Charlene Santos

Pharmacokinetics and efficacy of PEGylated liposomal doxorubicin in an intracranial model of breast cancer.

Introduction Breast cancer brain metastases (BCBM) are a challenging consequence of advanced BC. Nanoparticle agents, including liposomes, have shown enhanced delivery to solid tumors and brain. We compared pharmacokinetics (PK) and efficacy of PEGylated liposomal doxorubicin (PLD) with non-liposomal doxorubicin (NonL-doxo) in an intracranial model of BC. Methods Athymic mice were inoculated intracerebrally with MDA-MB-231-BR-luciferase-expressing cells. Tumor-bearing mice were administered PLD or NonL-doxo at 6mg/kg IV×1 and were euthanized prior to and 0.083, 1, 3, 6, 24, 72 and 96 h post-treatment. Samples were processed to measure sum total doxorubicin via HPLC. PLD and NonL-doxo were administered IV weekly as single agents (6 mg/kg) or in combination (4.5 mg/kg) with the PARP inhibitor, ABT-888, PO 25 mg/kg/day. Efficacy was assessed by survival and bioluminescence. Results Treatment with PLD resulted in approximately 1,500-fold higher plasma and 20-fold higher intracranial tumor sum total doxorubicin AUC compared with NonL-doxo. PLD was detected at 96 h; NonL-doxo was undetectable after 24 h in plasma and tumor. Median survival of PLD-treated animals was 32 days (d, [CI] 31–38), which was significantly longer than controls (26d [CI 25–28]; p = 0.0012) or NonL-doxo treatment (23.5d [CI 18–28], p = 0.0002). Combination treatment with PLD/ABT-888 yielded improved survival compared to NonL-doxo/ABT-888 (35d [CI 31–38] versus 29.5d [CI 25–34]; p = 0.006). Conclusions PLD provides both PK and efficacy advantage over NonL-doxo in the treatment of an in vivo model of BCBM. The results provide preclinical rationale to translate findings into early phase trials of PLD, with or without ABT-888, for patients with BCBM.

Effects of tumor microenvironment heterogeneity on nanoparticle disposition and efficacy in breast cancer tumor models.

Purpose: Tumor cells are surrounded by a complex microenvironment. The purpose of our study was to evaluate the role of heterogeneity of the tumor microenvironment in the variability of nanoparticle (NP) delivery and efficacy. Experimental Designs: C3(1)-T-Antigen genetically engineered mouse model (C3-TAg) and T11/TP53Null orthotopic syngeneic murine transplant model (T11) representing human breast tumor subtypes basal-like and claudin-low, respectively, were evaluated. For the pharmacokinetic studies, non-liposomal doxorubicin (NL-doxo) or polyethylene glycol tagged (PEGylated) liposomal doxorubicin (PLD) was administered at 6 mg/kg i.v. x1. Area under the concentration versus time curve (AUC) of doxorubicin was calculated. Macrophages, collagen, and the amount of vasculature were assessed by IHC. Chemokines and cytokines were measured by multiplex immunochemistry. NL-doxo or PLD was administered at 6 mg/kg i.v. weekly x6 in efficacy studies. Analyses of intermediary tumor response and overall survival were performed. Results: Plasma AUC of NL-doxo and PLD encapsulated and released doxorubicin was similar between two models. However, tumor sum total AUC of PLD was 2-fold greater in C3-TAg compared with T11 (P < 0.05). T11 tumors showed significantly higher expression of CC chemokine ligand (CCL) 2 and VEGF-a, greater vascular quantity, and decreased expression of VEGF-c compared with C3-TAg (P < 0.05). PLD was more efficacious compared with NL-doxo in both models. Conclusion: The tumor microenvironment and/or tumor cell features of breast cancer affected NP tumor delivery and efficacy, but not the small-molecule drug. Our findings reveal the role of the tumor microenvironment in variability of NP delivery and therapeutic outcomes. Clin Cancer Res; 20(23); 6083–95. ©2014 AACR.

Enhancer Remodeling during Adaptive Bypass to MEK Inhibition Is Attenuated by Pharmacologic Targeting of the P-TEFb Complex

Targeting the dysregulated BRAF-MEK-ERK pathway in cancer has increasingly emerged in clinical trial design. Despite clinical responses in specific cancers using inhibitors targeting BRAF and MEK, resistance develops often involving nongenomic adaptive bypass mechanisms. Inhibition of MEK1/2 by trametinib in patients with triple-negative breast cancer (TNBC) induced dramatic transcriptional responses, including upregulation of receptor tyrosine kinases (RTK) comparing tumor samples before and after one week of treatment. In preclinical models, MEK inhibition induced genome-wide enhancer formation involving the seeding of BRD4, MED1, H3K27 acetylation, and p300 that drives transcriptional adaptation. Inhibition of the P-TEFb-associated proteins BRD4 and CBP/p300 arrested enhancer seeding and RTK upregulation. BRD4 bromodomain inhibitors overcame trametinib resistance, producing sustained growth inhibition in cells, xenografts, and syngeneic mouse TNBC models. Pharmacologic targeting of P-TEFb members in conjunction with MEK inhibition by trametinib is an effective strategy to durably inhibit epigenomic remodeling required for adaptive resistance.Significance: Widespread transcriptional adaptation to pharmacologic MEK inhibition was observed in TNBC patient tumors. In preclinical models, MEK inhibition induces dramatic genome-wide modulation of chromatin, in the form of de novo enhancer formation and enhancer remodeling. Pharmacologic targeting of P-TEFb complex members at enhancers is an effective strategy to durably inhibit such adaptation. Cancer Discov; 7(3); 302-21. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235.

Efficacy of Carboplatin Alone and in Combination with ABT888 in Intracranial Murine Models of BRCA-Mutated and BRCA–Wild-Type Triple-Negative Breast Cancer

Patients with breast cancer brain metastases have extremely limited survival and no approved systemic therapeutics. Triple-negative breast cancer (TNBC) commonly metastasizes to the brain and predicts poor prognosis. TNBC frequently harbors BRCA mutations translating to platinum sensitivity potentially augmented by additional suppression of DNA repair mechanisms through PARP inhibition. We evaluated brain penetrance and efficacy of carboplatin ± the PARP inhibitor ABT888, and investigated gene-expression changes in murine intracranial TNBC models stratified by BRCA and molecular subtype status. Athymic mice were inoculated intracerebrally with BRCA-mutant: SUM149 (basal), MDA-MB-436 (claudin-low); or BRCA–wild-type (wt): MDA-MB-468 (basal), MDA-MB-231BR (claudin-low). TNBC cells were treated with PBS control [intraperitoneal (IP), weekly], carboplatin (50 mg/kg/wk, IP), ABT888 (25 mg/kg/d, oral gavage), or their combination. DNA damage (γ-H2AX), apoptosis (cleaved caspase-3, cC3), and gene expression were measured in intracranial tumors. Carboplatin ± ABT888 significantly improved survival in BRCA-mutant intracranial models compared with control, but did not improve survival in BRCA-wt intracranial models. Carboplatin + ABT888 revealed a modest survival advantage versus carboplatin in BRCA-mutant models. ABT888 yielded a marginal survival benefit in the MDA-MB-436, but not in the SUM149 model. BRCA-mutant SUM149 expression of γ-H2AX and cC3 proteins was elevated in all treatment groups compared with control, whereas BRCA-wt MDA-MB-468 cC3 expression did not increase with treatment. Carboplatin treatment induced common gene-expression changes in BRCA-mutant models. Carboplatin ± ABT888 penetrates the brain and improves survival in BRCA-mutant intracranial TNBC models with corresponding DNA damage and gene-expression changes. Combination therapy represents a potential promising treatment strategy for patients with TNBC brain metastases warranting further clinical investigation. Mol Cancer Ther; 14(4); 920–30. ©2015 AACR.

A Novel Monoclonal Antibody to Secreted Frizzled-Related Protein 2 Inhibits Tumor Growth

Secreted frizzled-related protein 2 (SFRP2) is overexpressed in human angiosarcoma and breast cancer and stimulates angiogenesis via activation of the calcineurin/NFATc3 pathway. There are conflicting reports in the literature as to whether SFRP2 is an antagonist or agonist of β-catenin. The aims of these studies were to assess the effects of SFRP2 antagonism on tumor growth and Wnt-signaling and to evaluate whether SFRP2 is a viable therapeutic target. The antiangiogenic and antitumor properties of SFRP2 monoclonal antibody (mAb) were assessed using in vitro proliferation, migration, tube formation assays, and in vivo angiosarcoma and triple-negative breast cancer models. Wnt-signaling was assessed in endothelial and tumor cells treated with SFRP2 mAb using Western blotting. Pharmacokinetic and biodistribution data were generated in tumor-bearing and nontumor-bearing mice. SFRP2 mAb was shown to induce antitumor and antiangiogenic effects in vitro and inhibit activation of β-catenin and nuclear factor of activated T-cells c3 (NFATc3) in endothelial and tumor cells. Treatment of SVR angiosarcoma allografts in nude mice with the SFRP2 mAb decreased tumor volume by 58% compared with control (P = 0.004). Treatment of MDA-MB-231 breast carcinoma xenografts with SFRP2 mAb decreased tumor volume by 52% (P = 0.03) compared with control, whereas bevacizumab did not significantly reduce tumor volume. Pharmacokinetic studies show the antibody is long circulating in the blood and preferentially accumulates in SFRP2-positive tumors. In conclusion, antagonizing SFRP2 inhibits activation of β-catenin and NFATc3 in endothelial and tumor cells and is a novel therapeutic approach for inhibiting angiosarcoma and triple-negative breast cancer. Mol Cancer Ther; 12(5); 685–95. ©2013 AACR.

A sensitive high performance liquid chromatography assay for the quantification of doxorubicin associated with DNA in tumor and tissues.

Doxorubicin, a widely used anticancer agent, exhibits antitumor activity against a wide variety of malignancies. The drug exerts its cytotoxic effects by binding to and intercalating within the DNA of tumor and tissue cells. However, current assays are unable to accurately determine the concentration of the intracellular active form of doxorubicin. Thus, the development of a sample processing method and a high-performance liquid chromatography (HPLC) methodology was performed in order to quantify doxorubicin that is associated with DNA in tumors and tissues, which provided an intracellular cytotoxic measure of doxorubicin exposure after administration of small molecule and nanoparticle formulations of doxorubicin. The assay uses daunorubicin as an internal standard; liquid-liquid phase extraction to isolate drug associated with DNA; a Shimadzu HPLC with fluorescence detection equipped with a Phenomenex Luna C18 (2μm, 2.0×100mm) analytical column and a gradient mobile phase of 0.1% formic acid in water or acetonitrile for separation and quantification. The assay has a lower limit of detection (LLOQ) of 10ng/mL and is shown to be linear up to 3000ng/mL. The intra- and inter-day precision of the assay expressed as a coefficient of variation (CV%) ranged from 4.01 to 8.81%. Furthermore, the suitability of this assay for measuring doxorubicin associated with DNA in vivo was demonstrated by using it to quantify the doxorubicin concentration within tumor samples from SKOV3 and HEC1A mice obtained 72h after administration of PEGylated liposomal doxorubicin (Doxil(®); PLD) at 6mg/kg IV x 1. This HPLC assay allows for sensitive intracellular quantification of doxorubicin and will be an important tool for future studies evaluating intracellular pharmacokinetics of doxorubicin and various nanoparticle formulations of doxorubicin.

Roles of chemokines CCL2 and CCL5 in the pharmacokinetics of PEGylated liposomal doxorubicin in vivo and in patients with recurrent epithelial ovarian cancer.

UNLABELLED Nanoparticles (NPs) are cleared by monocytes and macrophages. Chemokines CCL2 and CCL5 are key mediators for recruitment of these immune cells into tumors and tissues. The purpose of this study was to investigate effects of CCL2 and CCL5 on the pharmacokinetics (PKs) of NPs. Mice deficient in CCL2 or CCL5 demonstrated altered clearance and tissue distribution of polyethylene glycol tagged liposomal doxorubicin (PLD) compared to control mice. The PK studies using mice bearing SKOV3 ovarian cancer xenografts revealed that the presence of tumor cells and higher expression of chemokines were significantly associated with greater clearance of PLD compared to non-tumor bearing mice. Plasma exposure of encapsulated liposomal doxorubicin positively correlated with the total exposure of plasma CCL2 and CCL5 in patients with recurrent epithelial ovarian cancer treated with PLD. These data emphasize that the interplay between PLD and chemokines may have an important role in optimizing PLD therapy. FROM THE CLINICAL EDITOR The use of nanoparticles as drug delivery carriers is gaining widespread acceptance in the clinical setting. However, the underlying pharmacokinetics of these novel drugs has not really been elucidated. In this interesting article, the authors carried out experiments using mice deficient in CCL2 or CCL5 to study the clearance of liposomal system. They showed the important role the immune system played and would enable better designs of future drug delivery systems.

Gulp1 is associated with the pharmacokinetics of PEGylated liposomal doxorubicin (PLD) in inbred mouse strains

Nanoparticles (NP) including liposomes are cleared by phagocytes of the mononuclear phagocyte system. High inter-patient variability in pharmacokinetics of PEGylated liposomal doxorubicin (PLD) has been reported. We hypothesized that genetic factors may be associated with the variable disposition of PLD. We evaluated plasma and tissue disposition of doxorubicin after administration of PLD at 6mg/kg IV ×1 via tail vein in 23 different male inbred mouse strains. An approximately 13-fold difference in plasma clearance of PLD was observed among inbred strains. We identified a correlation between strain-specific differences in PLD clearance and genetic variation within a genomic region encoding GULP1 (PTB domain containing engulfment adapter 1) protein using haplotype associated mapping and the efficient mixed-model association algorithms. Our results also show that Gulp1 expression in adipose tissue was associated with PLD disposition in plasma. Our findings suggest that genetic variants may be associated with inter-individual pharmacokinetic differences in NP clearance.

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.

Abstract 3316: The effects of dopamine receptor 2 on breast cancer tumor initiating cells

Despite advances in early detection and treatment of breast cancers, metastatic disease remains a significant problem and a large cause of mortality in the US. Although tumors are characterized as a mass of uncontrolled cell division, it is thought that only small populations of cells, known as tumor initiating cells (TICs), are primarily responsible for metastasis. TICs are less differentiated than most tumor cells and many TICs show an epithelial-to-mesenchymal transition (EMT). Recently, the FDA-approved drug thioridazine, a dopamine receptor 2 (DRD2) antagonist, has been shown to inhibit pluripotency gene expression only in transformed stem cells. To test whether DRD2 can regulate TICs in breast cancer cells, two main assays were performed. The tumorsphere assay is used to measure TIC activity within a population of cells in vitro. In this assay, single cells are cultured in suspension, in the absence of serum. Cells must escape anoikis and grow without cell-cell contacts or the assortment of factors contained in serum. However, the gold standard in vivo assay to measure tumor initiation is the transplantation assay at limiting dilutions. Cells are injected into immunocompromised mice in decreasing numbers and the number of tumors each population of cells is capable of forming is measured. In this study the tumorsphere assay and transplantation assay both show that DRD2 supports TICs in breast cancers. Interestingly, our data suggest that DRD2 regulates TICs and cellular differentiation through the activity of the STAT3 transcription factor. Activation of STAT3 has been shown to induce gene expression changes that support TICs and upregulate the pluripotency factor OCT4. STAT3 activity can also induce EMT and resistance to some therapeutics. By inducing the expression of its own activator, IL-6, STAT3 can initiate a feed forward loop to maintain continual expression to promote TICs, EMT, and drug resistance. Halting the continual activation of STAT3 in these cells should be an important objective. Our data supports the concept that DRD2 may represent a new, and druggable target for therapies to decrease metastasis and/or recurrence by interfering with STAT3 and inhibiting TIC-like activities. Citation Format: Matthew Tegowski, Charlene Santos, Albert Baldwin. The effects of dopamine receptor 2 on breast cancer tumor initiating cells. [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 3316.