Christine S. Pien

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-Hodgkins lymphoma in this study, and merits further investigation in these populations.

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.

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.

Proteasome inhibition: A novel mechanism to combat asthma

BACKGROUND Nuclear factor-kappaB (NF-kappaB) is a critical transcription factor required for the regulation of many genes involved in inflammatory responses to noxious stimuli. On activation, NF-kappaB induces the transcription of numerous proinflammatory cytokines, enzymes, and cellular adhesion molecules. Blockade of the proteasome with selective inhibitors attenuates the effects of NF-kappaB, leading to suppression of the inflammatory response. OBJECTIVE We sought to determine whether proteasome inhibitors would be active in a model of asthma. METHODS The mouse delayed-type hypersensitivity model was used to screen a panel of compounds for in vivo activity. The proteasome inhibitor, PS-519, was shown to be the most active in this model and was selected for further development. Allergen-induced pulmonary eosinophilia in Brown Norway rats was used subsequently to determine anti-inflammatory activity in an animal model. RESULTS Direct administration of PS-519 into the lungs significantly reduced leukocyte numbers, particularly the selective increase in eosinophils. Because steroids are the mainstay anti-inflammatory therapy in asthma, and data is available to suggest their possible interaction to suppress the activation of NF-kappaB, rats were also treated by inhalation with combinations of a steroid and the proteasome inhibitor. In both the delayed-type hypersensitivity and the animal eosinophil model, low doses of proteasome inhibitors were shown to be effective when given with low doses of steroids. CONCLUSION Taken together, the present data suggest that proteasome inhibition may represent a novel strategy for the treatment of inflammatory lung diseases such as asthma.

INHIBITION OF NF-KAPPA B ACTIVATION IN VITRO AND IN VIVO : ROLE OF 26S PROTEASOME

It is becoming increasingly apparent that NF-kappa B plays a critical role in regulating the inflammatory response. Data obtained from studies in our laboratories demonstrate that the proteasome plays an important role in the inflammatory cascade by regulating the activation of NF-kappa B. Indeed, the availability of selective and orally active proteasome inhibitors should prove useful in delineating the roles of the proteasome and NF-kappa B in other pathophysiological conditions such as cancer and heart disease.

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-κB 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-κB 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.

Abstract 3912: MM-310, a novel EphA2-targeted docetaxel nanoliposome

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Taxanes are widely used to treat solid tumors either in the curative or palliative setting, in first or later lines of therapy. Analysis of docetaxel dose-response relationship strongly suggests that a higher dose would lead to high response, however will also lead to higher toxicity. This is likely related to the lack of organ and cellular specificity of docetaxel leading to high exposures in normal tissues and the relatively short circulation half-life which indirectly requires higher doses. With the goal of addressing the pharmacokinetic limitations of free docetaxel and the lack of cellular specificity, we developed a novel docetaxel-based nanoliposome (MM-310), targeted against Ephrin receptor A2 (EphA2) which is overexpressed in a wide range of tumors. MM-310 provides sustained release of docetaxel following accumulation in solid tumors. Preclinical models have demonstrated that MM-310 leverages tumor-specific accumulation through the enhanced permeability and retention effect, and cellular specificity through active targeting of EphA2 with specific scFv antibody fragments conjugated to the surface of the liposomes. Pharmacokinetic and biodistribution studies were performed in mice and rats to compare MM-310 to free docetaxel. Chronic tolerability studies were performed in rodent and non-rodent models with focus on overall animal health, as well as hematologic toxicities. Several cell-derived models of breast, lung and prostate xenografts were used to evaluate the differences betweenf MM-310 and free docetaxel. MM-310 had a significantly longer half-life than free docetaxel with prolonged exposure at the tumor site. In chronic tolerability studies, MM-310 was found to be 6-7 times better tolerated than free docetaxel with a maximum tolerated dose of at least 120 mpk, compared to 20 mpk for free docetaxel and no detectable hematological toxicity. At equitoxic dosing, MM-310 50 mpk showed greater activity than docetaxel 10 mpk in several breast, lung and prostate xenograft models. In conclusion, we developed a novel EphA2 targeted docetaxel nanoliposome with prolonged circulation time and slow and sustained drug release kinetics, to enable organ and cellular targeting. MM-310 was able to overcome hematologic toxicities observed upon treatment with free docetaxel in rodent and non-rodent models. MM-310 was also able to induce tumor regression or control tumor growth in several cell derived xenograft models, and was found to be more active than free docetaxel in most models. Citation Format: Dmitri B. Kirpotin, Suresh Tipparaju, Zhaohua Richard Huang, Walid S. Kamoun, Christine Pien, Tad Kornaga, Shinji Oyama, Ken Olivier, James D. Marks, Alexander Koshkaryev, Sarah S. Schihl, Gerald Fetterly, Birgit Schoeberl, Charles Noble, Mark Hayes, Daryl C. Drummond. MM-310, a novel EphA2-targeted docetaxel nanoliposome. [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 3912.

Abstract 871: Nanoliposomal targeting of ephrin receptor A2 (EphA2): Preclinical in vitro and in vivo rationale

Ephrins receptors are cell to cell adhesion molecules that mediate signaling and are implicated in neuronal repulsion, cell migration and angiogenesis. Ephrin receptor A2 (EphA2) is part of the ephrin family and was shown to be overexpressed in several solid tumors including ovarian, gastric and lung cancer and is associated with poor prognosis. MM-310 is a novel EphA2 targeted docetaxel nanoliposome. Similar to other nanoparticles, MM-310 leverages organ specificity through enhanced permeability and retention, but can also leverage cellular specificity through its EphA2 targeting arm. Binding of targeted liposomes to cells in vitro was assessed using flow cytometry in a large panel cell lines. 3D spheroids were used to assess targeting as well as liposome penetration. In order to test the effect of targeting on liposome microdistribution in vivo, primary and metastatic tumor bearing animals were injected with a mixture of EphA2-targeted liposome (EphA2-Ls) and non-targeted liposome (NT-Ls) labeled with two different lipophilic fluorescent dyes. Tissues were assessed using fluorescent microscopy. In order to evaluate the contribution of EphA2-targeting to efficacy, four gastric xenograft models were treated with either MM-310 or a non-targeted version of the drug NT-310, and compared to free docetaxel. In cell suspension models, we observed a high level of specificity for EphA2-Ls, with more than one hundred-fold increase in liposome cell association. 3D spheroid assays showed that EphA2-Ls binds and penetrates EphA2+ spheroids, while non-targeted liposomes show minimal penetration. Tissue microdistribution analysis in triple negative breast and esophageal tumor models following injection of the EphA2-Ls/NT-Ls mixtures showed a target mediated shift in the microdistribution of liposomes. EphA2-Ls penetrated deeper within the lesions while the NT-Ls deposited at high levels in areas close to the microvasculature. The target mediated shift in microdistribution was also observed in lung metastasis model, with a pattern of distribution that potentially matches disseminated tumor cells. In the same animals, targeting did not affect microdistribution in normal organs such as liver, spleen and skin. Four models of gastric and esophageal cancers were used to test the potential link between cell targeting and tumor growth control. While NT-310 and MM-310 were both able to control tumor growth leading to regression in most models, in three out of four models there was a statistically significant difference between MM-310 and NT-310 at 25 mpk. Biomarker analysis is underway to evaluate the key parameters necessary to mediate targeting. In conclusion, the data suggests clear evidence of targeting in 2D cell suspension, 3D spheroids, and in primary as well as metastatic tumor models in vivo. In vivo efficacy data showed evidence of the contribution of EphA2 targeting to tumor growth control and regression in several gastric cancer models. Citation Format: Walid S. Kamoun, Lia Luus, Christine Pien, Tad Kornaga, Shinji Oyama, Zhaohua Richard Huang, Suresh Tipparaju, Dmitri B. Kirpotin, James D. Marks, Alexander Koshkaryev, Melissa Geddie, Lihui Xu, Alexey Lugovosky, Daryl C. Drummond. Nanoliposomal targeting of ephrin receptor A2 (EphA2): Preclinical in vitro and in vivo rationale. [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 871.

Abstract 3096: Mechanisms of synergy of carboplatin and an EphA2-targeted docetaxel antibody-directed nanotherapeutic

Platinum-taxane combinations are widely used to treat solid tumors either in first or later lines of therapy. While effective in many settings, platinum-taxane combinatorial regimens are limited by toxicities. We have recently developed an antibody directed nanotherapeutic (MM-310) encapsulating a docetaxel prodrug, targeted to Ephrin receptor A2 (EphA2). Preclinical investigation of MM-310 revealed that the liposomal formulation leads to prolonged docetaxel exposure of the tumor with decreased exposure of normal tissues leading to a shift in toxicity profile and potentially enabling more safe and effective combinations with platinum-based chemotherapeutics. In this study, we evaluated the activity of MM-310 in combination with carboplatin in several xenograft tumor models and compared it to the activity of free docetaxel in combination with carboplatin at equitoxic dosing. Tolerability of MM-310 in combination with carboplatin in mice was evaluated, including assessing hepatotoxicity. Biodistribution, microdistribution, in vivo tumor growth, and mouse survival studies were performed in lung and ovarian cell line-derived (CDX) and patient-derived xenograft (PDX) models. MM-310 in combination with carboplatin was found to be well tolerated, enabling dosing of both drugs at high doses with maximum tolerability when the drugs were dosed three days apart. Carboplatin increased nanotherapeutic delivery to the tumor in a CDX model of triple negative breast cancer and in a PDX model of ovarian cancer. In vivo studies in lung and ovarian cancer xenograft models showed significant synergy between MM-310 and carboplatin when compared to the monotherapies, as well as when compared to free docetaxel with carboplatin, leading to a significant increase in tumor growth delay and survival (docetaxel/ carboplatin vs. MM-310/carboplatin, 0 vs 50% complete tumor regression, 24 vs 80 days median time to regrowth). Additionally, in the same studies, MM-310 and carboplatin was better tolerated than free docetaxel and carboplatin. In conclusion, we found that MM-310 in combination with carboplatin was significantly better tolerated and more effective than free docetaxel in combination with carboplatin. Mechanistically, the synergistic anti-tumor activity of MM-310 with carboplatin may be partially due to a carboplatin mediated enhancement of nanotherapeutic delivery. The increased preclinical activity of the MM-310/carboplatin combination, together with the high tolerability following scheduling optimization tested in mice, makes this combination a promising regimen that warrants evaluation in clinical trials. Citation Format: Walid S. Kamoun, Andrew J. Sawyer, Christine Pien, Alexander Koshkaryev, Lia Luus, Samantha Merrigan, Gang Sun, Sergey Kozin, Zhaohua Richard Huang, Suresh K. Tipparaju, Dmitri B. Kirpotin, Hannah Xu, Vasileios Askoxylakis, Patrick C. Reynolds, Daryl C. Drummond. Mechanisms of synergy of carboplatin and an EphA2-targeted docetaxel antibody-directed nanotherapeutic [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 3096. doi:10.1158/1538-7445.AM2017-3096