Stephan Klinz

HER2-targeted liposomal doxorubicin displays enhanced anti-tumorigenic effects without associated cardiotoxicity

Anthracycline-based regimens are a mainstay of early breast cancer therapy, however their use is limited by cardiac toxicity. The potential for cardiotoxicity is a major consideration in the design and development of combinatorial therapies incorporating anthracyclines and agents that target the HER2-mediated signaling pathway, such as trastuzumab. In this regard, HER2-targeted liposomal doxorubicin was developed to provide clinical benefit by both reducing the cardiotoxicity observed with anthracyclines and enhancing the therapeutic potential of HER2-based therapies that are currently available for HER2-overexpressing cancers. While documenting the enhanced therapeutic potential of HER2-targeted liposomal doxorubicin can be done with existing models, there has been no validated human cardiac cell-based assay system to rigorously assess the cardiotoxicity of anthracyclines. To understand if HER2-targeting of liposomal doxorubicin is possible with a favorable cardiac safety profile, we applied a human stem cell-derived cardiomyocyte platform to evaluate the doxorubicin exposure of human cardiac cells to HER2-targeted liposomal doxorubicin. To the best of our knowledge, this is the first known application of a stem cell-derived system for evaluating preclinical cardiotoxicity of an investigational agent. We demonstrate that HER2-targeted liposomal doxorubicin has little or no uptake into human cardiomyocytes, does not inhibit HER2-mediated signaling, results in little or no evidence of cardiomyocyte cell death or dysfunction, and retains the low penetration into heart tissue of liposomal doxorubicin. Taken together, this data ultimately led to the clinical decision to advance this drug to Phase I clinical testing, which is now ongoing as a single agent in HER2-expressing cancers.

Preclinical Activity of Nanoliposomal Irinotecan Is Governed by Tumor Deposition and Intratumor Prodrug Conversion

A major challenge in the clinical use of cytotoxic chemotherapeutics is maximizing efficacy in tumors while sparing normal tissue. Irinotecan is used for colorectal cancer treatment but the extent of its use is limited by toxic side effects. Liposomal delivery systems offer tools to modify pharmacokinetic and safety profiles of cytotoxic drugs. In this study, we defined parameters that maximize the antitumor activity of a nanoliposomal formulation of irinotecan (nal-IRI). In a mouse xenograft model of human colon carcinoma, nal-IRI dosing could achieve higher intratumoral levels of the prodrug irinotecan and its active metabolite SN-38 compared with free irinotecan. For example, nal-IRI administered at doses 5-fold lower than free irinotecan achieved similar intratumoral exposure of SN-38 but with superior antitumor activity. Tumor response and pharmacokinetic modeling identified the duration for which concentrations of SN-38 persisted above a critical intratumoral threshold of 120 nmol/L as determinant for antitumor activity. We identified tumor permeability and carboxylesterase activity needed for prodrug activation as critical factors in achieving longer duration of SN-38 in tumors. Simulations varying tumor permeability and carboxylesterase activity predicted a concave increase in tumor SN-38 duration, which was confirmed experimentally in 13 tumor xenograft models. Tumors in which higher SN-38 duration was achieved displayed more robust growth inhibition compared with tumors with lower SN-38 duration, confirming the importance of this factor in drug response. Overall, our work shows how liposomal encapsulation of irinotecan can safely improve its antitumor activity in preclinical models by enhancing accumulation of its active metabolite within the tumor microenvironment.

Impact of Tumor HER2/ERBB2 Expression Level on HER2-Targeted Liposomal Doxorubicin-Mediated Drug Delivery: Multiple Low-Affinity Interactions Lead to a Threshold Effect

Numerous targeted nanotherapeutics have been described for potential treatment of solid tumors. Although attention has focused on antigen selection and molecular design of these systems, there has been comparatively little study of how cellular heterogeneity influences interaction of targeted nanoparticles with tumor cells. Antigens, such as HER2/ERBB2, are heterogeneously expressed across different indications, across patients, and within individual tumors. Furthermore, antigen expression in nontarget tissues necessitates optimization of the therapeutic window. Understanding the performance of a given nanoparticle under different regimens of antigen expression has the ability to inform patient selection and clinical development decisions. In this work, HER2-targeted liposomal doxorubicin was used as a model-targeted nanoparticle to quantitatively investigate the effect of HER2 expression levels on delivery of doxorubicin to the nucleus. We find quantitatively greater nuclear doxorubicin delivery with increasing HER2 expression, exhibiting a threshold effect at approximately 2 × 105 HER2 receptors/cell. Kinetic modeling indicated that the threshold effect arises from multiple low-affinity interactions between the targeted liposome and HER2. These results support previous data showing little or no uptake into human cardiomyocytes, which express levels of HER2 below the threshold. Finally, these results suggest that HER2-targeted liposomal doxorubicin may effectively target tumors that fall below traditional definitions of HER2-positive tumors, thereby expanding the potential population of patients that might benefit from this agent. Mol Cancer Ther; 12(9); 1816–28. ©2013 AACR.

Correlation between Ferumoxytol Uptake in Tumor Lesions by MRI and Response to Nanoliposomal Irinotecan in Patients with Advanced Solid Tumors: A Pilot Study

Purpose: To determine whether deposition characteristics of ferumoxytol (FMX) iron nanoparticles in tumors, identified by quantitative MRI, may predict tumor lesion response to nanoliposomal irinotecan (nal-IRI). Experimental Design: Eligible patients with previously treated solid tumors had FMX-MRI scans before and following (1, 24, and 72 hours) FMX injection. After MRI acquisition, R2* signal was used to calculate FMX levels in plasma, reference tissue, and tumor lesions by comparison with a phantom-based standard curve. Patients then received nal-IRI (70 mg/m2 free base strength) biweekly until progression. Two percutaneous core biopsies were collected from selected tumor lesions 72 hours after FMX or nal-IRI. Results: Iron particle levels were quantified by FMX-MRI in plasma, reference tissues, and tumor lesions in 13 of 15 eligible patients. On the basis of a mechanistic pharmacokinetic model, tissue permeability to FMX correlated with early FMX-MRI signals at 1 and 24 hours, while FMX tissue binding contributed at 72 hours. Higher FMX levels (ranked relative to median value of multiple evaluable lesions from 9 patients) were significantly associated with reduction in lesion size by RECIST v1.1 at early time points (P < 0.001 at 1 hour and P < 0.003 at 24 hours FMX-MRI, one-way ANOVA). No association was observed with post-FMX levels at 72 hours. Irinotecan drug levels in lesions correlated with patients time on treatment (Spearman ρ = 0.7824; P = 0.0016). Conclusions: Correlation between FMX levels in tumor lesions and nal-IRI activity suggests that lesion permeability to FMX and subsequent tumor uptake may be a useful noninvasive and predictive biomarker for nal-IRI response in patients with solid tumors. Clin Cancer Res; 23(14); 3638–48. ©2017 AACR.

Abstract 2065: Magnetic resonance imaging with an iron oxide nanoparticle demonstrates the preclinical feasibility of predicting intratumoral uptake and activity of MM-398, a nanoliposomal irinotecan (nal-IRI)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Sustained intratumoral delivery of cytotoxic agents is a major challenge for effective cancer treatment, and motivated the development of MM-398, a stable nanoliposomal irinotecan (nal-IRI) with an extended plasma half-life and greater tumor deposition than free irinotecan. By using a systems pharmacology approach, we have previously shown that tumor deposition of nal-IRI and the subsequent conversion of irinotecan to the active metabolite, SN-38, by carboxylesterases are important determinants for nal-IRI activity in vivo. Ferumoxytol (FMX) is a 30nm iron-oxide, super-paramagnetic nanoparticle, known to be taken up by macrophages (as is nal-IRI), and for exhibiting magnetic resonance imaging properties. Since the size of a nanoparticle affects the rate of transcapillary transport significantly, we hypothesized that nal-IRI tumor biodistribution may be predicted by FMX-based MRI (Fe-MRI). Biodistribution and imaging studies were performed in mice bearing cell-line derived (A2780, HT29, A549) and patient-derived (pancreatic adenocarcinoma) tumor xenografts. The protocol consisted of a baseline MRI scan, i.v. injection of FMX (20mg/kg), and then i.v. injection of fluorescently labeled nal-IRI (10mg/kg) 24hr later. Mice were sacrificed 24hr and 72hr after nal-IRI injection, and irinotecan and SN-38 concentrations were determined in plasma, tumor, and tissues by HPLC analysis. The presence of FMX did not interfere with nal-IRI PK or biodistribution. Cellular distribution of liposomes within tumors was also not affected by FMX at up to 50mg/kg as measured by flow cytometry. Furthermore, immunohistochemistry showed that both liposomes and FMX were co-localized with tumor-associated macrophages. The drug metabolite measurements from tissue samples showed that the xenograft tumor models display wide ranges of nal-IRI deposition capacity (irinotecan concentrations at 24hr: ∼2,104 to 20,096ng/g). A2780 tumors displayed highest concentration of both iron (3.92 μg/ml) and irinotecan (9,466 ng/g) at 72hr after nal-IRI injection, whereas A549 tumors displayed lowest levels of both iron (0.23 μg/ml ) and irinotecan (436 ng/g). We observed a correlation between the tumor Fe-MRI signal and intratumoral levels of irinotecan 72hr after nal-IRI injection (R2=0.9, p<0.001). Furthermore, in vivo activity studies confirmed that xenograft models having higher intratumoral levels of irinotecan and SN-38 at 72hr showed greater tumor growth inhibition. In summary, preclinical studies demonstrate the potential of utilizing Fe-MRI as a potential diagnostic tool to identify patients with higher tumor permeability. Based on encouraging preclinical data, a pilot study in patients with advanced solid tumors with extensive Fe-MRI scanning and paired tumor biopsies (NCT # 01770353) is being conducted. Citation Format: Ashish V. Kalra, Joseph Spernyak, Jaeyeon Kim, Arnold Sengooba, Stephan Klinz, Nancy Paz, Jason Cain, Walid Kamoun, Ninfa Straubinger, Yang Qu, Sheryl Trueman, Eliel Bayever, Ulrik Nielsen, Daryl Drummond, Jonathan Fitzgerald, Robert Straubinger. Magnetic resonance imaging with an iron oxide nanoparticle demonstrates the preclinical feasibility of predicting intratumoral uptake and activity of MM-398, a nanoliposomal irinotecan (nal-IRI). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2065. doi:10.1158/1538-7445.AM2014-2065

Abstract CT224: Pilot study in patients with advanced solid tumors to evaluate feasibility of ferumoxytol (FMX) as tumor imaging agent prior to MM-398, a nanoliposomal irinotecan (nal-IRI)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction MM-398, a stable nanoliposomal irinotecan (nal-IRI), is designed to exploit leaky tumor vasculature for enhanced drug delivery to tumors. Tumor deposition of nal-IRI and subsequent irinotecan conversion by CES enzymes in both neoplastic cells and tumor associated macrophages (TAM) may positively correlate with activity. Predictive biomarkers to measure tumor deposition could identify patients likely to benefit from nal-IRI. FMX is a 30nm iron-oxide, superparamagnetic nanoparticle with MRI contrast properties. The particle size, its propensity for uptake by TAMs and similar distribution patterns to nal-IRI in preclinical models led to a clinical study evaluating the feasibility of correlating FMX-based MRI (Fe-MRI) acquisition with tissue drug metabolite levels and other biomarkers to estimate drug delivery to tumor. Patients and methods Eligible patients (n=12) with refractory solid tumors with at least two metastatic lesions >2cm accessible for a percutaneous biopsy were enrolled from one institution. Fe-MRI scans were performed on a 1.5T MRI using T2* iron sensitive sequences prior to and following FMX infusion (0.5h, 24h, 72h). MR images were used to direct biopsies at 72h to FMX high or low regions, permitting intra- and inter-patient comparisons of FMX and nal-IRI tumor levels. Patients continued on nal-IRI at 80mg/m2 q2w until progression. Tissue iron and TAM distribution were assessed by IHC, tissue-bound metabolite levels by mass-spectrometry. T2* signal was used to calculate FMX levels in total lesions along with FMX estimates on biopsy images derived from fused MRI-CT biopsy images. The first 9 patients (2M 7F; median age 57 years, range 28-71 years) are reported. Results There were no safety-related or other potential interactions with nal-IRI and FMX. Adverse events of nal-IRI were consistent with previous studies. FMX levels, quantified in 36 tumor lesions from the first 9 subjects, showed mean FMX accumulation of 37.9 mcg/mL [3.3-101.2 mcg/mL] and 13.2 mcg/mL [0.1-41.0 mcg/mL] at 24h and 72h, respectively. Lesions were localized mostly in liver (67%) and lymph nodes/peritoneal sites (25%). A mechanistic PK model indicated that tissue permeability to FMX contributed to Fe-MRI signals at 24h, while FMX binding contributed at 72h. Levels of irinotecan and SN-38 were 3.59mcg/g [2.29-4.89mcg/g] and 11.43ng/g [4.04-18.8ng/g], respectively, at 72h in biopsies from the first 6 patients. Conclusions This study is one of the first to measure active metabolite SN-38 levels in patient tumors. FMX can be used as a tumor contrast agent prior to nal-IRI treatment. T2* MRI sequences allowed for quantitation of FMX concentrations in tumor and reference tissue. A mechanistic model provided an estimation of FMX tumor tissue permeability and binding that may be useful as a predictive biomarker of nanotherapeutics such as nal-IRI. Citation Format: Ramesh K. Ramanathan, Ronald L. Korn, Jasgit C. Sachdev, Gerald J. Fetterly, Katie Marceau, Vickie Marsh, John M. Neil, Ronald G. Newbold, Natarajan Raghunand, Joshua Prey, Stephan G. Klinz, Eliel Bayever, Jonathan B. Fitzgerald. Pilot study in patients with advanced solid tumors to evaluate feasibility of ferumoxytol (FMX) as tumor imaging agent prior to MM-398, a nanoliposomal irinotecan (nal-IRI). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT224. doi:10.1158/1538-7445.AM2014-CT224

Abstract C90: HER2-targeted liposomal doxorubicin MM-302 has a favorable cardiosafety profile in preclinical models.

Introduction: Anthracyclines have historically been the backbone of anticancer therapy for decades; however, the clinical observation of permanent cardiotoxicity has limited their broader use. In the HER2 positive breast cancer setting, the combination of trastuzumab with anthracyclines was associated with an unacceptable risk of clinical cardiotoxicity. This appears to be related to 1) doxorubicin-induced cardiomyocyte stress and 2) inability of the cardiomyocytes to respond to the stress due to the trastuzumab-related impairment of HER2 signaling. Encapsulation of doxorubicin into liposomes (Doxil®) has been associated with a reduced risk of doxorubicin-associated cardiotoxicity relative to free doxorubicin. MM-302 is a HER2-targeted liposomal doxorubicin-based agent designed to target doxorubicin to HER2-overexpressing cancer cells, while limiting uptake into non-target cells. Since HER2-mediated signaling plays an important role in cardiac repair, the purpose of this work was to support clinical development of MM-302 by investigating whether HER2-targeting of liposomal doxorubicin would adversely affect the cardiac safety profile relative to untargeted liposomal doxorubicin. Methods: MM-302, untargeted liposomal doxorubicin and free doxorubicin were compared for their uptake into HER2-overexpressing cancer cells and two models of human stem cell-derived cardiomyocytes. High-content microscopy was used to determine the effects of exposure to specific cellular targets in cardiomyocytes. Mouse biodistribution studies were used to assess the total and nuclear accumulation of doxorubicin in mouse cardiac tissue upon treatment with either MM-302, untargeted liposomes or free doxorubicin. Quantitative immunofluorescence was used to quantify the HER2 expression on human normal and diseased heart specimens. Kinetic computational modeling was applied to interpret study results and to make predictions on human heart exposure to doxorubicin based on the experimental mouse data. Results: Human stem cell-derived cardiomyocytes showed nuclear accumulation of doxorubicin followed by cell death upon free doxorubicin treatment but not upon MM-302 or untargeted liposomes treatment. Similarly, MM-302 resulted in a significantly lower nuclear accumulation of doxorubicin compared to free doxorubicin treatment in mouse cardiac tissue. HER2 expression levels on normal and diseased human heart tissue were shown to be ≤100,000 receptors/cell, in concordance with the HER2 levels on the stem cell-derived cardiomyocytes found in vitro. Computational modeling predicts liposomal encapsulation to significantly protect the human heart from exposure to doxorubicin, consistent with clinical findings. Conclusions: MM-302 is not taken up by human cardiomyocytes via HER2, but selectively increases doxorubicin delivery to human HER2-overexpressing tumor cells. MM-302 can potentially improve the clinical efficacy demonstrated by conventional anthracyclines and maintain the cardiac safety profile of untargeted liposomal doxorubicin in patients with HER2 overexpressing cancers. Clinical evaluation of this patient population is currently in Phase I. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C90.

Abstract P5-01-06: Characterization of metastatic breast cancer lesions with ferumoxytol MRI and treatment response to MM-398, nanoliposomal irinotecan (nal-IRI)

Introduction Irinotecan has known activity in metastatic breast cancer (MBC). MM-398, nanoliposomal irinotecan (nal-IRI), is designed to exploit leaky tumor vasculature for enhanced drug delivery to tumors. Tumor deposition of nal-IRI and subsequent conversion to SN-38 in both neoplastic cells and tumor associated macrophages (TAM) may positively correlate with activity. Predictive biomarkers to measure tumor deposition could identify patients likely to benefit from nal-IRI. Ferumoxytol (FMX), an iron-oxide superparamagnetic nanoparticle with MRI contrast properties, is taken up by TAMs with similar distribution patterns to nal-IRI in preclinical models. Our previous work has shown the feasibility of quantitative FMX MRI (Fe-MRI) of tumor lesions, and we developed a quantitative mechanistic PK model of FMX deposition (AACR 2014, abstract #CT224). Here we report nal-IRI activity and FMX levels in MBC patients on the study. Patients and methods Patients (n=15) with refractory solid tumors and at least two metastatic lesions >2 cm accessible for percutaneous biopsy were enrolled in a Phase 1 study. Fe-MRI scans were performed using T2* iron sensitive sequences prior to and following FMX infusion (1 h, 24 h, 72 h). T2* signal was used to calculate FMX levels in total lesions by comparison to a standard curve. Comparison of quantified FMX lesion uptake with a mechanistic PK model previously indicated that tissue permeability to FMX contributed to early Fe-MRI signals at 1 h and 24 h, while FMX binding contributed at 72 h. Patients then received nal-IRI (80 mg/m2 q2w) until progression. Core biopsies were obtained 72 h after both FMX and nal-IRI infusions. RECIST evaluation was done by CT every 8 weeks. Results FMX was well tolerated, and adverse events to nal-IRI were consistent with previous studies. Three of the 13 patients receiving nal-IRI had ER/PR+ MBC (median # of prior Rx: 8 compared to 4 for all study patients). Thirteen liver lesions (4-5/pt) were evaluated by FMX-MRI and CT for these 3 patients. Average lesion size: 26.9±11.2 mm diameter and 8.1±11.3 cm3 (median 4.7 cm3). Time on treatment for the 3 patients was 57, 126 and 256 days (study median 57 days). Best overall response was 1 stable disease (SD) and 1 partial response (PR) in these 3 patients. The patient with a PR had an average lesion size reduction of 44.5%, while the patient with SD had an average lesion size increase of 12.5% at final evaluation. Lesions that shrank after nal-IRI showed higher early levels of FMX compared to the study median (median 39.6 vs. 32.6 mcg/mL at 1 h; median 37.7 vs. 34.5 mcg/mL at 24 h). This relationship between lesion response and FMX levels was consistent with the lesion behavior in the full data set (n=31 lesions/9 patients across 7 indications) of the study. Conclusions Clinical activity of nal-IRI was observed in a subset of heavily treated ER/PR+ MBC patients. The relationship between FMX levels in tumor lesions and nal-IRI activity suggests that lesion permeability to FMX may be a useful biomarker for nal-IRI deposition and tumor response in MBC and potentially other indications. A multi-institution expansion of this study in HER2-negative MBC is planned to confirm these findings. Citation Format: Jasgit C Sachdev, Ramesh K Ramanathan, Natarajan Raghunand, Jaeyeon Kim, Stephan G Klinz, Eliel Bayever, Jonathan B Fitzgerald, Ronald L Korn. Characterization of metastatic breast cancer lesions with ferumoxytol MRI and treatment response to MM-398, nanoliposomal irinotecan (nal-IRI) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-01-06.

Abstract C293: Irinotecan sucrosofate liposome injection, MM-398, demonstrates superior activity and control of hypoxia as measured through longitudinal imaging using [18F]FAZA PET compared to free irinotecan in a colon adenocarcinoma xenograft model.

Tumor hypoxia is strongly linked to aggressive disease progression and resistance to therapy. Positron emission tomography (PET) imaging with hypoxia tracers such as [18F]fluoroazomycin arabinoside (FAZA) allows for non-invasive quantification of tumor hypoxia during treatment. We and others have previously shown by immunohistochemical methods that treatments with longer lasting camptothecin formulations reduce tumor hypoxia after either single or multiple treatment cycles. Here we evaluated the kinetics and magnitude of hypoxia changes in tumors after treatment with irinotecan sucrosofate liposome injection (MM-398) which has shown an extended plasma half-life and higher intratumoral deposition in animal models relative to free pro-drug and compare it to the effects of free irinotecan at equivalent exposure levels. FAZA-PET/CT was used for longitudinal monitoring of tumor hypoxia changes in the HT29 mouse colon cancer xenograft model over a 21-day period following weekly chemotherapy administrations of either MM-398 (5 & 10mg/kg) or free irinotecan (50mg/kg). These dosages were predicted to result in comparable SN-38 exposure in either plasma or tumor based on a mechanistic pharmacokinetic model of MM-398 and free irinotecan that was developed using a systems pharmacology approach. Baseline levels of FAZA uptake in tumors were similar across treatment groups. Significant differences in tumor FAZA uptake were observed between these groups as early as Day 7 post treatment initiation, with increased FAZA uptake seen in tumors treated with free irinotecan. In contrast, differences in tumor volume only became statistically significant on Day 16. MM-398 at 10mg/kg was the most effective treatment for control of tumor volume and also minimized changes in FAZA uptake at all time-points. Background FAZA levels in the muscle were consistent over time across all treatment groups (0.78±0.18 %ID/g, 0.81±0.11 %ID/g and 0.71±0.20 %ID/g). However, normalization with muscle signal did not improve quantification of FAZA uptake differences in tumors. Tumor-specific hypoxia status at the study end point was confirmed by co-staining for CA9 and EF5 levels, which were, as expected, highly correlated. Average EF5 intensity/tumor area was lowest in the MM-398 (10mg/kg) treatment group, while being highest in the irinotecan (50mg/kg) treatment group. This study demonstrated the feasibility of performing longitudinal and repeated tumor hypoxia assessment using FAZA-PET imaging. Treatment with MM-398, but not free irinotecan, led to significant changes in the tumor microenvironment as measured by reduced hypoxia levels that occurred far earlier than anatomical changes assessed by tumor volume. Imaging of hypoxia levels after anti-cancer therapy with MM-398 has the potential to allow early assessment of treatment activity. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C293. Citation Format: Stephan G. Klinz, Jinzi Zheng, Raquel De Souza, Michael Dunne, Jason Cain, Jaeyeon Kim, Nancy Paz, Ashish Kalra, David Jaffray, Jonathan Fitzgerald. Irinotecan sucrosofate liposome injection, MM-398, demonstrates superior activity and control of hypoxia as measured through longitudinal imaging using [18F]FAZA PET compared to free irinotecan in a colon adenocarcinoma xenograft model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C293.

Abstract A6: Sustained intratumoral activation of MM-398 results in superior activity over irinotecan demonstrated by using a systems pharmacology approach

MM-398 is a stable nanotherapeutic encapsulation of the prodrug irinotecan with an extended plasma half-life and higher intratumoral deposition compared with free-irinotecan. MM-398 is currently in multiple clinical trials, including a phase 3 trial for patients with advanced gemcitabine-resistant pancreatic cancer (NAPOLI-1). Pancreatic cancer has been described as being notoriously difficult to treat, potentially due to inadequate drug penetration through the dense stroma, or because the hypoxic tumor microenvironment suppresses cytotoxic activity. We sought to better understand how MM-398, a relatively large (100nm) liposomal nanotherapeutic, could potentially treat pancreatic cancer by determining the relative roles of systemic vs. local tumor activation of irinotecan in contributing to the activity of MM-398. Using a systems pharmacology approach, we developed a mechanistic pharmacokinetic (PK) model of MM-398 and free-irinotecan to predict both plasma and intratumoral levels of irinotecan and SN-38. The model was trained with PK and biodistribution data from mice bearing HT-29 xenografts, which were administered intravenously with varying doses of MM-398 or free-irinotecan. Model simulations predicted that MM-398 resulted in equivalent SN-38 exposure (area under curve, AUC) in tumor at a fivefold lower dose than free-irinotecan. However, an in vivo animal activity study showed that 15-fold lower dose of MM-398 was sufficient to yield equal growth inhibition of HT-29 xenografts, which reveals the limit of relating simple AUC-based exposure to in vivo tumor response. While intratumoral SN-38 exposure from free-irinotecan was limited to the first 48 hours after dosing, MM-398 maintained high levels of SN-38 throughout the week-long time window. Further analysis of the exposure-response identified that the duration of intratumoral SN-38 levels above the threshold was a valid predictive marker for xenograft tumor response. Identifying the source of intratumoral SN38 is confounded by the fact that the mouse species has an additional carboxylesterase (CES) that can convert irinotecan to SN-38 in serum. The serum SN-38/irinotecan ratio in mice is tenfold higher than that observed in humans. In order to translate this preclinical observation into the clinic, it is critical to identify the role of mouse-specific serum CES on intratumoral SN-38 exposure. Thus, we performed a PK study with knockout mice lacking the Ces1c gene, which encodes serum CES, and then retrained our mechanistic PK model. Serum SN-38 levels in the Ces1c knockout mice were measurably decreased by ˜85% in the central compartment. In contrast, simulating the effect of knock-out of either serum CES or tumor CES, predicts that the duration of intratumoral residence of SN-38 is significantly affected by tumor CES, rather than serum CES. This suggests that local activation to SN-38 by tumor CES as the main driver for SN-38 tumor residence, which in turn drives response. In summary, we applied a systems pharmacology approach to identify the importance of tumor CES (local SN-38 generation) as one of the determinants of MM-398 response. Liposomal encapsulation of irinotecan dramatically alters the pharmacokinetic profile of SN-38 in the tumor, as well as tumor response, by maintaining SN-38 levels above the response threshold. Local, sustained activity of this active irinotecan metabolite could result in prolonged cytotoxic and tumor microenvironment modifications with beneficial effects on treatment of pancreatic cancer and other solid tumors. Citation Format: Jaeyeon Kim, Eliel Bayever, Peter Laivins, Clet Niyikiza, Ulrik Nielsen, Jonathan Fitzgerald, Ashish Kalra, Milind Chalishazar, Stephan Klinz, Nancy Paz, Bart Hendriks, Daryl Drummond, Dmitri Kirpotin, Victor Moyo. Sustained intratumoral activation of MM-398 results in superior activity over irinotecan demonstrated by using a systems pharmacology approach [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr A6.