Discovery of a Novel Cabazitaxel Nanoparticle-Drug Conjugate (CRLX522) with Improved Pharmacokinetic Properties and Anticancer Effects Using a ß-Cyclodextrin-PEG Copolymer Based Delivery Platform.

Novel nanoparticle-drug conjugates (NDCs) containing diverse, clinically relevant anticancer drug payloads (docetaxel, cabazitaxel, and gemcitabine) were successfully generated and tested in drug discovery studies. The NDCs utilized structurally varied linkers that attached the drug payloads to a ß-cyclodextrin-PEG copolymer to form self-assembled nanoparticles. In vitro release studies revealed a diversity of release rates driven by linker structure-activity relationships (SARs). Improved in vivo pharmacokinetics (PK) for the cabazitaxel (CBTX) NDCs with glycinate-containing (1c) and hexanoate-containing linkers (2c) were demonstrated, along with high and sustained tumor levels (>168 h of released drug in tumor tissues). This led to potent efficacy and survival in both taxane- and docetaxel-resistant in vivo anticancer mouse efficacy models. Overall, the CBTX-hexanoate NDC 2c (CRLX522), demonstrated optimal and improved in vivo PK (plasma and tumor) and efficacy profile versus those of the parent drug, and the results support the potential therapeutic use of CRLX522 as a new anticancer agent.

Preclinical Efficacy of Bevacizumab with CRLX101, an Investigational Nanoparticle-Drug Conjugate, in Treatment of Metastatic Triple-Negative Breast Cancer.

VEGF pathway-targeting antiangiogenic drugs, such as bevacizumab, when combined with chemotherapy have changed clinical practice for the treatment of a broad spectrum of human cancers. However, adaptive resistance often develops, and one major mechanism is elevated tumor hypoxia and upregulated hypoxia-inducible factor-1α (HIF1α) caused by antiangiogenic treatment. Reduced tumor vessel numbers and function following antiangiogenic therapy may also affect intratumoral delivery of concurrently administered chemotherapy. Nonetheless, combining chemotherapy and bevacizumab can lead to improved response rates, progression-free survival, and sometimes, overall survival, the extent of which can partly depend on the chemotherapy backbone. A rational, complementing chemotherapy partner for combination with bevacizumab would not only reduce HIF1α to overcome hypoxia-induced resistance, but also improve tumor perfusion to maintain intratumoral drug delivery. Here, we evaluated bevacizumab and CRLX101, an investigational nanoparticle-drug conjugate containing camptothecin, in preclinical mouse models of orthotopic primary triple-negative breast tumor xenografts, including a patient-derived xenograft. We also evaluated long-term efficacy of CRLX101 and bevacizumab to treat postsurgical, advanced metastatic breast cancer in mice. CRLX101 alone and combined with bevacizumab was highly efficacious, leading to complete tumor regressions, reduced metastasis, and greatly extended survival of mice with metastatic disease. Moreover, CRLX101 led to improved tumor perfusion and reduced hypoxia, as measured by contrast-enhanced ultrasound and photoacoustic imaging. CRLX101 durably suppressed HIF1α, thus potentially counteracting undesirable effects of elevated tumor hypoxia caused by bevacizumab. Our preclinical results show pairing a potent cytotoxic nanoparticle chemotherapeutic that complements and improves concurrent antiangiogenic therapy may be a promising treatment strategy for metastatic breast cancer. Cancer Res; 76(15); 4493-503. ©2016 AACR.

Tumor Selective Silencing Using an RNAi-Conjugated Polymeric Nanopharmaceutical.

Small interfering RNA (siRNA) therapeutics have potential advantages over traditional small molecule drugs such as high specificity and the ability to inhibit otherwise "undruggable" targets. However, siRNAs have short plasma half-lives in vivo, can induce a cytokine response, and show poor cellular uptake. Formulating siRNA into nanoparticles offers two advantages: enhanced siRNA stability against nuclease degradation beyond what chemical modification alone can provide; and improved site-specific delivery that takes advantage of the enhanced permeability and retention (EPR) effect. Existing delivery systems generally suffer from poor delivery to tumors. Here we describe the formation and biological activity of polymeric nanopharmaceuticals (PNPs) based on biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) conjugated to siRNA via an intracellular cleavable disulfide linker (PLGA-siRNA). Additionally, these PNPs contain (1) PLGA conjugated to polyethylene glycol (PEG) for enhanced pharmacokinetics of the nanocarrier; (2) a cation for complexation of siRNA and charge compensation to avoid high negative zeta potential; and (3) neutral poly(vinyl alcohol) (PVA) to stabilize the PNPs and support the PEG shell to prevent particle aggregation and protein adsorption. The biological data demonstrate that these PNPs achieve prolonged circulation, tumor accumulation that is uniform throughout the tumor, and prolonged tumor-specific knockdown. PNPs employed in this study had no effect on body weight, blood cell count, serum chemistry, or cytokine response at doses >10 times the effective dose. PNPs, therefore, constitute a promising solution for achieving durable siRNA delivery and gene silencing in tumors.

CRLX101, an investigational camptothecin-containing nanoparticle-drug conjugate, targets cancer stem cells and impedes resistance to antiangiogenic therapy in mouse models of breast cancer.

Antiangiogenic therapies inhibit the development of new tumor blood vessels, thereby blocking tumor growth. Despite the advances in developing antiangiogenic agents, clinical data indicate that these drugs have limited efficacy in breast cancer patients. Tumors inevitably develop resistance to antiangiogenics, which is attributed in part to the induction of intra-tumoral hypoxia and stabilization of hypoxia-inducible factor 1α (HIF-1α), a transcription factor that promotes tumor angiogenesis, invasion, metastasis, and cancer stem cell (CSC) self-renewal. Here, we tested whether inhibiting HIF-1α can reverse the stimulatory effects of antiangiogenic-induced hypoxia on breast CSCs. Breast cancer cells grown under hypoxic conditions were treated with the dual topoisomerase-1 (TOPO-1) and HIF-1α inhibitor camptothecin and assessed for their CSC content. In a preclinical model of breast cancer, treatment with bevacizumab was compared to the combination treatment of bevacizumab with CRLX101, an investigational nanoparticle-drug conjugate with a camptothecin payload or CRLX101 monotherapy. While exposure to hypoxia increased the number of breast CSCs, treatment with CPT blocked this effect. In preclinical mouse models, concurrent administration of CRLX101 impeded the induction of both HIF-1α and CSCs in breast tumors induced by bevacizumab treatment. Greater tumor regression and delayed tumor recurrence were observed with the combination of these agents compared to bevacizumab alone. Tumor reimplantation experiments demonstrated that the combination therapy effectively targets the CSC populations. The results from these studies support the combined administration of dual TOPO-1- and HIF-1α-targeted agents like CRLX101 with antiangiogenic agents to increase the efficacy of these treatments.

Translational impact of nanoparticle-drug conjugate CRLX101 with or without bevacizumab in advanced ovarian cancer.

PURPOSE: Increased tumor hypoxia and hence elevated hypoxia-inducible factor-1α (HIF1α) is thought to limit the efficacy of vascular endothelial growth factor (VEGF) pathway-targeting drugs by upregulating adaptive resistance genes. One strategy to counteract this is to combine antiangiogenic drugs with agents able to suppress HIF1α. One such possibility is the investigational drug CRLX101, a nanoparticle-drug conjugate (NDC) containing the payload camptothecin, a known topoisomerase-I poison. EXPERIMENTAL DESIGN: CRLX101 was evaluated both as a monotherapy and combination with bevacizumab in a preclinical mouse model of advanced metastatic ovarian cancer. These preclinical studies contributed to the rationale for undertaking a phase II clinical study to evaluate CRLX101 monotherapy in patients with advanced platinum-resistant ovarian cancer. RESULTS: Preclinically, CRLX101 is highly efficacious as a monotherapy when administered at maximum-tolerated doses. Furthermore, chronic low-dose CRLX101 with bevacizumab reduced bevacizumab-induced HIF1α upregulation and resulted in synergistic efficacy, with minimal toxicity in mice. In parallel, initial data reported here from an ongoing phase II clinical study of CRLX101 monotherapy shows measurable tumor reductions in 74% of patients and a 16% RECIST response rate to date. CONCLUSIONS: Given these preclinical and initial clinical results, further clinical studies are currently evaluating CRLX101 in combination with bevacizumab in ovarian cancer and warrant the evaluation of this therapy combination in other cancer types where HIF1α is implicated in pathogenesis, as it may potentially be able to improve the efficacy of antiangiogenic drugs.

Correlating preclinical animal studies and human clinical trials of a multifunctional, polymeric nanoparticle.

Nanoparticles are currently being investigated in a number of human clinical trials. As information on how nanoparticles function in humans is difficult to obtain, animal studies that can be correlative to human behavior are needed to provide guidance for human clinical trials. Here, we report correlative studies on animals and humans for CRLX101, a 20- to 30-nm-diameter, multifunctional, polymeric nanoparticle containing camptothecin (CPT). CRLX101 is currently in phase 2 clinical trials, and human data from several of the clinical investigations are compared with results from multispecies animal studies. The pharmacokinetics of polymer-conjugated CPT (indicative of the CRLX101 nanoparticles) in mice, rats, dogs, and humans reveal that the area under the curve scales linearly with milligrams of CPT per square meter for all species. Plasma concentrations of unconjugated CPT released from CRLX101 in animals and humans are consistent with each other after accounting for differences in serum albumin binding of CPT. Urinary excretion of polymer-conjugated CPT occurs primarily within the initial 24 h after dosing in animals and humans. The urinary excretion dynamics of polymer-conjugated and unconjugated CPT appear similar between animals and humans. CRLX101 accumulates into solid tumors and releases CPT over a period of several days to give inhibition of its target in animal xenograft models of cancer and in the tumors of humans. Taken in total, the evidence provided from animal models on the CRLX101 mechanism of action suggests that the behavior of CRLX101 in animals is translatable to humans.

Inhibition of melanoma tumor growth by a pharmacological inhibitor of MetAP-2, PPI-2458.

Over the past few decades, melanoma has shown the fastest growing incidence rate of all cancers. This malignancy is clinically defined by its potential to rapidly metastasize, and advanced metastatic melanomas are highly resistant to existing therapeutic regimens. Here, we report that PPI-2458, a novel, orally active agent of the fumagillin class of irreversible methionine aminopeptidase-2 (MetAP-2) inhibitors, potently inhibited the proliferation of B16F10 melanoma cells in vitro, with a growth inhibitory concentration 50% (GI50) of 0.2 nM. B16F10 growth inhibition was correlated with the inhibition of MetAP-2 enzyme, in a dose-dependent fashion, as determined by a pharmacodynamic assay, which measures the amount of uninhibited MetAP-2 following PPI-2458 treatment. Prolonged exposure of B16F10 cells to PPI-2458 at concentrations of up to 1 microM, 5,000-fold above the GI50, did not alter their sensitivity to PPI-2458 growth inhibition and no drug resistance was observed. Moreover, prolonged exposure to this agent induced melanogenesis, concomitant with the elevated expression of the melanocyte-specific enzymes tyrosinase and tyrosinase-related proteins (TRP) 1 and 2, a morphological feature associated with differentiated melanocytes. PPI-2458, when administered orally (p.o.), significantly inhibited B16F10 tumor growth in mice in a dose-dependent fashion, with a maximum inhibition of 62% at 100 mg/kg. This growth inhibition was directly correlated to the amount of irreversibly inhibited MetAP-2 (80% at 100 mg/kg PPI-2458) in tumor tissue. These data demonstrate that PPI-2458 has potent antiproliferative activity against B16F10 cells in vitro and in vivo, and that both activities are directly correlated with levels of MetAP-2 enzyme inhibition. This antiproliferative activity, coupled with additional observations from studies in vitro (absence of detectable resistance to PPI-2458 and induction of morphological features consistent with differentiated melanocytes), provides a rationale for assessing the therapeutic potential of PPI-2458 in the treatment of melanoma.

A methionine aminopeptidase-2 inhibitor, PPI-2458, for the treatment of rheumatoid arthritis.

The hallmark of rheumatoid arthritis (RA) is the progressive destruction of articular joints, characterized by invasive synovial hyperplasia and pathological neovascularization. Here we report that PPI-2458, a member of the fumagillin class of irreversible methionine aminopeptidase-2 (MetAP-2) inhibitors, potently inhibits the proliferation of human fibroblast-like synoviocytes (HFLS-RA), derived from RA patients, with a growth inhibitory concentration 50 (GI(50)) of 0.04 nM and a maximum inhibition of >95% at 1 nM. Human umbilical vein endothelial cells (HUVEC) are similarly inhibited in proliferation by PPI-2458 (GI(50), 0.2 nM). We developed a method to measure the level of MetAP-2 enzyme inhibition after exposure to PPI-2458 and demonstrate that growth inhibition of PPI-2458-sensitive HFLS-RA and HUVEC is linked to MetAP-2 enzyme inhibition, in a dose-dependent fashion. The secretion of several inflammatory mediators such as IL-6 and vascular endothelial growth factor from activated HFLS-RA was not inhibited by PPI-2458. The CNS toxicity profile of PPI-2458, determined by the incidence of seizures, is significantly improved over that of the parental compound TNP-470. In the rat model of peptidoglycan-polysaccharide-induced arthritis, PPI-2458 significantly attenuated paw swelling when therapeutically administered after the onset of chronic disease. We suggest that the mechanism of PPI-2458 action, highly selective and potent anti-proliferative activity on HFLS-RA and HUVEC in vitro, a significantly improved CNS toxicity profile, and marked attenuation of chronic disease in the rat peptidoglycan-polysaccharide arthritis model in vivo, positions this compound as a drug for the treatment of RA.