Tumor Disposition Research Articles

Integrated PK/PD Modeling Relates Smoothened Inhibitor Biomarkers to The Heterogeneous Intratumor Disposition of Cetuximab in Pancreatic Cancer Tumor Models

Therapeutic antibodies have shown little efficacy in the treatment of pancreatic ductal adenocarcinomas (PDAC). Tumor desmoplasia, hypovascularity, and poor perfusion result in insufficient tumor cell exposure, contributing to treatment failure. Smoothened inhibitors of hedgehog signaling (sHHi) increase PDAC tumor permeability, perfusion, and drug delivery, and provide a tool to develop a quantitative, mechanistic understanding as to how the temporal dynamics of tumor priming can impact intratumor distribution of monoclonal antibodies (mAb). A linked pharmacokinetic (PK)/pharmacodynamic (PD) model was developed to integrate the plasma and tumor PK of a sHHi priming agent with its effects upon downstream stromal biomarkers Gli1, hyaluronic acid, and interstitial fluid pressure in PDAC patient-derived xenograft (PDX) tumors. In parallel, in situ tumor concentrations of cetuximab (CTX: anti-epidermal growth factor receptor; EGFR) were quantified as a marker for tumor delivery of mAb or antibody-drug conjugates. A minimal, physiologically-based pharmacokinetic (mPBPK) model was constructed to link sHHi effects upon mechanistic effectors of tumor barrier compromise with the intratumor distribution of CTX, and CTX occupancy of EGFR in tumors. Integration of the mPBPK model of mAb deposition and intratumor distribution with the PK/PD model of tumor responses to priming not only identified physiological parameters that are critical for tumor antibody distribution, but also provides insight into dosing regimens that could achieve maximal tumor disposition of therapeutic antibodies under conditions of transient PDAC tumor permeability barrier compromise that mechanistically-diverse tumor priming strategies may achieve.

Familial pancreatic cancer syndrome

In contrast to many other malignant entities the risk factors for pancreatic cancer are mostly unknown. Hereditary factors are causative in about 3-6% of all pancreatic cancers. In addition to hereditary tumor disposition syndromes and hereditary pancreatitis, the familial pancreatic cancer syndrome accounts for about 75% of all hereditary pancreatic cancers. Afamilial pancreatic cancer syndrome is present when at least two first-degree relatives in afamily have histologically proven pancreatic cancer and there are no criteria for another hereditary syndrome. An underlying uniform monogenetic gene defect in familial pancreatic cancer syndrome is currently unknown and agermline mutation in the BRCA2 gene is so far the most frequently identified genetic alteration. Patients at risk for familial pancreatic cancer should be offered participation in registry studies with associated early detection programs (e.g., the FaPaCa registry). These enable the detection of high-grade neoplasms and pancreatic cancer in the early stages, which appears to reduce mortality; however, the detected pancreatic lesions are often incorrectly diagnostically classified, and patients undergo complex pancreatic resection despite benign findings. Finally, studies have so far not been able to clearly clarify whether patients at risk benefit from an early detection program. According to current data, prophylactic operations are not indicated for familial pancreatic cancer syndrome and should only be performed if there is evidence of asuspicious finding that requires resection.

Pre-treatment of oncolytic reovirus improves tumor accumulation and intratumoral distribution of PEG-liposomes.

PEGylated liposomes (PEG-liposomes) are a promising drug delivery vehicle for tumor targeting because of their efficient tumor disposition profiles via the enhanced permeability and retention (EPR) effect. However, tumor targeting of PEG-liposomes, particularly their delivery inside the tumors, is often disturbed by physical barriers in the tumor, including tumor cells themselves, extracellular matrices, and interstitial pressures. In this study, B16 melanoma tumor-bearing mice were injected intravenously with oncolytic reovirus before administration of PEG-liposomes to enhance PEG-liposomes' tumor disposition. Three days after reovirus administration, significant expression of reovirus sigma 3 protein, elevation of apoptosis-related gene expression, and activation of caspase 3 in the tumors were found. Apoptotic cells were found inside the tumors. These data indicated that reovirus efficiently replicated in the tumors and induced apoptosis of tumor cells. The tumor disposition levels of PEG-liposomes were approximately doubled by reovirus pre-administration, compared with a PBS-pretreated group. PEG-liposomes were widely distributed in the tumors of reovirus-pretreated mice, whereas in the PBS-pretreated group, PEG-liposomes were found mainly around or inside the blood vessels in the tumors. Pre-treatment with reovirus also improved the tumor accumulation of PEG-liposomes in human pancreatic BxPC-3 tumors. 3D imaging analysis of whole BxPC-3 tumors demonstrated that pretreatment with reovirus led to the enhancement of PEG-liposome accumulation inside the tumors. Combination treatment with reovirus and paclitaxel-loaded PEG-liposomes (PTX-PEG-liposomes) significantly suppressed B16 tumor growth. These results provide important information for clinical use of combination therapy of reovirus and nanoparticle-based drug delivery system (DDS).

The Impact of Next-Generation Sequencing on Medical Genetic Diagnostics and Counseling

Next-generation sequencing is a modern diagnostic high-throughput method (multi-gene analysis) that can be used to better diagnose both hereditary cancers (tumor disposition syndromes, germline diagnostics) and somatic mutations in tumors. The broad usage of this technology in daily clinical practice demonstrates the real interindividual genetic variability. This method has great importance for the investigation of heterogeneous genetic diseases (e. g. tumors, neurodegenerative and neuromuscular diseases). Further indications are pharmacogenetics and non-invasive prenatal diagnosis. It can be expected that this diagnostic tool will find wide clinical application. With the rapid increase and complexity of genetic data information, the correct interpretation and transmission of the results in the human genetic counseling (germline diagnostics) is of great importance. The genetic counseling must be realigned and adapted accordingly in daily clinical practice.

A New Class of Tunable Acid-Sensitive Linkers for Native Drug Release Based on the Trityl Protecting Group.

Core-cross-linked polymeric micelles (CCPMs) are a promising nanoparticle platform due to favorable properties such as their long circulation and tumor disposition exploiting the enhanced permeability and retention (EPR) effect. Sustained release of covalently linked drugs from the hydrophobic core of the CCPM can be achieved by a biodegradable linker that connects the drug and the core. This study investigates the suitability of trityl-based linkers for the design of acid-triggered native active pharmaceutical ingredient (API) release from CCPMs. Trityl linker derivatives with different substituent patterns were synthesized and conjugated to model API compounds such as DMXAA-amine, doxorubicin, and gemcitabine, and their release kinetics were studied. Hereafter, API release from CCPMs based on mPEG-b-pHPMAmLac block copolymers was investigated. Variation of the trityl substitution pattern showed tunability of the API release rate from the trityl-based linker with t1/2 varying from <1.0 to 5.0 h at pH 5.0 and t1/2 from 6.5 to >24 h at pH 7.4, all at 37 °C. A clear difference in release kinetics was found between gemcitabine and doxorubicin, with gemcitabine showing no detectable release for 72 h at pH 5.0 and doxorubicin showing a t1/2 of less than 1 h. Based on these findings, we show that the reaction mechanism of trityl deprotection plays an important role in the API release kinetics. The first step in this mechanism, which is protonation of the trityl-bound amine, is pKa-dependent, which explains the difference in release rate. In conclusion, acid-sensitive and tunable trityl linkers are highly promising for the design of linker–API conjugates and for their use in CCPMs.

Impact of charge patches on tumor disposition and biodistribution of therapeutic antibodies

This study explores the impact of antibody surface charge on tissue distribution into various tissues including tumor. Tumor-bearing mice were dosed intravenously with a mixture comprising three antibodies engineered to carry negative charge patches, a balanced charge distribution, or positive patches, respectively (cassette dosing). Tissue levels were analyzed with a specific LC-MS/MS method. In addition, the antibody mix was administered to non-tumor bearing mice. Muscle and skin interstitial fluid were obtained by centrifugation and analyzed by LC-MS/MS. An in vitro endothelium model was explored for its feasibility to mimic the observed distribution differences.A balanced charge distribution was optimal in terms of total tumor exposure, while in other tissues, negatively charged and balanced charged antibodies gave similar results. In contrast, positive charge patches generally resulted in increased serum clearance but markedly enhanced tumor and organ uptake, leading to higher tissue-to-serum ratios. The uptake and availability in the interstitial space were confirmed by specific assessment of antibody levels in the interstitial fluid of the muscle and skin, with similar charge impact as in total tissue. The in vitro model was able to differentiate the transport propensity of this series of antibody variants. In summary, our results show the differential effects of charge patches on an antibody surface on biodistribution and tumor uptake. These insights may help in the design of molecules with biodistribution properties tailored to their purpose, and an optimized safety profile.

Dynamic Contrast-Enhanced Magnetic Resonance Imaging for the Prediction of Monoclonal Antibody Tumor Disposition.

The prediction of monoclonal antibody (mAb) disposition within solid tumors for individual patients is difficult due to inter-patient variability in tumor physiology. Improved a priori prediction of mAb pharmacokinetics in tumors may facilitate the development of patient-specific dosing protocols and facilitate improved selection of patients for treatment with anti-cancer mAb. Here, we report the use of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), with tumor penetration of the contrast agent gadobutrol used as a surrogate, to improve physiologically based pharmacokinetic model (PBPK) predictions of cetuximab pharmacokinetics in epidermal growth factor receptor (EGFR) positive xenografts. In the initial investigations, mice bearing Panc-1, NCI-N87, and LS174T xenografts underwent DCE-MRI imaging with the contrast agent gadobutrol, followed by intravenous dosing of an 125Iodine-labeled, non-binding mAb (8C2). Tumor concentrations of 8C2 were determined following the euthanasia of mice (3 h–6 days after 8C2 dosing). Potential predictor relationships between DCE-MRI kinetic parameters and 8C2 PBPK parameters were evaluated through covariate modeling. The addition of the DCE-MRI parameter Ktrans alone or Ktrans in combination with the DCE-MRI parameter Vp on the PBPK parameters for tumor blood flow (QTU) and tumor vasculature permeability (σTUV) led to the most significant improvement in the characterization of 8C2 pharmacokinetics in individual tumors. To test the utility of the DCE-MRI covariates on a priori prediction of the disposition of mAb with high-affinity tumor binding, a second group of tumor-bearing mice underwent DCE-MRI imaging with gadobutrol, followed by the administration of 125Iodine-labeled cetuximab (a high-affinity anti-EGFR mAb). The MRI-PBPK covariate relationships, which were established with the untargeted antibody 8C2, were implemented into the PBPK model with considerations for EGFR expression and cetuximab-EGFR interaction to predict the disposition of cetuximab in individual tumors (a priori). The incorporation of the Ktrans MRI parameter as a covariate on the PBPK parameters QTU and σTUV decreased the PBPK model prediction error for cetuximab tumor pharmacokinetics from 223.71 to 65.02%. DCE-MRI may be a useful clinical tool in improving the prediction of antibody pharmacokinetics in solid tumors. Further studies are warranted to evaluate the utility of the DCE-MRI approach to additional mAbs and additional drug modalities.

Betulinic acid and its spray dried microparticle formulation: In vitro PDT effect against ovarian carcinoma cell line and in vivo plasma and tumor disposition.

The race against ovarian cancer continue to motivate the research worldwide. It is known that many antitumor drugs have limited penetration into solid tumor tissues due to its microenvironment, thus contributing to their low efficacy. Therapeutic modalities have been exploited to elicit antitumor effects based on microenvironment of tumor, including Photodynamic therapy (PDT). Prospection of natural small molecules and nanotechnology are important tools in the development of new ways of obtaining photoactive compounds that are biocompatible. The Betulinic acid (BA) has shown potential biological effect as bioactive drug, but it has low water solubility. Thus, in the present study, owing to the poor solubility of the BA, its free form (BAF) was compared to a spray dried microparticle betulinic acid/HP-β-CD formulation (BAC) aiming to assess the BAF and BAC efficacy as a photosensitizer in PDT for application in ovarian cancer. BAF and BAC were submitted to assays in the presence of LED (λ=420nm) under different conditions (2.75J/cm2, 5.5J/cm2, and 11J/cm2) and in absence of irradiation, after 5min or 4h of contact with ovarian carcinoma cells (A2780) or fibroblast murine cells (3T3). Furthermore, HPLC-MS/MS and MALDI-MSI methods were developed and validated in plasma and tumor of mice proving suitable for in vivo studies. The results found a greater photoinduced cytotoxic effect for the BAC at low concentration for A2780 when irradiated with LED with similar results for fluorescence microscopy. The results motivate us to continue the studies with the BA as a potential antitumor bioactive compound.

Rare tumors as leading symptom of hereditary tumor syndromes

Monogenic hereditary tumor syndromes or tumor disposition syndromes (TDS) are based on germline/constitutional mutations in key genes of carcinogenesis. Early onset and aclustering of tumors belonging to atypical spectrum in the personal or family history are indicators for ahereditary form. In particular, rare specific tumors occur relatively frequently in the context of TDS. Based on aliterature search the current article presents information on which TDS should be considered for differential diagnosis (DD) in the presence of arare tumor. The identification of acausal germline mutation in the index patient is important for the DD, the evaluation of recurrence risks, and predictive testing of asymptomatic at-risk family members. In TDS with autosomal dominant inheritance, it is often possible to identify several high-risk individuals in the affected families. Early detection and correct classification are of high clinical relevance as the patients and persons at risk can often be offered effective preventive procedures (surveillance, prophylactic operations), and in some cases, special therapeutic options exist. TDS are paradigmatic for an extremely successful concept of preventive oncology and personalized medicine. The introduction of new methods of high-throughput sequencing (next generation sequencing) enables amore effective genetic diagnosis, but also poses achallenge for the interpretation of findings and counseling. Referral to multidisciplinary expert centers is useful for care of the families.

Effect of Size on Solid Tumor Disposition of Protein Therapeutics.

In this study, we evaluated the effect of size on tumor disposition of protein therapeutics, including the plasma and tumor pharmacokinetics (PK) of trastuzumab (∼150 kDa), FcRn-nonbinding trastuzumab (∼150 kDa), F(ab)2 fragment of trastuzumab (∼100 kDa), Fab fragment of trastuzumab (∼50 kDa), and trastuzumab scFv (∼27 kDa) in both antigen (i.e., HER2)-overexpressing (N87) and antigen-nonexpressing (MDA-MB-468) tumor-bearing mice. The observed data were used to develop the maximum tumor uptake versus molecular weight and tumor-to-plasma area under the curve (AUC) ratio versus molecular weight relationships. Comparison of the PK of different sizes of FcRn nonbinding molecules in target-expressing tumor showed that ∼100 kDa is an optimal size to achieve maximum tumor uptake and ∼50 kDa is an optimal size to achieve maximum tumor-to-plasma exposure ratio of protein therapeutics. The PK data were also used to validate a systems PK model for tumor disposition of different-sized protein therapeutics. The PK model was able to predict a priori the PK of all five molecules in both tumor types reasonably well (within 2- to 3-fold). In addition, the model captured the bell-shaped relationships observed between maximum tumor uptake and molecular weight and between tumor-to-plasma AUC ratio and molecular weight. Our results provide an unprecedented insight into the effect of size and target engagement on the tumor PK of protein therapeutics. Our results also provide further validation of the tumor disposition model, which can be used to support discovery, development, and preclinical-to-clinical translation of different sizes of protein therapeutics. SIGNIFICANCE STATEMENT: This article highlights the importance of molecular size and target engagement on the tumor disposition of protein therapeutics. Our results suggest that ∼100 kDa is an optimal size to achieve maximum tumor uptake and ∼50 kDa is an optimal size to achieve maximum tumor-to-plasma exposure ratio for non-FcRn-binding targeted protein therapeutics. We also demonstrate that a systems pharmacokinetics model developed to characterize tumor disposition of protein therapeutics can predict a priori the disposition of different-sized protein therapeutics in target-expressing and target-nonexpressing solid tumors.

Complex effects of tumor microenvironment on the tumor disposition of carrier-mediated agents.

Major advances in carrier-mediated agents, including nanoparticle, conjugates and antibody-drug conjugates, have created revolutionary drug delivery systems in cancer over the past two decades. While these agents provide several advantages, such as greater duration of exposure and solubility, compared with their small-molecule counterparts, there is substantial variability in delivery of these agents to tissues and especially tumors. This review provides an overview of tumor microenvironment factors that affect the pharmacokinetics and pharmacodynamics of carrier-mediated agents observed in preclinical models and patients.

Abstract 4075: Effect of PEG chain length on antibody-drug conjugate tumor and tissue distribution in tumor bearing xenograft mice

Abstract Antibody-drug conjugates (ADCs) are a promising new therapeutic modality having demonstrated clinical efficacy in the treatment of cancer. While ADCs have better exposures than their constitutive chemotypes, it is important that they maintain the favorable pharmacokinetics of their antibody backbones. Previous work incorporating PEG into the drug-linker as a sidechain helped to mask the hydrophobicity of the chemotypes, resulting in improved ADC exposures in rats compared to ADCs with non-PEGylated drug-linkers. The current studies were aimed at understanding the effect of PEG chain length on ADC efficacy, tumor and tissue distribution in tumor bearing mice. Efficacy and biodistribution of radiolabeled ADCs with 2, 4, 8, 12 and 24 PEG units in the drug-linker as a side chain were investigated in SCID mice bearing a subcutaneous xenograft of L540cy tumor cells. A non-PEGylated ADC was used as control. Antibody based radioactivity in plasma, tumor, liver, lung, kidney, spleen and brain were measured over 14 days. MMAE, the free drug released from the ADC, was measured by mass spectrometry. Reduction in tumor weight was determined as a measure of efficacy. Compartmental analysis of the plasma exposure data was conducted to understand the change in rate constants with change in PEG chain length. ADCs with side chain PEGylated drug-linkers had greater plasma and tumor exposures than the non-PEGylated control ADC. Increasing PEG chain length in the linker led to increased plasma and tumor exposures, and lower plasma clearances. Tumor distribution was binary with ADCs with 2 and 4 PEG units in the linker showing similar tumor exposures while the ADCs with 8, 12 and 24 PEG units providing similar but significantly higher tumor exposures. ADCs with 8, 12 and 24 PEG units in the linker had significantly higher tumor to plasma exposure ratios than ADCs with 2 and 4-PEG units in the drug-linker. The reduction in tumor weights was also binary. ADCs with 2 and 4 PEG units provided a 35-45% decrease in tumor weights while ADCs with 8, 12 and 24 PEG units in the linker provided 75-85% reduction in tumor weights. The non-PEGylated control ADC decreased the tumor weights by 11%. In general, PEGylated ADCs increased the extent of tissue distribution, but the tissue to plasma ratios remained the same indicating that the increased tissue exposures was a function of increased plasma exposures. However, PEGylated ADC tumor exposure increased beyond that expected based solely on the increase in plasma exposure. Incorporation of PEG as a side chain into the drug-linker improved the tumor disposition of ADCs with little alteration in tissue disposition. ADCs with 8, 12 and 24 PEG units in the drug- linker showed preferential uptake into tumor, but not the tissues as evidenced by tumor (or tissue) to plasma exposure ratios. Over all, the ADCs with 8, 12 and 24 PEG units in the linker provided the optimal combination of tumor distribution, efficacy and tissue distribution. Citation Format: Nagendra V. Chemuturi, Martha Anderson, Julia Cochran, Thomas Pires, Josh Hunter, David Meyer, Jason Neale, Alice Chin, Paul Pittman, Cindy Balasubramanian, Francisco Zapata, Jessica Simmons, Robert Lyon, Steve Alley. Effect of PEG chain length on antibody-drug conjugate tumor and tissue distribution in tumor bearing xenograft mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4075. doi:10.1158/1538-7445.AM2017-4075

Application of a PK-PD Modeling and Simulation-Based Strategy for Clinical Translation of Antibody-Drug Conjugates: a Case Study with Trastuzumab Emtansine (T-DM1).

Successful clinical translation of antibody-drug conjugates (ADCs) can be challenging due to complex pharmacokinetics and differences between preclinical and clinical tumors. To facilitate this translation, we have developed a general pharmacokinetic-pharmacodynamic (PK-PD) modeling and simulation (M&S)-based strategy for ADCs. Here we present the validation of this strategy using T-DM1 as a case study. A previously developed preclinical tumor disposition model for T-DM1 (Singh and Shah, AAPSJ. 2015; 18(4):861-875) was used to develop a PK-PD model that can characterize in vivo efficacy of T-DM1 in preclinical tumor models. The preclinical data was used to estimate the efficacy parameters for T-DM1. Human PK of T-DM1 was a priori predicted using allometric scaling of monkey PK parameters. The predicted human PK, preclinically estimated efficacy parameters, and clinically observed volume and growth parameters for breast cancer were combined to develop a translated clinical PK-PD model for T-DM1. Clinical trial simulations were performed using the translated PK-PD model to predict progression-free survival (PFS) and objective response rates (ORRs) for T-DM1. The model simulated PFS rates for HER2 1+ and 3+ populations were comparable to the rates observed in three different clinical trials. The model predicted only a modest improvement in ORR with an increase in clinically approved dose of T-DM1. However, the model suggested that a fractionated dosing regimen (e.g., front loading) may provide an improvement in the efficacy. In general, the PK-PD M&S-based strategy presented here is capable of a priori predicting the clinical efficacy of ADCs, and this strategy has been now retrospectively validated for all clinically approved ADCs.

Profiling the relationship between tumor-associated macrophages and pharmacokinetics of liposomal agents in preclinical murine models

The mononuclear phagocyte system (MPS) has previously been shown to significantly affect the clearance, tumor delivery, and efficacy of nanoparticles (NPs). This study profiled MPS cell infiltration in murine preclinical tumor models and evaluated how these differences may affect tumor disposition of PEGylated liposomal doxorubicin (PLD) in models sensitive and resistant to PLD. Significant differences in MPS presence existed between tumor types (e.g. ovarian versus endometrial), cell lines within the same tumor type, and location of tumor implantation (i.e. flank versus orthotopic xenografts). Further, the differences in MPS presence of SKOV-3 ovarian and HEC1A endometrial orthotopic cancer models may account for the 2.6-fold greater PLD tumor exposure in SKOV-3, despite similar plasma, liver and spleen exposures. These findings suggest that profiling the presence of MPS cells within and between tumor types is important in tumor model selection and in tumor types and patients likely to respond to NP treatment.

Preclinical to Clinical Translation of Antibody-Drug Conjugates Using PK/PD Modeling: a Retrospective Analysis of Inotuzumab Ozogamicin.

A mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model was used for preclinical to clinical translation of inotuzumab ozogamicin, a CD22-targeting antibody-drug conjugate (ADC) for B cell malignancies including non-Hodgkin's lymphoma (NHL) and acute lymphocytic leukemia (ALL). Preclinical data was integrated in a PK/PD model which included (1) a plasma PK model characterizing disposition and clearance of inotuzumab ozogamicin and its released payload N-Ac-γ-calicheamicin DMH, (2) a tumor disposition model describing ADC diffusion into the tumor extracellular environment, (3) a cellular model describing inotuzumab ozogamicin binding to CD22, internalization, intracellular N-Ac-γ-calicheamicin DMH release, binding to DNA, or efflux from the tumor cell, and (4) tumor growth and inhibition in mouse xenograft models. The preclinical model was translated to the clinic by incorporating human PK for inotuzumab ozogamicin and clinically relevant tumor volumes, tumor growth rates, and values for CD22 expression in the relevant patient populations. The resulting stochastic models predicted progression-free survival (PFS) rates for inotuzumab ozogamicin in patients comparable to the observed clinical results. The model suggested that a fractionated dosing regimen is superior to a conventional dosing regimen for ALL but not for NHL. Simulations indicated that tumor growth is a highly sensitive parameter and predictive of successful outcome. Inotuzumab ozogamicin PK and N-Ac-γ-calicheamicin DMH efflux are also sensitive parameters and would be considered more useful predictors of outcome than CD22 receptor expression. In summary, a multiscale, mechanism-based model has been developed for inotuzumab ozogamicin, which can integrate preclinical biomeasures and PK/PD data to predict clinical response.

Evolution of Antibody-Drug Conjugate Tumor Disposition Model to Predict Preclinical Tumor Pharmacokinetics of Trastuzumab-Emtansine (T-DM1).

A mathematical model capable of accurately characterizing intracellular disposition of ADCs is essential for a priori predicting unconjugated drug concentrations inside the tumor. Towards this goal, the objectives of this manuscript were to: (1) evolve previously published cellular disposition model of ADC with more intracellular details to characterize the disposition of T-DM1 in different HER2 expressing cell lines, (2) integrate the improved cellular model with the ADC tumor disposition model to a priori predict DM1 concentrations in a preclinical tumor model, and (3) identify prominent pathways and sensitive parameters associated with intracellular activation of ADCs. The cellular disposition model was augmented by incorporating intracellular ADC degradation and passive diffusion of unconjugated drug across tumor cells. Different biomeasures and chemomeasures for T-DM1, quantified in the companion manuscript, were incorporated into the modified model of ADC to characterize in vitro pharmacokinetics of T-DM1 in three HER2+ cell lines. When the cellular model was integrated with the tumor disposition model, the model was able to a priori predict tumor DM1 concentrations in xenograft mice. Pathway analysis suggested different contribution of antigen-mediated and passive diffusion pathways for intracellular unconjugated drug exposure between in vitro and in vivo systems. Global and local sensitivity analyses revealed that non-specific deconjugation and passive diffusion of the drug across tumor cell membrane are key parameters for drug exposure inside a cell. Finally, a systems pharmacokinetic model for intracellular processing of ADCs has been proposed to highlight our current understanding about the determinants of ADC activation inside a cell.

Efficient anti-tumor effect of photodynamic treatment with polymeric nanoparticles composed of polyethylene glycol and polylactic acid block copolymer encapsulating hydrophobic porphyrin derivative

To develop potent and safer formulation of photosensitizer for cancer photodynamic therapy (PDT), we tried to formulate hydrophobic porphyrin derivative, photoprotoporphyrin IX dimethyl ester (PppIX-DME), into polymeric nanoparticles composed of polyethylene glycol and polylactic acid block copolymer (PN-Por). The mean particle size of PN-Por prepared was around 80nm and the zeta potential was determined to be weakly negative. In vitro phototoxicity study for PN-Por clearly indicated the significant phototoxicity of PN-Por for three types of tumor cells tested (Colon-26 carcinoma (C26), B16BL6 melanoma and Lewis lung cancer cells) in the PppIX-DME concentration-dependent fashion. Furthermore, it was suggested that the release of PppIX-DME from PN-Por would gradually occur to provide the sustained release of PppIX-DME. In vivo pharmacokinetics of PN-Por after intravenous administration was evaluated in C26 tumor-bearing mice, and PN-Por exhibited low affinity to the liver and spleen and was therefore retained in the blood circulation for a long time, leading to the efficient tumor disposition of PN-Por. Furthermore, significant and highly effective anti-tumor effect was confirmed in C26 tumor-bearing mice with the local light irradiation onto C26 tumor tissues after PN-Por injection. These findings indicate the potency of PN-Por for the development of more efficient PDT-based cancer treatments.

Novel potential nanostructures for photodynamic therapy

To develop potent and safer formulation of photosensitizer for cancer photodynamic therapy (PDT), we tried to formulate hydrophobic porphyrin derivative, photoprotoporphyrin IX dimethyl ester (PppIX-DME), into polymeric nanoparticles composed of polyethylene glycol and polylactic acid block copolymer (PN-Por). The mean particle size of PN-Por prepared was around 80 nm and the zeta potential was determined to be weakly negative. In vitro phototoxicity study for PN-Por clearly indicated the significant phototoxicity of PN-Por for three types of tumor cells tested (Colon-26 carcinoma (C26), B16BL6 melanoma and Lewis lung cancer cells) in the PppIX-DME concentration-dependent fashion. Furthermore, it was suggested that the release of PppIX-DME from PN-Por would gradually occur to provide the sustained release of PppIX-DME. In vivo pharmacokinetics of PN-Por after intravenous administration was evaluated in C26 tumor-bearing mice, and PN-Por exhibited low affinity to the liver and spleen and was therefore retained in the blood circulation for a long time, leading to the efficient tumor disposition of PN-Por. Furthermore, significant and highly effective anti-tumor effect was confirmed in C26 tumor-bearing mice with the local light irradiation onto C26 tumor tissues after PN-Por injection. These findings indicate the potency of PN-Por for the development of more efficient PDT-based cancer treatments.

Augmented EPR effect by photo-triggered tumor vascular treatment improved therapeutic efficacy of liposomal paclitaxel in mice bearing tumors with low permeable vasculature

The effects of photo-triggered tumor vascular treatment (PVT) on the structural and functional properties of tumor vasculature were assessed in Colon-26 (C26) and B16/BL6 (B16) tumor-bearing mice. Furthermore, anti-tumor efficacy of subsequently injected PEG liposomal paclitaxel (PL-PTX) was also evaluated. As a photosensitizer, a hydrophobic porphyrin derivative was used and formulated in polymeric nanoparticle composed of polyethylene glycol-block-polylactic acid to avoid its non-specific in vivo disposition. In the mice bearing C26 with high permeable vasculature, the prominent anti-tumor activity was confirmed by PVT alone, but the subsequently injected PL-PTX did not show any additive effect. PVT itself initially induced apoptotic cell death of tumor vascular endothelial cells and platelet aggregation, which would have subsequently induced apoptosis of C26 tumor cells surrounding the vasculature. On the other hand, in the mice bearing B16 with low permeable vasculature, PVT enhanced the anti-tumor activity of subsequently injected PL-PTX, which would be attributed to the tumor disposition amount and area of PEG liposomes enhanced by PVT. These results clearly indicated that the treatment would have made it possible to provide more efficient extravasation of PL-PTX, leading to its more potent anti-tumor effect.

Abstract 4241: Preclinical studies of brain/brain tumor disposition and antitumor efficacy of the aromatase inhibitor letrozole

Abstract In this study, we investigated the potential role of aromatase (CYP19) as a target for the treatment of CNS malignancies, as well as brain disposition and anti-tumor efficacy of letrozole, an aromatase inhibitor, in Sprague Dawley rats. Cytotoxicity and aromatase activity of letrozole against human glioma cell lines were measured using MTT assay and Enzyme Immunoassay respectively. For brain and brain tumor PK of letrozole, rats with and without orthotopic implantation of C6 glioma received letrozole (4 - 12 mg/kg; i.v. andor oral). Dual probe intracerebral microdialysis was performed to determine the unbound extracellular fluid (ECF) letrozole concentrations. Serial ECF and blood samples were simultaneously collected over 8 hrs. µPET/CT imaging was performed using 18F FDG to evaluate changes in active tumor volumes pre- and post-treatment of letrozole. Brain tissues were collected at the end of the experiment for histological evaluations. All glioma cell lines included in this study expressed CYP19 and letrozole exerted marked cytotoxicity against these cells ( IC50s 0.1 - 3.5 μM). The relative brain distribution coefficients, measured as the ratio of the observed AUC in ECF and AUC of unbound letrozole in plasma (AUCecf/AUCp,ub) ranged from 0.31 - 0.98. Furthermore, the tumoral ECF levels of letrozole was 1.5 - 2 fold higher relative to tumor-free region of the brain, resulting in tumoral ECF Cmax values that were 10 and 35-fold higher than the observed IC50 value of 0.1 µM against C6 gliomas cells in culture. µPET/CT imaging showed a marked reduction of active tumor volume (75-90%) after 8-10 days of letrozole treatment (N=7). Oral administration of letrozole showed similar brain disposition and efficacy of letrozole. Immunohistochemical analysis of aromatase demonstrated selectivelymarkedly higher aromatase expression in tumoral regions of the brain as well asand a clear considerable reduction in aromatase expression in letrozole the treated rats relative to the treatment group as compared to the control group. Thus, employing multifaceted and cutting edge in vitro and in vivo methods, we conclude: a) aromatase is abundantly expressed in glioma cell lines examined, b) letrozole exerts marked cytotoxicity in these cells presumably due to aromatase inhibition, c) letrozole has selectively higher accumulation in tumoral region of the brain and d) In vivo µPET/CT studies show marked efficacy of letrozole on C6 glioma in a preclinical rat model. Note: This abstract was not presented at the meeting. Citation Format: Nimita Dave, Pankaj B. Desai, Gary A. Gudelsky, Kathleen LaSance, Lionel M.L. Chow, Xiaoyang Qi. Preclinical studies of brain/brain tumor disposition and antitumor efficacy of the aromatase inhibitor letrozole. [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 4241. doi:10.1158/1538-7445.AM2014-4241