Phase 1 study of the ATR inhibitor berzosertib in combination with cisplatin in patients with advanced solid tumours.

BACKGROUND: Berzosertib (formerly M6620, VX-970) is a highly potent and selective, first-in-class ataxia telangiectasia-mutated and Rad3-related protein kinase (ATR) inhibitor. We assessed the safety, tolerability, pharmacokinetics, and preliminary efficacy of berzosertib plus cisplatin. METHODS: Adult patients with advanced solid tumours refractory or resistant to standard of care therapies received ascending doses of cisplatin (day 1) and berzosertib (days 2 and 9) every 3 weeks (Q3W). RESULTS: Thirty-one patients received berzosertib (90-210 mg/m2) and cisplatin (40-75 mg/m2) across seven dose levels. The most common grade ≥3 treatment-emergent adverse events were neutropenia (20.0%) and anaemia (16.7%). There were two dose-limiting toxicities: a grade 3 hypersensitivity reaction and a grade 3 increase in alanine aminotransferase. Berzosertib 140 mg/m2 (days 2 and 9) and cisplatin 75 mg/m2 (day 1) Q3W was determined as the recommended Phase 2 dose. Cisplatin had no apparent effect on berzosertib pharmacokinetics. Of the 31 patients, four achieved a partial response (two confirmed and two unconfirmed) despite having previously experienced disease progression following platinum-based chemotherapy. CONCLUSIONS: Berzosertib plus cisplatin is well tolerated and shows preliminary clinical activity in patients with advanced solid tumours, warranting further evaluation in a Phase 2 setting. CLINICAL TRIALS IDENTIFIER: NCT02157792.

Phase 1 study of the ATR inhibitor berzosertib (formerly M6620, VX-970) combined with gemcitabine ± cisplatin in patients with advanced solid tumours.

BACKGROUND: Berzosertib (formerly M6620, VX-970) is a highly potent and selective, first-in-class inhibitor of ataxia telangiectasia and Rad3-related protein kinase (ATR). We assessed multiple ascending doses of berzosertib + gemcitabine ± cisplatin in patients with resistant/refractory advanced solid tumours. METHODS: We evaluated the safety, tolerability, pharmacokinetics (PK) and preliminary efficacy of intravenous berzosertib + gemcitabine ± cisplatin using a standard 3 + 3 dose-escalation design. The starting doses were berzosertib 18 mg/m2, gemcitabine 875 mg/m2 and cisplatin 60 mg/m2. RESULTS: Fifty-two patients received berzosertib + gemcitabine and eight received berzosertib + gemcitabine + cisplatin. Four patients receiving berzosertib + gemcitabine had a total of seven dose-limiting toxicities (DLTs) and three receiving berzosertib + gemcitabine + cisplatin had a total of three DLTs. Berzosertib 210 mg/m2 (days 2 and 9) + gemcitabine 1000 mg/m2 (days 1 and 8) Q3W was established as the recommended Phase 2 dose (RP2D); no RP2D was determined for berzosertib + gemcitabine + cisplatin. Neither gemcitabine nor cisplatin affected berzosertib PK. Most patients in both arms achieved a best response of either partial response or stable disease. CONCLUSIONS: Berzosertib + gemcitabine was well tolerated in patients with advanced solid tumours and showed preliminary efficacy signs. CLINICAL TRIAL IDENTIFIER: NCT02157792.

Nanoliposome targeting in breast cancer is influenced by the tumor microenvironment.

MM-302 is an anti-HER2 antibody-targeted pegylated liposomal doxorubicin designed to deliver doxorubicin specifically to HER2-expressing solid tumors. The delivery and activity of MM-302 were evaluated in orthotopic, transgenic, and intravenous breast cancer models expressing varying levels of HER2 that metastasize to some of the most common sites of dissemination for breast cancer, namely, lung, liver, and brain. Metastatic burden was quantified by gross evaluation, immunohistochemistry (IHC), and bioluminescent imaging. Liposome delivery was quantified by IHC and ex vivo fluorescent imaging. Unlike its non-targeted counterpart, pegylated liposomal doxorubicin (PLD), MM-302 showed activity at controlling both primary and metastatic tumor burden in all models tested. The effect of HER2-targeting was greatest in the lung where lymphatic vessel density and MM-302 delivery were highest. Our data indicate that the therapeutic advantage of actively targeting a nanoliposome with an antibody is influenced by both target expression and the tumor microenvironment.

Safety and pharmacokinetics of MM-302, a HER2-targeted antibody-liposomal doxorubicin conjugate, in patients with advanced HER2-positive breast cancer: a phase 1 dose-escalation study.

BACKGROUND: This phase 1 dose-escalation trial studied MM-302, a novel HER2-targeted PEGylated antibody-liposomal doxorubicin conjugate, in HER2-positive locally advanced/metastatic breast cancer. METHODS: Patients were enrolled in four cohorts: MM-302 monotherapy (8, 16, 30, 40, and 50 mg/m2 every 4 weeks [q4w]); MM-302 (30 or 40 mg/m2 q4w) plus trastuzumab (4 mg/kg q2w); MM-302 (30 mg/m2) plus trastuzumab (6 mg/kg) q3w; MM-302 (30 mg/m2) plus trastuzumab (6 mg/kg) and cyclophosphamide (450 mg/m2) q3w. RESULTS: Sixty-nine patients were treated. The most common adverse events (AEs) were fatigue and nausea. Grade 3/4 AEs of special interest included neutropenia, fatigue, mucosal inflammation, anemia, thrombocytopenia, febrile neutropenia, and palmar-plantar erythrodysesthesia. The MTD was not reached. With MM-302 ≥ 30 mg/m2, overall response rate (ORR) was 13% and median progression-free survival (mPFS) 7.4 months (95% CI: 3·5-10·9) in all arms. In 25 anthracycline-naïve patients, ORR was 28·0% and mPFS 10·9 months (95% CI: 1·8-15·3). Imaging with 64Cu-labeled MM-302 visualized tumor-drug penetrance in tumors throughout the body, including the brain. CONCLUSION: MM-302 monotherapy, in combination with trastuzumab, or trastuzumab plus cyclophosphamide, was well tolerated and showed promising efficacy. The selected phase 2 MM-302 dose was 30 mg/m2 plus 6 mg/kg trastuzumab q3w.

Liposomal Irinotecan Achieves Significant Survival and Tumor Burden Control in a Triple Negative Breast Cancer Model of Spontaneous Metastasis.

Triple negative breast cancer (TNBC) represents a significant therapeutic challenge due to its highly aggressive nature and lack of effective treatment options. Liposomal irinotecan (nal-IRI, ONIVYDE) was approved in 2015 (by the Food and Drug Administration, European Medicines Agency, and Therapeutic Goods Administration) and is a topoisomerase inhibitor indicated, in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy. This study investigates the potential therapeutic benefit of nal-IRI for the treatment of advanced TNBC in a clinically relevant mouse model of spontaneous metastasis (LM2-4). Female SCID mice were orthotopically inoculated with TNBC LM2-4-luc cells in the lower mammary fat pad. Following primary tumor resection, bioluminescence imaging (BLI) was used to monitor both metastasis formation and spread as well as response to treatment with nal-IRI. Weekly treatment with 10 mg/kg of nal-IRI provided a 4.9-times longer median survival compared to both 50 mg/kg irinotecan treated and untreated animals. The survival benefit was supported by a significant delay in the regrowth of the primary tumor, effective control, and eventual regression of metastases assessed using longitudinal BLI, which was confirmed at the study end point with magnetic resonance (MR) imaging and post-mortem observation. This preclinical investigation demonstrates that, at a five-times lower dose compared to the free drug, liposomal irinotecan provides significant survival benefit and effective management of metastatic disease burden in a clinically relevant model of spontaneous TNBC metastases. These findings support the evaluation of nal-IRI in patients with advanced and metastatic TNBC.

Liposomal Irinotecan Accumulates in Metastatic Lesions, Crosses the Blood-Tumor Barrier (BTB), and Prolongs Survival in an Experimental Model of Brain Metastases of Triple Negative Breast Cancer.

PURPOSE: The blood-tumor barrier (BTB) limits irinotecan distribution in tumors of the central nervous system. However, given that the BTB has increased passive permeability we hypothesize that liposomal irinotecan would improve local exposure of irinotecan and its active metabolite SN-38 in brain metastases relative to conventional irinotecan due to enhanced-permeation and retention (EPR) effect. METHODS: Female nude mice were intracardially or intracranially implanted with human brain seeking breast cancer cells (brain metastases of breast cancer model). Mice were administered vehicle, non-liposomal irinotecan (50 mg/kg), liposomal irinotecan (10 mg/kg and 50 mg/kg) intravenously starting on day 21. Drug accumulation, tumor burden, and survival were evaluated. RESULTS: Liposomal irinotecan showed prolonged plasma drug exposure with mean residence time (MRT) of 17.7 ± 3.8 h for SN-38, whereas MRT was 3.67 ± 1.2 for non-liposomal irinotecan. Further, liposomal irinotecan accumulated in metastatic lesions and demonstrated prolonged exposure of SN-38 compared to non-liposomal irinotecan. Liposomal irinotecan achieved AUC values of 6883 ± 4149 ng-h/g for SN-38, whereas non-liposomal irinotecan showed significantly lower AUC values of 982 ± 256 ng-h/g for SN-38. Median survival for liposomal irinotecan was 50 days, increased from 37 days (p<0.05) for vehicle. CONCLUSIONS: Liposomal irinotecan accumulates in brain metastases, acts as depot for sustained release of irinotecan and SN-38, which results in prolonged survival in preclinical model of breast cancer brain metastasis.

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

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 gradient-loadable (64)Cu-chelator for quantifying tumor deposition kinetics of nanoliposomal therapeutics by positron emission tomography.

Effective drug delivery to tumors is a barrier to treatment with nanomedicines. Non-invasively tracking liposome biodistribution and tumor deposition in patients may provide insight into identifying patients that are well-suited for liposomal therapies. We describe a novel gradient-loadable chelator, 4-DEAP-ATSC, for incorporating (64)Cu into liposomal therapeutics for positron emission tomographic (PET). (64)Cu chelated to 4-DEAP-ATSC (>94%) was loaded into PEGylated liposomal doxorubicin (PLD) and HER2-targeted PLD (MM-302) with efficiencies >90%. (64)Cu-MM-302 was stable in human plasma for at least 48h. PET/CT imaging of xenografts injected with (64)Cu-MM-302 revealed biodistribution profiles that were quantitatively consistent with tissue-based analysis, and tumor (64)Cu positively correlated with liposomal drug deposition. This loading technique transforms liposomal therapeutics into theranostics and is currently being applied in a clinical trial (NCT01304797) to non-invasively quantify MM-302 tumor deposition, and evaluate its potential as a prognostic tool for predicting treatment outcome of nanomedicines.

Single-cell quantitative HER2 measurement identifies heterogeneity and distinct subgroups within traditionally defined HER2-positive patients.

Human epidermal growth factor receptor 2 (HER2) is an important biomarker for breast and gastric cancer prognosis and patient treatment decisions. HER2 positivity, as defined by IHC or fluorescent in situ hybridization testing, remains an imprecise predictor of patient response to HER2-targeted therapies. Challenges to correct HER2 assessment and patient stratification include intratumoral heterogeneity, lack of quantitative and/or objective assays, and differences between measuring HER2 amplification at the protein versus gene level. We developed a novel immunofluorescence method for quantitation of HER2 protein expression at the single-cell level on FFPE patient samples. Our assay uses automated image analysis to identify and classify tumor versus non-tumor cells, as well as quantitate the HER2 staining for each tumor cell. The HER2 staining level is converted to HER2 protein expression using a standard cell pellet array stained in parallel with the tissue sample. This approach allows assessment of HER2 expression and heterogeneity within a tissue section at the single-cell level. By using this assay, we identified distinct subgroups of HER2 heterogeneity within traditional definitions of HER2 positivity in both breast and gastric cancers. Quantitative assessment of intratumoral HER2 heterogeneity may offer an opportunity to improve the identification of patients likely to respond to HER2-targeted therapies. The broad applicability of the assay was demonstrated by measuring HER2 expression profiles on multiple tumor types, and on normal and diseased heart tissues.

Exploratory Food Effect Assessment in Patients in Early Clinical Development of Oncology Drugs.

Instructions for administration with regard to food are a key aspect of how patients experience oral drugs. Through potential effects on pharmacokinetics, the food condition can influence safety and efficacy, and thereby is one of many dimensions of dose optimization. Regulatory guidance from major health authorities advocates for the early investigation of food effect (FE) in clinical development. In oncology, exploratory FE (eFE) evaluation is often incorporated into the first-in-human (FIH) studies in patients to inform food condition of later clinical studies. However, the design aspects of such exploratory assessments are generally under-reported and barely described, and yet complex, due to uniqueness of FIH study design and drug development process in oncology. Herein, we review literature of eFE assessment study design in oncology in patients, and present the Novartis experience in the design, execution, and impact of eFE in FIH oncology studies from 2014 to 2021. Based on this, we propose a roadmap for eFE assessment in early clinical drug development for oncology drugs in patients, including a framework for common study design options with a focus on study- and patient-level timing for typical scenarios. We also provide a broad spectrum of decision-making factors which should be evaluated to drive the design and implementation of eFE assessment, spanning from clinical development strategy, FIH study design, to compound-specific features.

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.

DataPflex: a MATLAB-based tool for the manipulation and visualization of multidimensional datasets.

MOTIVATION: DataPflex is a MATLAB-based application that facilitates the manipulation and visualization of multidimensional datasets. The strength of DataPflex lies in the intuitive graphical user interface for the efficient incorporation, manipulation and visualization of high-dimensional data that can be generated by multiplexed protein measurement platforms including, but not limited to Luminex or Meso-Scale Discovery. Such data can generally be represented in the form of multidimensional datasets [for example (time x stimulation x inhibitor x inhibitor concentration x cell type x measurement)]. For cases where measurements are made in a combinational fashion across multiple dimensions, there is a need for a tool to efficiently manipulate and reorganize such data for visualization. DataPflex accepts data consisting of up to five arbitrary dimensions in addition to a measurement dimension. Data are imported from a simple .xls format and can be exported to MATLAB or .xls. Data dimensions can be reordered, subdivided, merged, normalized and visualized in the form of collections of line graphs, bar graphs, surface plots, heatmaps, IC50's and other custom plots. Open source implementation in MATLAB enables easy extension for custom plotting routines and integration with more sophisticated analysis tools. AVAILABILITY: DataPflex is distributed under the GPL license (http://www.gnu.org/licenses/) together with documentation, source code and sample data files at: http://code.google.com/p/datapflex. SUPPLEMENTARY INFORMATION: Supplementary data available at Bioinformatics online.

Population pharmacokinetics of ATR inhibitor berzosertib in phase I studies for different cancer types.

PURPOSE: Berzosertib (formerly M6620) is the first-in-class inhibitor of ataxia-telangiectasia and Rad3-related protein, a key component of the DNA damage response, and being developed in combination with chemotherapy for the treatment of patients with advanced cancers. The objectives of this analysis were to characterize the pharmacokinetics (PK) of berzosertib across multiple studies and parts, estimate inter-individual variability, and identify covariates that could explain such variability. METHODS: A population PK analysis was performed using the combined dataset from two phase I clinical studies (NCT02157792, EudraCT 2013-005100-34) in patients with advanced cancers receiving an intravenous infusion of berzosertib alone or in combination with chemotherapy. The analysis included data from 240 patients across 11 dose levels (18-480 mg/m2). Plasma concentration data were modeled with a non-linear mixed-effect approach and clinical covariates were evaluated. RESULTS: PK data were best described by a two-compartment linear model. For a typical patient, the estimated clearance (CL) and intercompartmental CL were 65 L/h and 295 L/h, respectively, with central and peripheral volumes estimated to be 118 L and 1030 L, respectively. Several intrinsic factors were found to influence berzosertib PK, but none were considered clinically meaningful due to a very limited effect. Model simulations indicated that concentrations of berzosertib exceeded p-Chk1 (proximal pharmacodynamic biomarker) IC50 at recommended phase II doses in combination with carboplatin, cisplatin, and gemcitabine. CONCLUSIONS: There was no evidence of a clinically significant PK interaction between berzosertib and evaluated chemo-combinations. The covariate analysis did not highlight any need for dosing adjustments in the population studied to date. CLINICAL TRIAL INFORMATION: NCT02157792, EudraCT 2013-005100-34.

Longitudinal PET Imaging to Monitor Treatment Efficacy by Liposomal Irinotecan in Orthotopic Patient-Derived Pancreatic Tumor Models of High and Low Hypoxia.

PURPOSE: Hypoxia is linked to aggressiveness, resistance to therapy, and poor prognosis of pancreatic tumors. Liposomal irinotecan (nal-IRI, ONIVYDE®) has shown potential in reducing hypoxia in the HT29 colorectal cancer model, and here, we investigate its therapeutic activity and ability to modulate hypoxia in patient-derived orthotopic tumor models of pancreatic cancer. PROCEDURES: Mice were randomized into nal-IRI treated and untreated controls. Magnetic resonance imaging was used for monitoring treatment efficacy, positron emission tomography (PET) imaging with F-18-labelled fluoroazomycinarabinoside ([18F]FAZA) for tumor hypoxia quantification, and F-18-labelled fluorothymidine ([18F]FLT) for tumor cell proliferation. RESULTS: The highly hypoxic OCIP51 tumors showed significant response following nal-IRI treatment compared with the less hypoxic OCIP19 tumors. [18F]FAZA-PET detected significant hypoxia reduction in treated OCIP51 tumors, 8 days before significant changes in tumor volume. OCIP19 tumors also responded to therapy, although tumor volume control was not accompanied by any reduction in [18F]FAZA uptake. In both models, no differences were observable in [18F]FLT uptake in treated tumors compared with control mice. CONCLUSIONS: Hypoxia modulation may play a role in nal-IRI's mechanism of action. Nal-IRI demonstrated greater anti-tumor activity in the more aggressive and hypoxic tumor model. Furthermore, hypoxia imaging provided early prediction of treatment response.

Synergistic drug-cytokine induction of hepatocellular death as an in vitro approach for the study of inflammation-associated idiosyncratic drug hepatotoxicity.

Idiosyncratic drug hepatotoxicity represents a major problem in drug development due to inadequacy of current preclinical screening assays, but recently established rodent models utilizing bacterial LPS co-administration to induce an inflammatory background have successfully reproduced idiosyncratic hepatotoxicity signatures for certain drugs. However, the low-throughput nature of these models renders them problematic for employment as preclinical screening assays. Here, we present an analogous, but high-throughput, in vitro approach in which drugs are administered to a variety of cell types (primary human and rat hepatocytes and the human HepG2 cell line) across a landscape of inflammatory contexts containing LPS and cytokines TNF, IFN gamma, IL-1 alpha, and IL-6. Using this assay, we observed drug-cytokine hepatotoxicity synergies for multiple idiosyncratic hepatotoxicants (ranitidine, trovafloxacin, nefazodone, nimesulide, clarithromycin, and telithromycin) but not for their corresponding non-toxic control compounds (famotidine, levofloxacin, buspirone, and aspirin). A larger compendium of drug-cytokine mix hepatotoxicity data demonstrated that hepatotoxicity synergies were largely potentiated by TNF, IL-1 alpha, and LPS within the context of multi-cytokine mixes. Then, we screened 90 drugs for cytokine synergy in human hepatocytes and found that a significantly larger fraction of the idiosyncratic hepatotoxicants (19%) synergized with a single cytokine mix than did the non-hepatotoxic drugs (3%). Finally, we used an information theoretic approach to ascertain especially informative subsets of cytokine treatments for most highly effective construction of regression models for drug- and cytokine mix-induced hepatotoxicities across these cell systems. Our results suggest that this drug-cytokine co-treatment approach could provide a useful preclinical tool for investigating inflammation-associated idiosyncratic drug hepatotoxicity.

Multiple effects of acetaminophen and p38 inhibitors: towards pathway toxicology.

The majority of drug-related toxicities are idiosyncratic, with little pathophysiological insight and mechanistic understanding. Pathway toxicology is an emerging field of toxicology in the post-genomic era that studies the molecular interactions between toxicants and biological pathways as a way to bridge this knowledge gap. Using two case studies--acetaminophen and p38 MAPK inhibitors--this review illustrates how a pathway-based perspective has advanced our understanding of compound and target-based toxicities. The advancement of pathway toxicology will be dependent on integrated applications of techniques from basic sciences and a fundamental understanding of the interdependence of multiple biological pathways in living organisms.

Analysis of mechanistic pathway models in drug discovery: p38 pathway.

Mechanistic models of signal transduction have emerged as valuable tools for untangling complex signaling networks and gaining detailed insight into pathway dynamics. The natural extension of these tools is for the design of therapeutic strategies. We have generated a novel computational model of lipopolysaccharide-induced p38 signaling in the context of TNF-alpha production in inflammatory disease. Using experimental measurement of protein levels and phospho-protein time courses, populations of model parameters were estimated. With a collection of parameter sets, reflecting virtual diversity, we step through analysis of the p38 signaling pathway model to answer specific drug discovery questions regarding target prioritization, inhibitor simulation, model robustness and co-drugging. We demonstrate that target selection cannot be assessed independently from inhibitor mechanism of action and is also linked with robustness to cellular variability. Finally, we assert that in the face of parameter uncertainty one can still uncover consistent findings that can guide drug discovery efforts.

Companion Diagnostic 64Cu-Liposome Positron Emission Tomography Enables Characterization of Drug Delivery to Tumors and Predicts Response to Cancer Nanomedicines.

Deposition of liposomal drugs into solid tumors is a potentially rate-limiting step for drug delivery and has substantial variability that may influence probability of response. Tumor deposition is a shared mechanism for liposomal therapeutics such that a single companion diagnostic agent may have utility in predicting response to multiple nanomedicines. Methods: We describe the development, characterization and preclinical proof-of-concept of the positron emission tomography (PET) agent, MM-DX-929, a drug-free untargeted 100 nm PEGylated liposome stably entrapping a chelated complex of 4-DEAP-ATSC and 64Cu (copper-64). MM-DX-929 is designed to mimic the biodistribution of similarly sized therapeutic agents and enable quantification of deposition in solid tumors. Results: MM-DX-929 demonstrated sufficient in vitro and in vivo stability with PET images accurately reflecting the disposition of liposome nanoparticles over the time scale of imaging. MM-DX-929 is also representative of the tumor deposition and intratumoral distribution of three different liposomal drugs, including targeted liposomes and those with different degrees of PEGylation. Furthermore, stratification using a single pre-treatment MM-DX-929 PET assessment of tumor deposition demonstrated that tumors with high MM-DX-929 deposition predicted significantly greater anti-tumor activity after multi-cycle treatments with different liposomal drugs. In contrast, MM-DX-929 tumor deposition was not prognostic in untreated tumor-bearing xenografts, nor predictive in animals treated with small molecule chemotherapeutics. Conclusions: These data illustrate the potential of MM-DX-929 PET as a companion diagnostic strategy to prospectively select patients likely to respond to liposomal drugs or nanomedicines of similar molecular size.

Liposomal 64Cu-PET Imaging of Anti-VEGF Drug Effects on Liposomal Delivery to Colon Cancer Xenografts.

Liposomes (LP) deliver drug to tumors due to enhanced permeability and retention (EPR). LP were labeled with 64Cu for positron emission tomography (PET) to image tumor localization. Bevacizumab (bev), a VEGF targeted antibody, may modify LP delivery by altering tumor EPR and this change can also be imaged. Objective: Assess the utility of 64Cu-labeled LP for PET in measuring altered LP delivery early after treatment with bev. Methods: HT-29 human colorectal adenocarcinoma tumors were grown subcutaneously in SCID mice. Empty LP MM-DX-929 (Merrimack Pharmaceuticals, Inc. Cambridge, MA) were labeled with 64CuCl2 chelated with 4-DEAP-ATSC. Tumor-bearing mice received ~200-300 µCi of 64Cu-MM-DX-929 and imaged with microPET. All mice were scanned before and after the treatment period, in which half of the mice received bev for one week. Scans were compared for changes in LP accumulation during this time. Initially, tissues were collected after the second PET for biodistribution measurements and histological analysis. Subsequent groups were divided for further treatment. Tumor growth following bev treatment, with or without LP-I, was assessed compared to untreated controls. Results: PET scans of untreated mice showed increased uptake of 64Cu-MM-DX-929, with a mean change in tumor SUVmax of 43.9%±6.6% (n=10) after 7 days. Conversely, images of treated mice showed that liposome delivery did not increase, with changes in SUVmax of 7.6%±4.8% (n=12). Changes in tumor SUVmax were significantly different between both groups (p=0.0003). Histology of tumor tissues indicated that short-term bev was able to alter vessel size. Therapeutically, while bev monotherapy, LP-I monotherapy, and treatment with bev followed by LP-I all slowed HT-29 tumor growth compared to controls, combination provided no therapeutic benefit. Conclusions: PET with tracer LP 64Cu-MM-DX-929 can detect significant differences in LP delivery to colon tumors treated with bev when compared to untreated controls. Imaging with 64Cu-MM-DX-929 is sensitive enough to measure drug-induced changes in LP localization which can have an effect on outcomes of treatment with LP.