Potent PI3Kδ Inhibitor Research Articles

Abstract 2665: High dose intermittent scheduling of AZD8835, a novel potent and selective inhibitor of PI3Kα and PI3Kδ, identifies potential treatment strategies for PIK3CA-dependent cancers

Abstract The PIK3CA gene, encoding the p110 catalytic unit of PI3Kα, is one of the most frequently mutated oncogenes described in human cancer. Hence PI3Kα is a target subject to intensive efforts in identifying inhibitors and evaluating their therapeutic potential. To date most studies with PI3K inhibitors have used a continuous (daily) dosing schedule and although clinical responses have been reported the overall activity observed has been moderate. This may in part be due to suboptimal pathway inhibition which is capped by normal tissue toxicities such as rash, diarrhoea and hyperglycaemia. Furthermore, additional dose reduction may be required when such agents are used in combination with other therapies. Therefore in our efforts to optimise inhibition of PI3K pathway signalling, we have explored high dose intermittent scheduling as an alternative to continuous dosing. Here we describe pre-clinical studies that exemplify such concepts, centred around use of AZD8835, a PI3K inhibitor currently in Phase 1 clinical evaluation. AZD8835 is a novel and potent inhibitor of PI3Kα and PI3Kδ, with selectivity vs. PI3Kβ, PI3Kγ(IC50s of 6nM, 6nM, 431nM and 90nM respectively in enzyme assays) and other kinases. AZD8835 preferentially displays activity in tumour models with a mutant PIK3CA background, such as ER+ve breast cancer models. Such models were used in our investigations, in both cell culture and in mouse xenograft contexts. We demonstrate that a high dose intermittent schedule of single agent AZD8835 achieves greater pathway inhibition yielding significant anti-tumour responses. In the sensitive BT474 xenograft model, a dose of 100mg/kg AZD8835 BID on days 1 and 4 in a weekly schedule delivered -36% tumour regression, accompanied by a strongly elevated rapid onset apoptosis signal with 4-16% cells staining positively for cleaved-caspase3. We also evaluated AZD8835 in combination with other targeted therapeutic agents, in MCF7, BT474 and T47D breast models, observing increased sensitivity relative to single agent AZD8835; firstly with agents that target other nodes in the PI3K pathway; secondly with agents targeting parallel but interconnected driver pathways in breast disease (ER, CDK4/6). Overall the data indicate that high dose intermittent scheduling can deliver strong anti-tumour efficacy in a range of combination settings and provides a promising alternative to continuous dosing. Such schedules merit clinical evaluation. Citation Format: Kevin Hudson, Urs Hancox, Cath Trigwell, Phillippa Dudley, Lyndsey Hanson, Robert McEwen, Alys Jones, Marie Cumberbatch, Urszula Polanska, Rebecca Ellston, Oona Delpuech, Pablo Morentin Gutierrez, Lara Ward, Francisco Cruzalegui, Stephen Green. High dose intermittent scheduling of AZD8835, a novel potent and selective inhibitor of PI3Kα and PI3Kδ, identifies potential treatment strategies for PIK3CA-dependent cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2665. doi:10.1158/1538-7445.AM2015-2665

Abstract 2830: Discovery of AZD8835, a potent and selective inhibitor of PI3Kα and PI3Kδ for the treatment of PIK3CA-dependent cancers

Abstract Amplification and mutations of the PIK3CA gene encoding the p110α catalytic unit of PI3Kα occur frequently in many human cancers including ca. 25% of breast cancers. The three most common mutations (E542K, E545K and H1047R) in the PIK3CA gene have been confirmed as activating mutations. The strong evidence pointing at the oncogenic nature of the PIK3CA gene mutations and their high frequency have fuelled a strong interest in developing selective inhibitors of PI3Kα. During the lead generation phase of this project, we identified a series of 2-amino pyridines / pyrazines as potent inhibitors of PI3Kα both at the enzymatic and the cellular level. However, these initial leads displayed poor general kinase selectivity. We report the optimisation of this series, with a special focus on optimisation of general kinase selectivity. This work led to the identification of AZD8835, a potent inhibitor of PI3Kα (wild type, E545K and H1047R mutations) and PI3Kδ with excellent selectivity vs. PI3Kβ, PI3Kγ and an excellent general kinase selectivity. AZD8835 is a potent inhibitor of p-Akt in cells sensitive to PI3Kα inhibition (IC50 0.057 μM in PIK3CA mutant human breast ductal carcinoma BT474 cell line) and in cells sensitive to PI3Kδ inhibition (IC50 0.049 μM in JeKo-1 B cell line), but not to cells sensitive to PI3Kβ inhibition (IC50 3.5 μM in PTEN null breast adenocarcinoma MDA-MB-468 cell line) or PI3Kγ inhibition (IC50 0.53 μM in monocytic RAW264 cell line). Furthermore, AZD8835 displayed high metabolic stability and suitable physical properties for oral administration. In vivo, inhibition of p-Akt was observed at 30 minutes and 8 hours in the PIK3CA H1047R mutant SKOV3 tumour model in mice after chronic oral administration of AZD8835 (25 mg/kg b.i.d.). AZD8835 showed excellent tumour growth inhibition in this same model after chronic oral administration (25 mg/kg b.i.d.). Based on these results, AZD8835 was selected as a clinical candidate for the treatment of PIK3CA-dependant cancers and has recently entered phase I clinical trials. Citation Format: Bernard Barlaam, Sabina Cosulich, Benedicte Delouvrie, Martina Fitzek, Herve Germain, Stephen Green, Craig S. Harris, Kevin Hudson, Christine Lambert-van der Brempt, Maryannick Lamorlette, Le Griffon Antoine, Remy Morgentin, Gilles Ouvry, Ken Page, Georges Pasquet, Linette Ruston, Twana Saleh, Michel Vautier, Lara Ward. Discovery of AZD8835, a potent and selective inhibitor of PI3Kα and PI3Kδ for the treatment of PIK3CA-dependent cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2830. doi:10.1158/1538-7445.AM2015-2830

Abstract 4700: The novel and selective PI3Kδ inhibitor, RV1001, displays single agent biologic activity in spontaneous canine NHL

Abstract Background: Non-Hodgkin's lymphoma (NHL) is the most common cancer in dogs with over 70,000 new cases diagnosed each year. Evidence suggests that canine NHL is an excellent spontaneous large animal model of human NHL, particularly for the evaluation of new therapeutics. The purpose of this work was to establish the preclinical profile and clinical efficacy of the novel, potent, and selective PI3Kδ inhibitor RV1001 in canine NHL with ultimate goal of informing the development of such inhibitors in human NHL. Methods: Activity of RV1001 on individual PI3K isoforms was determined using a Homogenous Time Resolved Fluorescence assay (Millipore, Billerica, MA). Potency of the compound on the delta isoform was further corroborated in anti-FcϵR1 induced CD63 expression studies using human whole blood. Ability of RV1001 to arrest growth across several cell lines was determined by a MTT assay. Inhibition of endogenous pAKT in cell lines and primary canine NHL cells was determined by Western blotting. A phase I study of RV1001 was performed in dogs with T and B naïve and drug resistant NHL to assess safety, pharmacokinetic profile and response to therapy. Results: RV1001 demonstrated significant potency against PI3Kδ (39.3 nM) with several fold selectivity over the α (>250), β (>30), and γ (>20) isoforms. In addition, RV1001 inhibited anti-FcϵR1 induced CD63 expression, a marker of PI3Kδ signaling, in human whole blood basophils with an EC50 of 94.7 nM. Proliferation assays in leukemic cells indicated that the compound arrested growth at concentrations between 1.5 and 13 μM. Reduced PI3Kδ signaling was manifested by a reduction in pAKT with half-maximal inhibition noticed between 10 and 100 nM. Oral administration of 100 mg/kg RV1001 to healthy dogs over a 14-day period resulted in significant accumulation (Cmax = 70 μM on Day 1 versus 186 μM on Day 14; PI3Kδ = 69 μM on Day 14). To date, 9 dogs have been entered into the Phase I clinical trial beginning at a dose of 10 mg/kg SID using a standard 3×3 design. One dog with T cell NHL has had a durable complete response to therapy (10 mg/kg), and 3 additional dogs have had partial responses (15 mg/kg n = 2 and 25 mg/kg n = 1) for an objective response rate of 44%. Adverse events associated with RV1001 administration include grade 1 anorexia and grade 3 and 4 LFT elevation. Pharmacokinetic analyses revealed Cmax concentrations of 10-25 μM with AUC0-t ranging from 50 to 100 μM.hr with accumulation evident after 3-4 weeks of dosing. Exploration of alternative dosing regimens is ongoing to eliminate the drug accumulation and thus circumvent the observed hepatotoxicity. Conclusions: RV1001 exhibits potent activity against PI3Kδ with associated inhibition of AKT phosphorylation, excellent oral bioavailability, and strong single agent efficacy in dogs with both B and T NHL. Data generated from these studies will have broad applicability to the further development of isoform specific PI3K inhibitors in people with NHL. Citation Format: Cheryl A. London, Sarah B. Rippy, Misty D. Bear, Kim Cronin, Andrew H. Abbo, Kumar V. Penmetsa, Srikant Viswanadha, Swaroop Vakkalanka. The novel and selective PI3Kδ inhibitor, RV1001, displays single agent biologic activity in spontaneous canine NHL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4700. doi:10.1158/1538-7445.AM2015-4700

Synthesis and SAR study of potent and selective PI3Kδ inhibitors

2,3,4-Substituted quinolines such as (10a) were found to be potent inhibitors of PI3Kδ in both biochemical and cellular assays with good selectivity over three other class I PI3K isoforms. Some of those analogs showed favorable pharmacokinetic properties.▪

Discovery of (R)-8-(1-(3,5-difluorophenylamino)ethyl)-N,N-dimethyl-2-morpholino-4-oxo-4H-chromene-6-carboxamide (AZD8186): a potent and selective inhibitor of PI3Kβ and PI3Kδ for the treatment of PTEN-deficient cancers.

Several studies have highlighted the dependency of PTEN deficient tumors to PI3Kβ activity and specific inhibition of PI3Kδ has been shown activity against human B-cell cancers. We describe the discovery and optimization of a series of 8-(1-anilino)ethyl)-2-morpholino-4-oxo-4H-chromene-6-carboxamides as PI3Kβ/δ inhibitors, which led to the discovery of the clinical candidate 13, also known as AZD8186. On the basis of the lower lipophilicity of the chromen-4-one core compared to the previously utilized pyrido[1,2-a]pyrimid-4-one core, this series of compounds displayed high metabolic stability and suitable physical properties for oral administration. Compound 13 showed profound pharmacodynamic modulation of p-Akt in PTEN-deficient PC3 prostate tumor bearing mice after oral administration and showed complete inhibition of tumor growth in the mouse PTEN-deficient PC3 prostate tumor xenograft model. 13 was selected as a clinical candidate for treatment of PTEN-deficient cancers and has recently entered phase I clinical trials.

Evaluation of WO2014075392 and WO2014075393, Merck’s first PI3Kδ inhibitor filings

Introduction: There is considerable interest in the development of selective PI3Kδ inhibitors for the treatment of inflammatory diseases and haematological cancers. Merck has no previous filings in this field but licensed Exelixis' programme, including its lead compound XL-499, in December 2011.Areas covered: Both applications claim novel 9-alkyl-6,8-disubstituted purine derivatives as selective δ inhibitors for the treatment of asthma, obstructive airways disease, arthritis and cancer. The two applications differ in the range of exemplified substituents, the first focusing on 8-heteroaryl substituted purines, the second on 8-aminopurine derivatives. Many of the exemplified compounds have IC50 values < 10 nM against PI3Kδ with a number having sub-nanomolar potency.Expert Opinion: The compounds appear to be XL-499 derivatives, some of which are more potent than XL-499. The compounds claimed by Merck are some of the most potent PI3Kδ inhibitors yet described but it is unclear whether a development compound has been identified.

PP-055 EVALUATION THE RELATIONSHIP BETWEEN MTHFR GENE POLYMORPHISM AND PREGNANCY LOSS

Background: Idelalisib (IDELA) is a potent and selective inhibitor of PI3Kδ, which is critical for activation, proliferation and survival of B cells and their homing and retention in lymphoid tissues. An unmet need exists for effective therapies in patients with CLL positive for del(17p) and other adverse prognostic factors. This report describes the efficacy of IDELA in combination with rituximab (R) in such high-risk relapsed patients. Methods: Samples for del(17p), del(11q), TP53mut, IGHVmut, ZAP70 and CD38 expression, and β2-microglobulin were collected prospectively and tested using standard methods. Patients were stratified based on presence of del(17p) and/or TP53mut, and on IGHV mutational status. The endpoints evaluated in the high-risk subpopulations in the preplanned 1st interim analysis include progression-free-survival (PFS) and overall response rate (ORR). The primary study analysis was reported in NEJM 2014. Results: IDELA+R retained robust efficacy across all high-risk subpopulations. Importantly, IDELA+R achieved 76.5% ORR and PFS HR 0.13 in the highest risk patients who were positive for both del(17p) and TP53mut, compared to 80.4% ORR and PFS HR 0.17 in those who had neither present. Conclusions: These results confirm the retained robust efficacy of IDELA in high-risk CLL subpopulations and support IDELA as a potentially important novel treatment for patients with CLL positive for del(17p) and other adverse prognostic factors.

Abstract CT204: The pharmacokinetics and safety of idelalisib in subjects with severe renal impairment

Abstract Background: Idelalisib (IDELA) is a novel, potent inhibitor of PI3Kδ, which has been shown to be prominently expressed in cells of hematopoietic origin. Renal elimination plays a minor role on elimination of IDELA in humans (∼ 15% of dose excreted in urine). The objective of the present study was to evaluate the PK and safety of IDELA and GS-563117 (primary metabolite) in subjects with severe renal impairment (RI) following a single oral dose of IDELA. Methods: Subjects with severe RI (eGFR 15 - 29 ml/min) and healthy matched controls (eGFR ≥ 80 ml/min) received IDELA 150 mg single dose. Blood and urine samples were collected over 6 days and IDELA/GS-563117 levels were measured using a validated LC/MS/MS method. Geometric least-squares mean ratio and 90% confidence interval of PK exposure parameters in the RI group versus matched controls were calculated, with clinically relevant exposure change defined as ≥ two-fold increase. Safety assessments were performed throughout the study. Results: A total of 12 subjects were enrolled in the study. The majority of subjects were female, white, and of non-Hispanic/Latino ethnicity, and the median age was similar between the 2 groups. Study treatments were generally well tolerated. All AEs were Grade 1 in severity. No clinically significant abnormal changes were observed in vital signs and ECG results from baseline. The AUClast, AUCinf, and Cmax geometric least-squares mean ratio were 127%, 127%, and 105% for IDELA and 124%, 124%, and 96% for GS-563117, respectively, for subjects with severe renal impairment vs matched control subjects (Table 1). There were no relevant relationships between IDELA or GS-563117 exposures and baseline eGFR. Conclusion: There were no clinically relevant differences in the PK of IDELA or GS-563117 between subjects with severe RI vs matched healthy control subjects following a single oral dose of IDELA 150 mg. Dose adjustments for IDELA are not considered necessary in subjects with mild, moderate, or severe renal impairment. PK Parameters in Severe RI and Matched Healthy SubjectsPK ParameterMean (%CV)% Geometric Least Squares Means Ratio(90% CI)Matched Control Group(N = 6)Severe Renal Impairment Group(N = 6)IDELACmax (ng/mL)2530 (27)2680 (28)105 (77, 143)Tmax (h)2.50 (2.00, 3.00)3.00 (2.00, 3.00)t1/2 (h)9.48 (7.37, 10.2)12.1 (6.25, 23.1)AUClast (ng·h/mL)11700 (20)15500 (37)127 (91, 176)AUCinf (ng·h/mL)11800 (20)15700 (37)127 (92, 176)GS-563117Cmax (ng/mL)2590 (21)2570 (37)96 (69, 134)Tmax (h)4.00 (3.00, 4.00)4.00 (4.00, 6.00)t1/2 (h)11.4 (10.9, 12.1)15.8 (8.87, 21.7)AUClast (ng·h/mL)35800 (31)50300 (60)124 (71, 215)AUCinf (ng·h/mL)36000 (30)50800 (61)124 (72, 215) Tmax and T1/2 expressed as median (Q1, Q3) Citation Format: Feng Jin, Michelle Robeson, Huafeng Zhou, Grace Hisoire, Srini Ramanathan. The pharmacokinetics and safety of idelalisib in subjects with severe renal impairment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT204. doi:10.1158/1538-7445.AM2014-CT204

Abstract 4518: The PI3K-δ inhibitor TGR-1202 induces cytotoxicity and inhibits phosphorylation of AKT in 17p deleted and non-17p deleted CLL cells in vitro

Abstract Background: The PI3K pathway is a central pro-survival mechanism in chronic lymphocytic leukemia (CLL), with expression of the delta isoform of PI3K largely restricted to lymphocytes. Clinical evaluation of PI3K-δ inhibitors, such as idelalisib, in CLL patients has been promising. CLL patients with 17p deletion (17pdel) have displayed inferior responses to chemo-immunotherapy, and we hypothesized that inhibiting the PI3K/AKT pathway would be equally efficacious in patients with and without 17pdel. TGR-1202 is a novel PI3K-δ specific inhibitor with unique pharmacologic properties and demonstrated in-vitro activity. Herein, we evaluate the in vitro effect of TGR-1202 on CLL lymphocytes, specifically evaluating differences between 17pdel CLL and non-17pdel CLL samples. Methods: We collected blood from CLL patients seen at the Duke Center for CLL and enrolled in IRB approved protocols at the Duke University and Durham VA Medical Centers. CLL lymphocytes were isolated using negative selection yielding greater than 95% purity. Primary CLL cells were incubated with serial dilutions of TGR-1202 tested for apoptosis by activated caspase-3 and 7AAD staining measured by flow cytometry. Cytotoxicity was measured using the colorimetric MTS reagent. Phosphorylated AKT (S473) was measured by flow cytometry after incubation with TGR-1202 followed by incubation with anti-IgM or anti-IgD. AKT phosphorylation was quantified by median fluorescent intensity (MFI). Results: We have evaluated TGR-1202 in 15 CLL lymphocyte samples, 10 with normal non-17pdel cytogenetics, and 5 with 17pdel. TGR-1202 induced cytotoxicity and apoptosis in a dose-dependent manner in concentrations between 4 nM and 25 μM with no significant difference in results observed between normal CLL lymphocytes those that had 17pdel cytogenetics. The mean ED50 in 17pdel CLL cells was 326 nM, whereas the mean ED50 in non-17pdel CLL cells was 887 nM, although due to the small sample size, this difference was not statistically significant (p = 0.16, t-test). We also found that TGR-1202 significantly suppressed AKT phosphorylation in CLL lymphocytes. Conclusions: TGR-1202 is a potent PI3K-δ inhibitor that suppresses AKT phosphorylation and induces apoptosis-dependent cytotoxicity in primary CLL lymphocytes, both with favorable and adverse cytogenetics. We observed that higher concentrations of TGR-1202 were required to induce cytotoxicity in non-17pdel CLL lymphocytes on average, than in 17pdel CLL. Evaluation of additional CLL cell samples with 17pdel is ongoing to confirm initial findings. A Phase I trial with once-daily TGR-1202 is currently ongoing in patients with hematologic malignancies, including CLL. To date, TGR-1202 has been well tolerated, with no drug-related hepatic toxicities observed. Our initial observations suggest that clinical evaluation of TGR-1202 enriched in CLL patients with 17pdel may be warranted. Citation Format: Daphne R. Friedman, Tiffany Simms, Sallie D. Allgood, Danielle M. Brander, Peter Sportelli, Hari P. Miskin, Swaroop Vakkalanka, Srikant Viswanadha, J. Brice Weinberg, Mark C. Lanasa. The PI3K-δ inhibitor TGR-1202 induces cytotoxicity and inhibits phosphorylation of AKT in 17p deleted and non-17p deleted CLL cells in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4518. doi:10.1158/1538-7445.AM2014-4518

Abstract 4531: INCB40093 is a selective PI3Kδ inhibitor with potent antiproliferative activity against human B-cell tumors

Abstract Phosphatidylinositol 3-kinases (PI3Ks) belong to a family of lipid signaling kinases that phosphorylate phosphatidylinositol-4,5-bisphosphate, giving rise to phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 functions as a second messenger that controls a number of cellular processes, including growth, survival, adhesion and migration. The delta isoform of PI3K (PI3Kδ) plays an essential role in B-cell development and functions by mediating the signaling of key receptors on B cells including BCR, BAFF-R, IL-4R, CD40 and TLRs. In addition, increased malignant B cell proliferation and survival has been associated with aberrant activation of PI3Kδ, and data from recent clinical studies using idelalisib, a PI3Kδ specific inhibitor, indicate that the enzyme is a therapeutic target for the treatment of B cell lymphomas. INCB40093 is a novel, potent small molecule inhibitor of PI3Kδ with 74 to &amp;gt; 900-fold selectivity over the other PI3K family members. It also does not inhibit (&amp;lt; 30% inhibition) a broad panel of 196 other kinases when tested at a concentration of 1 μM. INCB40093 blocks the proliferation of primary B cells induced by the activation of PI3Kδ-mediated signaling through several key receptors (e.g. BCR, CD40). More importantly, INCB40093 is potent (IC50 values of ∼10-100 nM) in cell-based assays relevant to the pathogenesis of B cell malignancies, such as PI3Kδ-mediated signaling and growth of human B cell lines. This effect is not due to general cytotoxicity because 10 μM INCB40093 had no significant effect on the growth of non-lymphoid cell lines. Compared to the inhibition of primary B cell proliferation, INCB40093 is at least 50 times less potent in assays that measure human T cell or NK cell proliferation, and neutrophil and macrophage function, suggesting that the impact of INCB40093 on the human immune system will likely be restricted to B cells. In vivo, the effects of INCB40093 were evaluated in two human hematological cancer xenograft models. In the Pfeiffer model of non-Hodgkin's lymphoma, oral administration of INCB40093 inhibited tumor growth as a single agent. INCB40093 also potentiated the anti-tumor effects of the clinically relevant chemotherapeutic agent, bendamustine. The tumor growth inhibitory effects of INCB40093 were also demonstrated in a myeloma tumor xenograft model, INA-6.Tu1. Taken together, pharmacological data obtained in both in vitro and in vivo model systems support the potential utility of orally administered INCB40093 in the treatment of B cell malignancies. Citation Format: Niu Shin, Kathy Wang, Leslie Hall, Qian Wang, Gengjie Yang, Yanlong Li, Yun-Long Li, Maryanne Covington, Jordan Fridman, Robert Newton, Peggy Scherle. INCB40093 is a selective PI3Kδ inhibitor with potent antiproliferative activity against human B-cell tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4531. doi:10.1158/1538-7445.AM2014-4531

Abstract 4207: JAK inhibition reverses IL10-mediated resistance to B cell receptor (BCR) pathway inhibition in DLBCL

Abstract Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma, and can be classified into activated B-cell like (ABC) and germinal center B-cell like (GCB) DLBCL according to gene expression profiling. In both subtypes, dysregulation of the B cell receptor (BCR) signaling pathway plays a critical role in tumorigenesis and survival. BCR activation induces phosphorylation of CD79A/B by Src family tyrosine kinases, which leads to the activation of the BTK and PI3Kδ/Akt pathways. Oncogenic mutations have been identified in DLBCL impacting CD79A/B (BCR complex), PTEN (negative regulator of PI3K), CARD11 and MYD88 (NF-kB activating factors) function. The influence of these mutations on the DLBCL phenotype appears to be further modified by aberrant cytokine signaling and JAK/Stat pathway activation. High levels of JAK/Stat-activating cytokines such as IL6 and IL10 are present in DLBCL, and patients with high serum IL10 have been shown to have shorter event-free survival and a higher international Prognostic Index score. Therefore, concurrent inhibition of both the BCR and JAK/Stat pathways may provide an attractive therapeutic approach in DLBCL. We have developed and characterized a potent and selective PI3Kδ inhibitor INCB040093 and have established that DLBCL cells are sensitive to INCB040093 treatment. We demonstrate that IL10 activates the JAK/Stat signaling pathway in DLBCL cells, which renders the cells resistant to INCB040093 in cell proliferation assays. This resistance can be reversed by co-treatment with a JAK1/JAK2 inhibitor. Although INCB040093 induces apoptosis in DLBCL cells, concurrent activation of the JAK/Stat pathway by IL10 prevents INCB040093-induced apoptosis, an effect that can be abrogated by a JAK1/JAK2 inhibitor. Similar resistance to IL-10 is observed with the BTK inhibitor, ibrutinib, which can also be reversed by a JAK1/JAK2 inhibitor. Interestingly, a JAK1 selective inhibitor is as effective as a JAK1/JAK2 inhibitor in reversing the IL10-mediated resistance to PI3Kδ and BTK inhibition. Combination treatment blocks both PI3K/Akt and JAK/Stat signaling pathways and results in the down-regulation of pro-survival proteins, including Pim2 and c-myc. These data illustrate the functional cross-talk that exists in DLBCL between the BCR and JAK/Stat signaling pathways, and support the clinical evaluation of combined BCR and JAK inhibition as a novel approach to targeted therapy. Citation Format: Xuesong (Mike) Liu, Margaret Favata, Jun Li, Niu Shin, Kathy He Wang, Qian Wang, Yun-Long Li, Andrew Combs, Chu-Biao Xue, Robert Newton, Kris Vaddi, Peggy Scherle. JAK inhibition reverses IL10-mediated resistance to B cell receptor (BCR) pathway inhibition in DLBCL. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4207. doi:10.1158/1538-7445.AM2014-4207

Abstract 4529: The PI3Kδ inhibitor, idelalisib, inhibits transcription and translation through PI3K/Akt pathway in mantle cell lymphoma

Abstract PI3Kδ is the major PI3K family class I isoform expressed in B-cells that is known to promote cancer cell growth and survival. PI3Kδ pathway is activated through cell surface receptors, e.g. B-cell receptor signaling. This suggests that PI3Kδ is a therapeutic target for B-cell hematological malignancies. Idelalisib is a potent and selective PI3Kδ inhibitor (IC50=19nM) that is currently under investigation in Phase II/III trials in B-cell neoplasms. In solid tumors, it has been established that PI3K/Akt axis regulates transcription and translation processes. Based on the overexpression and hyperactivity of PI3Kδ in B-cell lymphoma such as mantle cell lymphoma (MCL), and PI3Kδ-specific inhibition by idelalisib, we hypothesized that idelalisib treatment will 1) impact the transcription and translation processes that are PI3K/Akt-dependent, 2) disrupt RNA and protein synthetic capacity, and 3) decrease short-lived oncogenic mRNA and proteins levels that are important for MCL cell survival. Idelalisib (0.5 to 5μM) induced modest levels of apoptosis (&amp;lt;15%) in MCL cell lines and primary cells. A reverse-phase protein array (RPPA) study suggested that idelalisib treatment resulted in a decrease in phosphorylation of transcription regulators (GSK-3αβ and c-Myc), and protein synthesis regulators (S6, p70S6K, 4E-BP1, eIF4 and mTOR) in MCL cell lines and primary cells. Immunoblot analysis suggests that a 4hr treatment with idelalisib (0.5 or 1μM) resulted in a decrease of phosphorylated Akt (T308 and S473) levels in both JeKo-1 and Mino cells following IgM stimulation. In parallel, phospho-levels of GSK-3β (S9), 4E-BP1 (S65) and S6 (S235/236) were also decreased. In IgM-stimulated MCL cell lines, moderate to significant decrease in global RNA synthesis (10-15% decrease in JeKo-1, 50% decrease in Mino) and protein synthesis (15% decrease in JeKo-1, &amp;gt;20% decrease in Mino) were detected with 72hr incubation of idelalisib (0.5 to 1μM). Consistent with cell line data, in B-cell lymphoma primary cells, transcription levels were decreased by 20% or &amp;gt;50% following a 24hr treatment with 1 or 5µM idelalisib, respectively; and a 40% decrease of translation level was detected with 5µM idelalisib. In Jeko-1, early response genes MCL1 mRNA level was reduced following 24hr idelalisib treatment, whereas the other mRNAs (PIM2, MYC, CCND1) were decreased to a lesser extent. Mcl-1 and cyclin D1 protein levels were decreased in JeKo-1 and Mino cells following short incubation of idelalisib (0.5 or 1µM). In summary, idelalisib was effective in reducing phosphorylation of Akt and its downstream effectors in MCL cell lines (GSK-3β, 4E-BP1 and S6). Idelalisib inhibits global RNA and protein synthesis, and decreases the levels of short-lived survival proteins. Collectively, these data imply that similar to solid tumors, PI3K regulates key factors involved in gene transcription and protein translation in MCL. Citation Format: Qingshan Yang, Lisa S. Chen, Sattva S. Neelapu, Varsha Gandhi. The PI3Kδ inhibitor, idelalisib, inhibits transcription and translation through PI3K/Akt pathway in mantle cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4529. doi:10.1158/1538-7445.AM2014-4529

The pharmacokinetics and safety of idelalisib in subjects with moderate or severe hepatic impairment.

2592 Background: Idelalisib (IDELA) is a potent inhibitor of PI3Kδ, which has been shown to be prominently expressed in cells of hematopoietic origin. IDELA is metabolized primarily by aldehyde oxi...

Drug interaction profile of idelalisib and its major metabolite, GS-563117.

2593 Background: Idelalisib (IDELA), a potent PI3Kδ inhibitor in Phase 3 studies for hematological malignancies, is metabolized mainly by aldehyde oxidase to GS-563117 and partially by CYP3A. In vitro, IDELA inhibits Pgp, OATP1B1, and 1B3 and GS-563117 is a time-dependent CYP3A inhibitor. Accordingly, the present study evaluated the effects of IDELA on these transporters/enzyme, and rifampin (RIF), a strong inducer, on IDELA pharmacokinetics (PK). Methods: Single oral dose of probe Pgp (digoxin; DIG), OATP1B1/1B3 (rosuvastatin; ROS), and CYP3A (midazolam; MDZ) substrates were given alone or with IDELA 150 mg BID. IDELA 150 mg single dose was given alone or with RIF 600 mg QD. PK of IDELA, GS-563117, and probes were determined (N = 11 to 12). Lack of PK interaction was defined as 90% confidence interval of the geometric mean ratio within 70-143% (combination vs alone). Safety was assessed throughout the study. Results: The study enrolled mainly white males (N=24) with median age of 38 years. Upon treatment, most common adverse events (AE) were headache and pyrexia; Gr 3 transaminase increases (5/24 subjects) were reversible. Two subjects had serious AEs after treatment completion. No clinically relevant changes in vital signs/ECGs were noted. IDELA did not affect DIG or ROS PK but increased MDZ plasma exposure, consistent with GS-563117 CYP3A inhibition in vitro. RIF reduced IDELA and GS-563117 exposures, indicating enhanced IDELA metabolism by CYP3A with a strong inducer. Conclusions: IDELA does not affect common drug transporters and is a moderate inhibitor of CYP3A due to GS-563117. Strong inducers of CYP3A decrease IDELA exposure. PK parameters (mean [%CV]). DIG DIG DIG + IDELA % Geometric least squares means ratio (90% CI) Cmax (ng/mL) 1.6 (18) 1.9 (19) 124 (115, 133) AUCinf (ng·h/mL) 38.0 (28) 37.0 (28) 100 (87, 115) ROS ROS ROS + IDELA Cmax (ng/mL) 1.6 (52) 1.9 (67) 115 (97, 137) AUCinf (ng·h/mL) 23.1 (32) 25.7 (59) 103 (83, 124) MDZ MDZ MDZ + IDELA Cmax (ng/mL) 16.6 (29) 38.1 (14) 238 (200, 283) AUCinf (ng·h/mL) 89.1 (36) 454 (24) 537 (456, 632) IDELA IDELA IDELA + RIF Cmax (ng/mL) 2,150 (24) 933 (41) 42 (36, 49) AUCinf (ng·h/mL) 9,600 (37) 2,290 (40) 25 (23, 27) GS-563117 Cmax (ng/mL) 1,990 (39) 675 (44) 32 (28, 37) AUCinf (ng·h/mL) 21,400 (59) 2,240 (49) 11 (9, 12)

Efficacy of idelalisib in CLL subpopulations harboring del(17p) and other adverse prognostic factors: Results from a phase 3, randomized, double-blind, placebo-controlled trial.

7011^ Background: Idelalisib (IDELA) is a potent and selective inhibitor of PI3Kδ, which is critical for activation, proliferation and survival of B cells and their homing and retention in lymphoid...

Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts: ACTIONS ON CYTOSKELETAL ORGANIZATION, SURVIVAL, AND RESORPTION

Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (β), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15-20 min to 65-75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kβ and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics.

The Potent PI3K-δ,γ Inhibitor, IPI-145, Exhibits Preclinical Activity In Murine and Human T-Cell Acute Lymphoblastic Leukemia

Abstract Phosphoinositide-3 kinases (PI3Ks) are key cellular signaling proteins that act as a central node for relaying signals from cell surface receptors to downstream mediators, such as AKT. The PI3K-δ and PI3K-γ isoforms are preferentially expressed in normal and malignant leukocytes where they play critical roles in cell differentiation, migration, and proliferation. Constitutive activity of the PI3K pathway is common in T-cell acute lymphoblastic leukemia (T-ALL) and frequently involves the deletion of PTEN, the phosphatase that negatively regulates the PI3K pathway. An important role for the PI3K-δ and PI3K-γ isoforms has been demonstrated in the Pten-deleted genetically engineered murine model of T-ALL in conjunction with PI3K-δ and/or PI3K-γ gene knock outs (Subramanian et al, Cancer Cell, 2012). IPI-145 is a potent inhibitor of PI3K-δ and PI3K-γ currently being studied in a Phase 1 trial (IPI-145-02) in patients with advanced hematologic malignancies, including T-ALL (ClinicalTrials.gov NCT01476657). We performed in vitro studies to address the sensitivity of human and murine T-ALL cell lines to IPI-145 and to additional PI3K inhibitors with defined isoform selectivity. The human T-ALL cells examined were from cell lines that lack PTEN protein expression (Loucy, MOLT-4, CCRF-CEM, CEM/C2, p12 Ichikawa, and Karpas-45) and cell lines that express PTEN protein (MOLT13 and MOLT16). In addition, two murine cell lines derived from a Pten-deleted model of T-ALL (LPN049 and LPN236) were studied. The expression levels of class I PI3K isoforms were determined by western blotting and quantitative RT-PCR, which revealed varying levels of protein and RNA expression across the cell lines. In vitro treatment of human T-ALL cells with IPI-145 resulted in variable degrees of growth inhibition, with the PTEN-deficient Loucy cell line demonstrating the greatest sensitivity with an IC50 of 245 nM. In the cell lines tested, growth inhibition to IPI-145 was only seen in PTEN-deficient human cell lines, whereas all PTEN-expressing human T-ALL cell lines were resistant to IPI-145 (IC50 &gt; 10 uM). However, not all PTEN-deficient human T-ALL cells demonstrated sensitivity to IPI-145 (e.g., CEM/C2), indicating that loss of PTEN does not confer sensitivity to PI3K inhibition in all settings. Inhibition of phospho-AKT (pAKT) correlated with growth inhibition, with an IC50 of 286 nM in the Loucy cell line. Studies to evaluate the mechanism of growth inhibition revealed that IPI-145 treatment resulted in apoptosis of sensitive cells as measured by 7-AAD and Annexin V staining. Cell lines derived from the Pten-deleted murine T-ALL model were also sensitive to IPI-145 with IC50s in the 300-600 nM range as measured by MTT assay. In addition, IPI-145 led to apoptosis, as measured by cleaved Caspase 3 and 7-AAD/Annexin V. Interestingly, Pten-deleted murine T-ALL cell lines showed down-regulation of pAKT and c-MYC expression with IPI-145 in a dose responsive manner that corresponded with increasing activated Caspase-3 expression. In NOTCH1-expressing murine T-ALL cell lines, down regulation of NOTCH1 and activated NOTCH1 was also observed in parallel with c-MYC down-regulation. To explore further the individual contributions of the varying PI3K isoforms on T-ALL cell growth, the effect of IPI-145 on tumor cell growth was compared with PI3K-δ, PI3K-δ,γ, and PI3K-β selective compounds in the Loucy PTEN-deficient T-ALL cell line. These experiments support an anti-leukemic effect for both PI3K-δ and PI3K-γ inhibition and indicate that the greatest effect is seen with combined PI3K-δ and PI3K-γ inhibition. A role for PI3K-β in T-ALL cell survival was not observed. Evaluation of the in vivo activity of IPI-145 on Loucy xenografts, as well as PTEN-expressing MOLT-13 xenografts, is ongoing. Together, these data provide a strong rationale for combined targeted inhibition of PI3K-δ and PI3K-γ in T-ALL. Disclosures: Huang: Infinity Pharmaceuticals, Inc.: Research Funding. Proctor:Infinity Pharmaceuticals, Inc.: Employment. Yang:Infinity Pharmaceuticals, Inc.: Research Funding. Gao:Infinity Pharmaceuticals, Inc.: Research Funding. Zhang:Infinity Pharmaceuticals, Inc.: Research Funding. Huang:Infinity Pharmaceuticals, Inc.: Research Funding. Changelian:Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment. McGovern:Infinity Pharmaceuticals, Inc.: Employment. You:Infinity Pharmaceuticals, Inc.: Research Funding.

PI3K-δ and PI3K-γ Inhibition by IPI-145 Abrogates Immune Responses and Suppresses Activity in Autoimmune and Inflammatory Disease Models

Phosphoinositide-3 kinase (PI3K)-δ and PI3K-γ are preferentially expressed in immune cells, and inhibitors targeting these isoforms are hypothesized to have anti-inflammatory activity by affecting the adaptive and innate immune response. We report on a potent oral PI3K-δ and PI3K-γ inhibitor (IPI-145) and characterize this compound in biochemical, cellular, and invivo assays. These studies demonstrate that IPI-145 exerts profound effects on adaptive and innate immunity by inhibiting B and Tcell proliferation, blocking neutrophil migration, and inhibiting basophil activation. We explored thetherapeutic value of combined PI3K-δ and PI3K-γ blockade, and IPI-145 showed potent activity in collagen-induced arthritis, ovalbumin-induced asthma, and systemic lupus erythematosus rodent models. These findings support the hypothesis that inhibition of immune function can be achieved through PI3K-δ and PI3K-γ blockade, potentially leading to significant therapeutic effects in multipleinflammatory, autoimmune, and hematologic diseases.

Identification of GNE-293, a potent and selective PI3Kδ inhibitor: Navigating in vitro genotoxicity while improving potency and selectivity

In an effort to identify potent and isoform selective inhibitors of PI3Kδ, GNE-293 (34) was identified. Inhibitor 2 was found to induce micronuclei formation in both the MNT and HCA in vitro assays. Compounds testing negative for genotoxicity were successfully identified through modifications of the 2-benzimidazole substituent and the methylene moiety to disrupt planarity. A variety of heteroatom linkers were explored to examine their effect on potency and isoform selectivity by restricting torsional angles to favor ligand interactions with PI3Kδ’s Trp760. These modifications also resulted in an improved in vivo pharmacokinetic profile.

Abstract 26: Evaluation of the safety, pharmacokinetics and pharmacodynamics of GS-9973, a novel SYK inhibitor, and GS-1101, a novel PI3Kδ inhibitor, when administered alone or in combination in humans.

Abstract Background: GS-9973 is a selective, reversible, small molecule spleen tyrosine kinase (Syk) inhibitor. GS-1101 is a potent and selective PI3Kδ inhibitor currently in Phase 3 studies. The safety, pharmacokinetics (PK), pharmacodynamics (PD) of GS-9973 given alone or together with GS-1101 were evaluated in healthy subjects. Methods: GS-9973 and GS-1101 were each given twice-daily (BID) with food for 4 days (Day 4: AM dose only) either alone or together as follows: Cohort 1: 200 mg and 100 mg, respectively; Cohort 2: 600 mg and 100 mg, respectively; Cohort 3: 600 mg and 150 mg, respectively. Along with PK, PD samples were assessed for functional inhibition of ex vivo αIgE-stimulated CD63 expression on basophils. Safety was assessed throughout the study. Results: Across cohorts, subjects were ≥ 83% white, 100% female, with mean ages of 20 to 57 years. Adverse events (AEs) were generally mild to moderate; one subject discontinued due to Grade 1 ALT/AST elevation during GS-1101 150 mg dosing. Most common AEs were headache and somnolence. GS-9973 exposures were higher with GS-1101 (51% to 64% for AUCtau; 74% to 96% for Ctau; Table 1). Minor changes in GS-1101 exposures were noted. GS-9973 (600 mg) plus GS-1101 (≥ 100 mg) dosing provided near complete inhibition of CD63 expression over the dosing interval, consistent with synergy demonstrated from in vitro studies. Conclusions: GS-9973 and GS-1101 were generally well tolerated over the dose range evaluated. Coadministration of GS-9973 and GS-1101 provided substantial PD response over the dosing interval; moderately higher GS-9973 exposures and minor changes in GS-1101 exposures were noted. These safety, PD, and PK results support further clinical evaluation of these agents in combination. Table 1. GS-9973 and GS-1101 PK PK Parameter Mean (% CV) % Geometric Least Squares Means Ratio (90% CI) GS-9973 GS-9973 200 mg (N = 8) GS-9973 200 mg + GS-1101 100 mg (N = 8) Combination vs Alone AUCtau 4150 (48) 6750 (47) 1.64 (1.41, 1.91) Cmax 618 (40) 915 (38) 1.50 (1.24, 1.80) Ctau 201 (66) 379 (62) 1.96 (1.61, 2.38) GS-9973 GS-9973 600 mg (N = 16) GS-9973 600 mg + GS-1101 100/150 mg (N = 14) AUCtau 12700 (69) 16000 (55) 1.51 (1.30, 1.75) Cmax 1570 (60) 1900 (55) 1.38 (1.20, 1.59) Ctau 832 (82) 1120 (72) 1.74 (1.48, 2.03) GS-1101 GS-1101 100 mg (N = 16) GS-1101 100 + GS-9973 200 or 600 mg (N = 16) AUCtau 8070 (22) 9510 (25) 1.17 (1.13, 1.22) Cmax 1660 (18) 1880 (18) 1.13 (1.07, 1.20) Ctau 127 (44) 170 (50) 1.33 (1.20, 1.48) GS-1101 GS-1101 150 mg (N = 7) GS-1101 150 mg + GS-9973 600 mg (N = 6) AUCtau 13800 (15) 15800 (20) 1.11 (1.00, 1.24) Cmax 2680 (11) 2870 (21) 1.06 (0.93, 1.21) Ctau 320 (43) 411 (29) 1.23 (1.03, 1.47) Units for AUCtau: ng*h/ml; Cmax and Ctau: ng/ml; all doses BID Citation Format: Feng Jin, Julie A. Di Paolo, Lixin Shao, Ori Yellin, Michael J. Hawkins, Srinivasan Ramanathan. Evaluation of the safety, pharmacokinetics and pharmacodynamics of GS-9973, a novel SYK inhibitor, and GS-1101, a novel PI3Kδ inhibitor, when administered alone or in combination in humans. [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 26. doi:10.1158/1538-7445.AM2013-26