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Extended topoisomerase 1 inhibition through liposomal irinotecan results in improved efficacy over topotecan and irinotecan in models of small-cell lung cancer

Leonard, Shannon C.a; Lee, Helena; Gaddy, Daniel F.a; Klinz, Stephan G.a; Paz, Nancya; Kalra, Ashish V.a; Drummond, Daryl C.a; Chan, Daniel C.b; Bunn, Paul A.b; Fitzgerald, Jonathan B.a; Hendriks, Bart S.a

doi: 10.1097/CAD.0000000000000545
PRECLINICAL REPORTS
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Liposomal irinotecan (irinotecan liposome injection, nal-IRI), a liposomal formulation of irinotecan, is designed for extended circulation relative to irinotecan and for exploiting discontinuous tumor vasculature for enhanced drug delivery to tumors. Following tumor deposition, nal-IRI is taken up by phagocytic cells followed by irinotecan release and conversion to its active metabolite, SN-38. Sustained inhibition of topoisomerase 1 by extended SN-38 exposure as a result of delivery by nal-IRI is hypothesized to enable superior antitumor activity compared with traditional topoisomerase 1 inhibitors such as conventional irinotecan and topotecan. We evaluated the antitumor activity of nal-IRI compared with irinotecan and topotecan in preclinical models of small-cell lung cancer (SCLC) including in a model pretreated with carboplatin and etoposide, a first-line regimen used in SCLC. Nal-IRI demonstrated antitumor activity in xenograft models of SCLC at clinically relevant dose levels, and resulted in complete or partial responses in DMS-53, DMS-114, and NCI-H1048 cell line-derived models as well as in three patient-derived xenograft models. The antitumor activity of nal-IRI was superior to that of topotecan in all models tested, which generally exhibited limited control of tumor growth and was superior to irinotecan in four out of five models. Further, nal-IRI demonstrated antitumor activity in tumors that progressed following treatment with topotecan or irinotecan, and demonstrated significantly greater antitumor activity than both topotecan and irinotecan in NCI-H1048 tumors that had progressed on previous carboplatin plus etoposide treatment. These results support the clinical development of nal-IRI in patients with SCLC.

aMerrimack Pharmaceuticals, Cambridge, Massachusetts

bDivision of Medical Oncology, University of Colorado Cancer Center, Aurora, Colorado, USA

Correspondence to Bart S. Hendriks, PhD, Merrimack Pharmaceuticals, 1 Kendall Square, B7201, Cambridge, 02139 MA, USA Tel: +1 617 441 4000; e-mail: bart.hendriks@emdserono.com Present address: Shannon C. Leonard: Rubius Therapeutics, Cambridge, Massachusetts, USA Present address: Helen Lee: Sanofi US, Cambridge, Massachusetts, USA Present address: Daniel F. Gaddy: CatalytIQ Insights, Cambridge, Massachusetts, USA Present address: Stephan G. Klinz: Ipsen Bioscience Inc., Cambridge, Massachusetts, USA Present address: Nancy Paz: Decibel Therapeutics, Boston, Massachusetts, USA Present address: Ashish V. Kalra: Scholar Rock, Cambridge, Massachusetts, USA Present address: Jonathan B. Fitzgerald: Torque, Cambridge, Massachusetts, USA Present address: Bart S. Hendriks: EMD Serono, Billerica, Massachusetts, USA

This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0/

Received March 27, 2017

Accepted June 24, 2017

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Introduction

Lung cancer is the leading cause of cancer-related death worldwide. Small-cell lung cancer (SCLC) accounts for about 15% of lung cancers overall and is a particularly aggressive neoplasia with a 5-year survival rate of less than 10%. Little progress has been made in improving the outcome of this malignancy over the last 30 years. Approximately 30% of patients with SCLC are diagnosed with limited-stage disease, and are typically treated with a combination of radiation and chemotherapy 1. With current treatments, only ~20% of patients with limited-stage disease maintain the potential to be cured 2. Extensive-stage disease remains incurable with an expected median survival of 7–9 months 1.

Extensive-stage disease is typically treated in the front-line setting with a combination of platinum (carboplatin or cisplatin) plus the topoisomerase 2 inhibitor etoposide in the USA and European Union 3,4. About 50–90% of patients with extensive-stage disease respond to initial treatment 1; however, patients typically relapse rapidly and develop resistance. Irinotecan, a prodrug for its active metabolite SN-38, has shown activity in front-line settings in patients with SCLC, and its combination with cisplatin is commonly used in Japan 5 and is included in the NCCN and ESMO guidelines 3,4. A number of trials have compared the combination of irinotecan plus cisplatin or carboplatin with etoposide plus cisplatin or carboplatin. Several of these trials demonstrated a significant advantage for the irinotecan combination, and meta-analyses of all trials showed significantly longer overall survival in the irinotecan combination relative to etoposide plus platinum combinations 6–9. Nevertheless, etoposide plus platinum combinations are used most frequently in North America and Europe. In the second-line setting, single-agent irinotecan has been evaluated in a few small studies (overall response rate, time to treatment progression, and overall survival in the range of 0–47%, 1.7–2.6 months, and 4.6–13.3 months, respectively) 10–12 with similar outcomes to that of topotecan 13. On the basis of these and other studies, irinotecan monotherapy is included in major treatment guidelines for the treatment of patients with relapsed SCLC 3,4.

Topotecan is the only agent approved for second-line therapy in the USA and Europe. The approved dose of intravenous topotecan is 1.5 mg/m2 given on days 1–5 every 3 weeks. Clinically, the administration of topotecan is often limited by its tolerability, and in practice many oncologists reduce both the starting dose and number of days when the drug is administered 14. Topotecan, like irinotecan, is a campthothecin derivative and topoisomerase 1 (TOP1) inhibitor that stabilizes the typically transient TOP1–DNA cleavable complex. This results in the accumulation of single-stranded breaks in DNA followed by conversion to replication-mediated DNA double-strand breaks and cell death.

There is evidence that the pharmacokinetics and tolerability of topotecan create an exposure problem that limits its activity. Topotecan is water soluble, contributing to rapid clearance from the bloodstream. Following administration, plasma levels peak at about 70 nmol/l and are maintained above a clinically relevant threshold of 10 nmol/l for about 2.6 h/daily infusion 10. However, in-vitro studies using cell lines of SCLC origin have reported that the inhibitory concentrations required to achieve 50% of maximal activity range from single-digit nanomolar up to micromolar concentration 15,16. In addition, the complex ionization chemistry of topotecan contributes to a relatively low cellular permeability 10,17. In a translational study, tumor TOP1 inhibition was assessed by measuring TOP1 protein levels in biopsies at day 4 or 5 from patients with gastric and esophageal cancers treated with 1.5 or 2.0 mg/m2 topotecan 18. At the 1.5 mg/m2 dose level, the degree of TOP1 inhibition measured 30 min after drug administration was only ~75%, indicating that topotecan is not able to fully inhibit its target. Thus, these data suggest that clinical administration of topotecan likely results in suboptimal peak levels and time of exposure in patients.

Multiple studies have demonstrated schedule-dependent efficacy of TOP1 inhibitors in preclinical models 19–21 in which increased antitumor activity is generally observed with prolonged exposure directed at the tumor. Indeed, for this drug class it is well understood that liposomal formulations can improve the therapeutic activity through a slow and controlled release of the payload and by providing sustained duration of tumor cell exposure. Stable and long-circulating liposomal formulations for irinotecan and topotecan with potent in-vivo efficacy against cancer xenograft models have been described 17,22. In particular, liposomal irinotecan (irinotecan liposome injection, nal-IRI) is designed for extended circulation relative to irinotecan and for exploiting leaky tumor vasculature for enhanced drug delivery to tumors 22. Liposomal encapsulation of irinotecan resulted in detectable levels of irinotecan and SN-38 in the plasma for over 50 h after administration in mice. By contrast, irinotecan and its metabolite SN-38 were cleared from circulation in under 8 h when dosed at similar levels 23. Nal-IRI has been demonstrated to mediate prolonged SN-38 exposure, subsequent DNA damage, and tumor cell death in models of colorectal and pancreatic cancer, as well as in other cancer models 23,24. On the basis of a demonstrated improvement in overall survival, nal-IRI, in combination with 5-fluorouracil and leucovorin, has recently been approved in the USA and European Union for the treatment of patients with metastatic pancreatic adenocarcinoma after disease progression following gemcitabine-based therapy 25.

It is hypothesized that nal-IRI may have utility in additional indications including SCLC. Greater than 80% of SCLC tumor lesions express detectable levels of vascular endothelial growth factor 2, which is involved in angiogenesis and vessel permeability and may indicate the ability to achieve effective liposome delivery into SCLC tumors. Following tumor deposition, nal-IRI is taken up by phagocytic cells followed by irinotecan release and conversion to SN-38 23. Macrophages are the primary phagocytic cell type that is believed to take up nal-IRI within the tumor tissue and express high levels of carboxylesterase (CES) 1 24,26, one of the enzymes needed to convert irinotecan to SN-38. CES in SCLC cell lines has been described by van Ark-Otte et al.27 who found that CES activity was significantly higher when compared with non-SCLC cell lines while showing similar sensitivity to SN-38. Further, staining of samples from patients with SCLC has indicated consistent macrophage infiltration, which was found to increase with tumor stage 28,29.

For the reasons outlined above, it is hypothesized that nal-IRI has the potential to greatly extend TOP1 inhibition in SCLC tumors relative to topotecan and provide improved antitumor activity. Here, we evaluate and compare nal-IRI, irinotecan, and topotecan in xenograft models of SCLC.

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Materials and methods

Materials

Nal-IRI was prepared as described previously 22. Irinotecan and SN-38 were obtained from Areva Pharmaceuticals (Elizabethtown, Kentucky, USA) and topotecan was obtained from Accord Healthcare (Durham, North Carolina, USA). SCLC cell lines (DMS-114, DMS-53, NCI-H1048, and NCI-H841) were obtained from ATCC and grown under recommended conditions. Solvents and reagents were obtained from Sigma Aldrich (St. Louis, Missouri, USA) and VWR (Radnor, Pennsylvania, USA). All irinotecan, topotecan, and nal-IRI concentrations and/or dose levels are described on the basis of their respective HCl salts.

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Cell proliferation assay

SCLC cell lines were transfected with NucLight Red Lentivirus (Essen BioScience Ann Arbor, Michigan, USA) and selected with puromycin treatment following the manufacturer’s instructions. For cell proliferation assay, NucLight Red-transfected cells were harvested using 0.25% trypsin and plated into 96-well plates at a density of 5000 cells/well. Cells were allowed to adhere overnight before addition of drugs (SN-38 or topotecan). Drug solutions were prepared in media containing no more than 1% of DMSO. Various concentrations (0.01–10 000 nmol/l) of drugs were added to each well. Plates were then transferred to the IncuCyte ZOOM (Essen Bioscience, USA) system (in a 37°C incubator, supplemented with 5% CO2) for an incubation period of 88 h. Images were acquired in three fields of view per well every 4 h. Cell numbers were quantified using IncuCyte ZOOM (Essen Bioscience, USA) software by segmenting cell nuclei based on the NucLight Red fluorescence. The total number of cells was determined by summing the number of cells in the three fields of view for each well.

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Animal studies

Cell line-derived xenograft models were developed in nonobese diabetic/severe combined immunodeficiency mice (5–7-week-old females) that were purchased from Charles River Laboratory (Wilmington, Massachusetts, USA), with the exception of NCI-H841 efficacy studies, which were performed in athymic nude mice, also from Charles River Laboratory. At the time of treatment, cell line-derived tumors averaged 200–300 mm3. Irinotecan and nal-IRI were administered once a week as an intravenous bolus injection. Topotecan was administered intraperitoneally once or twice weekly, as indicated. Body weight was recorded throughout the study and tumor volumes were measured using calipers. Before tumor collection, intracardial perfusion was performed to remove blood from the tumor tissue.

Patient-derived xenograft (PDX) models, used for assessment of drug delivery and CES activity, were established in nude mice (Harlan Laboratories, Washington, DC, USA) by Champions Oncology (Hackensack, New Jersey, USA) using their Champions Tumorgraft technology. PDX models of SCLC origin used in antitumor activity studies were derived from donor mice and engrafted subcutaneously into the flanks of 6–8-week-old Balb/c nude mice (Vital River Laboratories, Beijing, China) and run by GenenDesign (Shanghai, China). At the time of treatment, patient-derived tumors averaged 250–300 mm3 and were at passage 6 or less. Irinotecan and nal-IRI were administered once a week as an intravenous bolus injection. Topotecan was administered intraperitoneally twice a week. All treatments continued for a total of 6 weeks. Body weight was recorded throughout each study and tumor volumes were measured with calipers twice a week until tumors reached 1500 mm3, animals were in poor general health, or until 8 weeks after the first dose.

The care and treatment of animals was performed in accordance with Institutional Animal Care and Use Committee guidelines. Institutional requirements for humane endpoints were used as surrogates for survival.

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High-performance liquid chromatography quantification of irinotecan and SN-38

Tumor levels of irinotecan and SN-38 were quantified using high-performance liquid chromatography (HPLC) as previously described 23. In brief, tissues were homogenized for 2 min in 20% w/v water using a Tissue-Lyser (Qiagen, Hilden, Germany), extracted by mixing 0.1 ml homogenate with 0.9 ml 1% acetic acid/methanol, vortexed for 10 s, and placed at −80°C for 1 h. Samples were centrifuged at 10 000 rpm for 10 min at room temperature and supernatants were collected. Samples and standards were analyzed using HPLC (Dionex; Thermo Fisher, Sunnyvale, California, USA) with a C18 reverse-phase column [Synergi Polar-RP 80A 250×4.60 mm2 4-µm column from Phenomenex (Torrance, California, USA)]. Irinotecan and SN-38 were eluted using a gradient of 30% acetonitrile, 70% of 0.1% trifluoroacetic acid/H20 to 68% acetonitrile, and 32% of 0.1% trifluoroacetic acid/H20 over 13 min at 0.1 ml/min. Irinotecan and SN-38 peaks eluted at ~7.7 and 8.4 min, respectively, and were detected using an in-line fluorescence detector excited at 372 nm and emitting at 556 min.

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Irinotecan activation assay

Irinotecan activation in tissues was measured as previously described 23. Tumor tissues were homogenized in 6% w/v 0.1 mol/l Tris–HCl/1% Triton X-100 solution (pH 7.5) using a Tissue-Lyser for 2–4 min. A measure of 250 µg of protein lysate was mixed with an equal volume of 10 µmol/l irinotecan and incubated at 37°C for 24 h. Reactions were terminated by addition of an equal volume of 1% acetic acid/methanol. Samples were centrifuged at 10 000 rpm for 15 min and processed using HPLC as described above.

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Statistical analysis

Data were analyzed using GraphPad Prism, version 7.0 (GraphPad Software, La Jolla, California, USA). Results were analyzed using a nonparametric unpaired two-tailed Student’s t-test (Mann–Whitney), unless otherwise indicated.

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Results

Cell growth inhibition of TOP1 inhibitors increases with exposure

Although the mechanism of TOP1 inhibition has been extensively studied 30, we sought to specifically investigate the effect of dose and duration of inhibition. The activity of two TOP1 inhibitors, topotecan and SN-38 (the active metabolite of irinotecan), was tested in SCLC cell lines in in-vitro growth and viability assays. Cell growth inhibition kinetics of topotecan and SN-38 in the NCI-H1048 SCLC cell line over a time course of 88 h are shown in Fig. 1a and b. Effective cell growth inhibition was observed at ~10 nmol/l for both topotecan and SN-38, whereas cell killing was observed after prolonged incubation times at concentrations greater than or equal to 10 nmol/l. Concentration-dependent growth inhibition at selected time points is shown in Fig. 1c and d. Treatment with both topotecan and SN-38 decreased cell viability by greater than 90%. The IC50 values were variable and spanned several orders of magnitude depending on time of incubation. The human plasma Cmax for 1.5 mg/m2 topotecan is indicated in Fig. 1c and falls within the concentration required for cytotoxicity; however, the Cmax is observed shortly after administration and concentrations decline rapidly on a time scale of hours 14. The concentration range of SN-38 at which cell killing begins to occur coincides with the amount of SN-38 measured in tumor biopsies taken from patients with various solid tumors 72 h after administration of nal-IRI (range: 3–163 nmol/l 31), which is depicted in Fig. 1d as the gray shaded area. Similar results were observed in the DMS-114 cell line (Lee H, unpublished data). These experiments demonstrate that for both SN-38 and topotecan the concentration and time of exposure are important determinants of cytotoxicity.

Fig. 1

Fig. 1

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Dose and exposure are potentially limiting for topotecan

The antitumor activity of topotecan was evaluated for different doses and schedules in xenograft models. The dose used for mouse studies was based on published clinical dosing of topotecan in second-line treatment of SCLC 32 and calculated using standard surface area to weight ratios conversion per NCI guidelines. Using this method, the equivalent mouse dose corresponding to the clinical dose of 1.5 mg/m2 (days 1–5, every 3 weeks) is ~0.83 mg/kg/week and should be divided between days 1 and 2, repeating every 7 days to result in a similar exposure time as the clinical regimen (29%/cycle for preclinical vs. 24%/cycle for clinical).

To explore the effect of dose, a higher dose (1.66 mg/kg/week divided over days 1 and 2) was also evaluated in the NCI-1048 model in addition to the clinically relevant dose. As seen in Fig. 2a and b, topotecan administered at 0.83 mg/kg/week split over days 1 and 2 was well tolerated and had modest antitumor activity, eliciting some growth delay but failing to control tumor growth. Doubling the dose, using the same schedule, improved antitumor activity and resulted in longer growth delay, but ultimately failed to control tumor growth. To test the effect of schedule on antitumor activity, the same total dose delivered as a bolus on day 1 was also evaluated in the DMS-114 model. As seen in Fig. 2c and d, tumor growth curves for mice treated with topotecan at 0.83 mg/kg/week delivered as a bolus were indistinguishable from those of the control mice; however, a slight relative reduction in body weight was observed. Dividing the same total dose over 2 days demonstrated a trend toward improved antitumor activity and tolerability although the differences were not statistically significant and provided minimal delay in tumor growth.

Fig. 2

Fig. 2

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Nal-IRI effectively delivers irinotecan and SN-38 to SCLC tumors

The ability of nal-IRI to deposit in tumors through leaky vasculature and deliver irinotecan and SN-38 was evaluated in SCLC cell line-derived xenograft models (NCI-H1048, DMS-114, NCI-H841) relative to other cell line-derived and PDX models of other tumor types. Nal-IRI was administered intravenously to mice bearing xenograft tumors. At 24 h after administration, mice were sacrificed and tumors were harvested. Irinotecan and SN-38 in tumors were measured using HPLC. Figure 3a demonstrates that tumors derived from SCLC cell lines have similar or higher levels of nal-IRI deposition, as assessed by irinotecan content, compared with other tumor types. Further, analysis of SN-38 levels indicates that increased irinotecan delivery was associated with increased levels of SN-38 (Pearson correlation=0.62, P=0.02; Fig. 3b). These findings are consistent with a proposed mechanism of liposome deposition and local conversion of irinotecan to SN-38 within the tumor 23.

Fig. 3

Fig. 3

Irinotecan is converted by CES to its active metabolite, SN-38, which is 100–1000× more active than its parent compound 21. CES activity was measured by incubating tumor extracts with irinotecan and measuring SN-38 levels after 24 h using HPLC. Tumor grafts corresponding to the SCLC indication were extracted from the lung data set previously published 23 and are compared with colorectal and pancreatic tumor samples. As shown in Fig. 3c, SCLC tumors have CES activity comparable to that in gastrointestinal indications such as colorectal and pancreatic cancer, in which irinotecan has demonstrated clinical activity.

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Nal-IRI has dose-dependent anti-tumor activity

The activity of nal-IRI as a single agent was investigated in H841 SCLC subcutaneous flank xenografts in female nude mice. Dosing started at day 24 after inoculation. Irinotecan, dosed at 50 mg/kg, yielded a tumor growth delay (defined as the time for tumor volumes to reach 1000 mm3) of about 8 days compared with controls, whereas nal-IRI, dosed at 15 mg/kg, yielded a tumor growth delay of ~25 days compared with controls. After dosing nal-IRI at 30 or 50 mg/kg, the greatest reductions in tumor volume were reached at 16 days after the last dose of nal-IRI. Tumors in mice dosed with 30 mg/kg nal-IRI steadily increased in size after the nadir, whereas tumors in mice dosed with 50 mg/kg nal-IRI maintained a small residual volume that was sustained for the duration of the study up to 120 days (Fig. 4). No obvious toxicities or weight loss was noted in the nal-IRI-treated groups.

Fig. 4

Fig. 4

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Nal-IRI improves antitumor activity compared with irinotecan and topotecan

The activity of nal-IRI, irinotecan, and topotecan were directly compared at clinically relevant doses in three cell line-derived models and three PDX models. Using the standard surface area to weight ratio conversion method as described above, the proposed clinical dose of nal-IRI (90 mg/m2 free base, every 2 weeks) converted for mouse is 16 mg/kg salt every week. For irinotecan at a clinical dose of 300 mg/m2 every 3 weeks, the equivalent mouse dose is ~33 mg/kg/week. As discussed previously, for topotecan at a clinical dose of 1.5 mg/m2 (days 1–5, every 3 weeks), the equivalent mouse dose is ~0.83 mg/kg/week, divided between days 1 and 2, repeating every 7 days.

Figures 5 and 6 present tumor growth kinetics, body weight, and survival of mice bearing SCLC xenograft tumors treated weekly with nal-IRI, irinotecan, or topotecan at clinically relevant dose levels, described previously. In the DMS-53, DMS-114, and NCI-H1048 cell line-derived models, nal-IRI displayed significantly greater antitumor activity than topotecan (Fig. 5). In addition, comparison with irinotecan was also tested in DMS-114 and NCI-H1048, in which nal-IRI demonstrated significantly greater antitumor activity than irinotecan (P<0.0001 for DMS-114 on day 52 and P<0.0001 for NCI-H1048 on day 59; nonparametric t-test) and topotecan (P<0.0001 for DMS-114 on day 65 and P<0.0001 for NCI-H1048 on day 84; nonparametric t-test). Furthermore, 10 out of 10 NCI-H1048-bearing mice treated with nal-IRI experienced complete regressions of their tumors compared with 0 out of 10 mice treated with topotecan. All treatments were well tolerated in all models and survival data reflected observed differences in tumor growth curves in the DMS-53 and NCI-H1048 models. Nal-IRI, irinotecan, and topotecan were also evaluated in three PDX models with a similar experimental design (Fig. 6). In all three models, nal-IRI showed significantly greater antitumor activity than did topotecan (P<0.0001 for LUN-182 on day 87, P<0.0001 for LUN-081 on day 91, and P<0.0001 for LUN-164 on day 91; nonparametric t-test), with nal-IRI resulting in tumor regression in all models, whereas treatment with topotecan resulted in tumor stasis or modest growth delay. In two out of the three models, nal-IRI showed greater antitumor activity than irinotecan and resulted in tumor shrinkage and sustained tumor control past the final week of dosing (P<0.0001 for LUN-182 on day 90 and P<0.0001 for LUN-164 on day 102; nonparametric t-test). By contrast, treatment with irinotecan resulted in tumor stasis during treatment, and then tumor growth following the end of treatment. In the LUN-081 PDX model (Fig. 6d), nal-IRI and irinotecan had comparable antitumor activity, resulting in durable tumor regressions that persisted beyond the final week of treatment. In this model, topotecan also demonstrated the greatest control of tumor growth during the treatment period, suggesting that LUN-081 may be inherently more sensitive to TOP1 inhibitors. This was further reflected in the corresponding survival data in Fig. 6f. All treatments were well tolerated by the mice, as reflected in the body weight data. Survival data reflected observed differences in tumor growth. The LUN-164 model demonstrated the fastest tumor growth rates in the absence of any treatment and was also the least responsive to either topotecan or irinotecan; this was the only tumor model for which survival curves based on humane endpoints were able to differentiate treatments over the time scale of the experiment, whereas insufficient events were observed in the LUN-182 and LUN-081 models.

Fig. 5

Fig. 5

Fig. 6

Fig. 6

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Nal-IRI has activity after failure of other TOP1 inhibitors

We next sought to further evaluate the hypothesis that the antitumor activity of topotecan and irinotecan are limited by their ability to provide sustained inhibition of TOP1. Mice bearing DMS-114 tumor xenografts that had been treated with topotecan (0.83 mg/kg/week split over days 1–2 for 4-weekly cycles) and failed to respond to treatment were randomized to treatment with either irinotecan or nal-IRI. Despite not responding to TOP1 inhibition by topotecan and having a comparatively large average tumor size of ~1000 mm3, mice receiving nal-IRI showed significant tumor shrinkage whereas mice receiving irinotecan failed to respond (Fig. 7a). These data are consistent with the hypothesis that the antitumor activity of topotecan may be limited by insufficient delivery rather than by resistance to TOP1 inhibition. Similarly, nal-IRI was also evaluated in mice bearing DMS-114 tumor xenografts that had been first treated with irinotecan. As with topotecan, the tumors failed to respond to 4-weekly treatments with irinotecan, but showed significant tumor shrinkage following the switch to nal-IRI (Fig. 7b). These results are further consistent with the schedule-dependent efficacy of TOP1 inhibitors and support the reasoning that sustained delivery of SN-38 through nal-IRI may result in increased antitumor activity.

Fig. 7

Fig. 7

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Nal-IRI has activity in the second-line setting

Finally, the activities of nal-IRI, irinotecan, and topotecan were directly compared in a second-line setting of SCLC. Mice bearing NCI-H1048 SCLC tumors were treated with carboplatin plus etoposide, a first-line regimen in SCLC. Once the tumors escaped growth control by carboplatin plus etoposide (tumor average 1320 mm3), mice were randomized to either continue treatment with carboplatin plus etoposide or switched to second-line treatment with either nal-IRI, irinotecan, or topotecan. All treatment sequences were well tolerated as reflected by body weight, as shown in Fig. 7b. As shown in Fig. 7d, nal-IRI displayed significant antitumor activity compared with topotecan and irinotecan after tumors progressed on first-line treatment with carboplatin plus etoposide (P=0.0002 on day 70 and P=0.0002 on day 84 for topotecan and irinotecan, respectively). All treatments were well tolerated as reflected by body weight (Fig. 7e). The median survival from the initiation of second-line treatment was 7 days with vehicle control, 10 days with continued etoposide+carboplatin, 21 days with topotecan, and 59 days with irinotecan. All tumors treated with second-line nal-IRI completely regressed with a median time to complete regression of 24 days. Improved antitumor activity of nal-IRI versus irinotecan or topotecan also resulted in increased survival of mice [P<0.0001 and <0.0001 for comparison with irinotecan and topotecan, respectively; log-rank test (Mantel–Cox)], as shown in Fig. 7f.

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Discussion

In this study, we provide further evidence of the schedule-dependent efficacy of the TOP1 inhibitors topotecan and irinotecan with its active metabolite SN-38 in SCLC. The in-vitro data suggest that time of inhibition is as important as concentration in determining the extent of cytotoxicity. Moreover, we demonstrate that the in-vivo activity of topotecan appears to be limited by the dose level and pharmacokinetics that result in its inability to provide sustained inhibition of TOP1. By contrast, we hypothesize that nal-IRI is able to provide sustained TOP1 inhibition and subsequent significant antitumor activity through the extended circulation afforded by liposomal encapsulation, specific accumulation in tumors, and the local conversion of irinotecan to SN-38. We provide data demonstrating effective liposomal delivery of irinotecan to tumors of SCLC origin and subsequent conversion of irinotecan to SN-38. In direct head-to-head comparisons, taking careful consideration of scaling of doses from standard clinical doses, nal-IRI displayed significantly greater antitumor activity than both irinotecan and topotecan in cell line-derived and patient-derived xenograft models. Further, complete regressions and/or sustained antitumor activity of nal-IRI for multiple weeks beyond the last dose were observed in multiple cell line-derived and patient-derived xenograft models. The activity of nal-IRI after failure of traditional TOP1 inhibitors irinotecan and topotecan is consistent with the premise that the activity of these agents is, in part, limited by their ability to provide sustained inhibition of TOP1.

The dose level of topotecan used in this study was specifically chosen to mimic the clinically approved dosing and regimen of 1.5 mg/m2 administered on days 1–5, every 3 weeks. Given the shorter cycle length of 1 week used in mouse studies, the 0.83 mg/kg dose was divided over 2 days to provide more sustained exposure. The importance of dosing on multiple days has been observed by others 33 and is emphasized by the improved growth inhibition of DMS-114 tumors by 2-day versus single-day dosing Fig. 2. This is in contrast with other studies in which higher single-day doses were used (5–10 mg/kg) 34–36. Limits of detection of the analytical methods may further provide impetus for use of dose levels higher than those that are clinically relevant. Assuming a typical mouse blood volume of 1 ml, the single-day dose used in our studies has a theoretical Cmax of nearly 20 µmol/l and is still well above the 70 nmol/l Cmax observed in patients 14.

Human plasma Cmax values for topotecan 14 do not exceed the IC50 values for many cell lines 15,16. Coupled with rapid clearance, this suggests that many patient tumors would be unlikely to receive saturating doses of topotecan, which would thus result in conditions permissive for continued cell growth. Consequently, tumor relapse following treatment with topotecan may be a result of insufficient inhibition, in addition to, and potentially contributing to, possible evolution of resistance. The ability to achieve higher local concentrations of TOP1 inhibition by liposomal delivery and local conversion of irinotecan to SN-38 may enable killing of a wide spectrum of tumor cells, including those with greater intrinsic levels of drug resistance. This is supported by the observations of complete regressions and sustained antitumor activity after the cessation of dosing in multiple efficacy studies (Fig. 5). Additional work to explore the capacity of nal-IRI in killing specific cell subpopulations, including cancer stem cells, and study of resistance mechanisms are areas of future interest.

There is comparatively little clinical study of liposomal agents in patients with lung cancer. A small phase 2 study of pegylated liposomal doxorubicin (PLD) in patients with recurrent SCLC concluded that PLD had limited activity 37 and the combination of PLD with vincristine and cyclophosphamide had modest activity in a subsequent phase 2 study as well 38. Previous studies have suggested that doxorubicin-based combinations are rarely active after failure of platinum plus etoposide failure 37. These studies, however, leave unanswered the question of the potential contribution of liposomal delivery of a TOP1 inhibitor, such as irinotecan.

The preclinical findings presented here support the potential for effective liposomal drug delivery to human tumors in vivo. Clinical reports of macrophage infiltration in tumors from patients with SCLC 28,29 fit well with the proposed predominant mechanism of action of nal-IRI involving cellular uptake and drug conversion. Preclinical data demonstrating SN-38 levels in xenograft tumors of SCLC origin, presumably mediated by CES activity in macrophages, suggest sufficient capacity of SCLC tumors for liposome uptake and local production of SN-38. Collectively, these data provide mechanistic rationale and support for a clinical study of nal-IRI versus topotecan in patients with previously treated SCLC. Evaluation of a combination of nal-IRI with platinum agents for the treatment of patients with SCLC in the first-line setting is also warranted. Nal-IRI is not labeled for use in SCLC and the product is still investigational in that setting.

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Acknowledgements

The authors thank Istvan Molnar for insightful discussions, PharmaEngine for support of early studies in SCLC, and colleagues at Champions Oncology and GenenDesign for laboratory support.

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Conflicts of interest

There are no conflicts of interest.

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Keywords:

in-vivo pharmacology; irinotecan; small-cell lung cancer; topotecan

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