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Physiologic barriers to drug delivery and selection for drug resistance limit survival outcomes in cancer patients. In this study, we present preclinical evidence that a subtumoricidal photodynamic priming (PDP) strategy can relieve drug delivery barriers in the tumor microenvironment to safely widen the therapeutic window of a nanoformulated cytotoxic drug. In orthotopic xenograft models of pancreatic cancer, combining PDP with nanoliposomal irinotecan (nal-IRI) prevented tumor relapse, reduced metastasis, and increased both progression-free survival and 1-year disease-free survival. PDP enabled these durable improvements by targeting multiple tumor compartments to (i) increase intratumoral drug accumulation by >10-fold, (ii) increase the duration of drug exposure above a critical therapeutic threshold, and (iii) attenuate surges in CD44 and CXCR4 expression, which mediate chemoresistance often observed after multicycle chemotherapy. Overall, our results offer preclinical proof of concept for the effectiveness of PDP to minimize risks of tumor relapse, progression, and drug resistance and to extend patient survival.Significance: A biophysical priming approach overcomes key treatment barriers, significantly reduces metastases, and prolongs survival in orthotopic models of human pancreatic cancer. Cancer Res; 78(2); 558–71. ©2017 AACR.

Sub-tumouricidal PDP does not increase systemic toxicity of chemotherapy. Changes in mouse body weight were monitored longitudinally before and after treatment.

Supplementary Figure 4: Inverse correlation between sensitivity to DNA damaging agents and SLFN11 expression.

Purpose: To determine the pharmacokinetics and the antitumor activity in pediatric cancer models of MM-398, a nanoliposomal irinotecan (nal-IRI).Experimental Design: Mouse plasma and tissue pharmacokinetics of nal-IRI and the current clinical formulation of irinotecan were characterized. In vivo activity of irinotecan and nal-IRI was compared in xenograft models (3 each in nu/nu mice) of Ewing's sarcoma family of tumors (EFT), neuroblastoma (NB), and rhabdomyosarcoma (RMS). SLFN11 expression was assessed by Affymetrix HuEx arrays, Taqman RT-PCR, and immunoblotting.Results: Plasma and tumor concentrations of irinotecan and SN-38 (active metabolite) were approximately 10-fold higher for nal-IRI than for irinotecan. Two doses of NAL-IRI (10 mg/kg/dose) achieved complete responses maintained for >100 days in 24 of 27 EFT-xenografted mice. Event-free survival for mice with RMS and NB was significantly shorter than for EFT. High SLFN11 expression has been reported to correlate with sensitivity to DNA damaging agents; median SLFN11 mRNA expression was >100-fold greater in both EFT cell lines and primary tumors compared with NB or RMS cell lines or primary tumors. Cytotoxicity of SN-38 inversely correlated with SLFN11 mRNA expression in 20 EFT cell lines.Conclusions: In pediatric solid tumor xenografts, nal-IRI demonstrated higher systemic and tumor exposures to SN-38 and improved antitumor activity compared with the current clinical formulation of irinotecan. Clinical studies of nal-IRI in pediatric solid tumors (especially EFT) and correlative studies to determine if SLFN11 expression can serve as a biomarker to predict nal-IRI clinical activity are warranted. Clin Cancer Res; 21(5); 1139–50. ©2015 AACR.

Supplementary Table 1. Human EFT, RMS, and NB cell lines used for current study. Supplementary Table 2. Median Survival of xenograft mice bearing human pediatric cancer treated with irinotecan or nal-IRI. Supplementary Table 3. Pharmacokinetic variables for irinotecan and nal-IRI following intravenous injections in mice.

Supplementary Figure 1: Changes in body weight of mice treated with vehicle.

Supplementary Figure 3: Representative pictures of macrophage infiltration in tumors.

Supplementary Figure 2: The effect of plasma caroxylesterase activity on the conversion of CPT-11 to SN-38 in mouse and human plasma.

Supplemental Figures S1-S5, Tables S1-S2. This document includes supplemental figures and corresponding legends. Table S1. Differential equations for plasma PK and tumor deposition models. Figure S1. Pharmacokinetic profile of nal-IRI across various tissues. Figure S2. Precision of parameter estimates. Figure S3. Comparing the concentrations of free CPT-11 and total CPT-11 in plasma and tumors. Figure S4. Surrogates for tumor depostion and local activation of nal-IRI. Table S2. Concentrations of CPT-11, SN-38 and CES activity in the panel of xenograft models. Figure S5. In vitro activation of nal-IRI in U937 cell lines.

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. Cancer Res; 74(23); 7003–13. ©2014 AACR.

Supplemental Methods. This document contains information on pharmacokinetic model development

Con la intención de contribuir hacia una reflexión crítica en el campo de la educación el presente artículo de reflexión quiere mostrar desde lo macro hasta los espacios educativos que está sucediendo en la realidad concreta y poder presentar a través de exponentes de la pedagogía critica posibles caminos para una educación que permita tomar en cuenta las habilidades heterogéneas en un sistema que quiere homogenizar. Se reflexiona a través de educadores que han criticado la educación actual desde un punto de vista humano como son Mc Laren, Freire y Ken Robinson. Asimismo, se vive una educación que quiere estandarizar a los educandos y se enfoca en asignaturas concretas que a pesar de su importancia dejan a un lado aquellas que pueden incentivar la creatividad, imaginación e innovación. Por otro lado, se toma en cuenta tanto al discente como al docente en este acto educativo.

Physiologic barriers to drug delivery and selection for drug resistance limit survival outcomes in cancer patients. In this study, we present preclinical evidence that a subtumoricidal photodynamic priming (PDP) strategy can relieve drug delivery barriers in the tumor microenvironment to safely widen the therapeutic window of a nanoformulated cytotoxic drug. In orthotopic xenograft models of pancreatic cancer, combining PDP with nanoliposomal irinotecan (nal-IRI) prevented tumor relapse, reduced metastasis, and increased both progression-free survival and 1-year disease-free survival. PDP enabled these durable improvements by targeting multiple tumor compartments to (i) increase intratumoral drug accumulation by >10-fold, (ii) increase the duration of drug exposure above a critical therapeutic threshold, and (iii) attenuate surges in CD44 and CXCR4 expression, which mediate chemoresistance often observed after multicycle chemotherapy. Overall, our results offer preclinical proof of concept for the effectiveness of PDP to minimize risks of tumor relapse, progression, and drug resistance and to extend patient survival. Significance: A biophysical priming approach overcomes key treatment barriers, significantly reduces metastases, and prolongs survival in orthotopic models of human pancreatic cancer. Cancer Res; 78(2); 558–71. ©2017 AACR.

Purpose: To determine the pharmacokinetics and the antitumor activity in pediatric cancer models of MM-398, a nanoliposomal irinotecan (nal-IRI). Experimental Design: Mouse plasma and tissue pharmacokinetics of nal-IRI and the current clinical formulation of irinotecan were characterized. In vivo activity of irinotecan and nal-IRI was compared in xenograft models (3 each in nu/nu mice) of Ewing's sarcoma family of tumors (EFT), neuroblastoma (NB), and rhabdomyosarcoma (RMS). SLFN11 expression was assessed by Affymetrix HuEx arrays, Taqman RT-PCR, and immunoblotting. Results: Plasma and tumor concentrations of irinotecan and SN-38 (active metabolite) were approximately 10-fold higher for nal-IRI than for irinotecan. Two doses of NAL-IRI (10 mg/kg/dose) achieved complete responses maintained for >100 days in 24 of 27 EFT-xenografted mice. Event-free survival for mice with RMS and NB was significantly shorter than for EFT. High SLFN11 expression has been reported to correlate with sensitivity to DNA damaging agents; median SLFN11 mRNA expression was >100-fold greater in both EFT cell lines and primary tumors compared with NB or RMS cell lines or primary tumors. Cytotoxicity of SN-38 inversely correlated with SLFN11 mRNA expression in 20 EFT cell lines. Conclusions: In pediatric solid tumor xenografts, nal-IRI demonstrated higher systemic and tumor exposures to SN-38 and improved antitumor activity compared with the current clinical formulation of irinotecan. Clinical studies of nal-IRI in pediatric solid tumors (especially EFT) and correlative studies to determine if SLFN11 expression can serve as a biomarker to predict nal-IRI clinical activity are warranted. Clin Cancer Res; 21(5); 1139–50. ©2015 AACR.

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.

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. Cancer Res; 74(23); 7003–13. ©2014 AACR.

e16205Background: nal-IRI facilitates intratumoral drug deposition through enhanced permeability and retention, with pharmacokinetic and pharmacodynamic (PK/PD) analyses demonstrating an extended c...

335 Background: Liposomal irinotecan (nal-IRI, ONIVYDE) is approved in the US, EU and other countries in combination with 5-fluorouracil/leucovorin for treatment of patients with metastatic pancreatic cancer after disease progression following gemcitabine-based therapy. We report pharmacokinetic and extended pharmacodynamic effects of nal-IRI in pancreatic tumor models compared to non-liposomal irinotecan HCl. Methods: AsPC-1, BxPC-3 and CFPAC-1 tumors were grown in NOD-SCID mice. For efficacy animals were dosed q7d with 25-50 mg/kg irinotecan HCl or at 5x lower doses of nal-IRI. For PK-PD studies doses of 10-50 mg/kg of fluorescently-labeled nal-IRI were used; samples were collected up to 72 hr for irinotecan HCl and up to 168 hr for nal-IRI. Tumor samples were evaluated for liposome localization, macrophage and tumor markers, vessels, DNA damage and apoptosis. Results: nal-IRI yields sustained circulation and delivery of its payload to tumors compared to irinotecan HCl. This results in improved control of growth rates across a range of pancreatic tumor models even at 5x lower doses. DNA damage in BxPC-3 tumors has a comparable extent with both formulations, but is maximal at 6 hr after irinotecan HCl (50 mg/kg) and at 72 hr after nal-IRI (10 mg/kg). Liposomes deposit in tumors heterogeneously around functional vessels. Accumulation peaks at 6 - 24 hr with similar deposition patterns in cell- or patient-derived xenografts. Liposomes are predominantly taken up by macrophages and to a lesser extent by tumor or other stromal cells. DNA damage is mostly confined to tumor cells, a majority of which have not internalized liposomes. DNA damage and apoptosis are seen only minimally in non-tumor cells even when displaying high liposome uptake. Conclusions: nal-IRI improves tumoral deposition of its payload in pancreatic tumor models. Deposition is heterogeneous and restricted to perivascular areas. DNA damage predominantly in tumor cells outside of the liposomal deposition area suggests sufficient intratumoral levels of the SN-38 active metabolite, possibly after payload release by stromal macrophages and concomitant conversion. Effects of repeated dosing cycles should be explored.

In the present study we have characterized T cell-driven immune function in mice that are genetically deficient in PKC theta. In response to simple immunologic stimulation invoked by in vivo T cell receptor (TCR) cross-linking, these mice showed significantly depressed plasma cytokine levels for IL-2, IL-4, IFNgamma, and TNFalpha compared to wild-type (WT) mice. In parallel, spleen mRNA levels for these cytokines were reduced, and NF-kappaB activation was also reduced in PKC theta knockouts (KO). Injection of allogeneic cells into the footpad of PKC theta deficient mice provoked a significantly diminished local T cell response compared to WT mice similarly challenged. Unlike comparable cells from wild type mice, CD45RBhi T cells harvested from PKC theta deficient mice failed to induce colitis in the SCID-CD45RB cell transfer model of IBD. In another T cell-dependent model of inflammatory disease, PKC theta deficient animals developed far less severe neurologic signs and reduced spinal cord inflammatory cell infiltrate compared to WT controls in the MOG-induced EAE model. A fundamental role for PKC theta in T cell activation and in the development of T cell-mediated inflammatory diseases is indicated by these results.

336 Background: Nanoliposomal irinotecan (nal-IRI, MM-398) recently gained approval in combination with 5-fluorouracil/leucovorin (5-FU/LV) in post-gemcitabine metastatic pancreatic ductal adenocarcinoma (PDAC) based on the extended survival and manageable safety profile observed in the Phase 3 NAPOLI-1 trial. Preclinically, we have previously demonstrated the anti-tumor activity of nal-IRI with 5-FU and oxaliplatin, standard of care agents in first-line PDAC, and are currently investigating this combination in patients with previously untreated metastatic PDAC in a Phase 2 clinical trial (NCT02551991). Herein, we further evaluate nal-IRI as a potential backbone of first-line metastatic PDAC by assessing the preclinical anti-tumor activity of nal-IRI relative to, and in combination with, gemcitabine and nanoparticle albumin-bound-paclitaxel (nab-P). Methods: Nal-IRI tumor metabolite (CPT-11 and SN-38) levels were measured in mice treated with nal-IRI in combination with gemcitabine or nab-P. Anti-tumor activity and tolerability of nal-IRI, 5-FU, gemcitabine and nab-P monotherapies and combinations were evaluated using pancreatic cancer cell line (ASPC-1 and CFPAC-1)-derived xenograft models, as well as a panel of five patient-derived xenograft models. Results: Administration of gemcitabine or nab-P prior to or simultaneously with nal-IRI resulted in unchanged or increased nal-IRI deposition, as measured by tumor CPT-11 and SN-38 levels at 24 hours post-injection. Moreover, in both cell line-derived and patient-derived xenograft models of PDAC, nal-IRI monotherapy demonstrated comparable or improved anti-tumor activity relative to gemcitabine or nab-P monotherapies. Further, nal-IRI consistently improved tumor growth inhibition and survival when used in combination with either 5-FU, gemcitabine and/or nab-P, relative to the combination of gemcitabine plus nab-P. All treatments were well-tolerated in these preclinical models. Conclusions: These findings illustrate the compatibility and therapeutic potential of nal-IRI as a foundation of first-line PDAC combination regimens, and warrant clinical evaluation.

Introduction: Tumor hypoxia has been strongly linked to aggressive disease progression and resistance to therapy, especially in pancreatic cancer where the desmoplastic reaction is thought to also interfere with deposition of both small molecule drugs and nanotherapeutics. [18F]fluoroazomycin arabinoside (FAZA) is a radioactive tracer that allows for non-invasive quantification of tumor hypoxia during treatment by positron emission tomography (PET). We have previously shown that in the HT-29 cell-line derived xenograft model of colorectal cancer, nanoliposomal irinotecan (nal-IRI) achieves improved tumor growth control and is able to maintain a significantly lower level of tumor hypoxia as compared to non-liposomal irinotecan. Here, we evaluate the effects of nal-IRI on the kinetics and magnitude of hypoxia changes in an orthotopic patient-derived tumorgraft model of a pancreatic cancer (OCIP51) that is highly hypoxic. Experimental Procedures: Tumor growth of orthotopically implanted OCIP51 tumors was monitored using magnetic resonance imaging. Longitudinal FAZA-PET imaging of tumor hypoxia changes was performed over a 21-day period following weekly administration of nal-IRI at 20 mg/kg (n = 10) and compared to untreated controls (n = 5). Mean tumor FAZA uptake (%ID/g) and hypoxic fractions were calculated. In addition [18F]-fluorothymidine (FLT-) PET was conducted before treatment initiation and after the 3rd dosing cycle to assess tumor cell proliferation. Tumor levels of irinotecan and its active metabolite SN-38 were evaluated using an HPLC method in samples harvested 24 h after the last administration of nal-IRI and in a separate pharmacodynamic study component (n = 10) at 24 h and 72 h after administration of a single dose of nal-IRI at 10 mg/kg. Nal-IRI induced DNA damage was assessed using γH2AX immunohistochemistry. Results: nal-IRI treatment resulted in tumor growth inhibition of 71.6% compared to controls at study end. Tumor growth control was observable at Day 5 post treatment initiation. FAZA uptake in treated tumors decreased by 36% within the first treatment cycle, while average FAZA levels in control tumors remained unchanged during this period. Nal-IRI treatment resulted in statistically significant decreases in the FLTmax and FLTmean values compared to pre-treatment values. 100% of nal-IRI treated mice survived to study end compared to only 40% of controls. Tumor weights at study end were almost 4 times smaller in nal-IRI-treated mice compared to the controls. Tumors from treated mice were fluid-filled and showed extensive blood pooling, while tumors from untreated mice appeared to be much less vascularized. Irinotecan levels detected in the OCIP51 tumors were 8 times lower at 72 h after nal-IRI administration, while SN-38 levels were ~28 times lower when compared to previous findings in HT-29 tumors. Treatment with nal-IRI in the OCIP51 tumors significantly increased the frequency and intensity of γH2AX staining across tumor cell areas compared to that observed in the untreated tumors, which were characterized by only a scattered and sporadic γH2AX staining. Importantly, the stromal areas did not show γH2AX staining in either the treated or the control group. Conclusions: This study demonstrated the feasibility of performing longitudinal tumor hypoxia and proliferation assessments using FAZA- and FLT-PET imaging in a highly hypoxic orthotopic model of pancreatic cancer. Although this model showed reduced levels of liposomal drug deposition compared to cell-line derived xenograft models, treatment with nal-IRI led to effective tumor growth control, as well as significant changes in the tumor microenvironment as measured by reduced hypoxia levels compared to baseline and control tumors. Results from this study support the utility of FAZA-PET for evaluation of tumor hypoxia after anti-cancer therapy with nal-IRI as a means to provide early assessment of treatment activity. Citation Format: Stephan Klinz, Jinzi Zheng, Raquel De Souza, Manuela Ventura, Nancy Paz, David Hedley, David Jaffray, Jonathan Fitzgerald.{Authors}. Nanoliposomal irinotecan (nal-IRI) is an active treatment and reduces hypoxia as measured through longitudinal imaging using [18F]FAZA-PET in an orthotopic patient-derived model of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B47.

Nanoliposomal irinotecan (Nal-IRI, MM-398) recently gained approval in combination with 5-fluorouracil/leucovorin (5-FU/LV) in post-gemcitabine metastatic pancreatic cancer based on results of the Phase 3 NAPOLI-1 trial. Nal-IRI, in combination with 5-FU/LV, improved overall survival in gemcitabine-refractory metastatic PDAC relative to 5-FU/LV alone with a well-defined and manageable toxicity profile in pretreated patients. FOLFIRINOX (5-FU/LV, irinotecan and oxaliplatin) is a chemotherapy regimen active in first-line metastatic PDAC. Herein, we evaluate the preclinical anti-tumor activity of a nal-IRI + 5-FU + oxaliplatin regimen relative to the FOLFIRINOX regimen. Using pancreatic cancer cell lines, we demonstrate enhanced cell death when nal-IRI treatment is simulated using prolonged exposure of SN-38 (the active metabolite of irinotecan) in combination with 5-FU and oxaliplatin. In cell line-derived and patient-derived xenograft models of pancreatic cancer we demonstrate improved anti-tumor activity of nal-IRI relative to exposure-matched doses of unencapsulated irinotecan. Further, nal-IRI consistently improved tumor growth inhibition and survival relative to unencapsulated irinotecan in preclinical models, both as a monotherapy and in combination with 5-FU and oxaliplatin. The addition of nal-IRI to 5-FU and/or oxaliplatin did not exacerbate the baseline toxicities of these agents, including weight loss and neutropenia, and tolerability could be further improved by delaying the administration of oxaliplatin to 1 day post-MM-398. These findings illustrate the therapeutic potential of nal-IRI in combination with 5-FU/LV and oxaliplatin and support an ongoing Phase 2 trial (NCT02551991) of this triplet regimen in first-line PDAC. Citation Format: Daniel F. Gaddy, Helen Lee, Nancy Paz, Shannon C. Leonard, Ashish Kalra, Ninfa L. Straubinger, Robert M. Straubinger, Bryan M. Gillard, Michael T. Moser, Daryl C. Drummond, Stephan G. Klinz, Bart S. Hendriks, Jonathan B. Fitzgerald. Preclinical anti-tumor activity of nanoliposomal irinotecan (Nal-IRI, MM-398) + 5-FU + oxaliplatin in pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4830.

MM-398 is a stable nanotherapeutic encapsulation of the prodrug irinotecan (CPT-11) with longer plasma half-life and higher tumor deposition due to an enhanced permeability and retention effect. Pancreatic cancer has responded poorly to many therapeutics, largely because of inadequate drug penetration due to poor vascularization and the highly aggressive, hypoxic nature of the disease. We sought to better understand how MM-398, a relatively large (100nm) liposomal nanotherapeutic, could be used treat pancreatic cancer. We have tested MM-398 in several pancreatic xenograft models: BxPC3 (KRAS wild type), AsPC-1(KRAS G12D) , Panc-1 (KRAS G12D) and MiaPaCa (KRAS G12C). All models demonstrated complete tumor regression at 20 mg/kg or a human equivalent dose of 60-120 mg/m2. At this same dose, MM-398 suppresses tumor growth in a gemcitabine insensitive AsPC-1 xenograft. MM-398 functionally blocked AsPC-1 tumor cell proliferation as measured by ki-67 staining; however, gemcitabine administered at its maximum tolerated dose did not impact proliferation. MM-398 is currently in multiple clinical trials, including a phase 3 trial for patients with advanced gemcitabine-resistant pancreatic cancer (NAPOLI-1). In order to further understand mechanisms driving response to MM-398, we screened and ranked several cell lines for their ability to convert irinotecan into the active metabolite, SN38. BxPC3 and HT-29 tumors ranked highest in ability to convert irinotecan to SN-38, as measured by HPLC. In a BxPC3 pancreatic orthotopic model which spontaneously metastasizes, 10 mg/kg MM-398 significantly reduced both primary and metastatic tumor load as measured by ex vivo biophotonic imaging of BxPC3luc cells to spleen, lung, liver, diaphragm and GI associated lymph nodes (p In summary, MM-398 induces tumor regression in multiple mouse models of pancreatic cancer, including an orthotopic metastatic model. MM-398 activity may be driven in part by the ability to modify tumor microenvironment parameters, such as hypoxia and vascularization, both of which limit efficacy of chemotherapeutic agents in the treatment of pancreatic cancer. These data support the continued investigation of MM-398 in pancreatic cancer. Citation Format: Nancy Paz, Peter Laivins, Clet Niyikiza, Ulrik Nielsen, Jonathan Fitzgerald, Ashish Kalra, Milind Chalishazar, Stephan Klinz, Jaeyeon Kim, Daryl Drummond, Dmitri Kirpotin, Victor Moyo, Eliel Bayever. MM-398/PEP02, a novel liposomal formulation of irinotecan, demonstrates stromal-modifying anticancer properties. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A63.

Objective The NF-κB signaling pathway promotes the immune response in rheumatoid arthritis (RA) and in rodent models of RA. NF-κB activity is regulated by the IKK-2 kinase during inflammatory responses. To elucidate how IKK-2 inhibition suppresses disease development, we used a combination of in vivo imaging, transcription profiling, and histopathology technologies to study mice with antibody-induced arthritis. Methods ML120B, a potent, small molecule inhibitor of IKK-2, was administered to arthritic animals, and disease activity was monitored. NF-κB activity in diseased joints was quantified by in vivo imaging. Quantitative reverse transcriptase–polymerase chain reaction was used to evaluate gene expression in joints. Protease-activated near-infrared fluorescence (NIRF) in vivo imaging was applied to assess the amounts of active proteases in the joints. Results Oral administration of ML120B suppressed both clinical and histopathologic manifestations of disease. In vivo imaging demonstrated that NF-κB activity in inflamed arthritic paws was inhibited by ML120B, resulting in significant suppression of multiple genes in the NF-κB pathway, i.e., KC, epithelial neutrophil–activating peptide 78, JE, intercellular adhesion molecule 1, CD3, CD68, tumor necrosis factor α, interleukin-1β, interleukin-6, inducible nitric oxide synthase, cyclooxygenase 2, matrix metalloproteinase 3, cathepsin B, and cathepsin K. NIRF in vivo imaging demonstrated that ML120B treatment dramatically reduced the amount of active proteases in the joints. Conclusion Our data demonstrate that IKK-2 inhibition in the murine model of antibody-induced arthritis suppresses both inflammation and joint destruction. In addition, this study highlights how gene expression profiling can facilitate the identification of surrogate biomarkers of disease activity and treatment response in an experimental model of arthritis.

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 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

Introduction: Pharmacokinetic properties of irinotecan limit tumor exposure to the active metabolite SN-38. MM-398, a nanoliposomal irinotecan (nal-IRI) developed to attempt to enhance delivery of irinotecan and SN-38 to tumor cells is being tested in clinical trials. We assessed the pharmacokinetics and anti-tumor activity of nal-IRI in comparison to the current clinical formulation of irinotecan (free irinotecan) in preclinical models of pediatric cancer in relationship to expression of the putative DNA/RNA helicase SLFN11. Experimental Design: Plasma and tissue samples were analyzed by liquid chromatography-tandem mass spectrometry for irinotecan and SN-38. In vivo activity of free irinotecan and nal-IRI was compared in subcutaneous xenograft models (3 each) of Ewing's sarcoma family of tumors (EFT), neuroblastoma (NB), and rhabdomyosarcoma (RMS). SLFN11 mRNA expression (real-time RT-PCR) was correlated to in vitro cytotoxicity of SN-38 (by DIMSCAN) in 20 EWS, 13 RMS, and 20 NB cell lines. SLFN11 mRNA expression in primary tumors (10 EFT, 10 NB, and 39 RMS) was assessed by Affymetrix HuEx microarrays. Results: Plasma and tumor concentrations of irinotecan and SN-38 were ∼ 10-fold higher for nal-IRI than for free irinotecan at 4h, 6h, and 24h post-intravenous injection (p 100 days (88% EFS on day 120). EFS for mice with RMS (median 39 days) and NB (median 81 days) was significantly shorter (P 100-fold higher in EFT cell lines and > 10-fold in EFT primary tumors compared with NB or RMS cell lines or primary tumors (P Conclusions: Nal-IRI demonstrated higher anti-tumor activity than free irinotecan in pediatric solid tumor xenografts, presumably due to increased systemic and tumor exposures to the SN-38 active metabolite. The high SLFN11 expression reported in EFT cell lines and the strong nal-IRI activity we have observed in EFT xenografts warrants testing in correlative clinical studies to determine if SLFN11 can reliably be used as a biomarker to predict nal-IRI activity in patients. Citation Format: Min H. Kang, Michael M. Song, Monish Makena, Joo-Sang Lee, Connor P. Hall, Ashly Hindle, Winford Ko, Nancy Paz, Jonathan Fitzgerald, Daryl C. Drummond, Timothy J. Triche, C. Patrick Reynolds. Activity of MM-398, nanoliposomal irinotecan (nal-IRI), in Ewings family tumor xenografts is associated with high exposure of tumor to drug and high SLFN11 expression. [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 3972. doi:10.1158/1538-7445.AM2014-3972

Background: MM-398 is a novel nanoliposomal encapsulation of irinotecan (nal-IRI), a topoisomerase I inhibitor. In preclinical studies, nal-IRI has been shown to greatly modify the pharmacokinetics and biodistribution of CPT-11 and its active metabolite, SN-38, thereby improving its activity. In this report, we evaluate the in vivo activity of nal-IRI in the two xenograft models of lung cancer. A pharmacodynamics (PD) study was performed to measure the drug activation and deposition parameters in these tumor models. In addition we investigated the effects of nal-IRI on tumor growth, tumor-associated macrophages (TAM's), vasculature, cell proliferation, and apoptosis. Methods: Xenograft models of subcutaneous H157 squamous cell carcinoma (SCC) and H841 small cell lung cancer (SCLC) were established in mice. A short pharmacodynamics (PD) study was performed, wherein, 24 hours after a single dose, animals were euthanized and tumors collected. PD analysis included profiling for carboxylesterase (CES) levels, vasculature (CD31), macrophage (F4/80), and metabolite (CPT-11 and SN-38) levels. For the tumor activity study, animals (5 per group) were treated by weekly i.v. injections with placebo liposome, free irinotecan at 25 mg/kg/wk, or nal-IRI at 30 and 50 mg/kg/wk for three weeks. Tumor volumes were measured with digital calipers. IHC analysis was performed for TAM content, tumor proliferation (Ki67), apoptosis, and vasculature. Results: (1) The carboxylesterase enzyme (activation) and CPT-11 (deposition) tumor levels resulted in extended intratumor SN38 duration, as predicted by the model simulation; (2) nal-IRI suppressed H157 tumor growth in a dose dependent manner, much more efficiently than free irinotecan. On day 25, nal-IRI, at 30 and 50 mg/kg/wk, inhibited tumor growth by 92.6% and 96.3%, respectively, when compared with placebo liposome. In contrast, free irinotecan inhibited tumor growth by 55.7%; (3) For the SCLC H841 xenograft, on day 35, nal-IRI at 30 and 50 mg/kg/wk, also inhibited tumor growth by 84.9% and 93.4% respectively, greater than free irinotecan by 32.7%, when compared with placebo liposome. No obvious toxicities or weight loss were noted in the nal-IRI treated groups; (4) TAM levels were significantly (p Conclusion: nal-IRI inhibited tumor growth in lung tumor xenograft models, suggesting the treatment of human SCC and SCLC, in which there are high unmet medical needs, as a potential target for clinical investigation. Citation Format: Daniel CF Chan, Ashish Kalra, Zhiyong Zhang, Nancy Paz, Dmitri Kirpotin, Daryl Drummond, Ulrik Nielsen, Paul A. Bunn, Jonathan Fitzgerald. Evaluating the pharmacodynamics and pharmacokinetic effects of MM-398, a nanoliposomal irinotecan (nal-IRI) in subcutaneous xenograft tumor models of human squamous cell carcinoma and small cell lung cancers. [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 4626. doi:10.1158/1538-7445.AM2014-4626

We have combined molecular modeling and classical structure-function techniques to define the interactions between the ligand-binding domain (LBD) of the vitamin D nuclear receptor (VDR) and its natural ligand, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)]. The affinity analogue 1alpha,25-(OH)(2)D(3)-3-bromoacetate exclusively labeled Cys-288 in the VDR-LBD. Mutation of C288 to glycine abolished this affinity labeling, whereas the VDR-LBD mutants C337G and C369G (other conserved cysteines in the VDR-LBD) were labeled similarly to the wild-type protein. These results revealed that the A-ring 3-OH group docks next to C288 in the binding pocket. We further mutated M284 and W286 (separately creating M284A, M284S, W286A, and W286F) and caused severe loss of ligand binding, indicating the crucial role played by the contiguous segment between M284 and C288. Alignment of the VDR-LBD sequence with the sequences of nuclear receptor LBDs of known 3-D structure positioned M284 and W286 in the presumed beta-hairpin of the molecule, thereby identifying it as the region contacting the A-ring of 1alpha, 25-(OH)(2)D(3). From the multiple sequence alignment, we developed a homologous extension model of the VDR-LBD. The model has a canonical nuclear receptor fold with helices H1-H12 and a single beta hairpin but lacks the long insert (residues 161-221) between H2 and H3. We docked the alpha-conformation of the A-ring into the binding pocket first so as to incorporate the above-noted interacting residues. The model predicts hydrogen bonding contacts between ligand and protein at S237 and D299 as well as at the site of the natural mutation R274L. Mutation of S237 or D299 to alanine largely abolished ligand binding, whereas changing K302, a nonligand-contacting residue, to alanine left binding unaffected. In the "activation" helix 12, the model places V418 closest to the ligand, and, consistent with this prediction, the mutation V418S abolished ligand binding. The studies together have enabled us to identify 1alpha,25-(OH)(2)D(3)-binding motifs in the ligand-binding pocket of VDR.

A tetraphenylporphyrin-C11-beta-estradiol conjugate has been synthesized. Competitive binding assay of the conjugate with estrogen receptor (ER)-ligand-binding domain showed that the conjugate binds specifically to the protein with high affinity. Potential use of this conjugate to selectively deliver cytotoxic porphyrins to ER-positive cells in various carcinomas is discussed.

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.

MM-398 is a stable nanotherapeutic encapsulation of the pro-drug irinotecan with an extended plasma half-life and higher intratumoral deposition in animal models 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). By using a systems pharmacology approach, we have previously identified the important determinants for MM-398 activity differentiating it from free irinotecan. One of the critical parameters identified in the model is activity of the tumor carboxylesterase (CES) as an important correlate to the MM-398 preclinical response. Our data also shows that tumor associated macrophages (TAMs) are the dominant cell type for phagocytosis of MM-398 in the tumor microenvironment. To validate these findings we used a perturbation strategy involving manipulation of TAMs with the anti-CSF1 antibody 5A1. Depletion of macrophages with 5A1 resulted in a strong size reduction of CD11b+F4/80+ mature macrophage populations in HT29, MC38 and LLC tumor models. Furthermore, other myeloid and non-myeloid cell populations showed tumor model and mouse strain-dependent changes in population size and liposome uptake. The overall impact of 5A1-mediated macrophage depletion was a reduction of the total cellular liposomal load within the tumors. Anti-CSF1 treatment also modulated the systemic clearance of MM-398 due to changes in the macrophage populations in the liver and spleen of the animals. To capture this complex interplay of systemic and local depletion events we have expanded our mechanistically-based PK model to include separate tumor macrophage and tumor cell compartments. The computational model also identified tumor permeability to MM-398 as a rate-limiting factor for drug deposition and in vivo activity. To assess the degree of variability in tumor permeability, we measured the levels of irinotecan and SN-38 using HPLC in six different xenograft models. A high degree of variability in irinotecan and SN-38 levels was seen across these models and between tumors from different mice bearing xenografts of the same cell lines. We then investigated the inter-relationship between tumor permeability, macrophage content, CES enzyme activity and SN-38 accumulation by assessing the levels of these markers in multiple human-derived primary xenograft models across different indications. In summary, we demonstrated the impact of TAM modulation on overall tumor levels of irinotecan and SN-38 following treatment with liposomally encapsulated irinotecan. We outline differences in permeability of xenografts to MM-398 and the capacity to convert irinotecan into SN-38. Citation Format: Ashish Kalra, Jaeyeon Kim, Stephan Klinz, Nancy Paz, Joseph Reutt, Daryl Drummond, Dmitri Kirpotin, Victor Moyo, Eliel Bayever, Peter Laivins, Clet Niyikiza, Ulrik Nielsen, Jonathan Fitzgerald. The tumor microenvironment modulates the delivery and activation of liposomal encapsulated irinotecan, MM-398. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5622. doi:10.1158/1538-7445.AM2013-5622

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.

MM-398 is a stable nanotherapeutic encapsulation of the pro-drug irinotecan (CPT-11). Irinotecan, a topoisomerase inhibitor is currently being used in clinical practice for treatment of several indications; however, associated toxicities, mainly, neutropenia and gastrointestinal toxicity, have limited its clinical utility. Previously, we have demonstrated that MM-398 treatment resulted in significantly higher intratumor concentrations of both irinotecan (142-fold) and SN-38 (9-fold), thereby exhibiting enhanced anti-tumor activity compared to free irinotecan in different xenograft models. Multiple phase 1 and 2 studies have established a pharmacokinetic and safety profile. Recent data support continued clinical development for the drug in various indications, including pancreatic, gastric, colorectal and potentially other solid tumors. In order to further understand the enhanced anti-tumor activity of MM-398 we developed a mechanism-based PK model of MM-398 and free irinotecan designed to predict intratumor levels of SN-38. Based on this model, we evaluated the role of various determinants of response of MM-398. Sensitivity analysis revealed that the local activation of MM-398 was important for obtaining higher SN-38 intratumor levels compared to free irinotecan. To identify cell types responsible for local activation of MM-398 we investigated cellular liposome phagocytosis ability. In vitro studies demonstrated preferential uptake of MM-398 by phagocytic macrophages compared to tumor cells. FACS analysis of tumor samples (from subcutaneous xenografts) highlighted higher uptake of labeled liposomes by CD11b + and F4/80+ cells as compared to tumor cells. We are developing in vivo systems using engineered cell lines overexpressing growth factors to recruit macrophages (CSF1) or overexpressing irinotecan conversion enzymes to further investigate the role of macrophagesin local tumor conversion of MM-398 and to validate the model prediction that local activation is critical to MM-398 mechanism of action. The extent of tumor vascularization and permeability were also highlighted in the sensitivity analysis. To determine the effect of MM-398 on these parameters, we treated mice bearing HT29 (colorectal cancer) xenografts with a single dose of MM-398 and measured hypoxic markers (CAIX) and microvessel density (CD31) by immunohistochemistry. Tumors treated with MM-398 showed a greater degree of CD31 staining and lower CAIX staining, indicating that MM-398 may be able to affect the tumor microenvironment. We are currently evaluating how the ability of MM-398 to alter the tumor microenvironment affects the activity of other chemotherapeutic agents in combination therapy. These findings could support the use of MM-398 as a combination modality against tumors that may have acquired resistance to traditional chemotherapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5696. doi:1538-7445.AM2012-5696

MM-398 (aka PEP02) is a stable, nanotherapeutic encapsulation of the pro-drug CPT-11 (irinotecan) that is currently in clinical development. In preclinical experiments, treatment with MM-398 resulted in significantly higher intratumoral concentrations of both irinotecan (142-fold) and its major metabolite, SN-38 (9-fold) and exhibited superior anti-tumor activity compared to free irinotecan in multiple tumor xenografts. Subsequently, multiple phase 1 and 2 studies have established a pharmacokinetic and safety profile that supports continued clinical development, including in pancreatic, gastric, colorectal and potentially other cancers. Because current evidence suggests that resistance to pancreatic cancer is driven largely by inadequate drug penetration into these often poorly vascularized, stromally dense and hypoxic tumors, we sought to better understand how this relatively large (100nm) liposomal nanotherapeutic could potentially result in increased efficacy in very advanced gemcitabine-resistant pancreatic cancer and other cancer types. We developed a mechanism-based PK model of MM-398 and free irinotecan designed to predict intratumor SN-38 levels. Sensitivity analysis revealed that for MM-398 local activation of irinotecan to SN-38 was a far more important parameter than systemic activation. Through cellular uptake studies, we demonstrated in vitro that MM-398 was preferentially internalized by phagocytic macrophage/monocyte cell lines and, to a far lesser extent, by tumor cell lines. Furthermore, tumor microdistribution studies by flow cytometry and IHC showed uptake of MM-398 liposomes in both tumor cells and tumor-associated macrophages with more liposomal material being present in the macrophages. This distribution also suggests that macrophages may contribute to the postulated rate limiting process of irinotecan activation. The sensitivity analysis also suggested that tumor permeability and vascularization are important determinants of tumor-associated SN-38 levels for both free irinotecan and MM-398. To determine the effect of MM-398 on these parameters we treated mice bearing HT29 (colorectal cancer) xenografts with a single dose of MM-398 and measured hypoxic markers (CAIX) and microvessel density (CD31) by IHC. Tumors treated with MM-398 showed a greater degree of CD31 staining and lower CAIX staining, indicating that MM-398 may be able to affect tumor characteristics that traditionally have contributed to therapy resistance and limited the delivery of cancer therapeutics and resistance. In summary, encapsulation of irinotecan alters rate-limiting processes that determine tumoral SN-38 levels. Delivery of MM-398 is believed to alter tumor microvessel density and decrease hypoxia. These intriguing mechanisms of action findings support the continued clinical development of MM-398 as a differentiated therapeutic for several cancer types. 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 C207.