Br. J. Cancer (1990), 62, 591-594
`
`Br. J. Cancer (1990), 62, 591
`
`594
`
`19" Macmillan Press Ltd., 1990
`
`© Macmillan Press Ltd., 1990
`
`SHORT COMMUNICATION
`
`The polyoxyethylene castor oil Cremophor EL modifies multidrug
`resistance
`
`G.J. Schuurhuis, H.J. Broxterman, H.M. Pinedo, Th. H.M. van Heijningen, C.K. van Kalken,
`J.B. Vermorken, E.C. Spoelstra & J. Lankelma
`
`Department of Medical Oncology, Free University Hospital, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
`
`In vitro the presence of P-glycoprotein is associated with the
`possibility to reverse multidrug resistance (MDR) with com-
`pounds with different structural features (Bradley et al.,
`1988). Although many such compounds have been described,
`only a few can be expected to be active
`clinically
`at
`achievable concentrations: quinidine (Tsuruo et al., 1984),
`amiodarone (Chauffert et al., 1987), bepridil (Schuurhuis et
`al., 1987) and cyclosporin A (Slater et al., 1986; Twentyman,
`1988) are examples. During our investigations on the in vitro
`effects of resistance modifiers on daunorubicin and vincristine
`freshly
`accumulation
`in
`obtained human tumour cells
`(Schuurhuis et al., 1989b) we found that Cremophor EL,
`which is a polyethoxylated castor oil used as a solubiliser,
`e.g. of vitamins and of the immunosuppressant drug cyclo-
`sporin A, had effects on drug accumulation similar to other
`resistance modifiers used. In order to study whether this
`effect of Cremophor EL was related to MDR, we inves-
`tigated
`effects of Cremophor EL on anthracycline
`the
`accumulation and anthracycline and vincristine cytotoxicity
`in MDR and sensitive model cell lines and we compared the
`using
`cyclosporin A and
`results
`obtained
`with
`those
`verapamil. Both human squamous lung cancer cells (SW-
`1573, Keizer et al.,
`1989; Broxterman et al., 1989) and
`human myeloma cells (8226, Dalton et al., 1986, 1989b) were
`used. The latter ones are of special interest because of the
`increased in vivo sensitivity of myeloma to a regimen contain-
`ing vincristine, doxorubicin and dexamethasone (the VAD
`regimen) when verapamil is used as a modifier (Dalton et al.,
`1989a). Both types of MDR cells overexpress P-glycoprotein
`(Dalton et al., 1989b; Kuiper et al., 1990).
`Cells were cultured in Dulbecco's modified minimal essen-
`tial medium supplemented with 10% fetal bovine serum
`(Gibco, Paisley, Scotland). The human multiple myeloma
`8226Dox4 and 8226Dox40 cells were cultured in the presence
`of 40 and 400 nM doxorubicin (Adriablastina, Farmitalia,
`Milan, Italy) respectively. SW-1573/2R160 cells were derived
`from SW-1573/2R50 cells (Keizer et al., 1989; Broxterman et
`al., 1989) by continuous exposure to 160 nM doxorubicin.
`Experiments were performed on cells cultured for 1-2 weeks
`without doxorubicin. Cells were allowed to adhere (SW-1573
`cells) or equilibrate in suspension (8226 cells) in six-well
`tissue culture plates (Costar, Cambridge, MA, USA). Then
`they were incubated under 5% CO2 with doxorubicin or
`vincristine sulfate (Sigma Chemical Co., St Louis, MO, USA)
`with or without resistance modifiers for at least three cell
`doubling times. Cell doubling times were 22 h (SW-1573),
`45h (SW-1573/2R160), 36h (8226S), 42h (8226Dox4) and
`44 h (8226Dox40). Thereafter the cells were counted as des-
`cribed by Schuurhuis et al. (1987) using a Sigmex microcell
`counter model CC-I 10. With high concentrations of Cremo-
`
`Correspondence: G.J. Schuurhuis.
`Received 8 January 1990; and in revised form 17 April 1990.
`
`phor EL (>132 jg ml') or when fresh human plasma was
`used, the cells were co-incubated with doxorubicin and Crem-
`ophor EL for 2 h, post-incubated for 2 h with Cremophor
`EL only and further incubated in fresh medium as described
`above. Resistance modifiers used were verapamil.HCI (Sig-
`ma), cyclosporin A (Sandoz AG, Basel, Switzerland), cyclo-
`sporin A in Cremophor EL (Sandimmune, Sandoz, AG) and
`Cremophor EL (Sandoz AG). Cyclosporin A in Cremophor
`EL was used because in this form (Sandimmune) cyclosporin
`A is administered clinically. The choice of the concentration
`of the modifiers used in the cytotoxicity experiments in this
`study is based on other in vitro studies: 1-2I1M cyclosporin
`A and 4 tLM verapamil usually are effective in modulating
`MDR (Durie & Dalton, 1988; further reviewed in Twen-
`tyman, 1988 and Kaye, 1988). The choice of Cremophor EL
`concentrations is based on the amounts present in the cyclo-
`sporin A solutions which are administered in the clinic (as
`Sandimmune), e.g. a final dilution of 2 gM cyclosporin A
`contains 33 fig ml-' Cremophor EL.
`Cellular accumulation and efflux experiments were per-
`formed essentially as described earlier (Schuurhuis et al.,
`1987). Some 0.1-0.3 x 106 cells were incubated for 2 h at
`37C in 550 jil Dulbecco's medium, pH 7.4, lacking NaHCO3
`but containing 20 mM HEPES and 10% fetal bovine serum,
`to which '4C-doxorubicin (Amersham Laboratories, Amer-
`sham, UK) or '4C-daunorubicin (Amersham) was added with
`or without resistance modifiers. The final concentration of
`doxorubicin and daunorubicin was made 0.5 liM by adding
`unlabelled doxorubicin and daunorubicin (Specia,
`Paris,
`France). After two washes with ice-cold phosphate-buffered
`saline, the cells were transferred to liquid scintillation fluid.
`No corrections were made for direct binding of anthracyc-
`lines to the cells since binding was the same whether or not
`modifiers were present and was too low to affect the con-
`clusions (5-20% at maximum). For efflux experiments sen-
`cells were incubated with 0.5 lSM doxorubicin or
`sitive
`daunorubicin. Resistant cells were incubated with 2.5 JAM
`(SW-i 573/2R 160)
`daunorubcin
`doxorubicin
`1 JiM
`or
`(8226Dox4); this resulted in about the same intracellular drug
`amounts as in the sensitive cells in these experiments after 2 h
`After washing with ice-cold
`Dulbecco's
`of incubation.
`medium, the cells were resuspended in fresh ice-cold medium
`and incubated for 1 h at 37°C. After washing the cell-
`associated radioactivity was determined.
`In Table I it is shown that Cremophor EL (132figml-')
`partly reversed doxorubicin resistance in SW-1573/2R160
`cells (the dose modifying factor, DMF, was 6.3; resistance
`index = 77) while only a small effect was observed on the
`parent cell line. With concentrations higher than 132 fig ml-I
`higher dose modifying factors were found (> 10). At a con-
`Cremophor EL had a small
`centration
`of 33 lg ml-'
`although
`significant
`SW-1573/2R160
`cells
`effect
`in
`(DMF = 1.9, see Table I). Two JAM pure cyclosporin A had a
`DMF of 8.3 ± 1.5 (mean ± s.d.
`three
`experiments,
`in
`P<0.01), while 2 JM cyclosporin A (Sandimmune), which
`
`MYLAN - EXHIBIT 1132
`Mylan Pharmaceuticals Inc. et al. v. Allergan, Inc.
`IPR2016-01127, -01128, -01129, -01130, -01131, & -01132
`
`

`

`592
`
`G.J. SCHUURHUIS et al.
`
`Table I
`
`Effect of resistance modifiers on doxorubicin cytotoxicity in human squamous lung cancer and
`myeloma MDR and sensitive cells
`DMP
`
`Vp
`(4 pM)
`1.4±0.2
`5.4 ± O..5
`
`IC50 (nM)
`Vp
`CEL
`CEL
`Cycl. Al
`Cell line
`(control)
`(33 zg mt-') (132 jg ml-')
`(16 AM)
`1.6±0.4c
`22±_3c
`1.8±0.6
`SW-1573
`1.5±0.1
`1700±300[77]d
`6.3 ±0.1
`1.9±0.1'
`SW-1573/2R160
`16.8 ± 6.If
`11.0 ± 1.4'
`1.1 ± 0.1
`8226S
`1.4± 0
`12
`2
`1.2
`0.1
`1.1
`0.3
`7.1 ± 1.2'
`4.6 ± 0.2g
`95 ± 12 [7.9]
`5.4 ± 1.79
`8226Dox4
`0.8g
`4.9
`3.4 + 0.3
`2.6 ± 1.3f
`13.3 ± 2.8'
`5.6 ± 2.2
`8226Dox40
`540 + 110 [45]
`-
`8.4 + 2.7
`Vp, verapamil; Cycl. A, cyclosporin A; CEL, Cremophor EL; ICG, doxorubicin concentration resulting in
`50% growth inhibition. aDMF, dose modifying factor = IGC
`without resistance modifier/IC50 with
`resistance modifier. bCycl. A (Sandimmune): 2 laM for SW-1573 cells, 1 gM for 8226 cells (2 jAM cycl. A is
`dissolved in 33 ,lg ml-' CEL and 1 jAM cycl.A in 16.5 fig ml-' CEL). cValues are means ± s.d. from 2-5
`independent experiments. dValues within brackets: IC50 MDR cell line/IC50 parent cell line. eSignificantly
`different from I (P < 0.05, Student's t test). fSignificantly different from 1 (P < 0.02). gSignificantly different
`from I (P<0.01).
`
`contains 33 ytg ml-' Cremophor EL, had a DMF of 16.8
`(Table I). These results show that both compounds as such
`are able to sensitize SW-1573/2R160 cells to doxorubicin and
`that the effects are additive when cyclosporin A is given as
`Sandimmune.
`Also in the human myeloma cell line 8226Dox40 with a
`moderately high doxorubicin resistance index (45, see Table
`I) Cremophor EL (33 fLg ml-') had only a small effect (DMF
`of 2.6, see Table I). Like in SW-1573/2R160 cells, with higher
`concentrations of Cremophor EL the effects on doxorubicin
`cytotoxicity became more pronounced (Table I). On the
`other hand, in 8226Dox4 cells with a low doxorubicin resis-
`tance index (7.9, see Table I) 33 glg ml-' Cremophor EL
`largely reversed doxorubicin resistance (Table I and Figure
`1). One jAM cyclosporin A in Cremophor EL (16.5 yg ml-')
`16 pM) had no greater effect than
`or verapamil (4 or
`Cremophor EL (33 yg ml-') alone (Table I). Figure 1 shows
`the dose - response relationship for Cremophor EL on dox-
`orubicin cytotoxicity: even at concentrations of 4.1 and
`8.2 lAg ml-' (which correspond to dilutions of 1:256,000 and
`1:128,000
`respectively),
`signifcant
`effects were observed.
`These data show that in cells with low levels of MDR
`reversal of resistance with cyclosporin A may have been
`achieved at least partly due to the carrier (Cremophor EL)
`effects alone. This may have important implications for the
`design and interpretation of clinical trials with cyclosporin A
`as reversing agent.
`Interestingly,
`results
`that
`indicate
`cells
`with
`our
`intermediate to high levels of resistance, like 8226Dox40 and
`SW-1573/2R160 cells, may not be good models to predict the
`possible clinical usefulness of resistance modifiers in P-
`glycoprotein-containing tumours. This may be due to the fact
`that drug efflux from cells with low amounts of P-
`glycoprotein can be blocked more efficiently.
`
`Drug accumulation experiments confirmed the findings
`reported above. Cremophor EL significantly stimulated dox-
`orubicin and daunorubicin accumulation in 8226Dox40 and
`SW-1573/2R160 cells, respectively, but not in the sensitive
`cells (Table II). In SW-1573/2R160 cells daunorubicin was
`of doxorubicin,
`used
`instead
`since drug accumulation
`differences between sensitive and resistant cells and impor-
`tantly, effects of modifiers on drug accumulation in resistant
`cells, were much more pronounced for daunorubicin than for
`doxorubicin in these cells. Cremophor EL (132 jg ml-')
`stimulates anthracycline accumulation at least partly by in-
`
`.100.
`
`~40
`
`U 20
`
`0
`
`0.01 -.05 0.1
`Dxorublcpn (FiM)
`Figure 1
`Effect of Cremophor EL on doxorubicin cytotoxicity in
`8226Dox4 and 8226S cells. Myeloma cells were incubated with
`doxorubicin in the presence of increasing concentrations of
`Cremophor EL (CEL) as described in the text. Symbols represent
`means ± s.e. from 2-3 independent experiments, except CEL
`(16.5 fig ml-') (one experiment). 8226Dox4: 0-0, control;
`O 0, CEL (4.1 figml-'); A-A, CEL (8.2 jAgml-'); V-V,
`CEL (16.5Sjgml-'; 0-0, CEL (33figml-'); O-*, CEL
`(132 jg ml-'); x -x, Cycl. A (Sandimmune, 2 1AM). 8226S: 0---
`*, control; *---*, CEL (33jLgml-'); x---x, Cycl. A (2jAM).
`
`Table II
`
`Effect of resistance modifiers on anthracycline accumulation and retention in human squamous lung cancer and
`myeloma MDR and sensitive cells
`
`anthracyclinea
`AEFb
`Cycl.A
`accumulation
`Vp
`Cel
`(8 jLM)
`(132 og mlh')
`(16 jAM)
`(pmol per 106 cells)
`Cell line
`346±52'
`1.18±0.11'
`1.05±0.12
`0.99+0.08'
`SW-1573
`0.39h
`0.45h
`4.39 ± 0.88h
`56 ± 8 [6.2]f
`SW-1573/2R160
`3.09
`2.05
`0.94±0.19
`0.99±0.11
`1.05±0.18
`8226S
`147 17
`0.19h
`0.16'
`0.11h
`1.30
`20 [1.4]
`1.34
`1.24
`107
`8226Dox4
`1.27± 0.17h
`0.18'
`1.30
`1 [1.8]
`1.54
`0.26h
`8226Dox40
`84
`-
`Abbreviations as in Table I. aDrug accumulation (2 h at 37°C) was carried out with 0.5 gM daunorubicin for SW-1573 cells
`and with 0.5 jAM doxorubicin for 8226 cells. "AEF, accumulation enhancement factor = drug accumulation with modifier/drug
`accumulation without modifier. CAnthracycline retention was measured after 1 h of drug efflux; shown are means ( ± s.d.) of
`initial amounts. dREF, retention enhancement factor = drug retention with CEL (1321g ml- ')/drug retention without CEL.
`'Values are means ± s.d. from 2-6 independent experiments each performed in triplicate. fValues between brackets: drug
`accumulation in sensitive cells/drug accumulation in resistant cells. gSignificantly different from 1 (P < 0.05, Student's t test).
`hSignficantly different from 1 (P<0.01).
`
`REP
`1.12±0.17
`1.37 ± 0.09'
`0.98±0.04
`0.05'
`1.08
`
`Anthr.
`retentionc
`12e
`63
`34 ± 8
`72±5
`9
`67
`
`

`

`CREMOPHOR EL MODIFIES MULTIDRUG RESISTANCE
`
`593
`
`creasing its retention in the MDR cells (Table II), as seems to
`be the case for cyclosporin A (Nooter et al., 1989). No
`significant effects of Cremophor EL on anthracycline reten-
`tion were seen in the parent cells. The effects of the modifiers
`on drug cytotoxicity in MDR cells seems to be due for an
`important part to a change in intracellular drug distribution
`instead of to stimulation of drug accumulation as will be
`discussed later. In addition, stimulation of anthracycline
`accumulation by modifiers occurs in a dose-dependent way
`and therefore low concentrations of modifiers stimulate
`anthracycline accumulation only slightly. In order to show
`clearly that the resistance modifiers used stimulate drug
`accumulation in our MDR cells we have chosen higher con-
`centrations of modifiers for accumulation and retention
`experiments than for cytotoxicity experiments.
`We have also determined the effect of Cremophor EL on
`vincristine cytotoxicity in 8226Dox4 cells since vincristine is
`included in clinical protocols for myeloma patients. Figure 2
`shows that Cremophor EL is active in reversing vincristine
`resistance with dose modifying factors of 2.2, 3.2, 8.4 and
`28.7 for the concentrations of 8.2, 16.5, 33 and 132ligmlh',
`respectively. Since the resistance index was 15, this means a
`more than complete reversal of resistance at 132figml-'.
`Interestingly, the sensitive cells were affected too, although to
`limited
`extent (DMF: 2.2,
`Figure
`2). Two gM
`a
`see
`verapamil, a concentration which is ony achievable clinically
`with serious side-effects (Benson et al., 1985; Ozols et al.,
`1987) was less effective than Cremophor EL at a concentra-
`tion of 33 yg ml-' (see Figure 2).
`One major determinant of the efficacy of a drug in the
`clinic can be its ability to bind to proteins (Koch-Weser &
`Sellers, 1976). We have shown previously that an increase in
`the protein concentration signifcantly decreased the potency
`of resistance modifiers such as verapamil, bepridil, diltiazem
`and Ro 11-2933/001 to stimulate anthracycline accumula-
`tion in MDR cells (Broxterman et al., 1987). Table III shows
`that Cremophor EL at concentrations of 33 and 132 fig ml-
`largely retains its ability to reverse doxorubicin resistance in
`8226Dox4 cells at a high protein concentration (compare
`Tables I and III). At this protein concentration the dose
`modifying factors of verapamil, even at a concentration of
`16I1M, are somewhat lower than for Cremophor EL (Table
`III). In addition, when 8226Dox4 cells were incubated in
`fresh human plasma
`for
`2 h
`with
`doxorubicin
`and
`Cremophor EL (132 pg ml-'),
`followed by a 2 h post-
`incubation in plasma with Cremophor EL only, the effect
`was about the same as in control experiments using 10%
`fetal bovine serum in the same incubation protocol (DMF of
`2.5-3.5). These results indicate that proteins probably do not
`strongly interfere with the capacity of Cremophor EL to
`modulate MDR.
`Despite the many studies addressing the mechanism of
`action of resistance modifiers, the answers offered are not yet
`satisfactory. Resistance modifiers seem to act at least partly
`by binding to P-glycoprotein (Safa et al., 1986; Cornwell et
`al., 1986; Foxwell et al., 1989) and competing for drug efflux
`via P-glycoprotein (Bradley et al., 1988), thereby increasing
`drug accumulation in the cell. As an alterntive some resis-
`tance modifiers may act via their detergent effect on mem-
`
`=
`
`=
`
`1001-
`
`80
`
`60
`
`40
`
`20
`
`0
`
`0
`
`0.01
`
`-7-m.
`
`0.1
`Vincristine (IJ.M)
`
`1.0
`
`Figure 2
`Effect of Cremophor EL on vincristine cytotoxicity in
`8226Dox4 and 8226S cells. Cells were incubated with vincristine
`in the presence or absence of Cremophor EL (CEL) or verapamil.
`Each point represents mean ± s.e. of 2-4 independent experi-
`ments. 8226Dox4: 0-O, control; 0-0, CEL (8.2 jgml-');
`A-A, CEL (16.59lgml-'); V-V, CEL (33pgml-'); O-O,
`CEL (1329Agml1');
`U--U, verapamil (0.5I1M); A---A, verap-
`amil (I 9AM); V---V, verapamil (2 9aM). 8226S: *---@, control;
`*---*, CEL (1329fg ml ').
`
`branes as reported for Tween 80 (Carlsen et al., 1976). We
`have shown previously that the action of resistance modifiers
`may be due largely to their effects on the intracellular drug
`localisation instead of on drug accumulation: in cells with
`high levels of MDR doxorubicin is present mainly in the
`cytoplasm in the absence of resistance modifiers. However, in
`the presence of resistance modifiers doxorubicin is mainly in
`the nucleus, as is the situation in drug-sensitive cells (Willing-
`ham et al., 1986; Schuurhuis et al., 1989a; Broxterman et al.,
`1990). Cremophor EL also was able to produce a similar
`change in drug localisation (from mainly cytoplasmic to
`mainly nuclear) in SW-1573/2R160 cells as determined with
`fluorescence microscopy (results not shown). These observa-
`tions offer an explanation for the finding that modifiers such
`as cyclosporin A are able to reverse drug resistance to a large
`extent without strongly affecting drug accumulation (Slater et
`al., 1986; Schuurhuis et al., 1989a; this study).
`Since protein kinase C (PKC) activity has been associated
`with MDR and its reversal (Aquino et al., 1988; Fine et al.,
`1988; O'Brien et al., 1989; Ferguson & Cheng, 1987), it is of
`interest that Cremophor EL, like other resistance modifiers
`such
`verapamil,
`tamoxifen,
`cyclosporin A and
`as
`phenothiazines (Mori et al.,
`1980; O'Brian et al.,
`1985;
`Walker et al., 1989; Schatzman et al., 1981), strongly inhibits
`PKC activity at concentrations comparable to those used in
`this study (Zhao et al., 1989).
`In conclusion, our findings demonstrate that Cremophor
`EL is a potent modifier of MDR in human myeloma cells at
`protein concentrations which closely mimic the in vivo situa-
`tion. Clinical studies in myeloma with Cremophor EL as a
`resistance modifier thus seem warranted. Further, Cremophor
`
`Table III
`
`Reversal of doxorubicin resistance in 8226 MDR cells in protein-richa
`medium
`
`CEL
`(33 lAg ml-')
`
`DMFO
`IC50 (nM)
`CEL
`Vp
`Vp
`(132 ,Ag ml' )
`Cell line
`(control)
`16 gM
`4 "AM
`8226S
`26.3
`5.9c
`-
`1.0
`0.1
`0.3
`1.2
`250 + 71 [9.5]d
`6.2 + 1.f
`5.5 ± 0.7e
`3.5 ± 0.7'
`8226Dox4
`2.0 + 0.7
`Abbreviations as in Table I. aThe growth medium contained 4% bovine serum albumin
`(Sigma) in addition to 10% fetal calf serum. bDMF, dose modifying factor: IC50 minus
`resistance modifier/IC", plus resistance modifier. cValues are means ± s.d. from 2-3
`IC,o 8226Dox4 cells/ICM, 8226S cells.
`dValues between brackets:
`experiments.
`'Significantly different from I (P<0.05, Student's t test). 'Signficantly different from I
`(P<0.02).
`
`

`

`594
`
`G.J. SCHUURHUIS et al.
`
`EL may turn out to be useful in the treatment of other
`P-glycoprotein-containing tumours in addition to myeloma
`since we have found that in vitro the compound was active
`on other P-glycoprotein-containing human MDR cancer cells
`like squamous lung cancer cells (this paper) and ovarian
`cancer cells (submitted) as well as intrinsically resistant P-
`glycoprotein-containing human colon cancer cells (submit-
`ted).
`
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