Targeting EphA2 in Bladder Cancer Using a Novel Antibody-Directed Nanotherapeutic.

Ephrin receptor A2 (EphA2) is a member of the Ephrin/Eph receptor cell-to-cell signaling family of molecules, and it plays a key role in cell proliferation, differentiation, and migration. EphA2 is overexpressed in a broad range of cancers, and its expression is in many cases associated with poor prognosis. We recently developed a novel EphA2-targeting antibody-directed nanotherapeutic encapsulating a labile prodrug of docetaxel (EphA2-ILs-DTXp) for the treatment of EphA2-expressing malignancies. Here, we characterized the expression of EphA2 in bladder cancer using immunohistochemistry in 177 human bladder cancer samples and determined the preclinical efficacy of EphA2-ILs-DTXp in four EphA2-positive patient-derived xenograft (PDX) models of the disease, either as a monotherapy, or in combination with gemcitabine. EphA2 expression was detected in 80-100% of bladder cancer samples and correlated with shorter patient survival. EphA2 was found to be expressed in tumor cells and/or tumor-associated blood vessels in both primary and metastatic lesions with a concordance rate of approximately 90%. The EphA2-targeted antibody-directed nanotherapeutic EphA2-ILs-DTXp controlled tumor growth, mediated greater regression, and was more active than free docetaxel at equitoxic dosing in all four EphA2-positive bladder cancer PDX models. Combination of EphA2-ILs-DTXp and gemcitabine in one PDX model led to improved tumor growth control compared to monotherapies or the combination of free docetaxel and gemcitabine. These data demonstrating the prevalence of EphA2 in bladder cancers and efficacy of EphA2-ILs-DTXp in PDX models support the clinical exploration of EphA2 targeting in bladder cancer.

Formulation optimization of an ephrin A2 targeted immunoliposome encapsulating reversibly modified taxane prodrugs.

Ephrin A2 targeted immunoliposomes incorporating pH-sensitive taxane prodrugs were developed for sustained delivery of active drug to solid tumors. Here we describe the systematic formulation development and characterization of these immunoliposomes. We synthesized both paclitaxel and docetaxel prodrugs to formulate as ephrin A2-targeted liposomes stabilized in the aqueous core with sucroseoctasulfate (SOS). The optimized lipid formulation was comprised of egg-sphingomyelin, cholesterol, and polyethylene glycol distearoyl glycerol (PEG-DSG). The formulations examined had a high efficiency of prodrug encapsulation (as high as 114 mol% taxane per mole phospholipid) and subsequent stability (>3 years at 2-8 °C). The taxane prodrug was stabilized with extraliposomal citric acid and subsequently loaded into liposomes containing a gradient of SOS, resulting in highly stable SOS-drug complexes being formed inside the liposome. The internal prodrug and SOS concentrations were optimized for their impact on in vivo drug release and drug degradation. Cryo-electron microscope images revealed dense prodrug-SOS complex in the aqueous core of the immunoliposomes. Ephrin A2-targeted taxane liposomes exhibited sub-nanomolar (0.69 nM) apparent equilibrium dissociation constant toward the extracellular domain of the ephrin A2 receptor, long circulation half-life (8-12 h) in mouse plasma, a release rate dependent on intraliposomal drug concentration and stable long-term storage. At an equitoxic dose of 50 mg taxane/kg, ephrin A2-targeted liposomal prodrug showed greater antitumor activity than 10 mg/kg of docetaxel in A549 non-small cell lung, as well as MDA-MB-436 and SUM149 triple negative breast cancer xenograft models. The lead molecule entered a Phase I clinical trial in patients with solid tumors (NCT03076372).

Antitumour activity and tolerability of an EphA2-targeted nanotherapeutic in multiple mouse models.

Antibody-mediated tumour targeting and nanoparticle-mediated encapsulation can reduce the toxicity of antitumour drugs and improve their efficacy. Here, we describe the performance of a nanotherapeutic encapsulating a hydrolytically sensitive docetaxel prodrug and conjugated to an antibody specific for EphA2-a receptor overexpressed in many tumours. Administration of the nanotherapeutic in mice led to slow and sustained release of the prodrug, reduced exposure of active docetaxel in the circulation (compared with administration of the free drug) and maintenance of optimal exposure of the drug in tumour tissue. We also show that administration of the nanotherapeutic in rats and dogs resulted in minimal haematological toxicity, as well as the absence of neutropenia and improved overall tolerability in multiple rodent models. Targeting of the nanotherapeutic to EphA2 improved tumour penetration and resulted in markedly enhanced antitumour activity (compared with administration of free docetaxel and non-targeted nanotherapeutic controls) in multiple tumour-xenografted mice. This nanomedicine could become a potent and safe therapeutic alternative for cancer patients undergoing chemotherapy.

The selective Trk inhibitor AZ623 inhibits brain-derived neurotrophic factor-mediated neuroblastoma cell proliferation and signaling and is synergistic with topotecan.

BACKGROUND: TrkB expression is associated with poor prognosis for patients with neuroblastoma. AZ623 is a novel potent and selective inhibitor of the Trk family of tyrosine kinases. The authors hypothesized that AZ623 would inhibit TrkB-mediated signaling in neuroblastoma tumor cells and would be synergistic when combined with chemotherapy. METHODS: Neuroblastoma cell lines were screened for TrkB receptor mRNA expression and for their proliferation rates in response to brain-derived neurotrophic factor (BDNF). The effects of AZ623 on Trk receptor phosphorylation, signaling, and cell growth were evaluated in BDNF-treated neuroblastoma cells. Mice with human neuroblastoma xenograft tumors were treated with AZ623 alone and in combination with topotecan, and tumor growth rates were determined during and after treatment. RESULTS: Neuroblastoma cell lines expressed various levels of the TrkB receptor and demonstrated increased proliferation in response to BDNF. BDNF treatment stimulated TrkB phosphorylation and downstream signaling that could be inhibited by AZ623. Neuroblastoma cells demonstrated in vitro sensitivity to AZ623, with concentration that inhibits 50% (IC50) values between 0.8 to 7 µM. AZ623 treatment was found to inhibit BDNF-mediated neuroblastoma cell proliferation. Mice with human neuroblastoma xenograft tumors demonstrated tumor growth inhibition when treated with AZ623 and with AZ623 combined with topotecan. Limited tumor regrowth was noted in mice with tumors treated with AZ623 combined with topotecan after treatment discontinuation. CONCLUSIONS: AZ623 is a novel selective Trk inhibitor that inhibits BDNF-mediated signaling and neuroblastoma cell proliferation. AZ623 treatment inhibits the growth of human neuroblastoma xenograft tumors, and treatment with AZ623 combined with topotecan results in the prolonged inhibition of tumor regrowth. On the basis of these results, further preclinical development is warranted.

NPI-0052 enhances tumoricidal response to conventional cancer therapy in a colon cancer model.

PURPOSE: In the current study, we examine the effects of a novel proteasome inhibitor, NPI-0052 (salinosporamide A), on proteasome function and nuclear factor-kappaB activation and evaluate its ability to enhance treatment response in colon cancer xenografts when administered orally. EXPERIMENTAL DESIGN: The effects of treatment on nuclear factor-kappaB activation, cell cycle regulation, and apoptosis were determined. The pharmacodynamic effect of NPI-0052 on 20S proteasome function was assayed in vivo following oral and i.v. drug administration and compared with treatment with bortezomib. The effect of combined treatment with chemotherapy was determined in a colon cancer xenograft model. RESULTS: We found that NPI-0052 is a potent, well-tolerated proteasome inhibitor that has pharmacodynamic properties distinct from bortezomib in that it achieves significantly higher and more sustained levels of proteasome inhibition. When combined with chemotherapy, NPI-0052 increases apoptosis and shifts cells toward G2 cell cycle arrest. When added to chemotherapy in vivo [using combinations of 5-fluorouracil (5-FU), CPT-11, Avastin (bevacizumab), leucovorin, and oxaliplatin], NPI-0052 significantly improved the tumoricidal response and resulted in a 1.8-fold increased response to CPT-11, 5-FU, and leucovorin triple-drug combination (P=0.0002, t test), a 1.5-fold increased response to the oxaliplatin, 5-FU, and leucovorin triple-drug combination (P=0.013, t test), and a 2.3-fold greater response to the CPT-11, 5-FU, leucovorin, and Avastin regimen (P=0.00057). CONCLUSIONS: The high level of proteasome inhibition achieved by NPI-0052 is well tolerated and significantly improves the tumoricidal response to multidrug treatment in a colon cancer xenograft model. Further evaluation of this novel proteasome inhibitor in clinical trials is indicated.

Development of 17-allylamino-17-demethoxygeldanamycin hydroquinone hydrochloride (IPI-504), an anti-cancer agent directed against Hsp90.

Heat shock protein 90 (Hsp90) is an emerging therapeutic target of interest for the treatment of cancer. Its role in protein homeostasis and the selective chaperoning of key signaling proteins in cancer survival and proliferation pathways has made it an attractive target of small molecule therapeutic intervention. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), the most studied agent directed against Hsp90, suffers from poor physical-chemical properties that limit its clinical potential. Therefore, there exists a need for novel, patient-friendly Hsp90-directed agents for clinical investigation. IPI-504, the highly soluble hydroquinone hydrochloride derivative of 17-AAG, was synthesized as an Hsp90 inhibitor with favorable pharmaceutical properties. Its biochemical and biological activity was profiled in an Hsp90-binding assay, as well as in cancer-cell assays. Furthermore, the metabolic profile of IPI-504 was compared with that of 17-AAG, a geldanamycin analog currently in clinical trials. The anti-tumor activity of IPI-504 was tested as both a single agent as well as in combination with bortezomib in myeloma cell lines and in vivo xenograft models, and the retention of IPI-504 in tumor tissue was determined. In conclusion, IPI-504, a potent inhibitor of Hsp90, is efficacious in cellular and animal models of myeloma. It is synergistically efficacious with the proteasome inhibitor bortezomib and is preferentially retained in tumor tissues relative to plasma. Importantly, it was observed that IPI-504 interconverts with the known agent 17-AAG in vitro and in vivo via an oxidation-reduction equilibrium, and we demonstrate that IPI-504 is the slightly more potent inhibitor of Hsp90.

Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer.

PURPOSE: To determine the dose-limiting toxicity and maximum-tolerated dose of the proteasome inhibitor bortezomib administered intravenously weekly for 4 every 5 weeks; to determine the bortezomib pharmacokinetics and pharmacodynamics using plasma levels and an assay for 20S proteasome inhibition (PI) in whole blood; to correlate toxicity with bortezomib dose and degree of 20S PI; and to conduct a preliminary determination of the antitumor activity of bortezomib in patients with androgen independent prostate cancer (AIPCa). PATIENTS AND METHODS: Fifty-three patients (48 with AIPCa) received 128 cycles of bortezomib in doses ranging from 0.13 to 2.0 mg/m(2)/dose, utilizing a careful escalation scheme with a continuous reassessment method. Pharmacokinetic and pharmacodynamic studies were performed in 24 patients (at 1.45 to 2.0 mg/m(2)). RESULTS: A dose-related 20S PI was seen, with dose-limiting toxicity at 2.0 mg/m(2) (diarrhea, hypotension) occurring at an average 1-hour post-dose of >/= 75% 20S PI. Other side effects were fatigue, hypertension, constipation, nausea, and vomiting. No relationship was seen between body-surface area and bortezomib clearance over the narrow dose range tested. There was evidence of biologic activity (decline in serum prostate-specific antigen and interleukin-6 levels) at >/= 50% 20S PI. Two patients with AIPCa had prostate-specific antigen response and two patients had partial response in lymph nodes. CONCLUSION: The maximum-tolerated dose and recommended phase II dose of bortezomib in this schedule is 1.6 mg/m(2). Biologic activity (inhibition of nuclear factor-kappa B-related markers) and antitumor activity is seen in AIPCa at tolerated doses of bortezomib. This agent should be further explored with chemotherapy agents in advanced prostate cancer.

Novel IKK inhibitors: beta-carbolines.

Inhibitors of IkappaB kinase (IKK) have long been sought as specific regulators of NF-kappaB. A screening effort of the endogenous IKK complex allowed us to identify 5-bromo-6-methoxy-beta-carboline as a nonspecific IKK inhibitor. Optimization of this beta-carboline natural product derivative resulted in a novel class of selective IKK inhibitors with IC(50)s in the nanomolar range. In addition, we show that one of these beta-carboline analogues inhibits the phosphorylation of IkappaBalpha and subsequent activation of NF-kappaB in whole cells, as well as blocking TNF-alpha release in LPS-challenged mice.

Proteasome inhibition ablates activation of NF-kappa B in myocardial reperfusion and reduces reperfusion injury.

Both acute coronary occlusion and reperfusion of an infarct-related artery lead to significant myocardial cell death. Recent evidence has been presented that activation of the transcription factor nuclear factor-kappaB (NF-kappaB) plays a critical role in reperfusion injury. NF-kappaB is usually bound to its inhibitor, IkappaB, and classic activation of NF-kappaB occurs when the 20S proteasome degrades IkappaB that has been phosphorylated and ubiquitinated. In this study, activation of NF-kappaB was inhibited by systemic administration of a 20S proteasome inhibitor (PS-519) in a porcine model of myocardial reperfusion injury. The experimental protocol induced myocardial ischemia in the distribution of the left anterior descending coronary artery for 1 h with subsequent reperfusion for 3 h. A single systemic treatment with PS-519 reduced 20S proteasome activity; blocked activation of NF-kappaB induced by reperfusion; reduced creatine kinase, creatine kinase-muscle-brain fraction, and troponin I release from the myocardium; preserved regional myocardial function measured by segmental shortening; significantly reduced the size of myocardial infarction; and exhibited no acute toxicity. These data show that myocardial reperfusion injury can be inhibited by using proteasome inhibitors, which likely function through the inhibition of NF-kappaB activation.

Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies.

PURPOSE: To determine the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), and pharmacodynamics (PD) of the proteasome inhibitor bortezomib (previously known as PS-341) in patients with refractory hematologic malignancies. PATIENTS AND METHODS: Patients received PS-341 twice weekly for 4 weeks at either 0.40, 1.04, 1.20, or 1.38 mg/m(2), followed by a 2-week rest. The PD of PS-341 was evaluated by measurement of whole blood 20S proteasome activity. RESULTS: Twenty-seven patients received 293 doses of PS-341, including 24 complete cycles. DLTs at doses above the 1.04-mg/m(2) MTD attributed to PS-341 included thrombocytopenia, hyponatremia, hypokalemia, fatigue, and malaise. In three of 10 patients receiving additional therapy, serious reversible adverse events appeared during cycle 2, including one episode of postural hypotension, one systemic hypersensitivity reaction, and grade 4 transaminitis in a patient with hepatitis C and a substantial acetaminophen ingestion. PD studies revealed PS-341 induced 20S proteasome inhibition in a time-dependent manner, and this inhibition was also related to both the dose in milligrams per meter squared, and the absolute dose of PS-341. Among nine fully assessable patients with heavily pretreated plasma cell dyscrasias completing one cycle of therapy, there was one complete response and a reduction in paraprotein levels and/or marrow plasmacytosis in eight others. In addition, one patient with mantle cell lymphoma and another with follicular lymphoma had shrinkage of nodal disease. CONCLUSION: PS-341 was well tolerated at 1.04 mg/m(2) on this dose-intensive schedule, although patients need to be monitored for electrolyte abnormalities and late toxicities. Additional studies are indicated to determine whether incorporation of dose/body surface area yields a superior PD model to dosing without normalization. PS-341 showed activity against refractory multiple myeloma and possibly non-Hodgkin's lymphoma in this study, and merits further investigation in these populations.

Proteasome inhibitor PS-341 inhibits human myeloma cell growth in vivo and prolongs survival in a murine model.

The proteasome is a ubiquitous and essential intracellular enzyme that degrades many proteins regulating cell cycle, apoptosis, transcription, cell adhesion,angiogenesis, and antigen presentation. We have shown recently that the proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human myeloma cells in vitro. In this study, we examined the efficacy, toxicity, and in vivo mechanism of action of PS-341 using a human plasmacytoma xenograft mouse model. One hundred immunodeficient (beige-nude-xid) mice were used in two independent experiments. The mice were injected s.c. with 3 x 10(7) RPMI-8226 myeloma cells. When tumors became measurable (9.2 days; range, 6-13 days after tumor injection), mice were assigned to treatment groups receiving PS-341 0.05 mg/kg (n = 13), 0.1 mg/kg (n = 15), 0.5 mg/kg (n = 14), or 1.0 mg/kg (n = 14) twice weekly via tail vein, or to control groups (n = 13) receiving the vehicle only. Significant inhibition of tumor growth, even with some complete tumor regression, was observed in PS-341-treated mice. The median overall survival was also significantly prolonged compared with controls (30 and 34 days for high dose-treated mice versus 14 days for controls; P < 0.0001). PS-341 was well tolerated up to 0.5 mg/kg, but some mice treated at 1.0 mg/kg became moribund and lost weight. Analysis of tumors harvested from treated animals showed that PS-341 induced apoptosis and decreased angiogenesis in vivo. These studies therefore demonstrate that PS-341 has significant in vivo antimyeloma activity at doses that are well tolerated in a murine model, confirming our in vitro data and further supporting the early clinical promise of PS-341 to overcome drug resistance and improve patient outcome.

A phase I trial of the novel proteasome inhibitor PS341 in advanced solid tumor malignancies.

PURPOSE: The purpose of this study was to evaluate the toxicity and pharmacodynamic behavior of the novel proteasome inhibitor PS341 administered as a twice weekly i.v. bolus for 2 weeks, followed by a 1-week recovery period in patients with advanced solid tumor malignancies. EXPERIMENTAL DESIGN: In this Phase I trial, 43 patients were treated with PS341 in doses ranging from 0.13 to 1.56 mg/m2/dose. A standard Phase I design was used. Pharmacodynamic studies were performed to access 20S proteasome activity. RESULTS: Forty-three patients were treated with 89 cycles of PS341. Patients were heavily pretreated. Dose-limiting toxicities on this schedule were diarrhea and sensory neurotoxicity. Other side effects seen were fatigue, fever, anorexia, nausea, vomiting, rash, pruritus, and headache. There was no dose-limiting hematological toxicity. A dose-related inhibition of 20S proteasome activity with increasing dose of PS341 was seen. There was one major response in a patient with refractory non-small cell lung carcinoma. CONCLUSIONS: Given the results of this trial, it is safe and reasonable to recommend treatment with PS341 on the schedule used in this trial at 1.56 mg/m2/dose in Phase II trials. Particular care should be taken with patients with preexisting neuropathy. Further testing in Phase II trials is warranted.

Proteasome inhibition reduces superantigen-mediated T cell activation and the severity of psoriasis in a SCID-hu model.

There is increasing evidence that bacterial superantigens contribute to inflammation and T cell responses in psoriasis. Psoriatic inflammation entails a complex series of inductive and effector processes that require the regulated expression of various proinflammatory genes, many of which require NF-kappa B for maximal trans-activation. PS-519 is a potent and selective proteasome inhibitor based upon the naturally occurring compound lactacystin, which inhibits NF-kappa B activation by blocking the degradation of its inhibitory protein I kappa B. We report that proteasome inhibition by PS-519 reduces superantigen-mediated T cell-activation in vitro and in vivo. Proliferation was inhibited along with the expression of very early (CD69), early (CD25), and late T cell (HLA-DR) activation molecules. Moreover, expression of E-selectin ligands relevant to dermal T cell homing was reduced, as was E-selectin binding in vitro. Finally, PS-519 proved to be therapeutically effective in a SCID-hu xenogeneic psoriasis transplantation model. We conclude that inhibition of the proteasome, e.g., by PS-519, is a promising means to treat T cell-mediated disorders such as psoriasis.