LY2157299 Monohydrate Research Articles

Bone Marrow Stromal Cells Induce ALDH+ Stem Cell Phenotype and Chemotherapy Resistance in AML Cells through TGF-β1 Mediated Signaling: Reversal By TGFβ Inhibitor Galunisertib

Bone marrow (BM) microenvironment in acute myeloid leukemia (AML) consists of various cell types including mesenchymal stromal cells (MSCs), osteoprogenitor cells, osteoblasts, osteocytes, endothelial, immune and perivascular reticular cells that support AML cell growth and protect them from chemotherapy. We have recently reported that AML cells alter the BM microenvironment by inducing osteogenic differentiation in MSCs to gain growth advantage in the bone marrow (Battula et al, JCI Insight, 2017). However the stroma-induced functional alterations in AML cells are not well understood. Stromal cells induce chemo-resistance in AML cells and as chemo-resistance and high tumorigenic potential are the characteristics of leukemia stem cells, we hypothesize that stromal cells can induce stem cell characteristics in AML cells. To test our hypothesis we co-cultured AML cell lines including OCI-AML3 and HL60 cells with BM-derived MSCs for 3 or 5 days and determined aldehyde dehydrogenase (ALDH) enzyme activity (a well-established marker of hematopoietic and leukemia stem cells) and gene expression by RNA sequencing. We found that the percentage of ALDH+ cells in OCI-AML3 increased from 7% to 89.1% in 3 days and from 23% to 92.3% in 5 days of co-culture with MSCs. Similarly, ALDHwas increased in HL60 cells from 7% to 68% in 3 days and from 20.7% to 67.9% in 5 days of co-culture with MSCs suggesting that stromal cells can induce certain stem cell features in AML cells.Next, to better determine the mechanism, OCI-AML3 cells were co-cultured with BM-MSCs (isolated from 3 different donors) for 5 days, the OCI-AML3 cells were FACS sorted and gene expression was analyzed by RNA sequencing. Using R statistics we identified differentially expressed genes in OCI-AML3 cells and found that gene sets associated with stem cell function including ALDH2, Wnt5b, Wnt5a were up-regulated whereas inflammatory and innate immune response genes were down-regulated in OCI-AML3 cells co-cultured with MSCs compared to cells cultured alone. These genes were validated by qRT-PCR assay. Ingenuity pathway analysis® (IPA) revealed a TGFβ1 activated gene signature in OCI-AML3 cells co-cultured with MSCs compared to cells culture alone. To investigate the role of TGFβ1 in the stroma-induced stem cell phenotype in AML cells, OCI- AML3 and HL60 cells were treated with recombinant TGFβ1 (5ng/ml) for 3 or 5 days. Indeed, treatment with TGFβ1 induced ALDH positive cells from 27.7% to 89.5% in 3 days and from 3.4% to 92.5% in 5 days (OCI-AML3). Similarly, in HL60 cells treated with TGFβ1, ALDH+ cells increased from 9.8% to 25.1 in 3 days suggesting that TGFβ1 induces a stem cell phenotype in AML cells. Next, we measured levels of TGFβ1 in cell culture supernatants from MSCs by ELISA and found that MSCs secrete TGFβ1 up to 1ng/ml into the surrounding medium. In addition, to study the role of TGFβ1 in stroma-induced stem cell activity in AML cells, we treated AML cells with or without TGFβ1 type-1 receptor kinase inhibitor Galunisertib (LY2157299 monohydrate from Eli Lilly®) in the presence or absence of MSCs. Strikingly, treatment of galunisertib at 1µM completely inhibited MSCs-induced ALDH activity in OCI-AML3 and HL60 cells in 5 day co-culture setting. To determine if galunisertib sensitizes AML cells to chemotherapy in the presence of MSCs, we treated OCI-AML3 with galunisertib (1µM) or cytarabine (1µM) or in combination for 72hrs in the presence or absence of MSCs. As expected presence of MSCs inhibited cytarabine induced cell killing (positive for annexin-V and DAPI) from 47.1% to 28.3% cells. In turn, the combination of cytarabine and galunisertib negated this affect and induced cell killing in OCI-AML3 cells from 28% to 54% even in the presence of MSCs suggesting complete loss of MSC-mediated chemo-protection in OCI-AML3 cells upon inhibition of TGF-β signaling. In-vivo testing of galunisertib in combination with cytarabine and doxorubicin in AML PDX models in NSG mice is currently ongoing.Collectively, data indicate that stromal cells induce chemo-resistance in AML cell by inducing stem cells phenotype through TGF-beta mediated signaling (Fig 1). TGF-β inhibition by galunisertib, an agent in early clinical trials in solid tumors inhibits stem cell phenotype and BM microenvironment-induced chemo-resistance and facilitates cell killing of AML cells. [Display omitted] DisclosuresBattula:United Therapeutics Corporation: Research Funding.

Abstract 1268: The impact of P-glycoprotein, breast cancer resistance protein and CYP3A on pharmacokinetics and metabolism of galunisertib

Abstract Transforming growth factor-beta (TGF-β) signaling plays a pivotal role in tumorigenesis and contributes to the hallmarks of cancer, including tumor proliferation, invasion and metastasis, inflammation, angiogenesis, and escape of immune surveillance. Galunisertib (LY2157299 monohydrate) is an oral small-molecule inhibitor of the TGF-β receptor I kinase that specifically down-regulates the phosphorylation of SMAD2, abrogating activation of this canonical pathway. Galunisertib showed promising antitumor activity in tumor-bearing animal models for breast, colon, and lung cancers, and for hepatocellular carcinoma. Potential drug transport mediated by ATP-binding cassette (ABC) transporters and metabolism by CYP3A are of clinical and regulatory concern, as these could modulate the systemic exposure and/or organ distribution of substrate drugs, and thus affect their therapeutic efficacy and toxicity. We here aimed to investigate the roles of two ABC transporters (P-glycoprotein/ABCB1 and Breast Cancer Resistance Protein/ABCG2) and CYP3A enzyme in the pharmacokinetics and tissue distribution of galunisertib. Transepithelial drug transport was tested using polarized monolayers of Madin-Darby Canine Kidney (MDCK-II) parental cells and its subclones overexpressing human (h) ABCB1, hABCG2, or mouse (m) Abcg2 cDNA. The results suggest that galunisertib is an excellent transport substrate of hABCB1 and endogenous canine ABCB1. mAbcg2 could modestly transport galunisertib, but hABCG2 could not. Based on the in vitro transport experiments, we performed an in vivo pharmacokinetic study in wild-type, ABC transporter knockout (Abcb1a/1b;Abcg2-/-), and Cyp3a knockout (Cyp3a-/-) mice. Following oral administration (20 mg/kg galunisertib), plasma exposure (AUC) in mice was similar to that achieved in humans dosed at 300 mg once daily. Interestingly, the brain-to-plasma ratio was 26-fold higher in Abcb1a/1b;Abcg2-/- mice compared to wild-type mice. However, no significant differences were observed in other tested tissue distributions or in oral availability of galunisertib among these three mouse strains. Our data suggest that ABC transporters play an important role in restricting the brain penetration of galunisertib, but have little impact on oral availability and other relative tissue distribution. Mouse Cyp3a appears to have little, if any, effect on the pharmacokinetics and metabolism of galunisertib. Our results suggest a potential way to boost brain penetration of galunisertib by pharmacological inhibition of transporters in the BBB with chemical inhibitors, especially for patients with brain metastases. These insights might be used to optimize the clinical application of galunisertib. Citation Format: Wenlong Li, Matthijs Tibben, Yaogeng Wang, Maria C. Lebre, Hilde Rosing, Jos H. Beijnen, Alfred H. Schinkel. The impact of P-glycoprotein, breast cancer resistance protein and CYP3A on pharmacokinetics and metabolism of galunisertib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1268.

A pilot study of galunisertib (LY2157299 monohydrate) plus stereotactic body radiotherapy (SBRT) in advanced hepatocellular carcinoma (HCC).

270 Background: TGF-β, the strongest known immunosuppressive cytokine, modulates the hepatic immune response to various antigens and to ionizing radiation. When activated, TGF-β blocks the effector T-cell response to cellular destruction and the release of tumor-specific antigens. Preclinical data demonstrate that neutralizing TGF-β during radiation effectively generates a CD8+ T-cell response to multiple endogenous tumor antigens, thereby generating an in-situ vaccine against a tumor. We hypothesized that the combination of TGF-β receptor inhibition plus SBRT would produce a potent and clinically effective antitumor immune response against HCC. Methods: Patients received galunisertib (LY2157299 monohydrate) on days 1-14 of each 28 day cycle. SBRT was delivered in a single fraction of 18 Gy between days 15-28 of C1. Pre-treatment and on-treatment biopsies were obtained, as well as serial blood collections for CTCs, ctDNA and PBMCs. Results: 15 patients with advanced HCC and Child’s Pugh A cirrhosis were treated. 9/15 (60%) had a prior history of infectious hepatitis, three of whom had active viremia at time of enrollment. 6/15 (40%) had received prior systemic therapy for HCC and 12/15 (80%) had received prior liver directed therapy. The most common treatment-related toxicities were: fatigue (53%), nausea (47%), increased alkaline phosphatase (33%), abdominal pain (33%), vomiting (20%), decreased white blood cell count (20%). All drug-related toxicities were grade 1 or 2, with the exception of one patient who had grade 3 achalasia, thought possibly to be related to study drug. Median PFS was 3.68mo. There were two confirmed partial responses (PRs) and six additional patients had stable disease (SD) for at least four months, resulting in a disease control rate (DCR) of 53%. Both PRs included shrinkage of lesions that had not been radiated. Translational studies are currently underway. Conclusions: The combination of galunisertib with SBRT in patients with advanced HCC is a well-tolerated regimen. In this small pilot study, we observe a DCR of 53% including two PRs. These results warrant further study of this therapeutic combination in advanced HCC. Clinical trial information: NCT02906397.

LY2157299 Monohydrate, a TGF-βR1 Inhibitor, Suppresses Tumor Growth and Ascites Development in Ovarian Cancer.

Transforming growth factor beta (TGF-β) signaling has pleiotropic functions regulating cancer initiation, development, and metastasis, and also plays important roles in the interaction between stromal and cancer cells, making the pathway a potential therapeutic target. LY2157299 monohydrate (LY), an inhibitor of TGF-β receptor I (TGFBRI), was examined for its ability to inhibit ovarian cancer (OC) growth both in high-grade serous ovarian cancer (HGSOC) cell lines and xenograft models. Immunohistochemistry, qRT-PCR, and Western blot were performed to study the effect of LY treatment on expression of cancer- and fibroblast-derived genes. Results showed that exposure to TGF-β1 induced phosphorylation of SMAD2 and SMAD3 in all tested OC cell lines, but this induction was suppressed by pretreatment with LY. LY alone inhibited the proliferation, migration, and invasion of HGSOC cells in vitro. TGF-β1-induced fibroblast activation was blocked by LY. LY also delayed tumor growth and suppressed ascites formation in vivo. In addition, independent of tumor inhibition, LY reduces ascites formation in vivo. Using OVCAR8 xenograft specimens we confirmed the inhibitory effect of LY on TGF-β signaling and tumor stromal expression of collagen type XI chain 1 (COL11A1) and versican (VCAN). These observations suggest a role for anti-TGF-β signaling-directed therapy in ovarian cancer.

Preclinical assessment of galunisertib (LY2157299 monohydrate), a first-in-class transforming growth factor-β receptor type I inhibitor.

Transforming growth factor-β (TGFβ) is an important driver of tumor growth via intrinsic and extrinsic mechanisms, and is therefore an attractive target for developing cancer therapeutics. Using preclinical models, we characterized the anti-tumor activity of a small molecule inhibitor of TGFβ receptor I (TGFβRI), galunisertib (LY2157299 monohydrate). Galunisertib demonstrated potent and selective inhibition of TGFβRI with corresponding inhibition of downstream signaling via inhibition of SMAD phosphorylation (pSMAD). Galunisertib also inhibited TGFβ-induced pSMAD in vivo, which enabled a pharmacokinetic/pharmacodynamic profile in Calu6 and EMT6-LM2 tumors. Galunisertib demonstrated anti-tumor activity including inhibition of tumor cell migration and mesenchymal phenotype, reversal of TGFβ-mediated immune-suppression, and tumor growth delay. A concentration-effect relationship was established with a dosing schedule to achieve the optimal level of target modulation. Finally, a rat model demonstrated a correlation between galunisertib-dependent inhibition of pSMAD in tumor tissues and in PBMCs, supporting the use of PBMCs for assessing pharmacodynamic effects.Galunisertib has been tested in several clinical studies with evidence of anti-tumor activity observed in subsets of patients. Here, we demonstrate that galunisertib inhibits a number of TGFβ-dependent functions leading to anti-tumor activity. The enhanced understanding of galunisertib provides rationale for further informed clinical development of TGFβ pathway inhibitors.

Abstract 955: LY3200882, a novel, highly selective TGFβRI small molecule inhibitor

Abstract The transforming growth factor β (TGFβ) signaling pathway is a pleiotropic cellular pathway that plays a critical role in cancer. In fact, aggressive tumors are typically associated with high ligand levels and thus associated with poor prognosis in various tumor types. Cancer cells use autocrine and paracrine TGFβ signaling to modulate tumor cells and the tumor microenvironment leading to a highly invasive and metastatic phenotype, inducing and increasing tumor vascularization, modulating the extracellular matrix in the stroma, and inhibiting immune surveillance and antitumor immunity. Clinical studies with galunisertib (aka LY2157299 monohydrate), a small molecule inhibitor targeting the TGFβ pathway, have provided proof of concept data supporting the role of TGFβ in cancer and the utility of targeting the TGFβ pathway. Here we describe the identification of LY3200882, a next generation small molecule inhibitor of TGF-β receptor type 1 (TGFβRI). The molecule is a potent, highly selective inhibitor of TGFβRI embodied in a structural platform with a synthetically scalable route. It is an ATP competitive inhibitor of the serine-threonine kinase domain of TGFβRI. Mechanism of action studies reveal revealed that LY3200882 inhibits various pro-tumorigenic activities. LY3200882 potently inhibits TGFβ mediated SMAD phosphorylation in vitro in tumor and immune cells and in vivo in subcutaneous tumors in a dose dependent fashion. In preclinical tumor models, LY3200882 showed potent anti-tumor activity in the orthotopic 4T1-LP model of triple negative breast cancer and this activity correlated with enhanced tumor infiltrating lymphocytes in the tumor microenvironment. Durable tumor regressions in the orthotopic 4T1-LP model were observed and rechallenge of congenic tumors resulted in complete rejection in all mice. In in vitro immune suppression assays, LY3200882 has shown the ability to rescue TGFβ1 suppressed or T regulatory cell suppressed naïve T cell activity and restore proliferation. Therefore, LY3200882 shows promising activity as an immune modulatory agent. In addition, LY3200882 has shown anti-metastatic activity in vitro in migration assays as well as in vivo in an experimental metastasis tumor model (intravenous EMT6-LM2 model of triple negative breast cancer). Finally, LY3200882 shows combinatorial anti-tumor benefits with checkpoint inhibition (anti-PD-L1) in the syngeneic CT26 model. In conclusion, we have developed a novel potent and highly selective small molecule inhibitor of TGFβRI for the treatment of cancer. Citation Format: Huaxing Pei, Saravanan Parthasarathy, Sajan Joseph, William McMillen, Xiaohong Xu, Stephen Castaneda, Ivan Inigo, Karen Britt, Bryan Anderson, Gaiying Zhao, Scott Sawyer, Douglas Beight, Talbi Kaoudi, Chandrasekar Iyer, Huimin Bian, Amy Pappas, David Surguladze, David Schaer, Karim Benhadji, Michael Kalos, Kyla Driscoll. LY3200882, a novel, highly selective TGFβRI small molecule inhibitor [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 955. doi:10.1158/1538-7445.AM2017-955

Disposition and metabolism of [14C]-galunisertib, a TGF-βRI kinase/ALK5 inhibitor, following oral administration in healthy subjects and mechanistic prediction of the effect of itraconazole on galunisertib pharmacokinetics

1. The disposition and metabolism of galunisertib (LY2157299 monohydrate, a TGF-βRI Kinase/ALK5 Inhibitor) was characterized following a single oral dose of 150 mg of [14C]-galunisertib (100 µCi) to six healthy human subjects.2. The galunisertib plasma half-life was 8.6 h, while the 14C half-life was 10.0 h. Galunisertib was abundant in circulation (40.3% of the 14C AUC024 h), with 7 additional metabolites detected in plasma. Two metabolites LSN3199597 (M5, mono-oxidation), and M4 (glucuronide of M3) were the most abundant circulating metabolites (10.7 and 9.0% of the 14C AUC024 h respectively). The pharmacological activity of LSN3199597 was tested and found to be significantly less potent than galunisertib.3. The dose was recovered in feces (64.5%) and in urine (36.8%). Galunisertib was cleared primarily by metabolism, based on low recovery of parent in excreta (13.0% of dose). Due to the slow in vitro metabolism of galunisertib in suspended hepatocytes, a long term hepatocyte system was used to model the human excretion profile.4. Expressed cytochromes P450 and hepatocytes indicated clearance was primarily CYP3A4-mediated. Mechanistic static modeling that incorporated small non-CYP-mediated metabolic clearance and renal clearance components predicted an AUC ratio of 4.7 for the effect of itraconazole, a strong CYP3A4 inhibitor, on galunisertib.

Relative bioavailability of three formulations of galunisertib administered as monotherapy in patients with advanced or metastatic cancer.

ObjectiveGalunisertib (LY2157299 monohydrate), an inhibitor of the transforming growth factor β (TGFβ) pathway, is currently under investigation in several clinical trials involving multiple tumor types. The primary objective of this study was to assess relative bioavailability of two new galunisertib formulations developed using the roller compaction (RC) dry-milled (RCD) and RC slurry-milled (RCS) processes, compared with the existing formulation developed using the high-sheer wet granulation (HSWG) process. The secondary objective was to report the safety profile after a single dose of the three formulations.MethodsPatients with advanced or metastatic cancer were enrolled into this single-center, 3-period, 6-sequence crossover study. Patients were assigned sequentially to 1 of 6 sequences in blocks of 6 to ensure that all 6 sequences have the same number of completers. A patient entering a sequence received a different galunisertib formulation as a single 150 mg dose orally during each of the 3 periods. Each period was separated from the next by a washout interval of at least 48 hours. Pharmacokinetic (PK) parameters, including area under curve (AUC) and Cmax, were computed using standard non-compartmentalized methods of analysis. For comparison of exposures between formulations, log-transformed AUC and Cmax values were analyzed using a linear mixed-effects model. Safety assessments included adverse event monitoring, physical examinations, and laboratory tests.ResultsOf the 14 patients who entered and completed the study, 13 patients were included in the final statistical analysis. AUC(0-tlast), AUC(0–48 h), and AUC(0-∞) for the RC formulations and the HSWG formulation were similar. Cmax was reduced by approximately 22% and tmax was longer by at least 1.00 h for the RCD and RCS formulations compared with the HSWG formulation. The RC formulations demonstrated a safety profile after a single dose similar to the HSWG formulation.ConclusionsIn this relative bioavailability study comparing galunisertib formulations after a single dose, RCD and RCS formulations had similar exposure and safety profile compared with the HSWG formulation.

1102TiP - A phase 1b/2 dose escalation and cohort expansion study of the safety, tolerability and efficacy of a transforming growth factor-beta (TGF-&bgr;) receptor I kinase inhibitor (galunisertib) in combination with anti–PD-1 (nivolumab) in advanced refractory solid tumours

TGF-&bgr; signaling plays an important role in tumorigenesis and contributes to the hallmarks of cancer, including tumor proliferation, invasion and metastasis, inflammation, angiogenesis, and escape of immune surveillance. Galunisertib (LY2157299 monohydrate) is an oral small molecule inhibitor of the TGF-&bgr; receptor I kinase that specifically down-regulates the phosphorylation of SMAD2, abrogating activation of the canonical pathway. Programmed Cell Death-1 (PD-1) is expressed on activated T cells and can act to dampen the immune response. Tumor cells overexpress PD-1 ligand (PD-L1) and inhibit the local immune response. Nivolumab blocks the binding of PD-L1 to its PD-1 receptor, allowing the activated T cells to identify and attack cancer cells. Thus, blockade of both TGF-&bgr; and PD-1 could be expected to reverse the immune escape and to potentially provide immune restoration to improve immune response and induce tumor regression. Trial design: This study (NCT02423343) is a phase 1b/2 open-label study that will be conducted in 2 parts. The phase 1b is an open-label, dose-escalation assessment of the safety and tolerability of galunisertib administered in escalating doses over 4 cohorts ending with 150 mg BID in combination with nivolumab 3 mg/kg IV every 2 weeks in patients with advanced refractory solid tumors. The phase 2 will be disease restricted and includes 3 expansion cohorts of patients with non-small cell lung cancer (n = 25), hepatocellular carcinoma (n = 25), or glioblastoma (n = 25). Patients in the 3 cohorts will be assigned to treatment concurrently, and enrollment will be complete when all cohorts have reached the prespecified enrollment target. Enrollment of patients in phase 1b began on 09 October 2015; as of 27April2016, seven patients entered the study; one withdrew for a non-DLT reason, and one patient remains on treatment every 2 weeks. Clinical trial identification: NCT02423343 Legal entity responsible for the study: Eli Lilly and Company

Abstract 1076: Plasma microRNAs associated with overall survival in patients with hepatocellular carcinoma treated with galunisertib (LY2157299 monohydrate), an inhibitor of transforming growth factor-β receptor1

Abstract Background: Galunisertib, a selective transforming growth factor-β receptor1 inhibitor, is being investigated in clinical trials for hepatocellular carcinoma (HCC). MicroRNAs (miRs) are small (∼22 nucleotide) non-coding RNAs that regulate expression of targeted genes, and are secreted by cells into blood. miRs are differentially expressed during HCC progression, differentiation, and response to therapy. We hypothesize that circulating miRs may be useful to identify patients who benefit from galunisertib treatment. Patients and Methods: Plasma samples from HCC patients (n = 105) treated with galunisertib were analyzed for miR expression. Patients were enrolled in part A, of a multi-part single-arm study in 2nd-line HCC (phase II trial NCT01246986/JBAK). All patients had elevated alpha-fetoprotein (AFP) levels at baseline (AFP ≥1.5 x ULN). The median OS of this cohort of patients was 7.2 mo. Eighty percent of patients received prior sorafenib treatment. Plasma samples were collected during patient screening, cycle 1 day 1 (pretreatment), and cycle 2 day 14. The Exiqon RT microRNA PCR Human panel I+II was used to measure 752 miRs. Expression levels of detectable miRs and their association with overall survival (OS) were investigated. Results: Low plasma levels of miR-665 (HR = 0.50, p = 0.001, q = 0.13), miR-320d (HR = 0.44, p = 0.001, q = 0.13), miR-320a (HR = 0.47, p = 0.001, q = 0.13), and miR-130b-3p (HR = 0.44, p = 0.002, q = 0.13) are associated with better survival. Whereas, low plasma levels of miR-451a (HR = 2.0, p = 0.002, q = 0.13), miR-let7g-5p (HR = 2.3, p = 0.002, q = 0.13), and miR-18a-3p (HR = 2.0, p = 0.002, q = 0.13) are associated with poor survival. To assess within patient baseline biological variation of miR expression, a comparison of the expression of 369 miRs in patients (n = 42) with 2 pre-treatment samples was performed. The proportion of miRs attaining statistical significance was smaller than what we would expect by chance. Conclusions: Circulating miRs may serve as easily accessible markers to identify HCC patients who may benefit from galunisertib treatment, which requires confirmation in randomized controlled study. Given the low intra-patient variability measured at baseline for most of the miRs, circulating miRs may represent reliable molecular markers with prognostic and/or predictive utility. Citation Format: Shawn T. Estrem, Michael Man, Xuekui Zhang, Duytrac Nguyen, Danni Yu, Michael M. Lahn, Ann Cleverly, Durisala Desaiah, Sandrine Faivre, Gianluigi Gianneli, Karim A. Benhadji. Plasma microRNAs associated with overall survival in patients with hepatocellular carcinoma treated with galunisertib (LY2157299 monohydrate), an inhibitor of transforming growth factor-β receptor1. [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 1076.

Clinical development of galunisertib (LY2157299 monohydrate), a small molecule inhibitor of transforming growth factor-beta signaling pathway.

Transforming growth factor-beta (TGF-β) signaling regulates a wide range of biological processes. TGF-β plays an important role in tumorigenesis and contributes to the hallmarks of cancer, including tumor proliferation, invasion and metastasis, inflammation, angiogenesis, and escape of immune surveillance. There are several pharmacological approaches to block TGF-β signaling, such as monoclonal antibodies, vaccines, antisense oligonucleotides, and small molecule inhibitors. Galunisertib (LY2157299 monohydrate) is an oral small molecule inhibitor of the TGF-β receptor I kinase that specifically downregulates the phosphorylation of SMAD2, abrogating activation of the canonical pathway. Furthermore, galunisertib has antitumor activity in tumor-bearing animal models such as breast, colon, lung cancers, and hepatocellular carcinoma. Continuous long-term exposure to galunisertib caused cardiac toxicities in animals requiring adoption of a pharmacokinetic/pharmacodynamic-based dosing strategy to allow further development. The use of such a pharmacokinetic/pharmacodynamic model defined a therapeutic window with an appropriate safety profile that enabled the clinical investigation of galunisertib. These efforts resulted in an intermittent dosing regimen (14 days on/14 days off, on a 28-day cycle) of galunisertib for all ongoing trials. Galunisertib is being investigated either as monotherapy or in combination with standard antitumor regimens (including nivolumab) in patients with cancer with high unmet medical needs such as glioblastoma, pancreatic cancer, and hepatocellular carcinoma. The present review summarizes the past and current experiences with different pharmacological treatments that enabled galunisertib to be investigated in patients.

Brain perfusion and permeability in patients with advanced, refractory glioblastoma treated with lomustine and the transforming growth factor-β receptor I kinase inhibitor LY2157299 monohydrate.

Transforming growth factor-β (TGF-β) signaling is associated with tumor progression and vascularization in malignant glioma. In the present study, magnetic resonance imaging was used to evaluate changes in the size and vascularity of glioblastomas in 12 patients who were treated with lomustine and the novel inhibitor of TGF-β signaling, LY2157299 monohydrate. A response in tumor size was observed in 2 of the 12 patients; in 1 of these 2 patients, a reduction in vascular permeability and perfusion was also detected. The effect was observed following 4 cycles of treatment (~3 months). Changes in vascularity have not previously been attributed to treatment with lomustine; therefore, the effect may be associated with LY2157299 treatment. LY2157299 does not appear to have an anti-angiogenic effect when combined with lomustine, and hence may have a different mechanism of action profile compared with anti-angiogenic drugs.

Anti-tumor activity of the TGF-β receptor kinase inhibitor galunisertib (LY2157299 monohydrate) in patient-derived tumor xenografts.

PurposeThe transforming growth factor-beta (TGF-β) signaling pathway is known to play a critical role in promoting tumor growth. Consequently, blocking this pathway has been found to inhibit tumor growth. In order to achieve an optimal anti-tumor effect, however, it remains to be established whether blocking the TGF-β signaling pathway alone is sufficient, or whether the tumor microenvironment plays an additional, possibly synergistic, role.MethodsTo investigate the relevance of blocking TGF-β signaling in tumor cells within the context of their respective tissue microenvironments, we treated a panel of patient-derived xenografts (PDX) with the selective TGF-β receptor kinase inhibitor LY2157299 monohydrate (galunisertib) and assessed both the in vitro and in vivo effects.ResultsGalunisertib was found to inhibit the growth in an in vitro clonogenic assay in 6.3 % (5/79) of the examined PDX. Evaluation of the expression profiles of a number of genes, representing both canonical and non-canonical TGF-β signaling pathways, revealed that most PDX exhibited expression changes affecting TGF-β downstream signaling. Next, we subjected 13 of the PDX to an in vivo assessment and, by doing so, observed distinct response patterns. These results suggest that, next to intrinsic, also extrinsic or microenvironmental factors can affect galunisertib response. pSMAD2 protein expression and TGF-βRI mRNA expression levels were found to correlate with the in vivo galunisertib effects.ConclusionsFrom our data we conclude that intrinsic, tumor-dependent TGF-β signaling does not fully explain the anti-tumor effect of galunisertib. Hence, in vivo xenograft models may be more appropriate than in vitro clonogenic assays to assess the anti-tumor activity of TGF-β inhibitors such as galunisertib.Electronic supplementary materialThe online version of this article (doi:10.1007/s13402-014-0210-8) contains supplementary material, which is available to authorized users.

Defining a therapeutic window for the novel TGF-β inhibitor LY2157299 monohydrate based on a pharmacokinetic/pharmacodynamic model.

To identify prospectively a safe therapeutic window for administration of a novel oral transforming growth factor β (TGF-β) inhibitor, LY2157299 monohydrate, based on a pharmacokinetic/pharmacodynamic (PK/PD) model. Simulations of population plasma exposures and biomarker responses in tumour were performed for future trials of LY2157299 in glioblastoma and other cancer populations. The model was updated after completion of each cohort during the first-in-human dose (FHD) study. The flexible design allowed continuous assessment of PK variability by recruiting the required number of patients in each cohort. Based on 30% inhibition of TGF-β RI kinase phosphorylates (pSMAD), biologically effective exposures were anticipated to be reached from 160 mg onwards. The therapeutic window was predicted, based on animal data, to be between 160 and 360 mg. No medically significant safety issues were observed and no dose limiting toxicities were established in this study. Observed plasma exposures (medians 2.43 to 3.7 mg l⁻¹ h, respectively) with doses of 160 mg to 300 mg were within the predicted therapeutic window. Responses, based on the MacDonald criteria, were observed in these patients. A therapeutic window for the clinical investigation of LY2157299 in cancer patients was defined using a targeted PK/PD approach, which integrated translational biomarkers and preclinical toxicity. The study supports using a therapeutic window based on a PK/PD model in early oncology development.

Transforming Growth Factor-β as a Therapeutic Target in Hepatocellular Carcinoma

Hepatocellular carcinoma arises in patients as a consequence of long-standing preexisting liver illnesses, including viral hepatitis, alcohol abuse, or metabolic disease. In such preexisting liver diseases, TGF-β plays an important role in orchestrating a favorable microenvironment for tumor cell growth and promoting epithelial-mesenchymal transition (EMT). TGF-β signaling promotes hepatocellular carcinoma progression by two mechanisms: first, via an intrinsic activity as an autocrine or paracrine growth factor and, second, via an extrinsic activity by inducing microenvironment changes, including cancer-associated fibroblasts, T regulatory cells, and inflammatory mediators. Although there is an increasing understanding on how TGF-β signaling is associated with tumor progression in hepatocellular carcinoma, it is not clear whether TGF-β signaling is limited to a certain subgroup of patients with hepatocellular carcinoma or is a key driver of hepatocellular carcinoma during the entire tumorigenesis of hepatocellular carcinoma. Inhibitors of the TGF-β signaling have been shown to block hepatocellular carcinoma growth and progression by modulating EMT in different experimental models, leading to the clinical investigation of the TGF-β inhibitor LY2157299 monohydrate in hepatocellular carcinoma. Preliminary results from a phase II clinical trial have shown improved clinical outcome and also changes consistent with a reduction of EMT.

A phase 2 study of a novel transforming growth factor-beta (TGF-β1) receptor I kinase inhibitor, LY2157299 monohydrate (LY), in patients with advanced hepatocellular carcinoma (HCC).

LBA173 Background: TGF-β signaling is associated with HCC progression in moderate to poorly differentiated tumors overexpressing alpha-fetoprotein (AFP) levels. We report here the safety, antitumor activity, and overall survival (OS) in HCC patients with elevated AFP treated with LY. Methods: Patients with advanced HCC who progressed on sorafenib (SF) or are ineligible to receive SF, advanced Child-Pugh A/B7 HCC, AFP ≥1.5x ULN, ECOG PS ≤1, measurable disease (RECIST 1.1), and ≤1 prior systemic regimen were eligible. LY was administered as intermittent dosing of 14 days on/off (28 days=1 cycle). Patients were randomized to either 160 mg/day (Arm A) or 300 mg/day (Arm B) LY. Primary endpoints were time-to-progression (TTP) and biomarker changes (serum AFP, TGF-β and E-cadherin) for each dose. AFP responders were defined as >20% decline from baseline. Secondary endpoints included toxicity (CTCAE, V 4.0) and pharmacokinetics (PK). Available data from interims are presented. Results: 109 patients were enrolled (Arm A=37; B=72). Baseline characteristics were (Arm A/B): median age 61/63 years; PS=0, 60%/53%; Child-Pugh A 97%/88%; etiology: hepatitis C 30%/38%, hepatitis B 27%/24%, alcohol 24%/25%. Overall, 78%/83% of patients had received prior SF; 57%/53% had AFP >400 ng/mL. Median TTP was 12.0 weeks (90% CI: 6.6, 12.6) in the overall population (Arm A/B, 12.1/10.0 weeks). In SF-naive patients, TTP was 18.3 weeks (90% CI: 6.6-42.4). TTP was higher in the non-alcohol compared to alcohol-only etiology group (median 12.1 vs. 6.1 weeks). Median baseline serum TGF-β1 was 3.4 ng/mL (range: 1.4-3.7) and E-cadherin was 6.1 mg/mL (range: 1.9-17.3). AFP decline of >20% occurred in 24% of patients. Based on the latest interim, median OS was 36 weeks. In AFP responders, median OS was 93.1 weeks vs 29.6 weeks in non AFP responders (Log Rank p=0.0006). Four patients discontinued treatment due to AEs. Most common grade 3/4 related AEs were: neutropenia (n=3), fatigue (n=2), and anemia (n=3). Preliminary PK analysis (n=51) demonstrated moderate interpatient exposure variability (42%). Conclusions: Based on the manageable toxicity profile, the evidence for biomarker responses, analysis of the aggregate PK/PD data, the 300 mg/day dose was chosen for future studies in HCC. Both TTP and overall survival in AFP responding patients is promising.

Nonclinical Safety Evaluation of a Transforming Growth Factor β Receptor I Kinase Inhibitor in Fischer 344 Rats and Beagle Dogs

Abstract Objective: The transforming growth factor β (TGF-β) pathway regulates diverse cellular functions and plays a prominent role in diseases such as cancer, autoimmune disorders and cardiovascular disease. LY2157299 monohydrate (LY2157299) is a potent and selective inhibitor of TGF-β receptor I kinase that is under clinical evaluation for the treatment of advanced cancer. Methods: This paper characterizes the toxicity profile of LY2157299 in Fischer 344 rats and beagle dogs for up to six months of daily oral dosing. LY2157299 is well tolerated in the rat and dog for up to one month of daily dosing at doses of 150 and 20 mg/kg, respectively. Results: In the rat, LY2157299 is well tolerated after three months of 2 weeks on/2 weeks off intermittent dosing schedule at 50 mg/kg. Chronic (≥3 months) oral administration results in multiple target organ toxicities involving the cardiovascular, gastrointestinal, immune, bone/cartilage, reproductive, and renal systems. Conclusion: Defining the appropriate dose and schedule led to a better understanding of how to define safety margins and thus enable the clinical investigation of LY2157299 in cancer patients.

Abstract C261: Pharmacokinetics (PK) and pharmacodynamics (PD) of the oral transforming growth factor-beta (TGF-β) receptor I kinase inhibitor LY2157299 monohydrate (LY) in hepatocellular carcinoma (HCC) compared to glioma patients.

Abstract Background: TGF-β signaling is associated with HCC progression in moderate to poorly differentiated tumors. LY is a TGF-β receptor I kinase inhibitor currently in clinical testing in glioblastoma (GBM), hepatocellular, and pancreatic cancer. We report here the safety and PK/PD profile of LY in HCC patients. Methods: Patients with advanced HCC who progressed on or were ineligible to receive sorafenib (SF), advanced Child-Pugh A/B7 HCC, ECOG PS ≤1, measurable disease (RECIST 1.1), and ≤1 prior systemic regimen were eligible. LY was administered as intermittent dosing of 14 days on/14 days off (28 days = 1 cycle). Patients received either 160 mg/day or 300 mg/day. PK data were used to build a population PK model for HCC patients. This population PK model guided appropriate sampling and identification of potential demographic covariates. PD analysis included serum alpha-fetoprotein (AFP). AFP response was defined as at least 20% decrease. Results: Among 148 patients enrolled, 137 patients were evaluable for PK. There was dose-proportional increase in exposure (AUC0-∞ = 3.8 mg*h/L for 160 mg and AUC0-∞ = 7.0 mg*h/L for 300 mg dose). There were higher exposures in HCC patients than previously observed for exposures in GBM patients (AUC0-∞ = 2.1 mg*h/L for 160 mg and AUC0-∞ = 3.7 mg*h/L for 300mg dose)1. PK data suggested that total oral clearance (CL/F = 25.4 L/hr) of LY in HCC patients is lower than previously reported clearance in GBM patients (CL/F = 38.4 L/hr). Moderate between-patient variability on exposure was observed in HCC patients similar to GBM patients. LY appears to be fully eliminated at the end of the 14-days-off period of a cycle. Administration of 300 mg/day to patients with HCC may result in higher exposures compared to patients with GBM. These increased exposures did not result in increase of toxicities. Eight patients discontinued treatment due to a possibly drug-related adverse event (AE). LY was well tolerated, most adverse events being grade1-2. Most common grade 3/4 possibly drug-related AEs in patients were: neutropenia (n = 3), anemia (n = 3) and fatigue (n = 2). Thirty-nine percent of 69 patients with HCC who had baseline AFP of >200 ng/mL had AFP response. The median time-to-progression of responders vs. non-responders was 18 vs. 10 weeks (p<0.01), respectively. Conclusion: Clearance of LY in HCC patients with Child Pugh A/B7 liver function is lower than in GBM patients. LY had manageable safety profile in HCC patients and was associated with AFP responses. Rodon AJ., et al. First human dose (FHD) study of the oral transforming growth factor-beta receptor I kinase inhibitor LY2157299 in patients with treatment-refractory malignant glioma. Abstract #3011. 2011 ASCO Annual Meeting, Chicago, IL, USA. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C261. Citation Format: Eric Raymond, Sandrine Faivre, Armondo Santoro, Robin K. Kelley, Robin K. Kelley, Philippe Merle, Ed Gane, Jean-Yves Douillard, Dirk Waldschmidt, Mary Mulcahy, Charlotte Costentin, Beatriz Minguez, Luca Di Lena, Ivelina Gueorguieva, Colin Miles, Ann Cleverly, Michael M. Lahn, Sophie Ameryckx, Karim A. Benhadji, Gianluigi Giannelli. Pharmacokinetics (PK) and pharmacodynamics (PD) of the oral transforming growth factor-beta (TGF-β) receptor I kinase inhibitor LY2157299 monohydrate (LY) in hepatocellular carcinoma (HCC) compared to glioma patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C261.

Phase Ib study evaluating safety and pharmacokinetics (PK) of the oral transforming growth factor-beta (TGF-ß) receptor I kinase inhibitor LY2157299 monohydrate (LY) when combined with chemoradiotherapy in newly diagnosed malignant gliomas.

2039 Background: Based on preclinical data suggesting an additive antitumor effect of combining TGF-ß inhibitors with temozolomide based chemoradiation (TMZ/RT), a Phase 1b study was initiated to evaluate the safety and PK of LY combined with TMZ/RT in patients with newly diagnosed glioma. Methods: LY was evaluated sequentially in 2 doses (160 mg/day and 300 mg/day) combined with TMZ/RT. TMZ/RT was administered as approved and LY given as intermittent dosing (14 days on/14 days off=1 cycle). Toxicity was assessed using the CTCAE, version 4. The PK profile of LY in combination with TMZ/RT was determined. Results: 19 patients with glioma (16 World Health Organization Grade 4; 3 Grade 3 were treated with LY (160 mg/day, n=10; 300 mg/day, n=9) and TMZ. The median number of cycles was 5 (range 1-13). Regardless of relatedness to study treatment, the following treatment-emergent adverse events (TEAEs) were observed in ≥25% of patients: nausea (n=11), fatigue (n=11), thrombocytopenia (n=11), headache (n=9), vomiting (n=8), lymphopenia (n=6), anorexia (n=6), constipation (n=5), radiation skin injury (n=5), and alopecia (n=5). The following TEAEs were related specifically to at least LY: thrombocytopenia (n=3, 2 Gr 3 and 1 Gr 4), fatigue (n=2), maculopapular rash (n=2, Gr 3), dermatitis acne form (n=3, 1 Gr 3) and in 1 patient each: nausea, vomiting, hypertension, hypersensitivity and leucopenia (Gr 4). No change in the PK profile of LY was shown when LY was combined with TMZ/RT. In the combination therapy, area under the curve (0-∞) at steady state was observed geomean (%CV) to be 5.5 (48%) mg*h/L following 300 mg/day (n=9). Following monotherapy with LY, these exposures were similar with a median (20th-80thpercentiles) of 4.7 (2.5-8.8) mg*h/L (n=37). Conclusions: No dose-limiting toxicities and no clinically meaningful cardiotoxicities were observed; hence, the treatment of LY at 300 mg/day in combination with standard chemoradiation has a manageable toxicity profile. A Phase 2a trial has been initiated to relate the pharmacodynamic effects with overall survival. Clinical trial information: NCT01220271.

Phase Ib study evaluating safety and pharmacokinetics (PK) of the oral transforming growth factor-beta (TGF-ß) receptor I kinase inhibitor LY2157299 monohydrate (LY) when combined with gemcitabine in patients with advanced cancer.

2563 Background: Based on animal studies, the combination of a TGF-β inhibitor and gemcitabine is expected to enhance antitumor activity of gemcitabine. Hence, we started a combination study of LY with gemcitabine to assess the safety of this combination therapy. Methods: Gemcitabine was administered as approved and LY was given daily as intermittent dosing (14 days on/14 days off=1 cycle). A dose escalation consisting of 3 cohorts (80 mg/day, 160 mg/day, and 300 mg/day) evaluated the safety of LY in combination with gemcitabine. Toxicity was assessed using the CTCAE, version 4. Pharmacokinetic (PK) profile of LY in combination with gemcitabine was determined. Results: A total of 14 patients were evaluated (cohort 1 [n=5, 4 adenocarcinoma of colon, 1 non-small cell lung cancer, cohort 2 [n=4, adenocarcinoma, 1 each of esophageal, well differentiated, pancreas, lung], cohort 3 [n=5, 3 pancreatic, 2 rectal cancer]) in this study. The median number of cycles was 2 (range (1-6). Regardless of causality, the following treatment-emergent adverse events (TEAEs) were observed in ≥25% of patients: anemia (n=10), nausea (n=8), thrombocytopenia (n=6), neutropenia (n=6), vomiting (n=6), anorexia (n=5), fatigue (n=5), diarrhea (n=5) asthenia (n=5), pyrexia (n=4), AST increased (n=4), and constipation (n=4). Possibly related to LY, (Grade, Gr, 3/4 as mentioned) TEAEs observed were: nausea (n=5), asthenia (n=4), fatigue (n=3, 1 Gr 3), neutropenia (n=3, 1 Gr 3), anemia (n=3, 2 Gr 3 ), and in 2 patients each, thrombocytopenia (both Gr 3), headache, edema peripheral, rash, anorexia, diarrhea (1 Gr 3), mucosal inflammation, vomiting and reversible rhabdomyolysis (n=1, Gr 4). No change in the PK profile of LY was shown when LY was combined with gemcitabine. Conclusions: There were no dose limiting toxicities observed and no clinically meaningful cardiotoxicities were detected. Because of the observed manageable safety profile, LY at 300 mg/day has been advanced into a randomized Phase 2 trial in pancreatic cancer in 1st line setting to assess the antitumor activity of the combination. Clinical trial information: NCT01373164.