Alfredo C. Castro

Discovery of a potent and orally active hedgehog pathway antagonist (IPI-926).

Recent evidence suggests that blocking aberrant hedgehog pathway signaling may be a promising therapeutic strategy for the treatment of several types of cancer. Cyclopamine, a plant Veratrum alkaloid, is a natural product antagonist of the hedgehog pathway. In a previous report, a seven-membered D-ring semisynthetic analogue of cyclopamine, IPI-269609 (2), was shown to have greater acid stability and better aqueous solubility compared to cyclopamine. Further modifications of the A-ring system generated three series of analogues with improved potency and/or solubility. Lead compounds from each series were characterized in vitro and evaluated in vivo for biological activity and pharmacokinetic properties. These studies led to the discovery of IPI-926 (compound 28), a novel semisynthetic cyclopamine analogue with substantially improved pharmaceutical properties and potency and a favorable pharmacokinetic profile relative to cyclopamine and compound 2. As a result, complete tumor regression was observed in a Hh-dependent medulloblastoma allograft model after daily oral administration of 40 mg/kg of compound 28.

Semisynthetic cyclopamine analogues as potent and orally bioavailable hedgehog pathway antagonists.

Herein is reported the synthesis of a novel class of hedgehog antagonists derived from cyclopamine. The acid sensitive D-ring of cyclopamine was homologated utilizing a sequence of chemoselective cyclopropanation and stereoselective acid-catalyzed rearrangement. Further modification of the A/B-ring homoallylic alcohol to the conjugated ketone led to the discovery of new cyclopamine analogues with improved pharmaceutical properties and in vitro potency (EC 50) ranging from 10 to 1000 nM.

Recent patents for Hedgehog pathway inhibitors for the treatment of malignancy.

Background: There is increasing evidence suggesting that blocking aberrant Hedgehog (Hh) signaling can be a novel therapeutic avenue for the treatment of cancer. During the past decade, efforts from academic and industrial groups have led to the discovery of a variety of Hh pathway inhibitors. Objective: This review covers the patent literature related to Hh pathway inhibitors for the treatment of proliferative diseases, regardless of their modes of action. Methods: A comprehensive survey of the patent literature since 1999 is presented. Results/conclusion: Most reported Hh pathway inhibitors act on the key signaling transducer Smoothened (SMO). Screening of compound libraries using reporter and binding assays have identified a broad diversity of chemical structures that interact with SMO. These screening approaches, followed by conventional medicinal chemistry, have delivered important clinical drug candidates, such as GDC-0449 and XL-139. In addition, modification of the naturally occurring Veratrum alkaloid cyclopamine has resulted in various active analogues, including clinical drug candidate IPI-926. Although there are recent scientific literature reports of small molecules acting downstream of SMO, there is limited patent literature on this mode of Hh pathway inhibition.

New developments in the discovery of small molecule Hedgehog pathway antagonists

The Hedgehog (Hh) signaling pathway is crucial for normal embryonic development. Aberrant Hh signaling is implicated in numerous pathologic conditions including proliferative diseases such as cancer. During the past decade, academic and industrial research efforts have resulted in the discovery of a variety of Hh pathway antagonists. This review focuses on the most recent advances in this field with particular emphasis on the medicinal chemistry approaches used to discover these Hh antagonists. While most of the small molecule modulators of the Hh pathway were discovered through screening and subsequent medicinal chemistry, a number of them originated from rational design or natural products.

Structure-activity relationships of a peptide inhibitor of the human FcRn:human IgG interaction.

A family of five peptides was previously discovered by phage display techniques that binds to the human neonatal Fc receptor (FcRn) and inhibits the human IgG:human FcRn protein-protein interaction [Proc. Nat. Acad. Sci. U.S.A.2008, 105, 2337-2342]. The consensus peptide motif consists of the sequence GHFGGXY where X is preferably a hydrophobic amino acid, and also includes a disulfide bridge enclosing 11-amino acids in varying positions about the consensus sequence. We describe herein the structure-activity relationships of one of the five peptides in binding to FcRn using surface plasmon resonance and IgG:FcRn competition ELISA assays. Modifications of the peptide length, cyclization, and the incorporation of amino acid substitutions and dipeptide mimetics were studied. The most potent analogs exhibited a 50- to 100-fold improvement of in vitro activity over that of the phage-identified peptide sequence.

Discovery of a Selective Phosphoinositide-3-Kinase (PI3K)-γ Inhibitor (IPI-549) as an Immuno-Oncology Clinical Candidate

Optimization of isoquinolinone PI3K inhibitors led to the discovery of a potent inhibitor of PI3K-γ (26 or IPI-549) with >100-fold selectivity over other lipid and protein kinases. IPI-549 demonstrates favorable pharmacokinetic properties and robust inhibition of PI3K-γ mediated neutrophil migration in vivo and is currently in Phase 1 clinical evaluation in subjects with advanced solid tumors.

The pre-clinical absorption, distribution, metabolism and excretion properties of IPI-926, an orally bioavailable antagonist of the hedgehog signal transduction pathway

Abstract 1. IPI-926 is a novel semisynthetic cyclopamine derivative that is a potent and selective Smoothened inhibitor that blocks the hedgehog signal transduction pathway. 2. The in vivo clearance of IPI-926 is low in mouse and dog and moderate in monkey. The volume of distribution is high across species. Oral bioavailability ranges from moderate in monkey to high in mouse and dog. Predicted human clearance using simple allometry is low (24 L h−1), predicted volume of distribution is high (469 L) and predicted half-life is long (20 h). 3. IPI-926 is highly bound to plasma proteins and has minimal interaction with human α-1-acid glycoprotein. 4. In vitro metabolic stability ranges from stable to moderately stable. Twelve oxidative metabolites were detected in mouse, rat, dog, monkey and human liver microsome incubations and none were unique to human. 5. IPI-926 is not a potent reversible inhibitor of CYP1A2, 2C8, 2C9 or 3A4 (testosterone). IPI-926 is a moderate inhibitor of CYP2C19, 2D6 and 3A4 (midazolam) with KI values of 19, 16 and 4.5 µM, respectively. IPI-926 is both a substrate and inhibitor (IC50 = 1.9 µM) of P-glycoprotein. 6. In summary, IPI-926 has desirable pre-clinical absorption, distribution, metabolism and excretion properties.

Abstract 1891: Pharmacological target validation studies of fatty acid synthase in carcinoma using the potent, selective and orally bioavailable inhibitor IPI-9119.

Fatty acid synthase (FASN) is a key enzyme responsible for fatty acids synthesis de novo in mammals. Overexpression of FASN is common in many cancers including prostate, breast and colon cancer and elevated expression of FASN has been linked with poor prognosis and reduced disease-free survival. Experiments with RNAi and small molecule inhibitors suggest that FASN is a metabolic oncogene with an important role in tumor growth and survival and an appealing target for cancer therapy. However, studies utilizing small molecule FASN inhibitors like orlistat and C75 have been confounded by the lack of potency and selectivity, as well as the poor pharmacological properties of these inhibitors. Herein we report pharmacological target validation studies of FASN using a potent, selective and orally bioavailable FASN inhibitor IPI-9119. Building on previous experience with serine hydrolase inhibitors, a series of novel mechanism-based FASN inhibitors were designed based on a tetrazolone carboxamide scaffold. Like orlistat, these analogs are irreversible inhibitors that specifically target the FASN thioesterase domain. Tetrazolone carboxamide analogs were shown to potently inhibit cellular FASN using an occupancy assay and to completely block de novo palmitate synthesis in HCT-116 colon cancer cells using a 13 C-glucose incorporation assay. Lead optimization of the tetrazolone carboxamide series resulted in the identification of IPI-9119 as a tool for in vivo proof-of-concept studies. IPI-9119 is a potent FASN inhibitor in both biochemical (IC 50 ∼1nM) and cellular occupancy assays (IC 50 ∼10nM), and shows more than 400-fold selectivity against several additional serine hydrolases. Importantly, IPI-9119 is orally bioavailable and has pharmacokinetic (PK) properties suitable for in vivo pharmacology studies. IPI-9119 was tested for growth inhibition in cancer cell lines in vitro and tumor xenograft models in vivo. Unexpectedly, in contrast to the knock-down studies and to data reported for orlistat and C75, IPI-9119 failed to elicit anti-proliferative effects in multiple cancer cell lines in vitro. Similarly, PK/PD experiments demonstrated that a single oral dose of IPI-9119 at 200 mg/kg leads to complete and sustained blockade of FASN in HCT-116 tumor xenografts, but IPI-9119 failed to show any anti-tumor activity when dosed as a single agent at 200 mg/kg BID for 10 days. In summary, we identified IPI-9119 as a potent, selective and orally bioavailable FASN inhibitor. Preliminary target validation studies with IPI-9119 in cancer cell lines and an HCT-116 xenograft model suggest that FASN inhibition alone is not sufficient to affect cancer cell proliferation and tumor growth. Further studies exploring combination treatments with IPI-9119 are warranted. Citation Format: Erin Brophy, James Conley, Patrick O9Hearn, Mark Douglas, Culver Cheung, John Coco, Laura D9Anello, Andrew Wylie, Thomas Tibbitts, Gregg Keaney, Lawrence Chan, Adilah Bahadoor, Dan Snyder, Marta Nevalainen, Alfredo Castro, Vito Palombella, Massimo Loda, Stephane Peluso. Pharmacological target validation studies of fatty acid synthase in carcinoma using the potent, selective and orally bioavailable inhibitor IPI-9119. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1891. doi:10.1158/1538-7445.AM2013-1891

Abstract B029: The potent and selective phosphoinositide-3-kinase-gamma inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment

Introduction: The phosphoinositide-3-kinase (PI3K) lipid kinases are a family of kinase isoforms that transduce signals in response to various stimuli in different cell types. The PI3K-γ isoform is expressed in immune cells and has limited, if any, expression in epithelial cancer cells. Genetic deletion and kinase-dead knock-in studies highlight a key role for PI3K-γ in the development and function of myeloid-derived cells that constitute a key component of the immune suppressive tumor microenvironment (Joshi Mol Canc Res 2014; Schmid Canc Cell 2011). Targeting PI3K-γ in these tumor-associated myeloid cells could therefore inhibit the immune suppressive tumor microenvironment, enabling the immune system to attack tumor cells more effectively. To date, potent and selective PI3K-γ inhibitors with drug-like properties have not been available to test this hypothesis. We now report the structure, biochemical, cellular, and in vivo properties of a potent and selective, small molecule inhibitor of PI3K-γ, IPI-549, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-γ inhibition. Results: Discovery efforts identified a highly selective inhibitor of PI3K-γ, IPI-549, with pharmaceutical properties suitable for further development. Binding studies with IPI-549 revealed a KD value of 0.29 nM for PI3K-γ with >58-fold weaker binding affinity for the other Class I PI3K isoforms. Enzymatic assays utilizing physiological ATP concentrations (3 mM) confirmed the selectivity of IPI-549 for PI3K-γ (>200-fold) over other Class I PI3K isoforms. Cellular assays designed to assess individual Class I PI3K isoform activity demonstrated that IPI-549 is highly potent and specific for PI3K-γ (IC50 of 1.2 nM; >140-fold selectivity). Further selectivity screening revealed that IPI-549 is selective for PI3K-γ over other protein and lipid kinases, receptors, ion channels, and transporters. In vitro functional assays demonstrated that IPI-549 blocked bone marrow derived M2 murine macrophage polarization in response to IL-4 and MCSF1, but did not inhibit ConA-induced T-cell activation. These data indicate the potential for IPI-549 to block immune suppressive macrophage development but not T-cell activity. Pharmacokinetic studies in mice demonstrated IPI-549 to be orally bioavailable with a long plasma half-life enabling selective inhibition of the PI3K-γ isoform relative to the other Class I PI3K isoforms. In an in vivo PI3K-γ-dependent neutrophil migration murine model, IPI-549 blocked neutrophil migration in a dose dependent manner. To evaluate the effect of PI3K-γ inhibition on tumor growth in an immunocompetent animal, IPI-549 was tested in murine syngeneic solid tumor models. Mice treated with IPI-549 demonstrated significant tumor growth inhibition in multiple syngeneic models. Studies to elucidate the mechanism of tumor growth inhibition indicated that IPI-549 affects immune suppressive myeloid cell numbers and/or function, leading to an increase in cytotoxic T-cell activity. Studies in nude or CD8 T-cell depleted mice demonstrated the T-cell dependence of IPI-549 mediated tumor growth inhibition. Finally, in vivo studies with IPI-549 in combination with immune checkpoint inhibitors showed increased tumor growth inhibition compared to either monotherapy. Conclusions: IPI-549 is a potent and selective inhibitor of PI3K-γ with pharmaceutical properties that allow for the selective inhibition of PI3K-γ in vivo. Our findings provide evidence that targeted inhibition of PI3K-γ by IPI-549 can restore antitumor immune responses and inhibit solid tumor growth in preclinical models. IND-enabling studies with IPI-549 are ongoing to support its initial clinical exploration in the setting of solid tumors. Citation Format: Jeffery Kutok, Janid Ali, Erin Brophy, Alfredo Castro, Jonathan DiNitto, Catherine Evans, Kerrie Faia, Stanley Goldstein, Nicole Kosmider, Andre Lescarbeau, Tao Liu, Christian Martin, Karen McGovern, Somarajan Nair, Melissa Pink, Jennifer Proctor, Matthew Rausch, Sujata Sharma, John Soglia, Jeremy Tchaicha, Martin Tremblay, Vivian Villegas, Katherine Walsh, Kerry White, David Winkler, Vito Palombella. The potent and selective phosphoinositide-3-kinase-gamma inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B029.

Abstract A192: The potent and selective phosphoinositide-3-kinase-γ inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment

Introduction The phosphoinositide-3-kinase (PI3K) lipid kinases transduce signals in response to various stimuli in different cell types. PI3K-γ is expressed in immune cells and has limited expression in epithelial cancer cells. Genetic inactivation of PI3K-γ highlights its role in the development and function of myeloid-derived cells that constitute a key component of the suppressive tumor microenvironment (Schmid 2011). Targeting PI3K-γ in tumor-associated myeloid cells could potentially relieve tumor immune tolerance, enabling the immune system to attack tumor cells more effectively. To date, potent and selective PI3K-γ inhibitors with drug-like properties have not been available to test this hypothesis. We now report the structure, biochemical, cellular, and in vivo properties of a potent and selective, small molecule inhibitor of PI3K-γ, IPI-549, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-γ inhibition. Results Discovery efforts identified a highly selective inhibitor of PI3K-γ, IPI-549, with pharmaceutical properties suitable for further development. Binding studies with IPI-549 revealed a KD of 0.29 nM for PI3K-γ and enzymatic assays confirmed the selectivity of IPI-549 for PI3K-γ (>200-fold) over the other Class I PI3K isoforms. Comparison of IPI-549 to previously reported PI3K-γ inhibitors in this enzymatic confirmed its unique potency and selectivity for PI3K-γ. Cellular assays for individual Class I PI3K isoform activity demonstrated that IPI-549 is highly potent and specific for PI3K-γ (IC50 of 1.2 nM; >140-fold selectivity). In kinase screens, IPI-549 is selective for PI3K-γ over other kinases, receptors, ion channels, and transporters. In vitro assays demonstrated that IPI-549 blocked immune suppressive M2 murine macrophage polarization in response to IL-4 and MCSF1. Pharmacokinetic studies in mice demonstrated IPI-549 to be orally bioavailable with a long plasma half-life enabling selective inhibition of PI3K-γ relative to other Class I PI3K isoforms. To characterize IPI-5499s ability to inhibit PI3K-γ in vivo, mice with air pouches treated with IPI-549 showed dose responsive inhibition of PI3K-γ-dependent neutrophil migration. The effect of IPI-549 on tumor growth was tested in murine syngeneic solid tumor models. Mice treated with IPI-549 demonstrated significant tumor growth inhibition in multiple models. Studies to elucidate the mechanism of tumor growth inhibition indicated that IPI-549 affects suppressive myeloid cell numbers and/or function, leading to an increase in cytotoxic T-cell numbers and activity, as assessed by marker studies. Nude or CD8 T-cell depleted mice studies demonstrated a T-cell dependence of IPI-549-mediated tumor growth inhibition. Finally, in vivo studies with IPI-549 in combination with immune checkpoint inhibitors or following chemotherapy showed increased tumor growth inhibition compared to monotherapies. Conclusions IPI-549 is a potent and selective inhibitor of PI3K-γ with pharmaceutical properties that allow for the selective inhibition of PI3K-γ in vivo. Our findings provide evidence that targeted inhibition of PI3K-γ by IPI-549 can restore antitumor immune responses and inhibit solid tumor growth in preclinical models. Citation Format: Karen McGovern, Janid Ali, Erin Brophy, Alfredo Castro, Jonathan DiNitto, Catherine Evans, Kerrie Faia, Stanley Goldstein, Nicole Kosmider, Andre Lescarbeau, Tao Liu, Christian Martin, Somarajan Nair, Melissa Pink, Jennifer Proctor, Matthew Rausch, Sujata Sharma, John Soglia, Jeremy Tchaicha, Martin Tremblay, Vivian Villegas, Kerry White, David Winkler, Vito Palombella, Jeffery Kutok. The potent and selective phosphoinositide-3-kinase-γ inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A192.