Circulating Oncometabolite 2-Hydroxyglutarate Is a Potential Surrogate Biomarker in Patients with Isocitrate Dehydrogenase-Mutant Intrahepatic Cholangiocarcinoma

Abstract Purpose: Mutations in the IDH1 and IDH2 (IDH1/2) genes occur in approximately 20% of intrahepatic cholangiocarcinoma and lead to accumulation of 2-hydroxyglutarate (2HG) in the tumor tissue. However, it remains unknown whether IDH1/2 mutations can lead to high levels of 2HG circulating in the blood and whether serum 2HG can be used as a biomarker for IDH1/2 mutational status and tumor burden in intrahepatic cholangiocarcinoma. Experimental Design: We initially measured serum 2HG concentration in blood samples collected from 31 patients with intrahepatic cholangiocarcinoma in a screening cohort. Findings were validated across 38 resected patients with intrahepatic cholangiocarcinoma from a second cohort with tumor volume measures. Circulating levels of 2HG were evaluated relative to IDH1/2 mutational status, tumor burden, and a number of clinical variables. Results: Circulating levels of 2HG in the screening cohort were significantly elevated in patients with IDH1/2-mutant (median, 478 ng/mL) versus IDH1/2–wild-type (median, 118 ng/mL) tumors (P &amp;lt; 0.001). This significance was maintained in the validation cohort (343 ng/mL vs. 55 ng/mL, P &amp;lt; 0.0001) and levels of 2HG directly correlated with tumor burden in IDH1/2-mutant cases (P &amp;lt; 0.05). Serum 2HG levels ≥170 ng/mL could predict the presence of an IDH1/2 mutation with a sensitivity of 83% and a specificity of 90%. No differences were noted between the allelic variants IDH1 or IDH2 in regard to the levels of circulating 2HG. Conclusions: This study indicates that circulating 2HG may be a surrogate biomarker of IDH1 or IDH2 mutation status in intrahepatic cholangiocarcinoma and that circulating 2HG levels may correlate directly with tumor burden. Clin Cancer Res; 20(7); 1884–90. ©2014 AACR.</jats:p

The test-retest reliability of ride perception during treadmill and outdoor running

Subjective assessment of footwear experience during running is critical for recommendation and design. Recently, ride perception was quantified using an eight-dimension ratings scale. Ride varied by shoe and individual for treadmill running. However, it is unknown whether ride perception is a reliable measure in circumstances other than treadmill running and across sessions. The purpose of this study was to assess the intra-runner reliability of the previously developed ride rating 5-point semantic differential scale and the influence of surface on reliability ratings. We collected ride ratings in-person from nine runners in a controlled (lab) and semi-controlled (5 km outdoor paved path) environment and remotely from 177 runners in an uncontrolled collection. Post-run ride ratings were collected from each group following two separate running sessions on multiple surfaces using native shoes. We used intra-class correlation analysis to determine intra-runner reliability of ride ratings between separate runs in the same shoe on the same surface and in the same shoe on different surfaces. We used a reliability threshold of ≥0.70 as adequate. More than half of the in-person and slightly under half of the remote runners reached adequate reliability for the same surface runs. Reliability was lower for different surface runs for the remote runners but not the in-person runners. For in-person collection, firmness had adequate reliability during treadmill running, while awareness and sound level had good reliability outdoors. Weight and yield showed adequate and near adequate reliability for remote collections for same surface runs. Ride perception exhibits test-retest reliability in the assessment of subjective experience of footwear. Differences in ratings between surfaces indicate both the definition and construct of ride is captured with this subjective scale.</p

Longitudinal Pharmacokinetic/Pharmacodynamic Profile of AG-120, a Potent Inhibitor of the IDH1 Mutant Protein, in a Phase 1 Study of IDH1-Mutant Advanced Hematologic Malignancies

Abstract INTRODUCTION: Somatic IDH1/2 mutations occur in multiple solid and hematologic tumors, including acute myeloid leukemia. Mutant IDH1/2 proteins have novel enzymatic activity, catalyzing the reduction of α-ketoglutarate to produce the oncometabolite, D-2-hydroxyglutarate (2-HG), which drives multiple oncogenic processes including impaired cellular differentiation. AG-120 is a first-in-class, oral, potent, reversible and selective inhibitor of the mutated IDH1 protein, and has been shown to lower 2-HG levels and restore cellular differentiation in IDH1-mutant primary human blast cells cultured ex vivo. AG-120 is currently being assessed in a first-in-human, phase 1 study enrolling patients with IDH1-mutant, advanced hematologic malignancies (NCT02074839). Our objective was to further assess the longitudinal pharmacokinetic/pharmacodynamic (PK/PD) data from the ongoing phase 1 study, including dose proportionality assessment of AG-120 exposure after single and multiple doses over time, and the influence of patient-intrinsic factors. METHODS: The AG-120 phase 1, open-label, dose-escalation and expansion study includes evaluation of safety, tolerability, maximum tolerated dose, PK/PD (including 2-HG levels) and clinical activity. Single-agent AG-120 is administered orally once (QD) or twice (BID) daily in continuous 28-day cycles. Patients included in this analysis received doses of 100 mg BID, 300 mg QD, 500 mg QD, 800 mg QD and 1200 mg QD (N=39). Blood, bone marrow and urine samples were collected at multiple time points for determination of PK/PD using qualified LC-MS/MS-based methods. Analyses were performed using WinNonLin®. RESULTS: AG-120 exposure exceeding the predicted efficacious exposure level was demonstrated at all dose levels following oral administration. Following both single (Day-3) and multiple (Cycle 1 Day 15 [C1D15] and Cycle 2 Day 1) dose administration, mean plasma exposures of AG-120 increased less than proportionally to dose, although plasma exposures were quite variable within dose levels. Preliminary PK data revealed a mean half-life of 119 ± 104 hr. Following multiple doses, most patients achieved steady state in Cycle 1, with ~2 to 3-fold accumulation in plasma observed. Furthermore, pre-dose AG-120 trough levels were maintained above the predicted efficacious exposure level throughout treatment (up to 11 cycles). Following a single dose of AG-120, plasma 2-HG levels gradually reduced over 3 days. After multiple doses, plasma 2-HG levels were reduced to levels seen in healthy volunteers (up to 99.7% inhibition) at all dose levels tested. Steady state 2-HG inhibition was reached at approximately C1D15 in most patients, and was maintained over the course of treatment (up to 11 cycles). Mean bone marrow 2-HG levels were also substantially reduced following multiple doses of AG-120 at all dose levels tested (up to 99.9% reduction compared with baseline). Plasma 2-HG levels showed a positive correlation with levels in bone marrow (r2=0.882, p&lt;0.001) and urine (r2=0.528, p&lt;0.001), with a stronger correlation being observed with the former tissue. There was no clear effect of patient-intrinsic factors such as body weight or body surface area on Cmaxor AUC within the ranges tested, although the current sample size is small. Population PK and PK/PD assessments will be conducted to confirm these findings. These analyses are based on data as of 1 May 2015; updated analyses will be presented. CONCLUSION: AG-120 exposure increased less than proportionally to dose following oral administration, with long half-life and maintenance of pre-dose levels above the predicted efficacious exposure, supporting QD dosing. In patients with IDH1 mutations, AG-120 inhibited plasma 2-HG to within levels found in healthy volunteers, and also inhibited 2-HG in bone marrow. Disclosures Fan: Agios Pharmaceuticals: Employment, Equity Ownership. Le:Agios Pharmceuticals: Employment, Equity Ownership. Manyak:Agios Pharmaceuticals: Employment. Liu:Agios: Employment. Prahl:Agios Pharmaceuticals: Employment, Equity Ownership. Bowden:Agios Pharmaceuticals: Employment. Biller:Agios Pharmaceuticals: Employment, Equity Ownership; Arbutus BioPharma (formerly Tekmira): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Arvinas: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Denali: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Agresta:Agios: Employment, Equity Ownership. Yang:Agios Pharmaceuticals: Employment, Equity Ownership. </jats:sec

DRIVE PK: A Phase 2 Trial of AG-348 in Patients with Pyruvate Kinase Deficiency

Abstract INTRODUCTION: Pyruvate kinase (PK) deficiency is a congenital hemolytic anemia caused by deficient function of the PK enzyme. This condition is currently managed with supportive care, including transfusions, folate supplementation, splenectomy, and reducing iron overload, but there are no treatments specifically targeting the underlying metabolic defect. AG-348 is a novel, orally available, small molecule activator of wild-type and mutant red blood cell PK. METHODS: Results of single-ascending dose (SAD) and multiple-ascending dose (MAD) studies in healthy adult volunteers (Yang H et al. Blood 2014;124[21]:4007; Yang H et al. Haematologica 2015;100[s1]:Abs S138) indicated good safety and tolerability and predictable pharmacokinetics at doses that demonstrated significant pharmacodynamic responses of increased whole blood adenosine triphosphate (ATP) and decreased 2,3-diphosphoglycerate (2,3-DPG) levels. In pre-clinical studies AG-348 inhibited the enzyme aromatase, and sex hormone profiles in the MAD study showed signs of aromatase inhibition. RESULTS: DRIVE PK is a multicenter, international, randomized, open-label, two-arm dose ranging trial of AG-348 in adults with PK deficiency. DRIVE PK is currently open for enrollment. Eligible patients are randomized to either low dose (50 mg BID) or high dose (300 mg BID) arms and receive treatment for 6 months, with the option of extended treatment. Patients are stratified by genotype to allow for genotype-phenotype correlations. A third dose may be added based on the observations made in the two original arms. The primary objective is to evaluate the safety and tolerability of AG-348. Secondary objectives include evaluation of pharmacokinetics and pharmacodynamics, as measured by AG-348, ATP and 2,3-DPG levels. A range of biochemical markers of clinical benefit will be assessed, including hemoglobin levels, reticulocyte count, bilirubin, erythropoietin, ferritin, transferrin saturation, and haptoglobin. PK activity, glycolytic flux assay and PK protein levels in blood will be analyzed as exploratory endpoints. The trial population consists of adult patients with PK deficiency with hemoglobin levels at screening of ≤12.0 g/dL (men) or ≤11.0 g/dL (women), who have received no more than three units of red blood cells in the 12 months preceding the first dose of AG-348, and no transfusion within 4 months. Full inclusion/exclusion criteria can be found on www.clinicaltrials.gov, NCT02476916. CONCLUSION: DRIVE PK, an ongoing phase 2 trial of the PK activator, AG-348, is the first interventional trial to target the underlying metabolic defect in patients with PK deficiency. This flexible trial with two dose arms, and potential for a third dose arm, is designed to evaluate safety and tolerability, pharmacokinetic and pharmacodynamic responses, as well as hematological markers of red blood cell metabolism. Disclosures Barbier: Agios: Employment, Equity Ownership. Silver:Agios: Consultancy. Merica:Agios Pharmaceuticals: Employment, Equity Ownership. Cohen:Agios: Consultancy. Kung:Agios: Employment, Equity Ownership. Yang:Agios Pharmaceuticals: Employment, Equity Ownership. Grace:Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Agresta:Agios: Employment, Equity Ownership. </jats:sec

Evaluation of Pharmacokinetic-Pharmacodynamic (PKPD) Relationship of an Oral, Selective, First-in-Class, Potent IDH2 Inhibitor, AG-221, from a Phase 1 Trial in Patients with Advanced IDH2 Mutant Positive Hematologic Malignancies

Abstract Introduction: Isocitrate dehydrogenase (IDH) is a critical enzyme in the citric acid cycle, catalyzing the oxidative decarboxylation of isocitrate to produce alpha-ketoglutarate (a-KG). The mutant IDH are not catalytically inactive enzymes, but rather possess novel enzymatic activities, catalyzing the reduction of α-KG to the ‘oncometabolite' 2-hydroxyglutarate (2-HG), which has been found to be elevated in patients with several tumor types, including acute myelogenous leukemia (AML). AG-221 is an oral, selective, first-in class, potent inhibitor of the IDH2 mutant protein. The compound has been demonstrated to reduce 2-HG levels by &gt;90% and reverse histone and DNA hypermethylation in vitro, and to induce differentiation in leukemia cell models. In vivo pharmacokinetic/pharmacodynamic (PK/PD) studies in a U87MG IDH2 (R140Q) xenograft mouse model demonstrated robust plasma 2-HG lowering, and the correlation between PK (AG-221 exposure) and PD (the inhibition of 2-HG production) was used for human efficacious exposure projection. The PK/PD correlation was further confirmed in a primary human AML xenograft model in mice. These results are compared to early PK/PD results from the ongoing first-in-human Phase I study of AG-221 in patients with advanced IDH2 mutant positive hematologic malignancies [NCT01915498]. Methods: This first-in-man Phase I study of oral AG-221 was designed to evaluate the safety, PK, and PD, including 2-HG levels, as well as clinical activity. AG-221 was administered orally once (QD) or twice (BID) per day in continuous 28-day cycles. Sequential cohorts of patients were enrolled at higher dose levels. Patients included in this analysis were enrolled to doses of 30, 50, 75 mg BID and 100 mg QD (total N=21). Patients bearing the two dominant IDH2 mutations, R140Q (85.7%) or R172K (14.3%), were enrolled in the Phase I study. Blood was collected at multiple time points for determination of the PK and PD effects of AG-221. The concentrations of AG-221and 2-HG in plasma samples were determined using a qualified LC-MS/MS based method. PK and PK/PD analyses were performed using WinNonLin®. In addition, PK/PD relationships and efficacy of AG-221 was evaluated in a U87MG IDH2-R140Q xenograft mouse model and a primary human AML xenograft mouse model carrying the IDH2-R140Q mutation following oral doses. Results: Preliminary analysis of PK demonstrated excellent oral AG-221 exposure in humans. The mean plasma half-life is greater than 40 hours. Plasma 2-HG concentrations decreased rapidly; substantial and constant plasma 2-HG inhibition was achieved following multiple AG-221 doses in patients, and the inhibition was dose and drug exposure dependent. Based on exposure-response analyses with R140Q patients, the AG-221 AUC0-10hr value of 47.1 hr•ug/mL is estimated to result in sustained 90% plasma 2-HG inhibition in human (Figure1) which is associated with IC90 of 66 ng/mL. This is consistent with an in vivo IC90 in an AML xenograft model, U87MG IDH2-R140Q. In addition, up to 50% plasma 2-HG inhibition was observed in limited number of patients with R172K mutation. Figure 1. AG-221 plasma exposure and 2-HG inhibition correlation in patients with IDH2-R140Q mutation Figure 1. AG-221 plasma exposure and 2-HG inhibition correlation in patients with IDH2-R140Q mutation Conclusions: The pharmacokinetic profile for AG-221 supports QD dosing based on the high plasma exposure and long half-life observed in this study. AG 221 suppressed the production of 2-HG in plasma to the normal range found in healthy volunteers. 2-HG inhibition in R140Q mutation was translated well from mice to humans as well as from in vitro to in vivo. Disclosures Fan: Agios Pharmaceuticals: Employment, Stockholder Other. Chen:Agios Pharmaceuticals: Employment, Stockholder Other. Wang:Agios Pharmaceuticals: Employment, Stockholder Other. Yen:Agios: Employment. Utley:Agios Pharmaceuticals: Employment, Stockholder Other. Almon:Agios Pharmaceuticals: Employment, Stockholder Other. Biller:Agios Pharmaceuticals: Employment, Stockholder Other. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. Yang:Agios Pharmaceuticals: Employment, Stockholder Other. </jats:sec

Population Pharmacokinetics and Pharmacodynamics of AG-348 in Healthy Human Volunteers Guide Dose Selection for the Treatment of Pyruvate Kinase Deficiency

Abstract INTRODUCTION: Pyruvate kinase (PK) deficiency is a glycolytic enzymopathy that results in non-spherocytic hemolytic anemia with a variable clinical presentation, ranging from mild or fully compensated forms to life-threatening neonatal anemia and life-long chronic hemolytic anemia associated with severe, debilitating co-morbidities. PK deficiency is caused by mutations in the PKLR gene, which in the red blood cell (RBC) results in defective pyruvate kinase isoform R (PK-R). PK-R catalyzes the final, irreversible step in glycolysis, the process on which mature RBCs rely almost exclusively to generate the energy carrier molecule adenosine triphosphate (ATP). PK-deficient RBCs and their progenitors are characterized by changes in metabolism associated with defective glycolysis, including a build-up of phosphoenolpyruvate (PEP) and 2,3-diphosphoglycerate (2,3-DPG), and lowered ATP levels. AG-348 is an orally available, allosteric activator of PK-R. It is hypothesized that intervention with AG-348 restores glycolytic pathway activity and normalizes RBC metabolism in vivo (Kung C et al. Blood, 2013). Biochemical experiments demonstrate that AG-348 is a potent pan-activator of many PK-R alleles associated with PK deficiency. Treatment of PK-deficient patient RBCs ex vivo with AG-348 results in increased ATP levels, and reductions in PEP and 2,3-DPG, consistent with pharmacological activation of the PK-R enzyme. This analysis integrates the pharmacokinetic and pharmacodynamic (PK/PD) properties of AG-348 in healthy human volunteers using population PK/PD modeling and simulation. METHODS: PK/PD modeling using a non-linear mixed effects approach was performed to understand the pharmacokinetics of AG-348 and PK/PD relationship of AG-348 to 2,3-DPG and ATP in humans. The PK/PD model integrated data from two phase 1, single-center, randomized, double-blind, placebo-controlled, dose escalation studies (one single and one 14-day multiple ascending dose) that enrolled a total of 96 healthy volunteers (Yang H et al. EHA Learning Center, 2015). AG-348 dose level ranged from 15-2500 mg given once (QD) or twice (BID) daily. Blood was collected from all patients to assess AG-348 pharmacokinetics, and for determination of levels of ATP and 2,3-DPG. Population simulations using the final model were performed to examine the dose-exposure-biomarkers relationship at various dose levels and duration of dosing. RESULTS: AG-348 showed rapid absorption following oral administration. Plasma exposure of AG-348 increased in a dose-proportional manner following a single dose. A three-compartmental model with a non-linear absorption compartment and a saturable induced enzyme compartment best described the pharmacokinetics of AG-348. Time-varying clearance was added to describe the observed decrease in exposure over time with multiple dosing; this is consistent with pre-clinical data that AG-348 is a moderate inducer of CYP3A4, the major oxidation pathway of AG-348. The multiple-dose data were well described by a semi-mechanistic autoinduction model with an indirect model and a saturable induction compartment. The PK/PD relationship between plasma AG-348 to ATP and 2,3-DPG showed best fit with a turnover model where the drug effect was described by an Emax model. Model simulations predicted maximum enzyme induction and PD response 3 weeks after the first dose following BID dosing. Population PK/PD simulations further supported the choice of 50 mg and 300 mg BID doses for the phase 2 study (Fig 1 and 2). The proposed current model incorporating PK/PD data over a wide range of AG-348 exposures and time-varying changes in clearance provides a useful tool for prediction of AG-348 pharmacokinetics that can be used to optimize AG-348 dosing for PK deficiency treatment. Furthermore, the population PK/PD model of AG-348 to ATP and 2,3-DPG biomarkers in healthy volunteers provides a good foundation to facilitate the analysis and understanding of patient data in the ongoing phase 2 study. CONCLUSION: This study represents the first comprehensive longitudinal analysis of AG-348 and its PD activity in humans. This integrated PK/PD model, incorporating time-varying PK/PD properties, forms the basis for understanding the exposure-response relationship in the ongoing phase 2 and future clinical studies of AG-348, as well as providing guidance on dosing selection to optimize the treatment of PK deficiency. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Le: Agios Pharmceuticals: Employment, Equity Ownership. Cohen:Agios: Consultancy. Chen:Agios: Employment. Kim:Agios: Employment. Silver:Agios: Consultancy. Agresta:Agios: Employment, Equity Ownership. Merica:Agios Pharmaceuticals: Employment, Equity Ownership. Kung:Agios: Employment, Equity Ownership. Kosinski:Agios: Employment, Equity Ownership; General Electric: Equity Ownership; SDIX: Equity Ownership. Silverman:Agios: Employment, Equity Ownership. Biller:Agios Pharmaceuticals: Employment, Equity Ownership; Arbutus BioPharma (formerly Tekmira): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Arvinas: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Denali: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Yang:Agios Pharmaceuticals: Employment, Equity Ownership. </jats:sec

Phase I Single (SAD) and Multiple Ascending Dose (MAD) Studies of the Safety, Tolerability, Pharmacokinetics (PK) and Pharmacodynamics (PD) of AG-348, a First-in-Class Allosteric Activator of Pyruvate Kinase-R, in Healthy Subjects

Abstract INTRODUCTION: Pyruvate kinase deficiency (PKD) is an inborn error of metabolism affecting children and adults that results in lifelong hemolytic anemia and is associated with serious long-term comorbidities such as poor growth and development in children and chronic iron overload in adults. PKD is caused by a functional deficiency of the R-isoform of pyruvate kinase (PK-R) caused most often by a missense mutation. As a result of deficiency of this terminal enzyme in glycolysis, blood 2,3-DPG levels are elevated and ATP levels are low. AG-348 is a novel, first-in-class, small molecule allosteric activator of PK-R that directly targets the underlying metabolic defect in PKD. Preclinical studies demonstrated that AG-348 increases the activity of both wild type and various mutated PK-R enzymes. The key objectives of these first-in-human, Phase I, randomized, double-blind, placebo-controlled single (SAD) and multiple ascending dose (MAD) studies are to identify a safe and pharmacodynamically-active dose and schedule for AG-348 to be used in subsequent clinical studies in subjects with PKD [NCT02108106; NCT202149966]. METHODS: In the SAD, healthymen and women (non-childbearing potential) aged 18-60 years were randomized to receive a single oral dose of AG-348 or placebo (P). Six cohorts were evaluated, each containing 8 subjects (6 AG-348, 2 P), starting with 30 mg in cohort 1 followed by 120, 360, 700, 1400 and 2500 mg in cohorts 2-6, respectively. In the ongoing MAD, 2 cohorts (120 mg bid and 360 mg bid) of 8 subjects each (6 AG-348, 2 P) have completed 14 days of dosing and 2 weeks of follow-up. In both studies, safety assessments included adverse events (AEs), vital signs, ECG and clinical laboratory parameters. Serial blood samples are drawn for assessment of PK and PD parameters (2,3-DPG and ATP) pre-dose and at regular intervals thereafter at multiple doses in both the SAD and MAD studies. Preliminary results are reported here; final results will be available at the time of presentation. RESULTS: In the SAD, all 48 subjects enrolled completed the study (47 males and 1 female; mean age 40 years). A preliminary analysis of safety data indicated that 19/48 (39%) subjects receiving AG-348 or placebo under fasted and/or fed conditions experienced at least 1 treatment-emergent AE during the study. All AEs were mild or moderate (Grade 1 and 2) in severity, and the most common were nausea (n=5; 10%) and headache (n=7; 14%). In the 2 completed MAD cohorts (13 males; 3 females; mean age 44 years), 8/16 (50%) of subjects receiving AG-348 or P experienced 11 AEs. All AEs were mild (n=10) or moderate (n=1) and the most frequent were venipuncture bruises. Frequency of AEs by treatment group will be presented. There were no serious AEs, discontinuations due to AEs, or dose-limiting toxicities in either study. Maximum tolerated dose was not reached in the SAD and dose escalation continues in the MAD. In SAD cohorts 1-6, exposure to single doses of AG-348 increased in a dose-proportional manner (mean plasma Cmax, AUC0-12hr and AUC0-72hr) (Figure 1 SAD study). Absorption was rapid, with a median Tmax of 0.75–4.0 h. Expected changes in pharmacodynamic markers including decrease in 2,3-DPG concentration (SAD [Figure 2] and MAD studies) and increase in ATP concentration (MAD study) were observed and will be presented in greater detail. CONCLUSION: AG-348 had a favorable safety profile and was well tolerated in healthy subjects based on preliminary analysis of subjects receiving a single dose up to 2500 mg or multiple bid doses up to 360 mg for up to 14 days. AG-348 also demonstrated a desirable PK profile, with rapid absorption, low PK variability and dose-proportional exposure with PD effect as demonstrated on 2,3-DPG (Figure 2) and ATP. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Yang: Agios Pharmaceuticals: Employment, Stockholder Other. Merica:Agios Pharmaceuticals: Employment, Stockholder Other. Chen:Agios Pharmaceuticals: Employment, Stockholder Other. Cohen:Agios Pharmaceuticals: Consultancy. Goldwater:PAREXEL International: Employment. Hill:Agios Pharmaceuticals: Employment, Stockholder Other. Kim:Agios Pharmaceuticals: Employment, Stockholder Other. Kosinski:Agios Pharmaceuticals: Employment, Stockholder Other. Kung:Agios Pharmaceuticals: Employment, Stockholder Other. Silver:Agios Pharmaceuticals: Consultancy. Utley:Agios Pharmaceuticals: Employment, Stockholder Other. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. </jats:sec

AG-348 Activation of Pyruvate Kinase in Vivo Enhances Red Cell Glycolysis in Mice

Abstract Pyruvate kinase deficiency (PKD) is an autosomal recessive enzymopathy that is the most common cause of hereditary nonspherocytic hemolytic anemia (HNSHA). PKD is a rare disease characterized by a life-long chronic hemolysis with severe co-morbidities. It is hypothesized that insufficient energy production to maintain red cell membrane homeostasis promotes the chronic hemolysis. Treatment is generally palliative, focusing on the resultant anemia, and there are no approved drugs that directly target mutated pyruvate kinase. AG-348 is an allosteric activator of the red cell isoform of pyruvate kinase (PKR) that has recently entered Phase I clinical trials in normal healthy volunteers. AG-348 increases the catalytic efficiency and enhances the protein stability of a spectrum of recombinantly expressed PKR mutant proteins that have been associated with PKD. PKD red cells are characterized by changes in metabolism associated with defective glycolysis, including a build-up of the upstream glycolytic intermediate 2,3-DPG and deficiency in the PKR product adenosine triphosphate (ATP). PKR flux, e.g. the rate of carbon flow through the PKR enzyme reaction, was examined in PKD patient or wild type (WT) donor blood samples by incubation of whole blood with a stable isotope tracer, [U-13C6]-glucose. At various time points after the addition of [U-13C6]-glucose, metabolism was quenched and metabolites were extracted. Metabolite pool sizes and 13C label incorporation into glycolytic intermediates were monitored by LC/MS. The rate of label incorporation was found to be significantly slower in PKD patient red cells, consistent with decreased glycolytic activity. Treatment of PKD red cells with AG-348 ex-vivo induces changes in metabolism consistent with increased glycolytic activity including reduced 2,3-DPG levels, increased ATP levels, and increased PKR enzyme activity levels. The effect of AG-348 on red cell metabolism in vivo was evaluated in mice. C57/BL6 mice were dosed by oral gavage either with a single dose, or with multiple doses (BID) of AG-348 for 7 days. Dose levels tested were 1 mpk, 10 mpk, 50 mpk, and 150 mpk. Following the last dose, mice were bled to evaluate drug exposure and pharmacodynamic markers including 2,3-DPG and ATP levels, and PKR activity. AG-348 was demonstrated to be a well-behaved compound, with dose-proportional increase in exposure, both in the single-dose and multiple dose studies. A single dose of AG-348 resulted in a dose-dependent increase in PKR activity levels, concomitant with reduction in 2,3-DPG levels. There were no significant changes in ATP levels after a single administration of AG-348. In the multiple-dose studies, similar changes in PKR activity and 2,3-DPG levels were observed. In contrast to the single-dose study, ATP levels were observed to be robustly increased in a dose-dependent manner. The effect of AG-348 on PKR flux was assessed in whole blood from mice treated with AG-348. C57BL/6 mice were dosed by oral gavage with AG-348 (150 mg/kg twice daily [BID]) for 3 days. Whole blood was incubated with [U-13C6]-glucose and the metabolite pool sizes and rate of 13C label incorporation into glycolytic intermediates were assessed. The data were subsequently analyzed using a mathematical model to quantify flux through the PKR reaction and it was determined that AG-348 treatment significantly increased flux through the PKR reaction. Collectively, these data demonstrate that AG-348 not only potently binds to and activates the PKR enzyme in vivo, but this enzyme activation induces enhanced glycolytic pathway activity in red cells that results in profound changes in cellular metabolism, as reflected in dramatically increased ATP levels and reduced 2,3-DPG levels. As AG-348 has similar potency against the WT PKR enzyme as against tested mutant PKR enzymes in vitro, these data support the hypothesis that AG-348 treatment may similarly enhance glycolytic activity in PKD patients and thus correct the underlying pathology of PKD. Figure 1 Figure 1. Disclosures Kung: Agios Pharmaceuticals: Employment, Stockholder Other. Hill:Agios Pharmaceuticals: Employment, Stockholder Other. Chen:Agios Pharmaceuticals: Employment, Stockholder Other. Jha:Agios Pharmaceuticals: Employment, Stockholder Other. Kosinski:Agios Pharmaceuticals: Employment, Stockholder Other. Clasquin:Agios Pharmaceuticals: Employment, Stockholder Other. Si:Agios Pharmaceuticals: Employment, Stockholder Other. Kim:Agios Pharmaceuticals: Employment, Stockholder Other. Hixon:Agios Pharmaceuticals: Employment, Stockholder Other. Dang:A: Employment, Stockholder Other. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. Silverman:Agios Pharmaceuticals: Employment, Stockholder Other. Yang:Agios Pharmaceuticals: Employment, Stockholder Other. </jats:sec

Abstract 3116: AG-221 offers a survival advantage in a primary human IDH2 mutant AML xenograft model

Abstract Somatic point mutations in isocitrate dehydrogenase 1/2 (IDH1/2) confer a gain-of-function in cancer cells resulting in the accumulation and secretion of an onco-metabolite, R (-)-2-hydroxyglutarate (2HG). High levels of 2HG have been shown to inhibit α-KG-dependent dioxygenases including histone and DNA demethylases, which play a key role in regulating the epigenetic state of cells. Recently, ex vivo treatment with AGI-6780, a potent IDH2 R140Q inhibitor induced cellular differentiation of leukemic blast cells isolated from primary human AML patient samples harboring an IDH2 R140Q mutation. These data provided the first evidence that inhibition of mutant IDH2 can reverse the block in cellular differentiation conferred by high levels of 2HG and could provide a therapeutic benefit to patients. AG-221 is a potent and selective inhibitor of the IDH2 mutant enzyme and is currently being evaluated in a first-in-human study entitled: A Phase 1, Multicenter, Open-Label, Dose-Escalation, Safety, Pharmacokinetic, Pharmacodynamic, and Clinical Activity Study of Orally Administered AG-221 in Subjects with Advanced Hematologic Malignancies with an IDH2 Mutation. The compound has been demonstrated to reduce 2-HG levels by &amp;gt;90% and reverse histone and deoxyribonucleic acid (DNA) hypermethylation in vitro, and to induce differentiation in leukemia cell models. We evaluated the efficacy of AG-221 in a primary human AML xenograft model carrying the IDH2 R140Q mutation. This is an aggressive model with mortality from AML consistently occurring by day 80, following tail vein engraftment. Results show that AG-221 is able to potently reduce 2HG found in the bone marrow, plasma and urine of engrafted mice. Treatment also induced a dose dependent, statistically significant, survival benefit where all mice in the high dose treatment group survived to the end of study. We also saw a dose dependent proliferative burst of the human specific CD45+ blast cells followed by cellular differentiation as measured by the expression of CD11b, CD14 and CD15 and cell morphology. Furthermore, the onset of differentiation correlated with survival, whereas mice that died in the low dose groups failed to show signs of cellular differentiation. These data provide strong preclinical in vivo evidence that AG-221 may have clinical benefit for IDH2 mutant patients through the reduction of 2HG and the induction of blast differentiation. Citation Format: Kate Ellwood-Yen, Fang Wang, Jeremy Travins, Yue Chen, Hua Yang, Kim Straley, Sung Choe, Marion Dorsch, Sam Agresta, David Schenkein, Scott Biller, Michael Su. AG-221 offers a survival advantage in a primary human IDH2 mutant AML xenograft model. [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 3116. doi:10.1158/1538-7445.AM2014-3116</jats:p