Int. J. Cancer: 85, 131–141 (2000)
`娀 2000 Wiley-Liss, Inc.
`
`Publication of the International Union Against Cancer
`Publication de l’Union Internationale Contre le Cancer
`
`INCREASED INTRACELLULAR DRUG ACCUMULATION AND COMPLETE
`CHEMOSENSITIZATION ACHIEVED IN MULTIDRUG-RESISTANT SOLID
`TUMORS BY CO-ADMINISTERING VALSPODAR (PSC 833)
`WITH STERICALLY STABILIZED LIPOSOMAL DOXORUBICIN
`
`Rajesh KRISHNA1,2, Maryse ST-LOUIS3 and Lawrence D. MAYER1,2*
`1Department of Advanced Therapeutics, BC Cancer Agency, Vancouver, Canada
`2Division of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada
`3Biotechnology Laboratory, University of British Columbia, Vancouver, Canada
`
`We have previously demonstrated that liposome encapsu-
`lation of doxorubicin (DOX) can alleviate adverse interac-
`tions with non-encapsulated DOX and the cyclosporine multi-
`drug-resistant (MDR) modulator Valspodar. We have now
`investigated the behavior of different liposomal DOX formu-
`lations in MDA435LCC6/MDR-1 human breast cancer solid
`tumor xenograft models to identify liposome characteristics
`associated with enhanced therapeutic activity and the mecha-
`nism whereby increased chemosensitization is achieved. Tox-
`icity studies incorporating conventional phosphatidylcholine
`(PC)/cholesterol (chol) and sterically stabilized (polyethylene
`glycol 2000 [PEG]-containing) formulations of DOX indi-
`cated that whereas PC/Chol DOX was approximately 3-fold
`more toxic in the presence of Valspodar, PEG containing
`distearoylglycerophosphocholine (DSPC)/Chol DOX was
`minimally affected.
`In mice bearing MDR tumors, co-
`administration of Valspodar and egg phosphocholine (EPC)/
`Chol DOX resulted in modest MDR modulation and efficacy,
`whereas the sterically stabilized formulation induced reduc-
`tions in tumor growth equivalent to that achieved for drug-
`sensitive tumors treated with non-encapsulated DOX. Phar-
`macokinetic studies revealed a 2.5-fold increase in plasma
`DOX area under the curve (AUC) upon co-administration of
`Valspodar with EPC/Chol DOX whereas no such alterations
`were observed with the sterically stabilized liposomes. Com-
`pared to non-encapsulated DOX combined with Valspodar,
`improvements in efficacy and toxicity correlated with the
`extent to which liposomal DOX formulations were able to
`circumvent pharmacokinetic interactions. Confocal micros-
`copy demonstrated that Valspodar increased cell-associated
`DOX which correlated with the level of anti-tumor efficacy.
`Int. J. Cancer 85:131–141, 2000.
`娀 2000 Wiley-Liss, Inc.
`
`The development of second generation multidrug-resistant (MDR)
`reversing agents alleviated many of the problems caused by earlier
`PGP blockers which were pharmacological agents with their own
`inherent toxicities. However, co-administration of conventional
`anticancer drugs with many of these newer MDR modulators has
`been shown to elicit drug-modulator interactions by virtue of PGP
`blockade in normal tissues such as liver, kidney, intestine and brain
`(Keller et al., 1992a; Gatmaitan and Arias, 1993). It may not be
`surprising then that clearance of several anticancer drugs is
`inhibited by MDR modulators such as cyclosporine (CsA) (Speeg
`et al., 1992), verapamil (Nooter et al., 1987), Valspodar (Speeg and
`Maldonado, 1994, previously named PSC 833) and GW918 (Booth
`et al., 1998).
`The effects of MDR modulators on drug transport proteins that
`cause alterations in anticancer drug excretion often lead to in-
`creased anticancer drug exposure of healthy tissues and have
`necessitated dose reduction in many preclinical (Krishna and
`Mayer, 1997; Keller et al., 1992a; Nooter et al., 1987) and clinical
`(Boote et al., 1996; Sarris et al., 1996) studies. Although doses can
`be adjusted to equal levels of toxicity, it is unclear how such
`pharmacokinetic (PK) changes may impact therapeutic activity.
`These interactions have been postulated to play a role in limiting
`the therapeutic outcome in some patients (Wishart et al., 1994;
`Miller et al., 1994). While changes in anticancer drug dose and/or
`
`schedule may be able to address toxicity or efficacy alterations
`brought about by MDR modulators,
`this clearly represents a
`significant complication in applying PGP blockade strategies to
`cancer chemotherapy. This is due to the fact that most chemother-
`apy regimens utilize drug combinations, of which more than one
`are often PGP substrates. Consequently, an ability to avoid such
`anticancer drug clearance alterations may be considered to be a
`significant advantage in MDR modulation strategies.
`We earlier reported that liposome encapsulation of doxorubicin
`(DOX) can reduce non-encapsulated drug-Valspodar interactions,
`resulting in improved growth suppression of MDR murine solid
`tumors (Krishna and Mayer, 1997). The liposomal formulation
`used in these studies was composed of 120 nm diameter distearoyl-
`glycerophosphocholine (DSPC)/cholesterol (Chol) (55:45 molar
`ratio) that retains DOX for extended periods of time (Mayer et al.,
`1989). The enhanced antitumor activity observed in this study
`appeared to be a consequence of increased protection from
`Valspodar-mediated PK changes and toxicity exacerbation. How-
`ever, the mechanisms by which these effects were achieved are not
`fully understood, particularly since significant amounts of DOX are
`delivered to the liver by liposomes without notable toxicological
`consequences. In addition to alleviation of PK alterations, the
`increased delivery of DOX to MDR solid tumors using liposome
`delivery systems was associated with increased anti-tumor activity
`when co-administered with Valspodar compared with non-
`encapsulated drugs. These studies were unable, however,
`to
`distinguish the degree of DOX bioavailability (liposome entrapped
`vs. released drug) in the solid tumor. Therefore, the relative roles of
`PGP blockade and tumor drug levels remain unresolved.
`In order to address these questions, we compared here the
`toxicity, efficacy, pharmacokinetics, cellular distribution properties
`of egg phosphocholine (EPC)/Chol DOX (a system where over
`50% of the drug is released in the first hour) and a sterically
`stabilized PEG2000 (PEG) distearoylphosphoethanolamine (DSPE)/
`DSPC/Chol DOX formulation combined with the MDR modulator,
`Valspodar. The latter system was chosen on the basis of reports that
`incorporation of 5 mol% PEG-polymerized lipid in 100 nm
`DSPC/Chol vesicles results in increased circulation longevity,
`reduced liver uptake and increased tumor delivery (Papahadjopou-
`los et al., 1991). Therefore, the effects of altering the drug release,
`liver uptake and tumor accumulation properties of liposomal DOX
`on toxicological and therapeutic activity were compared to reveal
`the processes underlying the improvements in toxicity and efficacy
`achieved with liposomal systems in a human breast carcinoma
`
`Rajesh Krishna’s present address is: Department of Metabolism and
`Pharmacokinetics, Pharmaceutical Research Institute, Bristol-Myers Squibb
`Co, P.O. Box 4000, Princeton, NJ 08543, USA.
`
`*Correspondence to: Department of Advanced Therapeutics, BC Cancer
`Agency, 600 West 10th Avenue, Vancouver, BC, V5Z-4E6, Canada.
`Fax: ⫹1 604 877-6011.
`
`Received 10 May 1999; Revised 12 July 1999
`
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`

`132
`
`KRISHNA ET AL.
`
`MDR solid tumor xenograft model. Furthermore, these formula-
`tions reflect the 2 liposomal DOX products that are either approved
`(sterically stabilized) or pending approval (EPC/Chol) for wide-
`spread clinical use. Consequently, determining their pharmacologi-
`cal properties in the presence of MDR modulators would be of
`considerable clinical interest.
`
`Material
`
`MATERIAL AND METHODS
`
`DOX hydrochloride for injection (U.S.P.) was purchased from
`David Bull (Vaudreil, Canada) and its purity affirmed by high-
`performance liquid chromatography (HPLC; see below). Valspodar
`was a generous gift from Novartis (Dorval, Canada) and its purity
`affirmed by LC/MS-MS (Varian LC-MS-MS system; Fisons,
`Altrincham, UK). DOX metabolite standards were generous gifts
`from Pharmacia Carlo Erba (Milan, Italy). PEG2000-DSPE (⬎99%
`purity), EPC (⬎99% purity) and DSPC (⬎99% purity) were
`obtained from Northern Lipids (Vancouver, Canada) and Chol from
`Sigma (St. Louis, MO). Cholesteryl hexadecyl ether (3H), a
`non-exchangeable, non-metabolizable lipid marker was purchased
`from Amersham (Oakville, Canada). HPLC grade solvents were
`obtained from BDH (Toronto, Canada) and used without further
`purification. Female BDF1 mice were obtained from Charles River
`(St. Constant, Canada). Female SCID/RAG2 mice were bred
`in-house at the BC Cancer Agency animal facility. The MDA435/
`LCC6 and its transfected MDR-1 line were generously provided by
`Dr. R. Clarke (Georgetown University, Washington, DC). These
`cells were maintained in tissue culture in Dulbeco’s modified Eagle
`medium (StemCell Technologies, Vancouver, Canada).
`
`Liposome and drug preparation
`Liposomes composed of EPC/Chol (55:45), PEG2000-DSPE/
`DSPC/Chol (5:50:45) and DSPC/Chol (55:45; mol:mol) were
`prepared by initially dissolving the lipid mixtures in chloroform
`(100 mg lipid per milliliter) and hydrating the dried lipid film in a
`300-mM citric acid, pH 4.00, buffer. The resulting multilamellar
`vesicles (MLVs) were subjected to 5 freeze-thaw cycles followed
`by a 10-cycle extrusion through 2 stacked 100-nm polycarbonate
`filters (Nuclepore, Pleasanton, CA) using a Lipex Extruder (Lipex
`Biomembranes, Vancouver, Canada). 3H-Cholesterylhexadecyl ether
`was used as a non-exchangeable, non-metabolizable lipid marker
`(Derksen et al., 1987). The resulting large unilammelar vesicles
`exhibited a mean diameter between 110–120 nm as determined
`using a Nicomp 270 submicron particle sizer (Particle Sizing
`Systems, Santa Barbara, CA).
`DOX was encapsulated in the liposomes using the transmem-
`brane pH gradient loading procedure (interior acidic) employing
`sodium carbonate as the alkalinizing agent and a drug-to-lipid
`weight ratio of 0.2:1.0 (Mayer et al., 1989). Liposomal DOX
`preparations were diluted with saline as necessary prior to in vivo
`administration. Valspodar (for animal studies) was dissolved in a
`10:1 mixture of ethanol (95%):Tween 80 and administered in a
`corn oil vehicle by oral gavage of a 200-µl volume (Keller et al.,
`1992b). Non-encapsulated DOX was administered in sterile saline.
`
`Toxicity evaluation studies
`Toxicity of the indicated DOX and Valspodar dose regimens was
`evaluated in dose range-finding studies using normal (non-tumor-
`bearing) female BDF1 mice. Toxic dose range-finding studies in
`tumor-free female mice were performed using 3 mice per group
`with appropriate group repetitions as described below. Briefly,
`mice were administered increasing (each dose in different groups)
`doses of i.v. DOX (free or liposomal) via the tail vein and oral
`Valspodar at a fixed dose of 100 mg/kg (4 hr before DOX) on days
`1, 5 and 9. Liposomal DOX doses were changed by increments of 5
`mg/kg on the day 1, 5, 9 injection schedule. DOX dose escalation
`was stopped when weight loss exceeded 30% or toxicity-related
`mortality was observed. Survival and the percent change in body
`weight was monitored over a 21-day period. Animals were
`
`monitored for other toxicity signs such as scruffy coat, dehydration,
`lethargy, ataxia or labored breathing. Animals which demonstrated
`significant physical manifestations of distress/toxicity or exhibited
`a body weight loss in excess of 30% were terminated. At the end of
`the 21-day study period, mice were terminated by carbon dioxide
`asphyxiation. Necropsies were performed to identify abnormalities
`in the major organs. The dose at which the body weight loss (group
`mean value) was ⱕ15% and all mice survived for the duration of
`study was established as the maximum tolerated dose (MTD). A
`deviation in MTD of ⫾1.5% was allowed in cases where body
`weight loss between 15–16.5% lasted only 1 day and where 100%
`recovery of body weight loss was observed, with 100% survival.
`The results were collated from at least 2 independent experiments.
`Select groups were repeated as a quality control measure to ensure
`reproducibility.
`
`In vivo anti-tumor activity
`MDA435LCC6 cells were cultured in DMEM and passaged at
`least 3 times in medium. A cell aliquot containing 5 ⫻ 106 cells in
`0.5 ml HBSS was injected i.p. into 2 female SCID/RAG2 mice.
`After 20–25 days, ascites (cells) were removed from the mouse via
`the peritoneal wall using a 20g needle and placed in sterile 15-ml
`conical tubes containing 5 ml of HBSS without Ca and Mg salts.
`The cell suspension was then centrifuged at 1000 g for 5 min and
`the supernatant discarded. Using a 27g needle fitted on a 1-ml
`syringe, 50 µl of the cell suspension was injected into 2 mammary
`fat pads on each mouse (2 ⫻ 106 cells per pad). A period of 18–21
`days was needed for the tumors to become palpable and measurable
`for drug treatments to begin. The MDR MDA435LCC6-MDR-1
`cells were maintained and passaged identically to the WT cells.
`These MDR cells have had the MDR-1 cDNA introduced into the
`MDA435LCC6 cells (Leonessa et al., 1996). Cell passaging and
`inoculation were performed identical to the procedures used in the
`WT sensitive cell line.
`Tumor growth suppression experiments were conducted in
`SCID/RAG2 mice bearing orthotopic human breast carcinoma
`MDA435LCC6 (multidrug resistant, MDR-1 and sensitive, WT)
`solid tumors. After approximately 3 weeks, when the tumors
`(n ⫽ 8 per group) were established, treatment was initiated with
`dosage regimens incorporating i.v. non-encapsulated or liposomal
`DOX with or without p.o. Valspodar (given 4 hr before DOX) on
`days 1, 5 and 9. Experiments were repeated to ensure reproducibil-
`ity of tumor growth inhibition properties. Caliper measurements of
`the tumors were performed daily, and the tumor weights calculated
`according to the formula (Krishna and Mayer, 1997):
`
`Tumor weight (g) ⫽
`
`2
`
`length (cm) ⫻ [width (cm)]2
`
`This conversion formula was verified by comparing the calculation
`derived tumor weights to excised and weighed tumors. Animal
`weights and mortality were monitored daily. Animals bearing
`ulcerated tumors or where tumor weights exceeded 10% of the
`animals’ body weight were terminated. The weights of the bilateral
`tumors were averaged for each mouse and mean tumor weights for
`each treatment group ⫾ standard error of the mean were calculated.
`Statistical tests for these longitudinal data were performed using
`repeat measures ANOVA employing Statistica for Windows 4.0
`(StatSoft, Tulsa, OK) and statistical significance was set at p ⬍
`0.05.
`
`Pharmacokinetics and tissue distribution
`Female MDA435LCC6/MDR-1 solid tumor-bearing SCID/
`RAG2 mice received i.v. via the tail vein a single bolus of
`liposomal doxorubicin (3H EPC/Chol or PEG2000-DSPE/DSPC/
`Chol 0.2:1.0). Valspodar (100 mg/kg) was administered p.o. (in 0.2
`ml) and doxorubicin administered 4 hr later. After DOX dosing,
`groups of 3 mice per time point were anesthetized with 100 µl i.p.
`of ketamine/xylazine at 30 min, 1, 2, 4, 16, 24, 48 and 72 hr. Blood
`was collected by cardiac puncture and placed into EDTA-coated
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`COMPLETE REVERSAL OF MDR IN SOLID TUMORS
`
`133
`
`microtainer tubes. Terminal blood and selected tissues were
`collected from all animals and were processed to determine lipid
`(using radioactivity) and DOX concentrations (using HPLC). After
`blood collection, the kidneys, livers and tumors were removed
`from each animal. Tissues were rinsed in PBS, pat dried on
`absorbent paper and weighed in pre-weighed 16 ⫻ 100-mm tubes.
`Samples were stored in ⫺20°C pending analysis. A 10–30%
`homogenate in distilled water was prepared using a Polytron
`homogenizer (Kinematica, Littau, Switzerland). A 0.2-ml aliquot of
`the homogenate was digested with a tissue solubilizer, decolorized
`with peroxide and analyzed for the lipid label by scintillation
`counting. The specimens obtained from the control animals were
`used as background samples.
`DOX and its metabolites in plasma and tissue extracts were
`determined using HPLC. The HPLC assay of Andersen et al.
`(1993) was used to analyze DOX and its metabolites with minor
`modification. Briefly, sample extraction with acetonitrile was
`followed by isocratic elution from a Nova-Pak C18 3.9 ⫻ 150 mm
`(Millipore, Bedford, MA) analytical reverse phase column and
`quantified by endogenous fluorescence (emission wavelength of
`515 nm). The mobile phase consisted of a 16-mM ammonium
`formate buffer (pH 3.5)/acetone/isopropanol mixture (75:20:5)
`delivered at a rate of 1.0 ml/min. The column was maintained at
`40°C. A NEC (Boxborough, MA) Powermate SX Plus Computer
`and a Systems Interface Module (Waters, Milford, MA) were used
`for data handling.
`Using this system, the retention times of DOXol, DOX, DOXone
`and 7-deoxyDOXone were 3.6, 5.8, 7.5 and 12.6 min, respectively.
`Recoveries, using acetonitrile as the extraction solvent, from
`plasma over a concentration range of 0.05–10 µg/ml of DOX,
`DOXone, 7-deoxyDOXone and DOXol, were between 80–110%.
`To protect DOX and its metabolites from photodegradation, all
`procedures were shielded from direct exposure to light. In addition,
`DOX was found to be stable in the mobile-phase solvent mixture
`for at least 96 hr (40°C), on the HPLC autosampler tray for at least
`96 hr, in reconstituted form at 4°C for at least 10 days and for at
`least 4 freeze-thaw cycles.
`The plasma data were modeled using WinNONLIN Version 1.5
`PK software (Pharsight, Mountain View, CA) to calculate area
`under the curve (AUC), half-life (T1/2) and plasma clearance (CLp)
`according to standard equations (Gibaldi and Perrier, 1982). The
`trapezoidal rule was used to calculate the AUCs in tissue concentra-
`tion-time profiles employing a computer software AUC (program
`provided by Dr. W. Riggs, Faculty of Pharmaceutical Sciences,
`University of British Columbia). Tissue DOX levels were corrected
`for blood volume to account for material residing in the vasculature
`of the tissues, using previously published values (Bally et al.,
`1993).
`Select samples were assayed for free and liposome-associated
`DOX using Microcon-30 filters (Amicon, Oakville, Canada) using
`the equilibrium filtration method of Mayer and St. Onge (1995).
`Separation of free from liposomal and protein-bound drug was
`performed using Microcon-30 (0.5-ml capacity, Amicon) ultrafiltra-
`tion devices with a m.w. cut-off of 30 kDa. Microcon-30 samples
`were centrifuged at 4°C, 8,000 g for 20 min in a microcentrifuge
`(IEC Micromax Centrifuge; International Equipment, Needham
`Heights, MA). The ultrafiltrate was processed for DOX by HPLC
`(see above).
`
`Confocal microscopy and imaging studies
`For confocal
`imaging studies, SCID/RAG2 mice bearing
`MDA435LCC6/MDR-1 tumors were treated with non-encapsu-
`lated DOX, EPC/Chol DOX or PEG-DSPE/DSPC/Chol DOX (5
`mg/kg) in the presence and absence of Valspodar 100 mg/kg (4 hr
`before DOX). At the indicated times following DOX administra-
`tion, tissues were aseptically dissected, bathed in PBS and imaged
`fresh. Before imaging, thin pieces of tumors were placed on
`concave slides and observed under a 60⫻ oil immersion lens.
`These were then viewed under the confocal microscope to deter-
`
`mine DOX distribution characteristics. As controls, known amounts
`of non-encapsulated DOX or PEG-DSPE/DSPC/Chol DOX lipo-
`somes were infused into freshly isolated muscle tissues and viewed
`for DOX fluorescence.
`Confocal images were collected on an Optiphot 2 research
`microscope (Nikon, Tokyo, Japan) attached to a confocal laser
`scanning microscope (MRC-600, BioRad, Hercules, CA) using
`COMOS software (BioRad). The laser line on the krypton/argon
`laser was 488 nm. Filterblock BHS was used to detect DOX (488
`nm excitation, 515 nm emission). The numerical aperture was 0.75
`on the ⫻20 air objective and 1.2 on the ⫻60 oil objective. The
`images were captured such that the xyz dimensions were 0.4 µm
`cubed (⫻20) and 0.2 µm pixel (⫻60). NIH Image version 1.61 was
`used for image analysis, and all images were based on maximum
`intensity projection. Projections made in the NIH Image were
`saved in TIFF format, then imported to Adobe Photoshop version
`4.0 where the different fluorophore images were assigned to
`individual RGB channels and subsequently merged to provide the
`final image of the single or multiple sections.
`
`Toxicity
`
`RESULTS
`
`In order to identify the MTD for the day 1, 5 and 9 dosage
`regimen used in therapeutic experiments as well as to investigate
`the mechanisms of Valspodar-mediated increases in DOX toxicity,
`21-day dose range-finding toxicity studies were conducted with
`EPC/Chol, DSPC/Chol and PEG-DSPE/DSPC/Chol
`liposomal
`formulations of DOX as well as non-encapsulated drug in the
`presence and absence of p.o. Valspodar in non-tumor-bearing
`healthy mice. The results of this study are shown in Table I, where
`the body weight loss is presented, along with the MTDs (as defined
`in Material and Methods). The 3 liposomal DOX formulations
`yielded variable toxicity characteristics that depended on their
`respective abilities to retain DOX.
`In the absence of Valspodar, EPC/Chol DOX exhibited an MTD
`of 15 mg/kg, where the body weight loss was 1.7% with 100%
`survival (Table I). For the 2 saturated lipid formulations, DSPC/
`Chol and PEG-DSPE/DSPC/Chol DOX, the MTD was identical at
`25 mg/kg, representing a 1.7-fold increase in MTD compared with
`the more leaky EPC/Chol formulation. When EPC/Chol DOX was
`
`TABLE I – TOXICITY AS A FUNCTION OF DOX DOSE FOR NON-ENCAPSULATED
`AND LIPOSOMAL FORMULATIONS1
`
`Group
`
`Non-encapsulated
`DOX
`
`MTD
`
`EPC/Chol
`DOX
`
`MTD
`
`DSPC/Chol
`DOX
`
`MTD
`
`PEG-DSPE/DSPC/Chol
`DOX
`
`MTD
`
`Dose
`(mg/kg)
`
`2.5
`5
`7.5
`10
`
`5
`10
`15
`20
`
`15
`20
`25
`
`15
`20
`25
`30
`
`Day 10 weight loss (% survival)
`
`⫺PSC 833
`
`⫹PSC 833
`
`0 (100)
`3.5 (100)
`10.7 (100)
`29.2 (0)
`7.5 mg/kg
`
`2.8 (100)
`1.7 (100)
`20.4 (0)
`15 mg/kg
`
`10.8 (100)
`3.1 (100)
`16.0 (100)
`25 mg/kg
`
`7.3 (100)
`6.5 (100)
`9.3 (100)
`4.4 (67)2
`25 mg/kg
`
`10.2 (100)
`18.5 (0)
`28.2 (0)
`
`2.5 mg/kg
`
`0.9 (100)
`23.1 (0)
`
`24.4 (0)
`5 mg/kg
`
`12.9 (100)
`15.0 (100)
`18.5 (67)
`20 mg/kg
`
`9.8 (100)
`14.8 (100)
`14.8 (100)
`25.3 (0)
`25 mg/kg
`
`1Data are group mean values (n ⫽ 3 mice per group treated i.v. on
`days 1, 5 and 9).–2Body weight loss nadir is on day 19 (⫺10.2%) with
`67% survival.
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`134
`
`KRISHNA ET AL.
`
`combined with Valspodar, significant toxicity resulted, necessitat-
`ing a dose reduction by 3-fold, to 5 mg/kg. This is comparable to
`the 3-fold decrease in MTD caused by Valspodar for non-
`encapsulated drug administration (Table I). In contrast, Valspodar
`necessitated a small 1.2-fold decrease in DOX encapsulated in
`DSPC/Chol liposomes and no dose reduction was required for the
`PEG-containing sterically stabilized liposomes (Table I).
`
`Effıcacy
`The anti-tumor activity of the 3 types of liposomal DOX
`formulations was evaluated in vivo in the absence and presence of
`Valspodar using the MDA435LCC6 and PGP-overexpressing
`
`MDA435LCC6/MDR-1 human breast carcinoma xenograft solid
`tumor models. Figure 1 presents the tumor growth curves for mice
`treated with non-encapsulated drug (a), EPC/Chol DOX (b),
`DSPC/Chol DOX (c) and sterically stabilized PEG-DSPE/DSPC/
`Chol DOX (d ) in the presence and absence of Valspodar.
`When MDA435LCC6 cells (sensitive, WT or resistant, MDR)
`are inoculated in the mammary fat pads of SCID/RAG2 mice, solid
`tumors readily establish (tumor take rates ⬎95%). Figure 1 shows
`that both MDR and WT-untreated controls exhibit comparable
`tumor growth rates, with the exponential growth phase occurring
`between days 5 and 14 (slope of linear regression line ⫽ 0.067 for
`
`FIGURE 1 – Anti-tumor efficacy of free (a), EPC/Chol DOX (b), DSPC/Chol (c) and PEG-DSPE/DSPC/Chol (d) against MDA435LCC6 WT or
`MDR1 human xenograft solid tumors in the absence and presence of co-administered Valspodar. MDA435LCC6 tumors were grown on mammary
`fat pads of female SCID/RAG2 mice. Oral Valspodar (100 mg/kg) and i.v. DOX treatments were initiated once tumors were established (20–100
`mg) and were given on days 1, 5 and 9 at the indicated doses of free and liposomal DOX. Valspodar was administered 4 hr prior to DOX injection.
`Data are expressed as mean ⫾ standard error of the mean. For legends, see individual panels.
`
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`COMPLETE REVERSAL OF MDR IN SOLID TUMORS
`
`135
`
`WT vs. 0.056 for MDR tumors). Both groups were terminated on
`day 18, when tumor weights reached 0.84 ⫾ 0.05 and 0.76 ⫾ 0.05 g
`for the WT and MDR tumors, respectively. Treatment of WT
`tumors with non-encapsulated DOX at 7.5 mg/kg on days 1, 5 and 9
`(Fig. 1a) resulted in significant tumor growth suppression until day
`20, when tumors weighed 0.088 ⫾ 0.01 g. These tumors eventually
`grew to 0.68 ⫾ 0.09 g by day 40. However, administration of
`non-encapsulated DOX at 7.5 mg/kg on days 1, 5 and 9 in mice
`bearing MDR tumors did not cause any tumor growth suppression
`(Fig. 1a). When MDR tumor-bearing mice were treated with the
`MTD of non-encapsulated DOX combined with Valspodar (DOX
`dose of 3 mg/kg), there was partial tumor growth inhibition until
`day 11. After this time,
`tumor growth rates were similar to
`untreated MDR controls until day 18 when mice were terminated
`(Fig. 1a). Growth of MDR tumors treated with non-encapsulated
`DOX plus Valspodar was significantly different from both un-
`treated MDR tumors as well as MDR tumors treated with
`non-encapsulated drug alone between days 7 and 12 post-DOX
`administration. However, in comparison to WT tumors treated with
`non-encapsulated DOX 7.5 mg/kg, the MDR tumor growth inhibi-
`tion caused by non-encapsulated drug and Valspodar was transient
`(Fig. 1a).
`When MDR tumors were treated with liposomal DOX formula-
`tions in the presence and absence of Valspodar (Fig. 1b–d ), varying
`degrees of tumor growth suppression were observed. As seen in
`Figure 1b, EPC/Chol DOX alone (5 mg/kg) was unable to induce
`substantial inhibition of MDR tumor growth, with tumors weighing
`0.86 ⫾ 0.2 g on day 18. In the presence of Valspodar, however,
`EPC/Chol DOX at 3 mg/kg treatment closely resembled EPC/Chol
`DOX alone until day 12, after which tumor growth was decreased
`between days 12 and 20 (Fig. 1b). This indicated modest delayed
`anti-tumor activity. This MDR modulation caused by EPC/Chol
`DOX and Valspodar was significantly better than that caused by
`non-encapsulated DOX and Valspodar ( p ⬍ 0.05).
`Figure 1c illustrates the tumor growth inhibition of DSPC/Chol
`liposomal DOX in the presence and absence of Valspodar. In the
`absence of Valspodar, DSPC/Chol liposomal DOX caused a modest
`reduction in tumor growth. The tumor growth inhibition caused by
`DSPC/Chol DOX was significantly different from untreated con-
`trols until day 12, after which the tumor growth rate increased.
`Tumor weight for MDR tumors treated with DSPC/Chol DOX
`alone was 0.51 ⫾ 0.1 g on day 20. In comparison, Valspodar caused
`a significant increase in DSPC/Chol DOX tumor growth suppres-
`sion of the MDR solid tumors, where tumor weight was 0.25 ⫾
`0.05 g on day 20. The tumor growth inhibition resulting from
`DSPC/Chol DOX and Valspodar
`treatment was significantly
`( p ⬍ 0.05) greater than that observed for EPC/Chol DOX and
`Valspodar.
`PEG-DSPE/DSPC/Chol DOX formulations displayed superior
`anti-tumor activity in the presence and absence of Valspodar when
`compared with the 2 liposomal DOX formulations described above
`as well as non-encapsulated drug (Fig. 1d ). In the absence of
`Valspodar, PEG-DSPE/DSPC/Chol DOX caused a significant
`reduction in tumor growth until day 11, when tumor weight was
`0.1 ⫾ 0.01 g (compared with a tumor weight of 0.05 ⫾ 0.01 g on
`day 1), after which tumor growth rates increased. Tumor weight for
`this group was 0.39 ⫾ 0.02 g on day 20. In the presence of
`Valspodar, PEG-DSPE/DSPC/Chol DOX significantly inhibited
`tumor growth and this growth suppression was not significantly
`different from WT tumors treated with non-encapsulated DOX at
`its MTD. Specifically, the tumor weights on day 20 for PEG-DSPE/
`DSPC/Chol DOX in presence of Valspodar were 0.1 ⫾ 0.02 g
`compared with 0.088 ⫾ 0.01 g for WT tumors treated with
`non-encapsulated DOX. This suppression of tumor growth for
`PEG-DSPE/DSPC/Chol DOX and Valspodar was significantly
`different ( p ⬍ 0.05) from all other treatment groups for mice
`bearing MDR solid tumors.
`
`Pharmacokinetics and tissue distribution
`A comprehensive PK evaluation was performed to correlate
`toxicity and efficacy data with plasma and tissue (tumor and liver)
`DOX and DOX metabolite distribution properties determined using
`HPLC analysis. The comparison of DSPC/Chol DOX and non-
`encapsulated (free) DOX pharmacokinetics and tissue distribution
`properties in the presence and absence of Valspodar has been
`extensively characterized (Krishna and Mayer, 1997). Since the
`toxicity and efficacy properties of these formulations were very
`comparable in the current models,
`the results presented here
`focused on comparisons between a liposomal system which leaks a
`significant portion of entrapped drug into the circulation (EPC/
`Chol DOX), and a sterically stabilized system that exhibits
`negligible drug release in plasma compartment and increased
`circulation lifetimes (PEG-DSPE/DSPC/Chol DOX). As a refer-
`ence comparison, non-encapsulated DOX treatments (with or
`without Valspodar) were evaluated for DOX concentrations in
`plasma and the tumor. The goal of these comparisons was to reveal
`important DOX distribution and metabolism properties that dictate
`toxicity and efficacy behavior.
`
`Plasma drug kinetics
`Figure 2a presents the DOX plasma concentrations after i.v.
`administration of non-encapsulated DOX and the 2 liposomal DOX
`formulations at a DOX dose of 5 mg/kg. Following administration
`of non-encapsulated drug, DOX is rapidly eliminated from the
`circulation. Concentrations of DOX beyond 4 hr were below assay
`detection limits. The concentration-time profile was characterized
`by a Cmax of 1.5 ⫾ 0.1 µg/ml and an AUC of 4.4 µg.hr/ml (Fig. 2a).
`However, when Valspodar was co-administered with non-
`encapsulated DOX at 5 mg/kg, DOX elimination from plasma was
`characterized by a prolonged terminal elimination phase (Fig. 2a).
`Valspodar caused significant ( p ⬍ 0.05) increases in Cmax (3.9 ⫾ 0.5
`µg/ml) and AUC (48.1 µg.hr/ml) of non-encapsulated DOX com-
`pared to data obtained in the absence of the MDR modulator. This
`approximately 11-fold increase in DOX AUC caused by Valspodar
`is consistent with the 10-fold increase in DOX AUC for the
`non-encapsulated DOX-Valspodar combination observed in the
`P388/ADR solid tumor model described earlier (Krishna and
`Mayer, 1997).
`As shown in Figure 2a, DOX elimination from plasma for both
`EPC/Chol and PEG-DSPE/DSPC/Chol DOX systems exhibits a
`monophasic elimination profile characterized by a 1-compartment
`model with first-order elimination. While EPC/Chol DOX plasma
`concentration exhibits rapid elimination of the drug within 24 hr,
`PEG-DSPE/DSPC/Chol DOX displays a prolonged circulation
`life-time with over 20% remaining at 24 hr. In all cases, parent
`DOX was the only detectable entity with no indication of any
`metabolites present. In the presence of Valspodar, the elimination
`profile remained monophasic, however, DOX concentrations at
`earlier time points were significantly ( p ⬍ 0.05) increased for
`EPC/Chol liposomes whereas no such Valspodar effect was ob-
`served for PEG containing DSPC/Chol liposomes. This is contrast
`to observations for non-encapsulated DOX which demonstrated
`increases in the terminal elimination phase in the presence of
`Valspodar (Fig. 2a). Co-administration of Valspodar and EPC/Chol
`DOX increased the AUC of DOX by 2.6-fold and Cmax by 2.3-fold,
`accounting for the 40% reduction in plasma clearance (Table II;
`significant at p ⬍ 0.05). In contrast, Valspodar caused minor
`changes in the pharmacokinetics of DOX encapsulated in PEG-
`DSPE/DSPC/Chol liposomes, with small 36% and 25% increases
`in Cmax and AUC, respectively (Table II).
`Figure 2b shows the elimination of liposomal lipid from plasma.
`These data demonstrate that, similar to DOX pharmacokinetics,
`liposomal lipid elimination is monophasic, characterized by a
`1-compartment model with first-order elimination. The plasma
`clearance (CLp) for PEG-DSPE/DSPC/Chol liposomes was 2.4-
`fold lower than EPC/Chol, and the half-life (T1/2) was 2.2-fold
`higher for PEG-DSPE/DSPC/Chol liposomes compared to EPC/
`
`AMN1077
`Amneal Pharmaceuticals LLC v. Alkermes Pharma Ireland Limited
`IPR2018-00943
`
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`KRISHNA ET AL.
`
`Chol (Table II). Valspodar did not significantly alter the liposomal
`lipid elimination kinetics for either liposomal formulation, as
`demonstrated by the small changes in lipid ha