Topotecan and Doxorubicin Combination to Treat Recurrent

Published OnlineFirst January 9, 2013; DOI: 10.1158/1078-0432.CCR-12-2459

Clinical Cancer Cancer Therapy: Preclinical Research

Topotecan and Doxorubicin Combination to Treat Recurrent Ovarian Cancer: The Inﬂuence of Drug Exposure Time and Delivery Systems to Achieve Optimum Therapeutic Activity

Nilesh A. Patankar1,3,5, Julia Pritchard3, Mariska van Grinsven3, Maryam Osooly5, and Marcel B. Bally1,2,3,4

Abstract Purpose: To provide proof-of-concept data to support use of Doxil–liposomal topotecan (Topophore C) combinations to treat ovarian cancer. Experimental Design: ES-2, OVCAR-3, and SKOV-3 ovarian cancer cell lines were treated with doxorubicin–topotecan combinations by exposing the cells to drugs from 1 to 72 hours. Pharmacokinetic analysis was conducted following administration of liposomal formulations of these drugs alone and in combination. Efﬁcacy assessments were completed in ES-2 and SKOV-3 ovarian cancer models. Results: On the basis of drug doses capable of achieving 50% reduction in cell viability over 72 hours, doxorubicin–topotecan combinations were additive in SKOV-3 but highly synergistic in ES-2 and OVCAR-3 cells. Favorable drug–drug interactions increased with increased drug exposure time. Topophore C pharmacokinetic remained unaffected when co-administered with Doxil. In the ES-2 model, Doxil at maximum tolerated dose (MTD 7.5 mg/kg) in combination with free topotecan (MTD 15 mg/kg) did not enhance median survival time (MST) over that achieved with topotecan alone. In contrast, MST was increased to 52 days with combination of Topophore C (MTD 2.5 mg/kg) and Doxil (7.5 mg/kg) compared with untreated animals (MST 18 days) or those treated with Topophore C alone (MTD 5 mg/kg, MST 40 days). In the SKOV-3 model, combination treatments showed better therapeutic efﬁcacy than the individual drugs. Conclusions: Topotecan–doxorubicin combinations produced additive or synergistic effects which were best achieved when the tumor cells were exposed to drugs over extended time. Doxil–Topophore C combinations are therapeutically superior as judged in two ovarian cancer models. Clin Cancer Res; 1–13. 2012 AACR.

Introduction signaling pathways uniquely expressed in the patients with Ovarian cancer is among the most common gynecologic ovarian cancer (4). While these more specific therapeutic cancers and is the leading cause of cancer-related deaths agents are offering benefits in many cancers, they are not among female patients with cancer (1, 2). Platinum-refrac- replacing the use of existing cytotoxic drugs. In fact, the tory ovarian cancer is considered an incurable disease and targeted drugs often exhibit poor therapeutic effects when the treatment options available at present are primarily used alone and their therapeutic value is achieved primarily palliative in nature (3). There is a need to develop improved in the combination setting. Thus, there remains a strong treatment options for these patients, and a great deal of rationale for exploring the use of strategies that can enhance hope has been placed on the identification and develop- the effects of existing cytotoxic drugs when given alone and ment of molecularly targeted drugs; drugs that can affect in combination (5–7). Combination chemotherapy is an effective strategy that has shown promise in the treatment of ovarian cancer. The concept of developing drug combina- Authors' Affiliations: 1Faculty of Pharmaceutical Sciences, 2Department tions selected on the basis of synergistic drug–drug inter- of Pathology & Laboratory Medicine, University of British Columbia; actions has been promoted (5, 6). Synergy can be defined as 3Experimental Therapeutics, B.C. Cancer Agency; 4Centre for Drug Research and Development, Vancouver, British Columbia, Canada; and an interaction that results in therapeutic effects that are 5Merrimack Pharmaceuticals, Cambridge, Massachusetts greater than that which could be expected from single-agent Note: Supplementary data for this article are available at Clinical Cancer activities (7–9). An alternative perspective is that a syner- Research Online (http://clincancerres.aacrjournals.org/). gistic drug combination can achieve therapeutic effects Corresponding Author: Nilesh A. Patankar, Merrimack Pharmaceuticals, equal to that achievable with single agents, but at signifi- 1 Kendall Square, Suite B7201, Cambridge, MA 02139. Phone: 857-919- cantly lower, better tolerated, drug doses (5). It is not well 4819; Fax: 617-491-1386; E-mail: [email protected] understood what factors influence synergy; however, exist- doi: 10.1158/1078-0432.CCR-12-2459 ing evidence suggests that drug dose, drug–drug ratio, and 2012 American Association for Cancer Research. drug sequencing all influence combination interactions

www.aacrjournals.org OF1

Patankar et al.

combinations (24–26). This concept envisioned develop- Translational Relevance ment of novel combination products (2 drugs formulated Combination therapy in the treatment of recurrent in a single LNP composition) or the combination of 2 ovarian cancer can be promising if the combination is different LNP formulations. A number of groups, includ- designed to achieve therapeutic synergy between selected ing our own, have been pursuing the development of drugs with proven therapeutic value. As the therapeutic optimized LNP formulations for topotecan (27–30), and effects of anticancer drugs are highly dependent on drug many of these exhibit promising therapeutic potential concentration and exposure time, these variables are based on preclinical studies. The formulation recently critically important in achieving optimal therapeutic developed in our laboratory uses pH gradient encapsu- outcomes for the combinations. Drug carriers are impor- lation methods combined with drug complexing ability tant tools to achieve increased drug concentrations at of encapsulated copper to prepare a formulation that sites of tumor growth over extended time periods. exhibits improved drug retention in vivo and improved Our results showed that an approved liposomal therapeutic activity when compared with the free drug formulation of doxorubicin (Doxil), when used in com- (30). This formulation, referred to as Topophore C, is bination with a liposomal topotecan formulation, now undergoing extensive preclinical evaluations. In an achieved significantly better treatment outcomes against effort to guide the clinical development of Topophore C, models of ovarian cancer. This combination also our preclinical studies have placed emphasis on its use in achieved improved therapeutic effects at lower drug relapse ovarian cancer. In the studies described here, doses that are better tolerated, and hence will be ideally Topophore C combinations with Doxil were assessed in suited for use in the context of emerging targeted ther- an effort initiated around the hypothesis that drug–drug apies that will ultimately lead to a time when refractory interactions promoting synergy will be dependent on ovarian cancer is treated with curative intent and not exposure time for the drugs used. As LNP formulations merely as a palliative care. provide a means to enhance exposure time of drugs, combinations of Topophore C and Doxil should provide a means to achieve optimal drug combination effects in vivo. The results indicate that the synergistic effects (5, 10, 11). In this study, the role of drug exposure time on achieved when using topotecan and doxorubicin in com- drug–drug interaction is being explored with the goal of bination can increase when exposure time increases and defining an improved drug combination for use in patients the therapeutic effects of Topophore C in combination with relapsed ovarian cancer. Currently, the U.S. Food and with Doxil are significantly better than those obtained Drug Administration (FDA) has approved topotecan (12– when using combinations of topotecan and Doxil. 17) and a liposomal formulation of doxorubicin (Doxil) as single agents for use in second-line therapy for ovarian Materials and Methods cancer (18, 19). Topotecan is a camptothecin analogue that Materials and chemicals specifically targets DNA–topoisomerase I complex and Doxorubicin (Adriamycin), topotecan (Hycamtin), and induces DNA damage by stabilizing this complex and Doxil were purchased from the British Columbia Cancer thereby acts as a topoisomerase I (topo I) inhibitor (20). Agency Pharmacy (Vancouver, BC, Canada). ES-2, SKOV-3, Doxorubicin, an anthracycline analogue, acts by stabilizing and OVCAR-3 human ovarian carcinoma cells lines the DNA–topoisomerase II complex after it has broken the were obtained from the American Tissue Culture DNA chain for replication, preventing the DNA double Collection. SKOV-Luc-D3 cell line was obtained from Cal- helix from being resealed and thereby stopping the process iper Life Sciences. 1,2-Distearoyl-sn-glycero-3-phosphocho- of replication (21). Combination of topo I and II inhibitors line (DSPC) was purchased from Avanti Polar Lipids and have been evaluated and results suggest synergy between 3H-cholesteryl hexadecyl ether (3H-CHE) from PerkinElmer them when administered sequentially (18, 19, 22, 23). Life Sciences. 14C-sucrose and Pico-Fluor 40 scintillation Interestingly, the combinations of topo I and topo II inhi- cocktail were purchased from PerkinElmer Life Sciences. The bitors currently being pursued for treatment of patients with divalent cationic ionophore A23187 (calcimycin), HEPES, relapsed ovarian cancer actually involve the use of a free Sephadex G-50, cholesterol (CH), and all other chemicals topo I inhibitor (topotecan) and a liposomal nanoparticu- (Reagent grade) were purchased from Sigma-Aldrich. late (LNP) formulation of a topo II inhibitor (Doxil). However, considering pharmacokinetic differences, the Cell culture LNP formulation will exhibit remarkably different plasma ES-2 and SKOV-3 cells were grown in McCoy’s 5A medi- elimination rates and biodistribution behavior when com- um containing 1.5 mmol/L L-glutamine and 10% FBS. pared with free topotecan; therefore, the drug levels and the OVCAR-3 cells were grown in RPMI-1640 medium supple- drug–drug ratio at the sites of tumor growth will be variable mented with 20% FBS, 0.01 mg/mL bovine insulin, and 2.5 over time. mmol/L L-glutamine. ES-2 cells are representative of clear It has been shown that the pharmacokinetic and bio- cell carcinoma and in animal models grow aggressively. distribution behavior of drug combinations can be con- The SKOV-3 and OVCAR-3 cell lines represent an ovarian trolled better when using LNP formulations of drug cancer referred to as serous adenocarcinoma and in animal

OF2 Clin Cancer Res; 2012 Clinical Cancer Research

Topotecan–Doxorubicin Combination in Ovarian Cancer

models the SKOV-3 cell line grows very slowly. Cells were action between the 2 drugs results in synergistic (CI < 1), subcultured when 80% to 90% confluent by rinsing with additive [CI ¼ 1(0.2)], or antagonistic (CI > 1) effects. PBS and detached from flask with 0.25% trypsin. Once detached, cells were counted using a hemocytometer and Preparation of Topophore C diluted in media to the appropriate concentration (see Liposomes were prepared using DSPC and cholesterol below) before addition to 96-well microtiter Falcon plates. (CH; 55:45 molar ratio) by film hydration extrusion as Cells were maintained in culture for up to 20 passages. After described previously (30). Briefly, DSPC and CH were 20 passages, new cells were expanded from frozen stock weighed, dissolved in chloroform, and then mixed such vials and stored in liquid nitrogen. that the final mole ratio of the 2 lipids was 55:45, respectively. A nonexchangeable and nonmetabolizable lipid Cell viability assay marker 3H-CHE (5 mCi/100 mmol total lipid) was used to The MTT assay was used as a measure of cell viability. In label the liposomes. This solution was then dried to a thin brief, 100 mL of cell suspension containing required cell film with a gentle stream of nitrogen gas. The residual number (6 103 for ES-2 and SKOV-3 and 1 104 for chloroform was removed by placing the lipid film under OVCAR-3) was added to the wells of 96-well plates and high vacuum for at least 3 hours. Dried lipid films were incubated at 37 C in humidified air with 5% CO2.After hydrated at 65 C by mixing with 300 mmol/L CuSO4 24 hours, 100 mL of cell culture medium containing (unbuffered, pH 3.5). Following hydration, the sample was appropriate concentration of either topotecan hydrochlo- subjected to 5 freeze (liquid nitrogen) and thaw (65C) ride or doxorubicin hydrochloride (0–100,000 nmol/L) cycles. The multilamellar vesicles (MLV) obtained were was added to these cells. Following 72 hours of this extruded 10 times through stacked polycarbonate filters of treatment, 50 mL of MTT solution was added to the wells 0.1- and 0.08-mm pore size at 65C using an Extruder and plates were incubated at 37C for 3.5 hours. Super- (Northern Lipids). The size of the large unilamellar vesicles natant was aspirated before dissolving the formazan generated using this method was determined using Phase crystals in 150 mL dimethyl sulfoxide (DMSO). Plates Analysis Light Scattering (ZetaPALS, Brookhaven Instru- were agitated for 10 minutes, and the optical density of ments Corp.). The external buffer of large unilamellar each well was read at 570 nm using a microplate reader vesicles was exchanged with sucrose (300 mmol/L), HEPES (Thermo Multiskan Spectrum). Cell viability was deter- (20 mmol/L), and EDTA (15 mmol; SHE buffer) at pH 7.5 mined by comparing absorbance from treated wells by running the sample through a Sephadex G-50 column against that from control wells (cells treated with culture equilibrated with the buffer. Liposomal lipid concentration medium instead of drug). Concentrations of topotecan was determined by measuring 3H-CHE using liquid scintil- and doxorubicin needed to cause 50% loss in viability as lation counting (Packard 1900TR Liquid Scintillation Ana- judged by the MTT assay (IC50) were determined. To lyzer). To load topotecan, liposomes with encapsulated determine the effect of prolong exposure of drugs onto copper sulfate (unbuffered solution, pH 3.5) were sus- the cells, cells were treated with increasing concentrations pended in SHE buffer (pH 7.5). Subsequently, A23187 of topotecan or doxorubicin and incubated for different (0.5 mg per 1 mg lipid) was added to the liposomes which time periods (1, 4, 8, 24, 48, and 72 hours). Following were then incubated at 30C for 30 minutes. This mixture each time point, media-containing drug were aspirated and a reconstituted solution of topotecan were warmed off the cells and 200 mL fresh media was added and the separately at 60C for 5 minutes using a temperature bath. cells were incubated for an additional time frame such Immediately before addition of the drug to the liposomes a that the total time in culture was 72 hours. To determine sufficient volume of 1 N NaOH was added such that the final the activity of the topotecan–doxorubicin combinations, pH of the suspension was 7.0 to 7.5 after addition of fixed ratio of the 2 agents was generated on the basis of a topotecan. The final drug–liposome mixture was incubated ratio defined by the IC50 of doxorubicin and topotecan in in a water bath at 60 C for 60 minutes. Following loading, the indicated cell line. These ratios were then tested over a the mixture was brought to the room temperature, and broad range of effective doses for activity against the 3 cell unencapsulated topotecan was separated from liposomes lines using the MTT assay as described above. To deter- on a Sephadex G-50 column pre-equilibrated with PBS (pH mine the effect of exposure time, fixed ratio combinations 7.5). Lipid concentrations were measured by scintillation of the 2 agents was exposed to the cell lines for different counting of the lipid marker 3H-CHE. Topotecan concen- lengths of time as described above. trations were determined by measuring the absorbance at All in vitro assays were conducted in triplicate and mean 370 nm on UV–Vis spectrophotometer (Agilent/Hewlett values obtained from 3 separate experiments were used for Packard, model: 8453, Agilent Technologies) as described further analysis. The dose-dependent effects of the drugs previously (30). Briefly, a portion of the sample collected when used alone and in combination were analyzed using from the spin columns was adjusted to 100 mL followed by CompuSyn, a computer program that analyzes dose– addition of 900 mL Triton X-100 (1% v/v). This sample was response data according to the Chou and Talalay median heated in a 90C water bath until the cloud point of the effect principle (MEP; refs. 6, 8, 9). The program generates detergent was observed. Subsequently, the sample was combination index (CI) values from the dose–response cooled to room temperature and the absorbance was deter- curves and provides an indication as to whether the inter- mined and compared against topotecan standard curve.

www.aacrjournals.org Clin Cancer Res; 2012 OF3

Patankar et al.

Pharmacokinetic analysis of drug combinations perature was maintained at 4C, and the column temper- Doxil (7.5 mg/kg), a combination of free topotecan (15 ature was adjusted to 55C. Each sample was run for 14 mg/kg) and Doxil (7.5 mg/kg) or a combination of Doxil minutes at a flow rate of 1.0 mL/min using a gradient (7.5 mg/kg) and Topophore C (5 mg/kg) was adminis- method, where the amount of organic phase was tered intravenously to female Ncr-Nude mice (Taconic; increased from 12% to 40% over 8 minutes. Plasma area 20–25 g; 4 per time point). To administer a combination, under the curve (AUC) and half-life of topotecan were the specified doses of the respective treatment agents were determined from these data using noncompartmental mixed just before injection. Following administration, at pharmacokinetic (PK) model with the help of PK Solu- specified time intervals via cardiac puncture, the mice tions software (Summit Research Services). were terminated by CO2 asphyxiation, and blood was Doxorubicin concentrations in the plasma were deter- collected by cardiac puncture and placed into EDTA mined by a previously established method (31). Briefly, containing microtainers (Becton Dickinson). The blood plasma samples were mixed with 10% SDS and 10 mmol/L was stored on ice until they were centrifuged (2,500 rpm H2SO4 (1/1/1) and volume was adjusted to 1 mL with for 15 minutes; 500 g) to separate plasma from blood water. This was followed by organic extraction using iso- cells. Plasma topotecan concentrations were determined propanol–chloroform solution (1/1; organic phase to sam- by high-performance liquid chromatography (HPLC). ple ratio of 2:1). The samples were frozen at 80C for 48 HPLC assays were conducted using a Waters Alliance hours to facilitate the precipitation of proteins and then HPLC system equipped with a Waters Model 717 plus thawed at the room temperature. Doxorubicin-containing autosampler, a Model 600E pump, a controller and a organic phase was separated by centrifugation at 2,500 g Model 2474 Multi l Fluorescence Detector (Waters) set at for 10 minutes at room temperature. Doxorubicin equiva- an excitation wavelength of 360 nm and an emission lent fluorescence in the organic phase was determined using wavelength of 425 nm. Samples were prepared by dilut- a luminescence spectrophotometer (Perkin Elmer LS50B) ing in a acetonitrile:methanol mixture (50:50 v/v). About with an excitation wavelength of 470 nm (slit width ¼ 2.5) 10 mL of diluted sample was injected onto a Waters and an emission wavelength of 550 nm (slit width ¼ 10). Symmetry Shield RP C18 cartridge column (100 e´, par- The standard curve for doxorubicin was generated by ticle size 3.5 mm; 75 4.6 mm, Waters). Mobile phase extracting it into the organic phase using the procedure consisted of mobile phase "A" (1% triethyleneamine in described above, and fluorescence readings from the sam- water, pH 6.4 adjusted with glacial acetic acid) and ples were compared against the freshly prepared standard mobile phase "B" (100% acetonitrile). The sample tem- curve.

AB 120 120 1 h 100 4 h 100 8 h 24 h 80 48 h 80 72 h 60 60 40 40 1 h % Cell viability

20 % Cell viability 4 h 8 h Figure 1. Effect of exposure time on 20 24 h 0 48 h the cytotoxicity (MTT assay 72 h endpoint) of topotecan against (A) −20 0 1 10 100 1,000 10,000 0.1 1 10 100 1,000 10,000 ES-2, (B) OVCAR-3, and (C) SKOV- Concentration (nmol/L) Concentration (nmol/L) 3 cell lines. Values indicate mean CD SE of at least 3 individual experiments. D, effect of exposure 140 10,000 time on the IC50 of topotecan 1 h ES-2 120 4 h OVCAR-3 against ES-2, OVCAR-3, and 8 h SKOV-3 24 h SKOV-3 cells. 100 48 h 72 h 1,000 80 50

60 IC 100

% Cell viability 40

0 0 110100 1,000 10,000 0 204060 80 Concentration (nmol/L) Time (h)

OF4 Clin Cancer Res; 2012 Clinical Cancer Research

Topotecan–Doxorubicin Combination in Ovarian Cancer

Assessment of antitumor efficacy minutes (as accurately as possible) after luciferin injection. In vivo assessments of antitumor efficacy were completed Regions of interest covering the entire peritoneal cavity were in 2 ovarian cancer models. For ES-2 model development, selected for the determination of total photon counts emit- ES-2 cells (1 105/500mL) were inoculated intraperitone- ted per second. One week after tumor cell inoculation, mice ally (i.p.) into female NCr-Fox1nu mice (Taconic). Seven were treated i.v. (every 7 days 3) at the drug doses days after tumor cell inoculation, treatment groups were specified. Control mice groups were injected with saline. treated i.v. (every 7 days 3) at the indicated drug doses. Following administration of the first dose, treated and Control mice groups were injected with saline. The drug control mice were imaged once a week to monitor tumor doses were escalated to levels that were close to maximum progression. All animals were observed postinoculation at tolerated doses (MTD), and the health status of all animals least 2 times a day, more if deemed necessary, for signs of inoculated with tumor cells was monitored carefully. In the morbidity. The health status of mice was monitored as event that the health status was poor as judged using a described above. All animal studies described above were scoring method defined in a standard operating procedure, conducted according to a protocol approved by the Uni- mice were terminated by CO2 asphyxiation. A balance versity of British Columbia’s Animal Care Committee. between measurable signs (weight loss, food intake, water intake, and stool softness) and behavioral changes (activity) Statistical analysis as well as physical appearance (coat and eye appearance) The results were analyzed using ANOVA. Significant was taken into consideration for euthanasia. Survival times differences between groups were identified using Stu- of the mice were recorded for all groups, and the day of dent–Newman–Keul multiple comparison post hoc test death was reported as 1 day after the mice were euthanized (GraphPad Instat software - GraphPad). Survival curves because of poor health status. generated using Kaplan–Meier plots were compared for For the SKOV-3 tumor model, 5 106 SKOV-3-Luc-D3 statistical significance using log-rank (Mantel–Cox) test cells in 500 mL were inoculated intraperitoneally into female (GraphPad Prism software, GraphPad). Differences mice (Ncr-nude, 20–25 g). Tumor growth was monitored between the groups were considered significant if P < 0.05. one time per week noninvasively by bioluminescent imaging with an IVIS200 imager (Xenogen). The commercially Results available Living Image software (Xenogen) was used to Cell viability assays following treatment with obtain and analyze images. Before imaging, mice were topotecan and doxorubicin alone and in combination injected i.p. with 500 mL luciferin solution (15 mg/mL), Results from the MTT assays have been summarized in anesthetized with isoflurane. Animals were imaged 20 Supplementary Table SI and Figs. 1 (topotecan) and 2

AB 140 120 1 h 120 4 h 8 h 100 100 24 h 48 h 72 h 80 80

60 60

40 40 1 h % Cell viability % Cell viability 4 h 20 8 h Figure 2. Effect of exposure time on 20 24 h 0 48 h the cytotoxicity (MTT assay) of 72 h doxorubicin against (A) ES-2, (B) 0 OVCAR-3, and (C) SKOV-3 cell lines. 10 100 1,000 10,000 100,000 0.1 1 10 100 1,000 10,000 Values indicate mean SE of at least Concentration (nmol/L) Concentration (nmol/L) 3 individual experiments. D, effect of CD exposure time on the IC of 50 120 10,000 topotecan against ES-2, OVCAR-3, 1 h ES-2 4 h OVCAR-3 and SKOV-3 cells. 100 8 h SKOV-3 24 h 48 h 72 h 80 1,000

60 50 IC 40 100

% Cell viability 20

0 0 10 100 1,000 10,000 100,000 0 20 40 60 80 Concentration (nmol/L) Time (h)

www.aacrjournals.org Clin Cancer Res; 2012 OF5

Patankar et al.

(doxorubicin). Regardless of the cell line used, topotecan ovarian cancer cell lines which ranged from 15:1 to 6.5:1 was more potent than doxorubicin. As noted, the IC50 for (doxorubicin to topotecan). These combinations were test- topotecan ranged from a low of 18 nmol/L in the SKOV-3 ed over a broad range of effective doses and the dose– cell line to a high of 83 nmol/L in the ES-2 cells. Doxoru- response curves have been summarized in Fig. 3A (ES-2 bicin IC50 ranged from a low of 251 nmol/L in the SKOV-3 cells), B (OVCAR-3 cells), and C (SKOV-3 cells). In all cells to a high of 539 nmol/L in the ES-2 cells. Exposure examples, these 72-hour studies show that drug combina- studies showed for all 3 cell lines that drug concentration tion was as active (SKOV-3 cells) or more active than the required to achieve an effect level of 50% (fa ¼ 0.5) agents when used alone. It is difficult to interpret drug–drug decreased as the exposure time increased. The activity of interactions on the basis of the sigmoidal dose–response topotecan (Fig. 1) was much more exposure time–depen- curves shown in Fig. 3 and for this reason the results were dent than doxorubicin (Fig. 2). Using ES-2 cells (Fig. 1A) as analyzed using the MEP developed by Chou and Talalay (8, an example, the IC50 was about 780 nmol/L if the exposure 9, 11, 32, 33) and functionalized in the CompuSyn program time was 1 hour and this concentration decreased almost described in the Methods. CompuSyn analysis of the dose– 10-fold to 83 nmol/L when the exposure time was increased response curves (Fig. 3D) indicated that topotecan–doxo- to 72 hours. The exposure time dependency was even more rubicin combination produced CI values of 0.22 and 0.15 dramatic for the OVCAR-3 cells (Fig. 1C), where exposure against ES-2 and OVCAR-3 cell lines, respectively, and times of 1 to 8 hours was insufficient to achieve significant therefore appeared highly synergistic. The CompuSyn anal- impacts on cell viability regardless of the drug concentration ysis of the drug combination data obtained for the SKOV-3 used. The effect of exposure time on the IC50 of topotecan cells suggests that the interactions are additive (CI of 1.1). has been summarized for all 3 cell lines in Fig. 1D. There The activities of topotecan and doxorubicin were depen- were decreases in the IC50 of doxorubicin as exposure time dent on exposure time, and for this reason, it was assumed increased but the effect of exposure time was less than that that combinations of topotecan and doxorubicin would noted for topotecan, albeit a difference of 5- to 10-fold was exhibit increased activity as the exposure time increased obtained for the ES-2 cells (Fig. 2A) and the SKOV-3 cells from 1 to 72 hours. This is illustrated by the data (obtained (Fig. 2B). for cells exposed to drug for 72 hours) summarized in Fig. The activity of topotecan and doxorubicin used alone and 4A (ES-2 cells), B (OVCAR-3 cells), and C (SKOV-3 cells). To in combination was also assessed and these results have assess how exposure time influences the activity of the drugs been summarized in Figs. 3 and 4. Fixed ratio combinations when used in combination, the resultant dose–response of doxorubicin and topotecan that were used were gener- curves were then analyzed through use of the CompuSyn ated on the basis of the IC50 values observed in the three program and the CI values measured at Fa values of 0.5

AB 120 120 Topotecan Topotecan Doxorubicin Doxorubicin 100 Combination 100 Combination

80 80

60 60

40 40 Figure 3. Dose–response curves for % Cell viability % Cell viability topotecan and doxorubicin as 20 20 single agents and as combination when tested on ovarian cancer cell 0 0 100 1,000 10,000 100 1,000 10,000 lines: (A) ES-2, (B) OVCAR-3, and Doxorubicin concentration (nmol/L) Doxorubicin concentration (nmol/L) (C) SKOV-3. Values indicate mean CD SE of at least 3 individual experiments. D, CIs for topotecan– 120 1.2 Topotecan SKOV-3 doxorubicin combination (IC50: Doxorubicin ¼ 100 Combination 1.0 IC50) at 50% affect level (Fa 0.5) as calculated using CompuSyn 80 0.8 program.

60 0.6 CI at Fa=0.5 % Cell viability 40 0.4 ES-2 20 0.2 OVCAR-3

0 0.0 100 1,000 10,000 Doxorubicin concentration (nmol/L)

OF6 Clin Cancer Res; 2012 Clinical Cancer Research

Topotecan–Doxorubicin Combination in Ovarian Cancer

AB 120 120

100 100

80 80 60 60 40 1 h 40 1 h

% Cell viability 4 h 20 4 h % Cell viability Figure 4. Effect of exposure time on 8 h 8 h 24 h 20 24 h the cytotoxicity of topotecan and 0 48 h 48 h doxorubicin when used in 72 h 72 h combination against (A) ES-2, (B) 0 10 100 1,000 10,000 100,000 10 100 1,000 10,000 OVCAR-3, and (C) SKOV-3 cell lines. Doxorubicin concentration (nmol/L) Doxorubicin concentration (nmol/L) Values indicate mean SE of at least 3 individual experiments. D, CIs for CD topotecan–doxorubicin 140 1.6 combination (IC :IC ) at 50% 4 h 50 50 1.4 ¼ 120 48 h affect level (Fa 0.5) as calculated 72 h using CompuSyn program. 100 1.2 1.0 80 0.8 60 1 h 0.6 4 h CI at Fa=0.5 % Cell viability 40 8 h 24 h 0.4 20 48 h 72 h 0.2 0 10 100 1,000 10,000 100,000 0.0 Doxorubicin concentration (nmol/L) ES-2 OVCAR-3 SKOV-3

(IC50) were plotted for different exposure times (Fig. 4D). (P < 0.05) greater than those achieved when topotecan was For ES-2 and OVCAR-3 cells, increased exposure time administered alone. This is reflected in a small increase in was associated with decreases in the CI as measured by topotecan AUC0–24 h from 15 to 70 mg h/mL. The plasma CompuSyn. For ES-2 cells, the data collected following a 4- elimination of Topophore C was not changed when co- hour exposure suggested that the CI value was 1.1, whereas a administered with Doxil (Fig. 5B). When Doxil was admin- 72-hour exposure, the CI value was 0.2. The drug combi- istered as a single agent, the associated active ingredient nation produced additive effects when tested against the doxorubicin showed biphasic plasma elimination with an SKOV-3 cell line, regardless of the exposure time. initial t(1/2)distribution of 0.36 hours corresponding rapid tissue distribution. A second t(1/2)elimination of 36 hours Pharmacokinetic analysis of the combination corresponded to an extended terminal elimination phase To model increases in drug exposure time for combina- (Fig. 5C). These observations were consistent with the tions of topotecan and doxorubicin, LNP formulations of earlier reports (34). Co-administration of Doxil with either the drugs were used. Doxil, an LNP formulation of doxo- free topotecan or Topophore C resulted in faster first-phase rubicin, is already approved for use in relapsed ovarian elimination with 30% of initial doxorubicin concentration cancer. Topophore C, an LNP formulation of topotecan that remaining in the plasma after 1 hour compared with that of was recently described (30, 31), was selected for the in vivo 50% when administered as single agent (Fig. 5C). Doxil studies summarized below. Before evaluating efficacy, it was elimination rate after first hour was not affected when the important to assess whether co-administration of the var- drug was co-administered with the topotecan formulations. ious drug combinations selected affected the pharmacoki- It was possible that the more rapid initial elimination of netic of the drugs when compared with their use alone. The doxorubicin following administration of Doxil was a result combinations tested included free topotecan and Doxil as of topotecan-mediated increased doxorubicin release from well as Topophore C and Doxil. For this reason, plasma Doxil. To assess this, an in vitro assay was conducted to elimination was assessed (see Materials and Methods) fol- measure drug loss from the Doxil formulation following lowing a single i.v. dose of Doxil (7.5 mg/kg), free topotecan incubation with free topotecan or Topophore C (in PBS (5 mg/kg), or Topophore C (5 mg/kg). These data were buffer at 37C for 1 hour). These data (summarized in the compared with results obtained following administration insert to Fig. 5C) indicated that under the conditions used, of the drugs in combination (Fig. 5A–C). Results indicated there was no loss of LNP-associated doxorubicin when that co-administration of Doxil had a small impact on the Doxil was mixed with free topotecan or Topophore C; plasma elimination profile of free topotecan (Fig. 5A). therefore, the faster initial rate of doxorubicin elimination The measured drug levels at 1 and 4 hours were significantly was not caused by topotecan-mediated doxorubicin release.

www.aacrjournals.org Clin Cancer Res; 2012 OF7

Patankar et al.

AB 100 160 Free topotecan Topophore C Free topotecan + Doxil 140 Topophore C + Doxil 80 120 Figure 5. Plasma elimination proﬁle 60 100 of topotecan following i.v. administration of single dose of (A) 80 40 free topotecan (5 mg/kg) or free 60 topotecan þ Doxil (5 þ 7.5) mg/kg 40 and (B) Topophore C (5 mg/kg) 20 or Topophore C þ Doxil 20

Plasma topotecan conc. (µg/mL) þ

Plasma topotecan conc. (µg/mL) (5 7.5) mg/kg to female mice. 0 0 Values indicate mean SD, n ¼ 4. 0 5 10 15 20 25 0 5 10 15 20 25 C, plasma elimination of Time (h) Time (h) doxorubicin following i.v. C administration of single dose of 1000 Doxil (7.5 mg/kg) alone or in Doxil combination with either free Doxil + Topophore C Doxil + Free topotecan topotecan (15 mg/kg) or Topophore C (5 mg/kg) to female mice. Values indicate mean SD, n ¼ 4. Inserted ﬁgure indicates amount of doxorubicin retained by 100 doxorubicin LNP (Doxil) following incubation with free topotecan or Topophore C in PBS at 37C/1 hour. Plasma doxorubicin conc. (µg/mL)

10 0 5 10 15 20 25 30 Time (h)

In vivo efficacy in two models of ovarian cancer tolerated, 15 mg/kg was used for the drug combination Antitumor activity of single-agent or combination treat- studies with Doxil because it was therapeutically superior. ments was determined in vivo using 2 different pseudo- As noted previously (30), the MTD of Topophore C was orthotopic models of ovarian carcinoma. As indicated in the 7.5 mg/kg, at least 2-fold lower than the dose that can be Materials and Methods, ES-2 or SKOV-3-Luc-D3 cells were achieved with free drug. Treatment with Topophore C, inoculated i.p., and tumor progression (SKOV-3) or tumor- however, provided significantly better activity than free related morbidity (ES-2 cells) were monitored as a function topotecan when administered at equitoxic doses. The MST of time following treatment. Treatments were given i.v. of mice treated with Topophore C at 2.5 mg/kg was 39 days, using a schedule of every 7 days 3. Previous studies representing a 114% increase in median lifespan when from our laboratory and others have shown that LNP compared with controls and more than a 1.5-fold improve- formulations can enter the peritoneal cavity following i.v. ment in activity when compared with results obtained with administration in non–tumor-bearing animals (35) and free topotecan administered at 15 mg/kg. Similar to free that the amount of LNP localization at this site increases topotecan, there was no significant dose–response curve, substantially under conditions where there is a tumor and when Topophore C was administered at 7.5 mg/kg, (the burden. Dose–response curves were generated for treatment MTD) the MST was 42 days, representing a 127% increase in groups where overall survival (OS) was used as an indicator median lifespan over controls. The maximum efficacious of efficacy in the ES-2 model. ES-2 tumor growth and dose of Topophore C was 5 mg/kg, and this dose was associated ascites development were observed rapidly when considered appropriate for the drug combination studies. animals were left untreated. The median survival time It is notable that Doxil, when used as a single agent at (MST) for control animals was 18 days from the day of its MTD of 7.5 mg/kg, exhibited no measurable therapeutic tumor inoculation with more than 80% of mice terminated activity (Table 1). Insensitivity to Doxil is consistent by day 21 due to tumor progression (Table 1). Free topo- with previous reports indicating that clear cell carcinoma tecan administered at 5 mg/kg resulted in a 57% increase (e.g., ES-2) is an aggressive and chemorefractory subtype of in MST (to 29 days; Table 1). This increased to 76% at ovarian cancers (36–40). 15 mg/kg (MST of 32 days), and a further increase in For the drug combination studies, Doxil was adminis- topotecan dose to 20 mg/kg did not show any further tered at its MTD (7.5 mg/kg) and it was co-administered improvement in OS and decreases in health status were with increasing doses of free topotecan or Topophore C. noted. Although the 20 mg/kg free topotecan dose was The dose-escalation studies were conducted to establish

OF8 Clin Cancer Res; 2012 Clinical Cancer Research

Topotecan–Doxorubicin Combination in Ovarian Cancer

Table 1. Antitumor activity of free topotecan, ited significant therapeutic benefits when the animals were treated with well-tolerated doses. When treated with 0.625 Topophore C, and Doxil in nude mice bearing mg/kg Topophore C in combination with Doxil (7.5 mg/ ES-2 clear cell carcinoma xenografts following kg), the MST was 28 days. Escalation of the Topophore C i.v. administration of free topotecan, Topophore dose to 2.5 mg/kg resulted in significant improvements in C, or Doxil as single agents and as combination treatment outcomes (MST of 52 days). When Topophore C treatments was used alone at the 2.5 mg/kg dose, the MST was only 39 days. Given the lack of activity of Doxil used as a single Drug Median agent, this provides strong evidence that the combination of dose, survival, % the LNPs is synergistic. The efficacy results summarized Treatment mg/kg d ILSa in Table 1 have also been presented in the form of Single-agent Kaplan–Meier survival plot (Supplementary Fig. SI) to Saline (control) — 18.5 N/A highlight the improvements in treatment outcomes achiev- Free topotecan 5 29 57 able with this combination. Free topotecan 7.5 30.5 65 The tumor burden after inoculation of SKOV-3-Luc-D3 Free topotecan 15 32.5 76 cells was estimated by assessing increases in luminescent Free topotecan 20 28 51 light emitted (photos/s) by the luciferase-modified cells Topophore C 2.5 39.5 114 following luciferin injection (see Materials and Methods). Topophore C 5 44 138 Mice with established tumors were organized into treat- Topophore C 7.5 42 127 ment groups defined on the basis of data obtained using the Doxil 7.5 19 3 ES-2 tumor model and these results have been summarized Combination treatment in Fig. 6 which includes representative images for each Saline (control) — 20.3 N/A treatment group. The SKOV-3-Luc-D3 cells could be visu- Free topotecan þ Doxil 5 þ 7.5 31.5 55 alized 1 day after tumor cell inoculation. Treatment was Free topotecan þ Doxil 10 þ 7.5 37 82 initiated 7 days after cell inoculation, and the animals were Free topotecan þ Doxil 15 þ 7.5 37.3 84 imaged every 7 days. The results obtained 28 days after Topophore C þ Doxil 0.625 þ 7.5 28 38 tumor cell inoculation (the day when the last treatment Topophore C þ Doxil 1.25 þ 7.5 43 112 was provided) and 42 days after tumor inoculation are Topophore C þ Doxil 2.5 þ 7.5 52 156 provided. Control mice exhibited a steady increase in bio- Topophore C þ Doxil 5 þ 7.5 20 — luminescence over the 42-day time course, where the increase in signal intensity from day 1 to 42 was 580% (see a Percentage ILS (increase in lifespan): values were deter- histogram in Fig. 6). When the mice were treated with free mined from MSTs of treated and control (untreated) groups. topotecan, they exhibited, on average, a 131% increase in the bioluminescent signal, suggesting that topotecan was therapeutically active in this model. Topophore C (2.5 mg/ whether there was an increase in toxicity when the drugs kg)-treated mice showed only 39% increase in signal at day were used in combination. When Doxil was combined with 42. Doxil (7.5 mg/kg) exhibited good therapeutic effects, free topotecan given at the 5 mg/kg dose, the MST was 31 where there was only a 92% increase in bioluminescence days (Table 1), showing a small, but not significant, increase signal at day 42. When this effective dose of Doxil was in activity when compared with use of free topotecan alone. combined with free topotecan (15 mg/kg), the increase in Increases in MST were achievable when the dose of topo- signal at day 42 was comparable or slightly higher than that tecan was escalated to 15 mg/kg, where the MST was 37 days observed when animals were treated with free topotecan or when compared with 32 days achieved following admin- Doxil alone. Combination of Doxil and Topophore C istration of topotecan alone at the 15 mg/kg dose. There was proved to be most effective. Only a 6% increase in biolu- some evidence of enhanced toxicity when these drugs were minescence signal was noted at day 42. used in combination. For example, significant weight loss (10–15%) was noted following the third dose of the free Discussion topotecan–Doxil combination (15 and 7.5 mg/kg, respec- Approaches to define effective combinations in the clinic tively) but the mice recovered within 1 week. This was in have not taken into consideration pharmacokinetic factors dramatic contrast to studies completed with Doxil and that could influence drug–drug interactions and engender Topophore C, where significant increases in drug-related improvements in treatment outcomes (24, 41, 42). A variety morbidity were noted. For example, combinations of Topo- of in vitro approaches have been used to measure drug–drug phore C and Doxil were not tolerated when dosed at 7.5 and interactions in cell culture (11, 32, 43); however, the use of 5 mg/kg, respectively, and the combination produced a in vitro data to predict synergy in vivo remains challenging. greater-than-expected (synergistic) toxicity that was We believe that this is due to 2 reasons: (i) drug pharma- reflected in significant weight loss (up to 25%) and the cology and pharmacokinetics of drugs cannot be adequately need to terminate animals due to poor health status. The mimicked in the in vitro setting and (ii) in vitro conditions combination of Doxil and Topophore C, however, exhib- cannot be mimicked accurately in vivo. In vitro assays,

www.aacrjournals.org Clin Cancer Res; 2012 OF9

Patankar et al.

Doxil + free topotecan

Saline (7.5 + 15) mg/kg

Doxil + Topophore C (7.5 + 2.5) mg/kg 15 mg/kg Free topotecan

Day 1 Day 28 Day 42

Saline Free topotecan (15 mg/kg) Topophore C (2.5 mg/kg) 1,000.00 Doxil (7.5 mg/kg) Doxil + free topotecan (7.5 + 15) mg/kg Doxil + Topophore C (7.5 + 2.5) mg/kg 2.5 mg/kg

Topophore C 800.00 P < 0.001 P < 0.05

600.00 P < 0.05 P < 0.01 400.00

200.00 Doxil 7.5 mg/kg

% Increase in bioluminescence 0.00 28 42 Time (d) Day 1 Day 28 Day 42

Figure 6. Tumor progression as determined by increase in bioluminescent signal in mice bearing intraperitoneally grown SKOV-3 serous adenocarcinoma xenografts. Mice were inoculated with luciferase-modiﬁed SKOV-3 cells on day 0, and tumor measurements were conducted once a week following i.p. injection of luciferin solution. Treatment groups were administered intravenously on day 7 (every 7 days 3) with either free topotecan (15 mg/kg) or Topophore C (2.5 mg/kg) or Doxil (7.5 mg/kg) either as single agents or in combination as shown, and control mice were administered equivalent volume of saline. Data points represent mean SD (n ¼ 6).

although limited, have already shown that drug:drug drug ratio within the plasma compartment and the tumor synergy (or antagonism) is influenced by drug dose (as over time. However, these formulations also extend represented by measured effect level) and drug:drug ratio. the circulation lifetime of the associated drugs and benefits These findings have profound implications particularly as may arise simply as a result of extended exposure times. drugs are typically combined in vivo under conditions where This is an important point to consider, given that many drug dose and drug:drug ratio cannot be controlled. In in vitro assays rely on endpoints determined 3 to 5 days after recent studies from our group, it was observed that surpris- drug addition, yet in vivo, the drug exposure time may be ing improvements in therapeutic activity can be achieved by considerably shorter. The studies described in this report controlling the ratios of drugs in various combinations were designed to assess whether drug exposure time (irinotecan/floxuridine, daunorubicin/cytarabine, or cis- influenced treatment outcomes for a combination that platin/daunorubicin) in vivo through use of well-designed appears to have some therapeutic potential for treatment LNPs. Efficacy of select combinations could be achieved at of relapsed ovarian cancer. The cell-based screening studies doses that are far lower than those required to achieve summarized here for 3 ovarian cancer cell lines (ES-2, similar effects with the free drug (25). These formulations SKOV-3, and OVCAR-3) show that prolonged exposure to rely on use of drug delivery methods to control the drug: topotecan or doxorubicin (added as single agents) enhances

OF10 Clin Cancer Res; 2012 Clinical Cancer Research

Topotecan–Doxorubicin Combination in Ovarian Cancer

therapeutic effects (see Figs. 2 and 3). This can be attributed administered. It is premature to speculate on why Doxil to the cell-cycle–specific nature of the drugs used. Topote- pharmacokinetics changed; however, changes in Doxil can and doxorubicin are known to produce greater effects elimination were not due to topotecan-mediated release during the S-phase of the cell cycle (44–46), thus greater of doxorubicin from the liposomal carrier (Fig. 5C, inset). cytotoxicity observed on prolong exposure would be a The effect was observed for both free and LNP formula- consequence of a greater proportion of cells entering in the tions of topotecan, so it is likely due to topotecan- S-phase. mediated change in liposome elimination; however, our The enhanced treatment effects noted with increasing laboratory is not able to measure this for the Doxil. It exposure time were also observed with the drugs used in should also be noted that the enhanced therapeutic combination (see Fig. 4). These drug combination data effects of the LNP combination were also associated with were analyzed using the MEP developed by Chou and an increase in toxicity. This could be due to changes in Talalay (8) to determine whether synergistic interactions Doxil pharmacokinetics and biodistribution and/or due are increased or decreased as a function of exposure time. to synergism between the 2 drugs on normal proliferating It should be noted that the MEP methodology is built cell populations in the gastrointestinal (GI) tract and around the concept that combination effects need to be hematopoietic system. The studies presented provide studied at fixed drug ratios and that these effects must be preclinical evidence to support the use of combinations determined over a broad range of effective doses (6–8). comprising an LNP formulations of topotecan and Doxil, Studies summarized here used fixed molar ratio (IC50 of but the results also suggest that combinations of free doxorubicin/IC50 of topotecan, determined at 72 hours), topotecan and Doxil may provide limited if any thera- and this ratio was tested over a range of effective doses. peutic benefit when compared with the agents used alone. However, it is obvious from the data plotted in Figs. 1 This will eventually be determined in patients. and 2 that this ratio would likely change depending on theexposuretimeused.Thishighlights the complexity of Conclusion these studies; however, the ratio used can be justified on Cytotoxic activity of topotecan and doxorubicin against the basis of 2 points: (i) the accuracy of the MEP in ovarian cancer cell lines was observed to be exposure time– determining drug:drug interactions is greatest when the dependent. Synergistic interactions observed between topo- affect level measured is 0.5 (i.e., the IC50 dose) and (ii) tecan and doxorubicin in vitro were translated very well in the MTT viability assay was always completed 72 hours vivo and the results suggest that this synergy may best be after treatment was initiated. achieved under conditions when the tumor cells are LNP formulations can be used as an effective means to exposed to both drugs over extended time periods. Con- achieve extended drug exposure in vivo and this is reflected current administration of Doxil with Topophore C proved in increased levels of drug in the blood compartment over to be effective when used to treat ovarian cancer xenograft time as well as enhanced delivery to sites of tumor growth, models. These results provide proof-of-concept data to including tumor growth within the peritoneal cavity support the use of this combination for treatment of recur- (35, 47–49). Doxil and Topophore C formulations have rent ovarian cancer. previously shown these abilities and therefore were used in the studies here to increase the exposure of doxorubicin and Disclosure of Potential Conflicts of Interest topotecan to the tumor site. The approach used is reliant on No potential conflicts of interest were disclosed. a clinical development plan for Topophore C that would involve a phase II study comparing the effectiveness of Authors' Contributions Conception and design: N.A. Patankar, M.B. Bally topotecan versus Topophore C and subsequently a study Development of methodology: N.A. Patankar, M.B. Bally evaluating combinations of Doxil, which is already Acquisition of data (provided animals, acquired and managed patients, approved for use in the treatment of relapsed ovarian provided facilities, etc.): N.A. Patankar, J. Pritchard, M. Osooly Analysis and interpretation of data (e.g., statistical analysis, biosta- cancer, and Topophore C. tistics, computational analysis): N.A. Patankar, M.B. Bally Importantly, the drug combination effects observed Writing, review, and/or revision of the manuscript: N.A. Patankar, M.B. Bally when using combinations of topotecan and doxorubicin Administrative, technical, or material support (i.e., reporting or orga- in vitro were mirror in the in vivo efficacy studies using the nizing data, constructing databases): N.A. Patankar, M. van Grinsven, LNP formulations. Combination treatments involving M. Osooly Study supervision: N.A. Patankar, M.B. Bally LNPs showed superior therapeutic efficacy over single agents in both ovarian cancer models evaluated. Doxil/ Grant Support Topophore C combinations were therapeutically superior This research was supported by grants from the Canadian Institutes of to the other combinations tested. Importantly, the Health Research. N.A. Patankar was supported by fellowships received from changes in Doxil elimination observed in Fig. 5 did not BC Innovation Council and University of British Columbia. The costs of publication of this article were defrayed in part by the appear to have an adverse impact on therapy. This is payment of page charges. This article must therefore be hereby marked perhaps due to the lack of Doxil efficacy in the ES-2 advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate ovarian cancer model and may suggest that the effects this fact. in the SKOV-3 model could be better if the pharmacoki- Received August 29, 2012; revised December 7, 2012; accepted December netics of Doxil were unchanged when topotecan was co- 18, 2012; published OnlineFirst January 9, 2013.

www.aacrjournals.org Clin Cancer Res; 2012 OF11

Patankar et al.

References 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, et al. Cancer cancer: a systematic review and economic evaluation. Health Technol statistics, 2008. CA Cancer J Clin 2008;58:71–96. Assess 2006;10:1–132, iii–iv. 2. Kurman RJ, Shih Ie M. The origin and pathogenesis of epithelial ovarian 24. Harasym TO, Tardi PG, Johnstone SA, Mayer LD, Bally MB, Janoff AS. cancer: a proposed unifying theory. Am J Surg Pathol 2010;34:433–43. Fixed drug ratio liposome formulations of combination cancer thera- 3. Ahmad T, Gore M. Review of the use of topotecan in ovarian carci- peutics. In: Liposome technology. 3rd ed. New York, NY: Informa noma. Expert Opin Pharmacother 2004;5:2333–40. Healthcare; 2006. p. 25–48. 4. Agarwal R, Linch M, Kaye SB. Novel therapeutic agents in ovarian 25. Mayer LD, Harasym TO, Tardi PG, Harasym NL, Shew CR, Johnstone cancer. Eur J Surg Oncol 2006;32:875–86. SA, et al. Ratiometric dosing of anticancer drug combinations: con- 5. Ramsay EC, Dos Santos N, Dragowska WH, Laskin JJ, Bally MB. The trolling drug ratios after systemic administration regulates therapeutic formulation of lipid-based nanotechnologies for the delivery of fixed activity in tumor-bearing mice. Mol Cancer Ther 2006;5:1854–63. dose anticancer drug combinations. Curr Drug Deliv 2005;2:341–51. 26. Tardi P, Johnstone S, Harasym N, Xie S, Harasym T, Zisman N, et al. In 6. Waterhouse DN, Gelmon KA, Klasa R, Chi K, Huntsman D, Ramsay E, vivo maintenance of synergistic cytarabine:daunorubicin ratios greatly et al. Development and assessment of conventional and targeted drug enhances therapeutic efficacy. Leuk Res 2009;33:129–39. combinations for use in the treatment of aggressive breast cancers. 27. Dadashzadeh S, Vali AM, Rezaie M. The effect of PEG coating on in Curr Cancer Drug Targets 2006;6:455–89. vitro cytotoxicity and in vivo disposition of topotecan loaded liposomes 7. Chou T. Synergism and antagonism in chemotherapy. San Diego, CA: in rats. Int J Pharm 2008;353:251–9. Academic Press; 1991. 28. Drummond DC, Noble CO, Guo Z, Hayes ME, Connolly-Ingram C, 8. Chou TC. Theoretical basis, experimental design, and computerized Gabriel BS, et al. Development of a highly stable and targetable simulation of synergism and antagonism in drug combination studies. nanoliposomal formulation of topotecan. J Control Release 2010;141: Pharmacol Rev 2006;58:621–81. 13–21. 9. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: 29. Liu JJ, Hong RL, Cheng WF, Hong K, Chang FH, Tseng YL. Simple and the combined effects of multiple drugs or enzyme inhibitors. Adv efficient liposomal encapsulation of topotecan by ammonium sulfate Enzyme Regul 1984;22:27–55. gradient: stability, pharmacokinetic and therapeutic evaluation. Anti- 10. Zimmermann GR, Lehar J, Keith CT. Multi-target therapeutics: when cancer Drugs 2002;13:709–17. the whole is greater than the sum of the parts. Drug Discov Today 30. Patankar NA, Waterhouse D, Strutt D, Anantha M, Bally MB. Topo- 2007;12:34–42. phore C: a liposomal nanoparticle formulation of topotecan for 11. Zoli W, Ricotti L, Tesei A, Barzanti F, Amadori D. In vitro preclinical treatment of ovarian cancer. Invest New Drugs. 2012 May 22. [Epub models for a rational design of chemotherapy combinations in human ahead of print]. tumors. Crit Rev Oncol Hematol 2001;37:69–82. 31. Bally MB, Nayar R, Masin D, Hope MJ, Cullis PR, Mayer LD. Liposomes 12. Creemers GJ, Bolis G, Gore M, Scarfone G, Lacave AJ, Guastalla JP, with entrapped doxorubicin exhibit extended blood residence times. et al. Topotecan, an active drug in the second-line treatment of Biochim Biophys Acta 1990;1023:133–9. epithelial ovarian cancer: results of a large European phase II study. 32. Berenbaum MC. What is synergy? Pharmacol Rev 1989;41:93–141. J Clin Oncol 1996;14:3056–61. 33. Waterhouse DN, Kalra J. Nanotechnology as an enabling approach to 13. Gordon AN, Fleagle JT, Guthrie D, Parkin DE, Gore ME, Lacave AJ. the development of fixed-dose combination products for treating Recurrent epithelial ovarian carcinoma: a randomized phase III study of cancer. Valencia (CA): American Scientifi c Publishers; 2008. pegylated liposomal doxorubicin versus topotecan. J Clin Oncol 34. Gabizon A, Shmeeda H, Barenholz Y. Pharmacokinetics of pegylated 2001;19:3312–22. liposomal Doxorubicin: review of animal and human studies. Clin 14. Horowitz NS, Hua J, Gibb RK, Mutch DG, Herzog TJ. The role of Pharmacokinet 2003;42:419–36. topotecan for extending the platinum-free interval in recurrent ovarian 35. Bally MB, Masin D, Nayar R, Cullis PR, Mayer LD. Transfer of liposomal cancer: an in vitro model. Gynecol Oncol 2004;94:67–73. drug carriers from the blood to the peritoneal cavity of normal and 15. Ozols RF. Optimum chemotherapy for ovarian cancer. Int J Gynecol ascitic tumor-bearing mice. Cancer Chemother Pharmacol 1994;34: Cancer 2000;10:33–7. 137–46. 16. Swisher EM, Mutch DG, Rader JS, Elbendary A, Herzog TJ. Topotecan 36. Crotzer DR, Sun CC, Coleman RL, Wolf JK, Levenback CF, Gershen- in platinum- and paclitaxel-resistant ovarian cancer. Gynecol Oncol son DM. Lack of effective systemic therapy for recurrent clear cell 1997;66:480–6. carcinoma of the ovary. Gynecol Oncol 2007;105:404–8. 17. ten Bokkel Huinink W, Gore M, Carmichael J, Gordon A, Malfetano J, 37. Pather S, Quinn MA. Clear-cell cancer of the ovary-is it Hudson I, et al. Topotecan versus paclitaxel for the treatment of chemosensitive? Int J Gynecol Cancer 2005;15:432–7. recurrent epithelial ovarian cancer. J Clin Oncol 1997;15:2183–93. 38. Pectasides D, Pectasides E, Psyrri A, Economopoulos T. Treatment 18. D'Arpa P, Liu LF. Topoisomerase-targeting antitumor drugs. Biochim issues in clear cell carcinoma of the ovary: a different entity? Oncolo- Biophys Acta 1989;989:163–77. gist 2006;11:1089–94. 19. Swift LP, Rephaeli A, Nudelman A, Phillips DR, Cutts SM. Doxorubicin- 39. Sugiyama T, Kamura T, Kigawa J, Terakawa N, Kikuchi Y, Kita T, et al. DNA adducts induce a non-topoisomerase II-mediated form of cell Clinical characteristics of clear cell carcinoma of the ovary: a distinct death. Cancer Res 2006;66:4863–71. histologic type with poor prognosis and resistance to platinum-based 20. Dupont J, Aghajanian C, Andrea G, Lovegren M, Chuai S, Venkatraman chemotherapy. Cancer 2000;88:2584–9. E, et al. Topotecan and liposomal doxorubicin in recurrent ovarian 40. Goff BA, Sainz de la Cuesta R, Muntz HG, Fleischhacker D, Ek M, Rice cancer: is sequence important? Int J Gynecol Cancer 2006;16 Suppl LW, et al. Clear cell carcinoma of the ovary: a distinct histologic type 1:68–73. with poor prognosis and resistance to platinum-based chemotherapy 21. Ghesquieres H, Faivre S, Djafari L, Pautier P, Lhomme C, Lozahic S, in stage III disease. Gynecol Oncol 1996;60:412–7. et al. Phase I dose escalation study of pegylated liposomal doxorubicin 41. Hanahan D, Bergers G, Bergsland E. Less is more, regularly: metro- (Caelyx) in combination with topotecan in patients with advanced nomic dosing of cytotoxic drugs can target tumor angiogenesis in malignancies. Invest New Drugs 2006;24:413–21. mice. J Clin Invest 2000;105:1045–7. 22. Jonsson E, Fridborg H, Nygren P, Larsson R. Synergistic interactions 42. Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors. Nat of combinations of topotecan with standard drugs in primary cultures Rev Cancer 2002;2:727–39. of human tumor cells from patients. Eur J Clin Pharmacol 1998;54: 43. Carter WH Jr, Wampler GL. Review of the application of response 509–14. surface methodology in the combination therapy of cancer. Cancer 23. Main C, Bojke L, Griffin S, Norman G, Barbieri M, Mather L, et al. Treat Rep 1986;70:133–40. Topotecan, pegylated liposomal doxorubicin hydrochloride and pac- 44. Bailly C. Topoisomerase I poisons and suppressors as anticancer litaxel for second-line or subsequent treatment of advanced ovarian drugs. Curr Med Chem 2000;7:39–58.

OF12 Clin Cancer Res; 2012 Clinical Cancer Research

Topotecan–Doxorubicin Combination in Ovarian Cancer

45. Burden DA, Osheroff N. Mechanism of action of eukaryotic topoisom- induced by transmembrane ion gradients. Chem Phys Lipids erase II and drugs targeted to the enzyme. Biochim Biophys Acta 1988;47:97–107. 1998;1400:139–54. 48. Sadzuka Y, Hirotsu S, Hirota S. Effect of liposomalization on the 46. Tewey KM, Rowe TC, Yang L, Halligan BD, Liu LF. Adriamycin-induced antitumor activity, side-effects and tissue distribution of CPT-11. DNA damage mediated by mammalian DNA topoisomerase II. Science Cancer Lett 1998;127:99–106. 1984;226:466–8. 49. Drummond DC, Noble CO, Hayes ME, Park JW, Kirpotin DB. Phar- 47. Bally MB, Mayer LD, Loughrey H, Redelmeier T, Madden TD, Wong K, macokinetics and in vivo drug release rates in liposomal nanocarrier et al. Dopamine accumulation in large unilamellar vesicle systems development. J Pharm Sci 2008;97:4696–740.

www.aacrjournals.org Clin Cancer Res; 2012 OF13

Topotecan and Doxorubicin Combination to Treat Recurrent Ovarian Cancer: The Influence of Drug Exposure Time and Delivery Systems to Achieve Optimum Therapeutic Activity

Nilesh A. Patankar, Julia Pritchard, Mariska van Grinsven, et al.

Clin Cancer Res Published OnlineFirst January 9, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-12-2459

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2013/01/09/1078-0432.CCR-12-2459.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/early/2013/01/28/1078-0432.CCR-12-2459. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.