell leu-
`in
`the
`kemia cells (CEM/VLB100)
`presence of a series of concentrations
`of Cremophor. Levels of Cremophor
`were measured in plasma from 21
`patients after a 3-hour infusion of
`135 or 175 mg/m2 paclitaxel. Both
`dose
`levels were given
`following
`premedication with oral dexa-
`methasone, intravenous promethazine
`
`intravenous
`hydrochloride, and
`cimetidine. The Cremophor bioassay
`involved incubation of CEM/VLB100
`cells (5 X 105) for 1 hour with 2
`Hg/mL daunorubicin in 0.5 mL HL-1
`medium plus 0.5 mL plasma prior to
`flow cytometric analysis. Pretreat-
`ment plasma was used to derive a
`standard curve for
`the effect of
`Cremophor on equilibrium dauno-
`rubicin
`levels. All measurements
`were done in triplicate. Results: In
`vitro experiments indicated that, for
`maximal inhibition of P-glycoprotein
`activity, concentrations of Cre-
`mophor of 0.1% (vol/vol) were re-
`quired. At
`the end of a 3-hour
`infusion of paclitaxel, plasma levels
`of Cremophor in 19 of 21 patients
`were 0.1% or higher and 0.09% in
`the remaining two. Concentrations of
`5-20
`\iM paclitaxel dissolved
`in
`ethanol without Cremophor did not
`inhibit P-glycoprotein in this assay.
`Conclusion: The concentrations of
`Cremophor measured
`in plasma
`drawn from patients after a 3-hour
`infusion of paclitaxel at 135 or 175
`mg/m2 were found to be sufficient to
`inhibit P-glycoprotein activity
`in
`vitro. Implications: The efficacy of
`paclitaxel against some tumors may
`be aided by its administration in a
`vehicle solution containing Cre-
`mophor
`in quantities
`that
`reach
`concentrations in the plasma suffi-
`cient to reverse multidrug resistance
`of neoplastic cells. [J Natl Cancer
`Inst 85:1685-1690, 1993]
`
`Multidrug resistance is a mechanism
`of cellular resistance to chemotherapy
`in which tumor cells express elevated
`levels of a membrane transport protein,
`the P-glycoprotein, that actively pumps
`out of the cell a broad spectrum of
`structurally unrelated drugs (7). Bell et
`al.
`(2) first reported high levels of
`P-glycoprotein in tumor samples from
`two of five patients with advanced
`drug-resistant ovarian cancer. Subse-
`quently, the potential
`importance of
`multidrug resistance in clinical resist-
`
`*See "Notes" section following "References."
`
`Journal of the National Cancer Institute, Vol. 85, No. 20, October 20, 1993
`
`REPORTS 1685
`
`NEPTUNE GENERICS EX. 1033 00001
`
`

`

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`
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`
` at New York University on February 2, 2015
`
`kemia) (19) were maintained in the a-modifi-
`cation of Eagle's minimum essential medium
`with 10% newborn calf serum (Cytosystems,
`Castle Hill, Australia) and 100 ng/mL vinblastine
`(David Bull Laboratories, Melbourne). These
`cells had been designated as "R100 cells" in
`previous publications (4,12,14). Serum-free, phe-
`nol red-free HL-1 medium was obtained from
`Ventrex Laboratories, Portland, Maine. Cre-
`mophor EL was from BASF Fine Chemicals,
`Melbourne.
`The method for determining the activity of the
`P-glycoprotein drug efflux pump has been pre-
`viously described (12.14). Briefly, 5 X 103 cells
`in logarithmic growth were incubated at 37 °C in
`1 mL medium with 2
`jig/mL daunorubicin
`(David Bull Laboratories) for 1 hour, during
`which
`time
`intracellular drug
`levels
`reached
`equilibrium. The effect of Cremophor on the
`activity of the P-glycoprotein drug efflux pump
`was rapid, since preincubation with Cremophor
`had no additional
`effect
`on
`equilibrium
`daunorubicin levels (12). Cells were then ana-
`lyzed for intracellular daunorubicin fluorescence
`by flow cytometry using a FACStar Plus cell
`sorter
`(Becton Dickinson, Mountain View,
`Calif.). All measurements were done in triplicate.
`The units of fluorescence were arbitrary, and
`absolute values from different days cannot be
`directly compared. Studies were done to deter-
`mine the effect of serum on the activity of
`Cremophor, to ascertain whether plasma could be
`substituted for serum, and to assess the effect of
`freezing
`on plasma
`samples
`containing
`Cremophor.
`To measure Cremophor in plasma from pa-
`tients, we incubated CEM/VLB100 cells (5 X
`105) for 1 hour with 2 ng/mL daunorubicin in
`0.5 mL HL-1 medium plus 0.5 mL plasma. All
`measurements were done in triplicate. Pretreat-
`menl plasma was used to derive a standard curve
`for
`the effect of Cremophor on equilibrium
`daunorubicin
`levels. The components
`in each
`assay were added in the following order: plasma
`(Cremophor, diluted
`in HL-1 medium for the
`standard curves only), CEMfVLBl0O
`cells
`in
`HL-1 medium, and daunorubicin. The increase in
`equilibrium
`intracellular daunorubicin levels in
`assays containing post-treatment plasma gave a
`measure of the Cremophor concentration.
`
`Results
`Assay Development
`
`interacts with
`Because Cremophor
`components of human plasma (20), we
`investigated
`the effect of
`increasing
`amounts of serum and plasma on the
`Cremophor-induced increase in equili-
`brium
`intracellular
`levels of dauno-
`rubicin, a drug excluded by multidrug-
`resistant cells, such as
`the CEM/
`VLB100 cells used in this assay. When
`the artificial serum-free HL-1 medium
`(<30 (ig/mL of total protein) was used,
`the maximum increase in intracellular
`
`ance to chemotherapy has been demon-
`strated in other tumors, including hem-
`atologic malignancies
`{3,4), breast
`cancer (5), childhood sarcomas (6), and
`lung cancer (7,5).
`Paclitaxel (Taxol) is a microtubule-
`stabilizing drug that appears to be the
`most active single agent identified to
`date against a number of
`tumors
`[reviewed in (9)]. In phase II studies
`(9), response rates of 30% have been
`reported in previously treated ovarian
`cancer and up to 60% in advanced
`breast cancer. In addition, the activity
`of paclitaxel against untreated non-
`small-cell lung cancer is greater than
`that of other agents (9). Although the
`antitumor activity of Taxus brevifolia
`extracts was first identified in 1963 and
`the structure of the active component
`was reported in 1971 {JO), the develop-
`ment of this agent has been delayed in
`part because of
`its
`lack of clearly
`superior activity compared to the ac-
`tivity of other agents and because of its
`low aqueous
`solubility
`(9). In
`the
`current clinical formulation, paclitaxel
`is dissolved
`in a mixture of 50%
`ethanol and 50% Cremophor EL (Cre-
`mophor), such that most patients re-
`ceive more
`than 20 mL Cremophor
`with each dose of paclitaxel. Cre-
`mophor is a polyethoxylated castor oil
`that is used as a solubilizing agent for
`several other drugs
`including
`ten-
`iposide, miconazole, cyclosporine, and
`some vitamin preparations. Cremophor
`has been implicated as a cause of the
`hypersensitivity reactions seen in early
`clinical trials of paclitaxel {11). How-
`ever, prolongation of the infusion time
`and administration of prophylactic anti-
`allergy medications have reduced the
`occurrence of this side effect (77).
`Like a number of other polyethoxy-
`lated surfactants {12-14), Cremophor
`inhibits the drug efflux activity of the
`P-glycoprotein pump in vitro {14-16)
`and
`increases
`the
`sensitivity of a
`multidrug-resistant murine
`transplant-
`able tumor to doxorubicin in vivo (72).
`Paclitaxel
`is also one of the diverse
`group of drugs
`to which multidrug-
`resistant
`cells
`are
`cross-resistant
`{17,18). Therefore, paclitaxel
`formu-
`lated in a Cremophor solution consists
`of both
`the cytotoxic agent and a
`compound potentially capable of over-
`coming resistance to paclitaxel.
`
`inertness
`the chemical
`Because of
`and
`lack of spectral properties of
`Cremophor, we have developed a bio-
`assay that has enabled us to measure
`the concentration of Cremophor
`in
`plasma and
`to determine
`the
`levels
`required for reversal of multidrug re-
`sistance in vitro. The assay is based on
`the ability of Cremophor to act as a
`multidrug
`resistance-reversing
`agent
`and, thus, to prevent the exclusion of a
`fluorescent drug that is pumped out of
`cells by the P-glycoprotein. The assay
`was then used to measure Cremophor
`in plasma from patients with advanced
`ovarian cancer who received a 3-hour
`infusion of paclitaxel.
`
`Patients and Methods
`Study Design
`
`receive
`to
`Twenty-one patients eligible
`paclitaxel for ovarian cancer were studied (see
`Table 1). Patients were required to have histo-
`logically confirmed advanced ovarian carcinoma
`with measurable or evaluable disease and to have
`received at least one prior platinum-containing
`regimen. Other prior chemotherapy up
`to a
`maximum of three regimens and prior radio-
`therapy were permitted. Other eligibility criteria
`were age of 75 years or less, a performance
`status of 0-2 according to the Eastern Coopera-
`tive Oncology Group criteria, and adequate
`baseline values of hematologic (absolute neu-
`trophil count 5«2.0 X 109/L and platelet count
`*100 X 109/L), renal (serum creatinine level
`*1.5 X upper limit of normal), and hepatic
`(serum bilirubin
`level «1.25X upper limit of
`normal) function.
`Patients who had one or two previous chemo-
`therapy regimens received 175 mg/m2 paclitaxel
`(Taxol: Bnstol-Myers Squibb, Melbourne, Aus-
`tralia), and patients with three prior regimens
`received 135 mg/m2. Both dose
`levels were
`given as a 3-hour infusion following premedica-
`tion with oral dexamethasone, intravenous prom-
`ethazine hydrochloride,
`and
`intravenous
`cimetidine. Paclitaxel was supplied as a 6-mg/mL
`solution in 50% Cremophor and 50% ethanol,
`and the appropriate dose was diluted in a 1000-
`ml_ 5% dextrose solution for infusion.
`On the first cycle of paclitaxel, blood samples
`(10-20 mL) were drawn into tubes containing
`lithium heparin prior to commencement of the
`infusion and upon completion of the
`infusion.
`Plasma was separated and frozen at -70 °C until
`analysis. Written informed consent was obtained
`for administration of paclitaxel and taking of
`blood samples. The protocol was approved by
`the institutional ethics committee.
`
`Cremophor Bioassay
`
`(de-
`Multidrug-resistant CEM/VLB]0O cells
`rived from the human CCRF-CEM T-cell leu-
`
`1686 REPORTS
`
`Journal of the National Cancer Institute, Vol. 85, No. 20, October 20, 1993
`
`NEPTUNE GENERICS EX. 1033 00002
`
`

`

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`
`http://jnci.oxfordjournals.org/
`
` at New York University on February 2, 2015
`
`Table 1. Cremophor plasma levels in patients with advanced ovanan cancer immediately following a 3-hour infusion of paclitaxel
`
`Patient
`No.
`
`Age, y
`
`Previous
`
`therapy*
`
`Paclitaxel
`dose, mgt
`
`Cremophor
`dose, ml4
`
`Plasma
`Cremophor
`(% v/v)
`
`23.2
`20.3
`24.2
`22.5
`16.8
`27.0
`26.1
`23.3
`26.7
`23.1
`25.4
`25.3
`25.2
`23.8
`25.8
`25.5
`19.3
`18.0
`21.3
`23.1
`22.1
`
`0.20
`0.14
`0.14
`0.15
`0.17
`0.17
`0.14
`0.14
`0.11
`0.14
`0.15
`0.09
`0.15
`0.12
`0.09
`0.15
`0.11
`0.13
`0.11
`0.17
`0.18
`
`39
`48
`58
`60
`59
`54
`45
`55
`54
`30
`65
`61
`60
`52
`57
`57
`67
`43
`54
`55
`46
`
`Cisplat + Cyclo
`Cisplat + cyclo; Carbo + Cyclo, Carbo
`Cisplat + Epi
`Carbo
`Chloram: Carbo; Carbo + ADR
`Carbo + Cyclo; Cisplat + VP16
`Carbo/cyclo; Cisplat
`Melphalan; Cisplat + VP16 + Bleo
`Cisplat + Cyclo
`Carbo + Cyclo: HMM; Cisplat
`Carbo + Cyclo: Chloram
`Carbo + Cyclo; Carbo
`Cisplat + Cyclo; Carbo
`Carbo + Cyclo; Epi + Ifos
`Carbo + Cyclo
`Cisplat + Cyclo
`Cisplat + Cyclo; Mitox + MMC; Cisplat + Cyclo
`Carbo + Cyclo; Chloram
`Cisplat + Cyclo; Carbo
`Cisplat + Cyclo
`Cisplat + Cyclo, Cisplat + VP16
`
`278
`243
`290
`270
`202
`324
`313
`280
`320
`111
`305
`303
`302
`285
`310
`306
`232
`216
`256
`111
`265
`
`123456789
`
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`
`•ADR = doxorubicin; Bleo = bleomycin; Carbo = carboplatin; Chloram = chlorambucil, Cisplat = cisplatin; Cyclo = cyclophosphamide; Epi =
`epirubicin, HMM = altretamine, Ifos = lfosfamide; Mitox = mitoxantrone; MMC = mitomycin; and VP16 = etoposide.
`tThe paclitaxel dose was 175 mg/m2 except in four patients (2, 5, 17, and 18), who received 135 mg/m2.
`|The Cremophor dose was calculated from the paclitaxel dose administered.
`
`nuclear with or without Cremophor, the
`only difference being that intracellular
`fluorescence increased markedly in the
`multidrug-resistant cell type when Cre-
`mophor was present (data not shown).
`To determine whether paclitaxel it-
`self could alter daunorubicin fluores-
`cence
`in
`this assay, we dissolved
`
`paclitaxel (pure substance) in ethanol at
`0.5 mM and then diluted it in HL-1
`medium. Paclitaxel at 5, 10, or 20
`u-M—concentrations
`that are
`in
`the
`range of reported peak levels following
`6-hour infusions (22)—did not increase
`intracellular daunorubicin
`levels.
`In
`addition, plasma taken from three pa-
`
`1. Multidrug-
`Fig.
`resistant CEM/VLBIOO
`cells were
`incubated
`with
`d a u n o r u b i c in
`(DNR) plus a series of
`concentrations of Cre-
`mophor either
`in HL-1
`serum-free medium
`(o)
`or in HL-1 medium sup-
`plemented with 25%
`(•), 50% (•), or 75%
`( •)
`newborn
`calf
`serum. All samples were
`run in triplicate, and the
`vertical bars indicate the
`standard deviation of the
`mean of each point. If
`no bars are apparent, the
`standard deviation was
`less than the size of the
`point.
`
`1
`
`1
`y
`
`\
`
`1 1
`
`h
`
`i
`
`i
`
`i
`
`-
`
`1.0
`
`/
`
`/1\
`
`i
`
`i
`
`0.0001 0.001
`
`0.
`
`i 0
`
`CREMOPHOR
`
`EL
`
`0.1
`
`01
`[%
`v/v]
`
`-
`
`-
`
`-
`
`600
`
`500
`
`400
`
`300
`
`200
`
`100
`
`0
`
`u ESCE^
`
`o E
`
`n
`
`Q
`
`daunorubicin occurred at a Cremophor
`concentration of 0.1%, although 0.01%
`was almost equally effective (Fig. 1).
`When the HL-1 medium was supple-
`mented with 25% newborn calf serum,
`0.1% Cremophor was still required for
`the maximum effect, but 0.01% had
`little effect. Increasing the proportion
`of serum
`to 50% or 75% produced
`curves similar to those found
`in the
`presence of 25% serum. It was not
`possible to use 100% serum because of
`problems with viscosity. Although the
`maximum
`intracellular daunorubicin
`fluorescence for cells in HL-1 medium
`was higher in the absence of serum, the
`absolute percentage increase above the
`background fluorescence was similar
`under both conditions. Human serum
`and plasma gave essentially
`identical
`results (data not shown). For conven-
`ience, plasma was used in all further
`studies.
`The effect of Cremophor on intra-
`cellular daunorubicin
`levels was not
`related to altered distribution of
`the
`drug, as reported to be the case for
`multidrug-resistant cells
`treated with
`cyclosporine (21). The localization of
`daunorubicin in the CEM/VLB100 cells
`(and
`in
`its drug-sensitive parent,
`CCRF-CEM cells) remained principally
`
`Journal of the National Cancer Institute, Vol. 85, No. 20, October 20, 1993
`
`REPORTS 1687
`
`NEPTUNE GENERICS EX. 1033 00003
`
`

`

`intra-
`following
`immediately
`tients
`venous doxorubicin did not
`increase
`equilibrium daunorubicin levels above
`the background.
`For use as quality controls in subse-
`quent assays and for the investigation
`of
`the effects of
`freezing plasma
`containing Cremophor, normal plasma
`was spiked with three concentrations of
`Cremophor (0.02%, 0.06%, and 0.2%
`in
`the plasma, yielding
`final assay
`concentrations of 0.01%, 0.03%, and
`0.1%, respectively). The plasma was
`then frozen
`in aliquots. When com-
`pared
`to Cremophor standard curves
`prepared
`freshly using
`the
`same
`plasma, the plasma frozen with Cre-
`mophor gave daunorubicin fluorescence
`values that differed by - 8% to +16%
`for
`the 0.1% Cremophor
`samples.
`When plasma from six volunteers was
`tested
`against
`this
`standard curve,
`differences up to 30% were observed at
`the 0.1% Cremophor level. To control
`for this interindividual variability, it is
`necessary to construct standard curves
`using each individual's plasma.
`
`Cremophor Concentrations in
`Plasma From Paclitaxel-Treated
`Patients
`
`Four patients received 135 mg/m2
`paclitaxel over a 3-hour period, and 17
`patients received 175 mg/m2 paclitaxel
`over a 3-hour period. No hypersen-
`sitivity reactions were observed.
`Using the described assay, all pre-
`treatment plasma
`samples
`(without
`added Cremophor) gave consistently
`low
`intracellular daunorubicin
`levels
`equivalent
`to
`those observed with
`plasma from healthy volunteers. Pre-
`treatment plasma was used to construct
`a standard curve
`for each of
`the
`patients. Data from the first three such
`assays are presented in Fig. 2. Com-
`parison with Fig. 1 indicates that the
`concentration-dependent
`increases
`in
`intracellular daunorubicin were very
`similar for both 50% newborn calf
`serum and 50% human plasma. The
`Cremophor concentrations
`in plasma
`taken from these patients at the conclu-
`sion of the paclitaxel infusion could be
`read directly
`from
`their
`individual
`standard curves (Fig. 2). The actual
`Cremophor
`levels
`in
`the patients'
`plasma were twice the values read from
`
`the value read from the standard curve. All points are means and standard deviations of triplicate
`measurements.
`
`the standard curve, since the incubation
`mixtures always contain 0.5 mL plasma
`and 0.5 mL HL-1 medium. In these
`patients, post-treatment plasma samples
`diluted one
`fifth with pretreatment
`plasma were also included in the assay.
`The
`resulting values
`read off
`the
`standard curve were consistent with a
`one-fifth dilution of Cremophor (data
`not shown). Estimates of plasma Cre-
`mophor concentration for blood sam-
`ples
`taken at the conclusion of
`the
`paclitaxel infusion for 21 patients are
`presented in Table 1. At the end of the
`paclitaxel
`infusion,
`the plasma con-
`centrations of Cremophor were 1.0% or
`higher for 19 of the 21 patients and
`0.9% for the remaining two.
`
`Discussion
`
`The ability of Cremophor to inhibit
`P-glycoprotein
`in vitro has been pre-
`viously reported {12,14-16). Although
`the
`interaction of Cremophor with
`serum components increases the con-
`centration of Cremophor required for
`this reversal of multidrug resistance,
`we have demonstrated in vitro activity
`even in the presence of 75% serum.
`The
`interactions between Cremophor,
`plasma components, and cells are likely
`to be complex. However, as a deliber-
`ate oversimplification,
`these
`interac-
`
`should be described by
`tions
`following equations:
`
`the
`
`[Cremophor] + [plasma] *-*
`[Cremophor - plasma]
`
`[Cremophor] + [P-glycoprotein] <->•
`[Cremophor - P-glycoprotein]
`
`[Cremophor - plasma] +
`[P-glycoprotein] <->
`[Cremophor - P-glycoprotein] +
`[plasma]
`
`[Cremophor - plasma] +
`[P-glycoprotein] <-> [plasma -
`Cremophor - P-glycoprotein]
`
`[1]
`
`[2]
`
`[3]
`
`[4]
`
`In the above equations, "plasma"
`indicates probable multiple
`serum/
`plasma components that interact with
`Cremophor (20), and equilibria involv-
`ing the "P-glycoprotein" could involve
`either direct interactions between Cre-
`mophor and P-glycoprotein or indirect
`interactions mediated via effects on the
`structural organization of the cell mem-
`brane. The equilibria involved must be
`rapid because Cremophor-mediated in-
`hibition of
`the P-glycoprotein drug
`efflux pump is the same, regardless of
`whether the Cremophor had been al-
`lowed to form a complex with serum
`components for 1 hour before dauno-
`rubicin was added or whether
`the
`Cremophor was added at the same time
`
`1688 REPORTS
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`Journal of the National Cancer Institute, Vol. 85, No. 20, October 20, 1993
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`
`-
`
`-
`
`CO
`
`o C
`
`O
`
`CM
`ft
`
`O E
`
`-
`
`L
`
`400
`
`r
`
`a
`
`'/
`
`i
`
`i
`0
`
`300
`
`200
`
`100
`
`LUORESCENCE
`
`DNR
`
`i
`i
`i
`i
`i
`i
`i
`i
`i
`i
`0.02 0.04 0.06 0.08 0.1 5=
`o
`«
`
`CREMOPHOR EL [% v / v]
`
`Fig. 2. Percent of Cre-
`mophor versus equili-
`brium
`daunorubicin
`(DNR) fluorescence as
`measured by flow cyto-
`metry. CEM/VLBI OO
`cells were
`incubated
`with 2 p.g/mL dauno-
`rubicin in 0.5 mL me-
`dium
`and
`0.5 mL
`plasma. Three standard
`curves
`(open symbols)
`were prepared by adding
`Cremophor
`to pretreat-
`ment patient
`plasma.
`Plasma from
`these pa-
`tients taken at the end
`of a 3-hour paclitaxel
`infusion was also as-
`sayed
`(solid symbols).
`The Cremophor
`con-
`centration
`(contributed
`by 0.5 mL of plasma in
`each 1 mL incubation)
`can be read against the
`corresponding
`standard
`curve
`(arrows). Cre-
`mophor levels in patient
`plasma are
`thus
`twice
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`
`as the daunorubicin (72). Similarly, the
`effect of Cremophor on P-glycoprotein
`activity (and presumably its interaction
`with cells)
`is also readily reversible
`(72). The observation
`(Fig. 1)
`that
`25%, 50%, or 75% newborn calf serum
`(or plasma) has a similar effect on the
`concentration dependency of Cre-
`mophor on P-glycoprotein
`activity
`would suggest that there is an excess of
`the component(s)
`that
`interact with
`Cremophor in assays with even 25%
`serum. The equilibrium (equation 1) is
`thus driven toward complex formation
`at all the serum or plasma concentra-
`tions tested. In the absence of plasma
`(HL-1 medium only), most of
`the
`change
`in
`intracellular daunorubicin
`concentration occurs over a 100-fold
`change
`in Cremophor concentration.
`This observation would be consistent
`with an interaction
`that
`is primarily
`bimolecular (equation 2). By contrast,
`in the presence of serum, only a 10-
`fold change in Cremophor concentra-
`tion is required for the bulk of the
`change
`in
`intracellular daunorubicin
`levels
`to occur. While extrapolation
`from simple equilibria should be made
`with caution, this finding could suggest
`that,
`in
`the presence of serum,
`the
`Cremophor interaction with cells is of a
`cooperative nature
`(equilibrium
`in
`equation 4). However, whatever
`the
`nature of the interactions involved, our
`results suggest that a concentration of
`0.1% u.L/mL Cremophor in body fluids
`should be sufficient
`to
`inhibit
`the
`P-glycoprotein drug efflux pump
`in
`tumors
`expressing
`the multidrug-
`resistant phenotype.
`
`The bioassay described here allows
`the estimation of absolute values of
`Cremophor concentrations in plasma or
`serum. While serum components affect
`the concentration dependency of
`the
`Cremophor effect,
`the equilibrium
`amounts of intracellular daunorubicin
`as read from the assay curves relate
`directly to absolute amounts of Cre-
`mophor added to each assay standard.
`In samples taken from 21 patients who
`received a 3-hour paclitaxel
`infusion,
`there was a sufficiently high circulating
`level (close to or greater than 0.1%
`[vol/vol]) of Cremophor in plasma at
`the end of the infusion for the effective
`inhibition of action of the P-glycopro-
`tein drug efflux pump. These data do
`
`not directly address the question of the
`relative contribution of the multidrug
`resistance-reversing
`surfactant Cre-
`mophor in the paclitaxel formulation to
`the overall efficacy of paclitaxel. The
`pharmacokinetics of Cremophor in hu-
`mans are unknown. However, a study
`performed with rats showed that most
`of the Cremophor had been cleared by
`24 hours, the first time point used (23).
`The activity of paclitaxel when given
`as a 24-hour
`infusion, where Cre-
`mophor levels are likely to be much
`lower than following a 3-hour infusion,
`indicates that high plasma Cremophor
`levels are not absolutely required for
`clinical antitumor activity. Further in-
`vestigation is needed on the relation-
`ship between
`tumor expression of
`P-glycoprotein, Cremophor levels, and
`subsequent response to paclitaxel.
`to
`Taxotere
`is
`the second
`taxane
`enter clinical trials. Early clinical re-
`sults
`indicate
`that
`the spectrum of
`antitumor activity of taxotere is likely
`to be similar to that of paclitaxel (24).
`While not formulated
`in Cremophor,
`taxotere is dissolved in polysorbate 80
`(Tween 80), another polyethoxylated
`surfactant
`that can reverse multidrug
`resistance in vitro (12,16).
`The data presented here demonstrate
`that a Cremophor dose of 16.8-27 mL
`given over a 3-hour period can produce
`plasma
`levels between 0.09% and
`0.2%. These levels should be sufficient
`for in vivo reversal of drug exclusion
`in cells exhibiting
`the multidrug-
`resistant phenotype. Hence, unlike the
`results with other agents
`such as
`verapamil (25), which have been ad-
`ministered
`in conjunction with cyto-
`toxic agents in attempts to overcome
`multidrug resistance, sufficient circulat-
`ing levels of the multidrug resistance-
`reversing agent, Cremophor EL, can be
`achieved without dose-limiting
`toxic
`effects and without the potential haz-
`ards of using an
`immunosuppressive
`drug such as cyclosporine
`for
`this
`purpose
`(26). Cremophor,
`therefore,
`could be investigated as a multidrug
`resistance modulator with
`other
`cytotoxic agents in other schedules.
`
`References
`(/) Bradley G, Juranka PF, Ling V: Mechanism
`of multidrug resistance. Biochim Biophys
`Acta 948:87-128, 1988
`
`(2) Bell DR. Gerlach JH. Kartner N. et al: De-
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`(3) Salmon SE, Grogan TM, Miller T, et al:
`Prediction of doxorubicin resistance in vitro
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`J Am Chem Soc 93:2325-2327, 1971
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`(12) Woodcock DM, Linsenmeyer ME,
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`(13) Coon JS, Knudson W, Clodfelter K, et al:
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`(15) Schuurhuis GJ, Broxterman HJ, Pinedo
`HM, et al: The polyoxyethylene castor oil
`Cremophor EL modifies multidrug resist-
`ance. Br J Cancer 62:591-594, 1990
`(16) Friche E, Jensen PB, Sehested M, et al: The
`solvents Cremophor EL and Tween 80
`modulate daunorubicin
`resistance
`in
`the
`multidrug resistant Ehrlich ascites tumor.
`Cancer Commun 2:297-303, 1990
`(17) Horwitz SB, Lothstein L, Manfredi JJ, et al:
`Taxol: mechanisms of action and resistance.
`Ann N Y Acad Sci 466:733-744, 1986
`(18) Waud WR, Gilbert KS, Harrison SD Jr, et
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`Journal of the National Cancer Institute, Vol. 85, No. 20, October 20, 1993
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`REPORTS 1689
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`NEPTUNE GENERICS EX. 1033 00005
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`

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`(19) Beck WT, Mueller TJ, Tanzer LR: Altered
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`(21) Nassberger L, Bergstrand A, DePierre JW.
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`
`Notes
`Affiliation of authors: Peter MacCallum Can-
`cer Institute, Melbourne, Victoria, Australia.
`to- David M Woodcock,
`Correspondence
`Ph.D., Molecular Genetics Laboratory, Peter
`MacCallum Cancer Institute, 481 Little Lonsdale
`St., Melbourne, Victoria 3000, Australia.
`We thank research nurse Anne