Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of Multiple Doses of AG-519, an Allosteric Activator of Pyruvate Kinase-R, in Healthy Subjects

Abstract BACKGROUND Pyruvate kinase (PK) deficiency is a congenital hemolytic anemia caused by deficiency of the glycolytic enzyme red cell PK (PK-R) due to mutations in the PKLR gene. PK catalyzes the last enzymatic step in the glycolytic pathway and is the main source of adenosine triphosphate (ATP) production in red blood cells. PKLR mutations lead to defective proteins that are hypothesized to reduce ATP levels in red cells, leading to hemolysis. Small molecule allosteric activation of PK-R resulting in increases in ATP and decreases in 2,3-diphosphoglycerate (2,3-DPG) in healthy volunteers has been observed with an earlier molecule, AG-348, the first small molecule PK-R activator to enter clinical trials (Yang et al. EHA 2015, S138). AG-519 is the second small molecule PK-R activator to enter clinical trials. AG-519 is a potent, highly selective and orally bioavailable PK-R activator devoid of the aromatase inhibitory effects that were observed with AG-348. AIMS AG-519 is currently being tested in a randomized, double-blind, phase 1 study in healthy volunteers (NCT02630927), with the objective of identifying a safe and pharmacodynamically active dose and schedule to support potential ongoing development in patients with PK deficiency. Here we report the first 4 cohorts of the multiple ascending dose (MAD) phase of this study. The single ascending dose (SAD) phase of the study and the first two cohorts of the MAD phase of the study have been reported previously (Barbier et al. EHA 2016, P752). METHODS Healthymen and women (non-childbearing potential) aged 18-60 years who provided informed consent were eligible. The MAD phase of the study consisted of 5 dose cohorts. The dose levels administered were determined during interim data reviews of each completed MAD cohort, as well as data from completed SAD cohorts. At each dose level, 8 subjects were enrolled and randomized to receive AG-519 (n=6) or placebo (n=2) twice daily (BID; approximately every 12 hours) for 14 days. Safety assessments included adverse events (AEs), vital signs, electrocardiogram and clinical laboratory parameters. Serial blood samples were drawn to measure plasma concentrations of AG-519 and whole blood concentrations of 2,3-DPG and ATP for pharmacokinetic and pharmacodynamic (PD) assessments. RESULTS Data are available for 32 subjects enrolled across 4 dose cohorts in the MAD phase of the study: 8 subjects each in cohort 1 (125 mg BID), cohort 2 (375 mg BID), cohort 3 (25 mg BID), and cohort 4 (300 mg BID). Blinded safety reviews indicated that multiple doses up to 375 mg have been well tolerated with no serious AEs or dose-limiting toxicities reported to date. One case of probable drug-induced Grade 2 thrombocytopenia was previously reported in 1 subject in the 375 mg cohort; the event was rapidly reversible with no clinical sequelae. The protocol was amended to require daily monitoring of platelets in subsequent cohorts and no other subjects have developed thrombocytopenia during treatment. The preliminary analysis of free testosterone and estradiol confirmed the absence of aromatase inhibitory activity. AG-519 steady-state was reached the third day after the first dose based on trough concentration values. The clearance of AG-519 after multiple doses was similar to that observed after single doses in the SAD cohorts. Dose-dependent increases in ATP in blood (Figure 1) and decreases in 2,3-DPG in blood correlated with dose-dependent increases in exposure of AG-519, with a peak effect at or below 375 mg BID. ATP response at 25 mg appears to be greater than 50% of maximal response. Results from the fifth MAD cohort, which evaluated the PD results with 10 mg BID, will be presented. ATP = adenosine triphosphate; BID = twice daily CONCLUSION AG-519 is well tolerated in healthy subjects at doses ranging from 25 mg to 375 mg BID for 14 days. The robust dose-dependent changes in ATP and 2,3-DPG concentrations in blood from healthy volunteers are consistent with increased activity of PK-R, the expected PD effect of AG-519. These data support the hypothesis that AG-519 may be able to enhance glycolytic activity in red cells of patients with PK deficiency to address the underlying cause of the disease. Figure 1 Change of ATP concentration in blood from baseline Figure 1. Change of ATP concentration in blood from baseline Disclosures Barbier: Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Bodie:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Connor:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Merica:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kung:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Le:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yang:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kosinski:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Silverman:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yuan:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Bowden:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Cohen:Agios Pharmaceuticals, Inc.: Consultancy.

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Characterization of Metabolic Response to AG-348, an Allosteric Activator of Red Cell Pyruvate Kinase, in Healthy Volunteers and Pyruvate Kinase Deficiency Patients

Blood ◽

10.1182/blood.v128.22.2452.2452 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2452-2452

Author(s):

Victor Chubukov ◽

Kendall Johnson ◽

Penelope A Kosinski ◽

Michelle Clasquin ◽

Abhishek Jha ◽

...

Keyword(s):

Pyruvate Kinase ◽

Whole Blood ◽

Healthy Subjects ◽

Lactate Production ◽

Glycolytic Flux ◽

Red Cell ◽

Employment Equity ◽

Glycolytic Intermediates ◽

Equity Ownership ◽

2,3 Dpg

Abstract Pyruvate kinase (PK) deficiency is a glycolytic enzymopathy that causes lifelong chronic hemolytic anemia. AG-348 is an allosteric activator of the red cell isoform of pyruvate kinase (PK-R) that is in clinical development to treat PK deficiency. Phase 1 studies of AG-348 in healthy volunteers (NCT02108106, NCT02149966) have been completed, and a phase 2 study in patients with PK deficiency is in progress (DRIVE PK, NCT02476916). We have previously reported that in the healthy volunteer studies, AG-348 induced changes in levels of the metabolites adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) that are consistent with PK-R activation. A preliminary analysis of data showed that nine out of 18 DRIVE PK patients achieved a maximal increase in hemoglobin (Hb) levels of >1.0 g/dL. In this study, we characterize whole blood metabolism of healthy human subjects (from the multiple-ascending dose study NCT02149966) as well as PK-deficient patients (from DRIVE PK), before and after administration of AG-348, with a focus on the flux through the PK-R reaction. Whole blood taken from healthy subjects/DRIVE PK patients on each respective study was incubated with a stable isotope tracer, [U-13C6]-glucose. Glycolytic flux through PK-R was estimated by kinetic flux profiling based on the isotope labeling, as well as by the specific lactate production rate, with the two methods giving consistent results. Blood cells from healthy subjects exhibited classic red blood cell (RBC) metabolism, with the majority of glucose catabolized through glycolysis. AG-348 was shown to significantly increase the maximal PK-R protein activity in these subjects, and also to have metabolic effects consistent with PK-R activation, marked by decreased concentrations of glycolytic intermediates such as 2,3-DPG and phosphoenolpyruvate (PEP). Increases in ATP concentrations were also observed, with the magnitude and kinetics of the increase strongly suggesting enhanced adenosine salvage or synthesis. The overall glycolytic rates, however, did not change significantly after two weeks of AG-348 dosing, revealing the homeostatic regulation of RBC glycolysis in healthy blood. Five of the first 18 DRIVE PK patients underwent an extensive sampling protocol for metabolic analysis. This analysis revealed a number of distinct metabolic qualities in PK-deficient patients at baseline compared with healthy subjects. These included significantly reduced rates of lactate production and high concentrations of nucleotides, amino acids, and Krebs cycle intermediates. 13C labeling was observed in Krebs cycle intermediates, demonstrating significant respiratory metabolism in whole blood cells from PK-deficient patients, while incomplete labeling of glycolytic intermediates suggested the presence of a metabolically inactive cell subpopulation. These observations are most consistent with the hypothesis that PK-deficient whole blood is dominated metabolically by immature erythrocytes that retain residual mitochondrial activity. With AG-348 treatment, three of the five DRIVE PK patients had increases in Hb of >1.0 g/dL. In those three patients, we observed increased incorporation of 13C label into glycolytic intermediates such as 2,3-DPG, suggesting an increase in metabolically active erythrocytes. First order flux estimates based on either 2,3-DPG labeling kinetics or lactate production rates showed a >0.1 mmol/L/hr (>50%) increase in glycolytic flux. Neither of the two DRIVE PK patients that did not have an Hb increase of >1.0 g/dL showed significant metabolic changes. In conclusion, metabolic profiling and stable isotope tracing experiments in blood from healthy subjects treated with AG-348 revealed strong homeostatic regulation of glycolysis even in the presence of activated PK-R. Analysis of data from a small number (n=5) of PK-deficient patients treated with AG-348 for two weeks showed that the three patients with Hb increases >1.0 g/dL also had increased glycolytic flux. While the small number of patients makes these results preliminary, it is the first demonstration of a direct link between increased red cell glycolysis induced by the PK-R activator AG-348 and the resulting hematological response as assessed by increases in Hb levels. Updated analyses including additional patients will be presented as more data are collected in the ongoing study. Disclosures Chubukov: Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Johnson:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kosinski:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Clasquin:Agios Pharmaceuticals, Inc.: Other: former employee and stock holder; Pfizer, Inc.: Employment. Jha:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kim:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Roddy:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Merica:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Barbier:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Dang:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Silverman:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kung:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership.

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

Blood ◽

10.1182/blood.v126.23.3336.3336 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3336-3336 ◽

Cited By ~ 1

Author(s):

Kha Le ◽

Marvin Cohen ◽

Yue Chen ◽

Hyeryun Kim ◽

Bruce Silver ◽

...

Keyword(s):

Board Of Directors ◽

Pyruvate Kinase ◽

Time Varying ◽

Phase 2 ◽

Employment Equity ◽

Advisory Committees ◽

Healthy Human ◽

Population Pk ◽

Equity Ownership ◽

2,3 Dpg

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.

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Pharmacokinetic and Pharmacodynamic Properties of Romiplostim

Blood ◽

10.1182/blood.v124.21.1463.1463 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1463-1463

Author(s):

Karen Arkam ◽

Sameer Doshi ◽

Bing-Bing Yang

Keyword(s):

Healthy Volunteers ◽

Healthy Subjects ◽

Immune Thrombocytopenia ◽

Receptor Complex ◽

Platelet Response ◽

Employment Equity ◽

Platelet Production ◽

Platelet Counts ◽

Equity Ownership ◽

Chronic Immune Thrombocytopenia

Abstract Background: Chronic Immune thrombocytopenia (ITP) is characterized by low platelet counts, resulting from increased platelet destruction and inadequate platelet production. Romiplostim is a 59 kDa peptibody which binds to and activates the thrombopoietin (TPO) receptor on platelet precursors in the bone marrow, and increases platelet counts. This analysis integrates the pharmacokinetic (PK) and pharmacodynamic (PD) properties of romiplostim in animals, healthy volunteers and patients with ITP, and describes its intricate PK-PD inter-relationship. Methods and Results: In healthy subjects, over a wide range of doses examined, the PK and PD (platelet response) of romiplostim were dependent on both the dose administered and the baseline platelet counts. Following SC administration, platelet counts increased in a dose-dependent fashion after 4 to 9 days, peaking at 12 to 16 days (Wang Clin Pharmacol Ther. 2004;76:628-38). When romiplostim binds to the TPO receptor on megakaryocytes and platelets, the peptibody-receptor complex is internalized and degraded inside the cells. Therefore, as platelet counts increase, a higher number of free receptors are available to clear romiplostim (Wang AAPS J. 2010;12:729-40). Results from rodent studies suggest that as the dose increases, the TPO receptors become saturated and the contribution of the kidney to clearance increases. Additionally, proteolysis plays a role in the clearance of romiplostim; however, the cytochrome P450 enzymes are not involved in protein catabolism (Wang Pharm Res. 2011;28:1931-8), hence there are no known drug-drug interactions or dietary restrictions (Nplate Prescribing Information 2014). Following SC administration, serum concentrations of romiplostim were markedly lower, however, platelet response was similar after the same dose of intravenous (IV) and SC administration (Wang Clin Pharmacol Ther. 2004;76:628-38). This suggests that the PD response is driven by the length of time that the romiplostim concentrations remained above a threshold rather than by the magnitude of concentrations achieved. This effect was verified in a mechanistic PK-PD modeling study in animals (Krzyzanski Pharm Res. 2013;30:655-69). In patients with ITP receiving SC romiplostim at a dose of 1 mcg/kg, the peak platelet response was achieved at 18 days (range 8 to 43; Bussel N Engl J Med. 2006;355:1672-81). Pharmacodynamic model analysis showed that compared with healthy subjects, patients with ITP had a shorter platelet life span and a decreased rate of production of progenitor cells, but no major difference in the time to maturation of megakaryocytes. The PD response in this modeling analysis was not notably affected by age, body weight, sex, and race (Perez-Ruixo J Clin Pharmacol. 2012;52:1540-51). The frequency of once-weekly dosing was selected because once every 2 weeks dosing was determined to be inadequate to achieve and maintain platelet counts in the therapeutic range (Bussel N Engl J Med. 2006;355:1672-81). A mechanistic PK-PD model based on data from the healthy subjects further suggested that weekly dosing resulted in a sustained platelet response while dosing less frequently resulted in high fluctuation of platelet counts (Wang AAPS J. 2010;12:729-40). Large inter- and intra-individual variability in the PD response was observed at a given dose; therefore, dose adjustments should be made based on a patient's platelet counts, using a titrated dosing scheme to prevent having platelet counts over 400 x 109/L (Perez-Ruixo J Clin Pharmacol. 2012;52:1540-51). Conclusion: Romiplostim is a peptibody that binds and activates the TPO receptor, and consequently increases platelet production in individuals with chronic ITP. The peptibody-receptor complex is internalized and degraded inside the cells, without involvement of the liver. Romiplostim's PD response is driven by the length of time that its concentrations remained above a threshold rather than by the magnitude of concentrations achieved. Moreover, weekly dosing has demonstrated a sustained platelet response while less frequent dosing resulted in fluctuating platelet counts. Disclosures Arkam: Amgen Inc.: Employment, Equity Ownership. Off Label Use: Romiplostim is a thrombopoietin receptor agonist indicated for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenia (ITP) who have had an insufficient response to corticosteroids, immunoglobulins, or splenectomy. This abstract also describes PK data from healthy volunteers.. Doshi:Amgen Inc.: Employment, Equity Ownership. Yang:Amgen Inc.: Employment, Equity Ownership.

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AG-221, a Small Molecule Mutant IDH2 Inhibitor, Remodels the Epigenetic State of IDH2-Mutant Cells and Induces Alterations in Self-Renewal/Differentiation in IDH2-Mutant AML Model in Vivo

Blood ◽

10.1182/blood.v124.21.437.437 ◽

2014 ◽

Vol 124 (21) ◽

pp. 437-437 ◽

Cited By ~ 12

Author(s):

Alan H. Shih ◽

Kaitlyn R Shank ◽

Cem Meydan ◽

Andrew M. Intlekofer ◽

Patrick Ward ◽

...

Keyword(s):

Dna Methylation ◽

Clinical Trials ◽

Small Molecule ◽

Leukemia Cells ◽

Employment Equity ◽

Advisory Committees ◽

Self Renewal ◽

Equity Ownership ◽

The Impact

Abstract Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are observed in patients with acute myeloid leukemia (AML). Leukemia-associated IDH1/2 mutations result in aberrant accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). The observation that IDH1/2 mutations are mutually exclusive with TET2 mutations led to the finding that IDH1/2-mutant production of 2-HG inhibits TET2 function and induces changes in DNA methylation. These data suggested that small molecule inhibition of mutant IDH enzymes might reverse the aberrant epigenetic remodeling of IDH-mutant leukemia cells and restore normal hematopoietic differentiation. We therefore investigated the in vivo efficacy of AG-221, a potent and selective mutant IDH2 inhibitor in early-phase clinical trials, in murine models of IDH2-mutant leukemia. We first assessed the impact of AG-221 on 2-HG production in hematopoietic cells expressing mutant IDH2-R140Q. AG-221 treatment (10mg/kg or 100mg/kg bid) led to a reduction in 2-HG in vivo (96.7% below pre-treatment levels). Moreover, AG-221 treatment restored megakaryocyte-erythroid progenitor (MEP) differentiation that is suppressed by mutant IDH2 expression in vivo (mean MEP% mean, 39% Veh vs 50% AG-221). We next investigated the impact of mutant IDH2 inhibition with AG-221 on DNA methylation in vivo. We used eRRBS, a bisulfite-based next-generation sequencing platform, to assess the effect of AG-221 therapy on DNA methylation. AG-221 or vehicle therapy treated LSK stem cells (lin- Sca+ c-Kit+) were sorted from mice expressing IDH2-R140Q and evaluated by eRRBS. AG-221 therapy reversed the effects of mutant IDH2; we observed a significant reduction in DNA methylation, including 180 genes that had 20 or more hypomethylated differentially methylated cytosines (DMCs) following treatment. 84 of these genes had reduced methylation at 10 or more DMCs in the gene promoter with AG-221 therapy compared to vehicle. Mutant IDH2 inhibition with AG-221 reversed aberrant methylation at many genes with a known role in hematopoietic proliferation and differentiation, including the master transcriptional factor RUNX1. We next assessed in vivo effects of the small-molecule IDH2-R140Q inhibitor in a mouse model of IDH2-mutant leukemia. We generated mice that simultaneously expressed a constitutive Flt3ITD knock-in allele and a conditional mutant IDH2R140Q knock-in allele. As reported recently using retroviral/transgenic models, Mx1-Cre IDH2R140QFlt3ITD developed fully penetrant, transplantable AML with expansion of c-Kit+ positive blasts in the peripheral blood, and widespread leukemic infiltration. AG-221 inhibited the serial replating capacity of IDH2R140QFlt3ITD expressing cells in vitro. We competitively transplanted IDH2R140QFlt3ITD AML cells and normal bone marrow cells into secondary recipients, and then assessed the effect of AG-221 therapy on leukemia in vivo and on disease burden. AG-221 (100mg/kg bid) treatment of mice engrafted with Mx1-Cre IDH2R140QFlt3ITD AML cells markedly reduced 2HG levels consistent with on target inhibition in vivo. AG-221 therapy induced differentiation of leukemic cells, with an increase in the CD11b+ population and a decrease in the c-Kit+ population in the peripheral blood at 2wks. We next assessed the impact of treatment with both AG-221 therapy with AC220, a potent, specific Flt3 inhibitor in late phase clinical trials. Combined IDH2R140Q and Flt3ITD inhibition resulted in a marked decrease in leukemic burden to vehicle-treated mice, with a significant reduction in leukemic cell chimerism in vivo in the setting of combined inhibition at 2 wks, (mean 45.2 fraction 88% veh, 73% AG-221, p<.01). These data demonstrate that AG-221 inhibits mutant IDH2-mediated 2-HG production in vivo and reverses the effects of mutant IDH2 on DNA methylation in mutant stem/progenitor cells. AG-221 induces differentiation and impairs self-renewal of IDH2-mutant leukemia cells, effects that are further enhanced by simultaneous inhibition of Flt3ITD. Clinical trials combining IDH2 inhibitors with other targeted AML therapies are warranted in order to increase therapeutic efficacy. Disclosures Intlekofer: Foundation Medicine, Inc: Consultancy. Thompson:Agios: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Travins:Agios Pharmaceuticals: Employment, Stockholder Other. Straley:Agios: Employment, Equity Ownership. Gliser:Agios Pharmaceuticals: Employment, Stockholder Other. Yen:Agios: Employment, Equity Ownership. Levine:Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Population Pharmacokinetics and Pharmacodynamics of AG-519, a Pyruvate Kinase Activator for the Treatment of Pyruvate Kinase Deficiency, in Human Healthy Volunteers

Blood ◽

10.1182/blood.v128.22.1263.1263 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1263-1263

Author(s):

Kha Le ◽

Marvin Cohen ◽

Ann J Barbier ◽

Elizabeth Merica ◽

Charles Kung ◽

...

Keyword(s):

Enzyme Activity ◽

Pyruvate Kinase ◽

Phase 1 ◽

Dose Selection ◽

Multiple Dosing ◽

Employment Equity ◽

Emax Model ◽

Atp Levels ◽

Equity Ownership ◽

2,3 Dpg

Abstract INTRODUCTION Pyruvate kinase (PK) deficiency is a life-long chronic hemolytic anemia with a variable clinical presentation, ranging from mild to life-threatening, and is 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 and irreversible step in glycolysis, the process on which mature RBCs rely almost exclusively to generate the energy carrier molecule, adenosine triphosphate (ATP). PK-R is thus a key enzyme for maintaining RBC energy levels, and it has been proposed that ATP levels are critical for optimally maintaining RBC membrane integrity. 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-519 is a potent, highly selective and orally bioavailable PK-R activator shown preclinically to have none of the aromatase inhibitory effects that were observed with AG-348, the first small molecule PK-R activator to enter clinical trials. It is hypothesized that intervention with AG-519 restores glycolytic pathway activity and normalizes RBC metabolism. Treatment of PK-deficient patient RBCs ex vivo with AG-519 results in increased ATP levels, and reductions in 2,3-DPG, consistent with pharmacological activation of PK-R enzyme activity. This analysis integrates the pharmacokinetic and pharmacodynamic (PK/PD) properties of AG-519 in healthy 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-519 and PK/PD relationship of AG-519 to 2,3-DPG, ATP and PK-R enzyme activity in humans. The PK/PD model integrated data from a phase 1, single-center, randomized, double-blind, placebo-controlled study. Data in single and multiple ascending dose escalation arms were included in the analysis, which comprises a total of 88 human volunteers. AG-519 dose levels ranged from 10-1250 mg. Blood was collected from all patients to assess AG-519 pharmacokinetics and levels of ATP and 2,3-DPG in blood, as well as PK-R activity. Population simulations using the final model were performed to examine the dose-exposure-biomarkers relationship. RESULTS AG-519 showed rapid absorption kinetics following oral administration. Area under the concentration-time curve of plasma AG-519 increased in a dose-proportional manner following single and multiple dosing. A three-compartment model with non-linear absorption compartment and linear elimination best described the pharmacokinetics of AG-519. Systemic clearance appeared to be time-invariant and no auto-induction was observed with multiple dosing. The PK/PD relationship between plasma AG-519 and ATP or 2,3-DPG in human volunteers was best fitted by a turnover model where the drug effect was described by an Emax model. A direct Emax model best described the relationship between plasma AG-519 and PK-R enzyme activity. Evaluation of ATP and 2,3-DPG levels and PK-R activity confirmed the potent effect of AG-519 on PK-R. A direct comparison of AG-519 target engagement and biomarker response to AG-348 will also be presented. Population PK/PD model simulations suggested that the doses selected in the phase 1 study covered a large exposure-response range of the three biomarkers, and provided a systematic and integrated framework for the understanding of AG-519 pharmacokinetics and pharmacodynamics, as well as a rationale for dose selection in future trials. CONCLUSIONS This study represents a comprehensive longitudinal PK/PD analysis of AG-519 in humans. This integrated PK/PD model formed the basis for understanding the exposure-response relationship of the ongoing phase 1 study and provided guidance on dose selection to inform the future development of AG-519. Disclosures Le: Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Cohen:Agios Pharmaceuticals, Inc.: Consultancy. Barbier:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Merica:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kung:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kosinski:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Biller:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yang:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership.

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A Randomized, Double-Blind, Placebo-Controlled Phase 1 Study of the Pharmacokinetics and Pharmacodynamics of REGN3918, a Human Antibody Against Complement Factor C5, in Healthy Volunteers

Blood ◽

10.1182/blood-2018-99-112262 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1039-1039 ◽

Cited By ~ 6

Author(s):

Jonathan Weyne ◽

Yan Ni ◽

Richard DelGizzi ◽

Stephen Godin ◽

Lori Morton ◽

...

Keyword(s):

Healthy Volunteers ◽

Hemolytic Activity ◽

Multiple Dose ◽

Complete Suppression ◽

Complement Factor ◽

Loading Dose ◽

Employment Equity ◽

Dose Cohort ◽

Equity Ownership ◽

Dose Dependent

Abstract INTRODUCTION: Blockade of complement factor C5 has demonstrated benefit in Paroxysmal Nocturnal Hemoglobinuria (PNH), atypical Hemolytic Uremic Syndrome and Generalized Myasthenia Gravis. We developed a human IgG4P antibody, REGN3918, that binds with high affinity to wild-type and variant (R885H/C) human C5. REGN3918 was well-tolerated in monkey toxicology studies with up to 26 weeks of dosing at up to 100 mg/kg/week. This finding was supportive of conducting this first-in-human study of REGN3918 in healthy volunteers. OBJECTIVE: The primary objective of this ongoing study is to evaluate the safety and tolerability of single ascending intravenous (IV) and subcutaneous (SC) doses and a multiple dose regimen consisting of an IV loading dose plus multiple weekly SC doses of REGN3918 administered in healthy volunteers. The secondary objectives of the study are to assess the pharmacokinetic (PK) and pharmacodynamic (PD) profile of REGN3918. METHODS: 56 subjects were randomized to 4 sequential ascending IV dose cohorts plus 2 sequential ascending SC cohorts followed by 1 multiple dose cohort (consisting of an IV loading dose and weekly SC doses). Each cohort consisted of 8 subjects randomized to receive REGN3918 or placebo (6 active: 2 placebo). An adaptive design was implemented to allow for dose level and dosing interval adjustment utilizing in-study PK and PD measures. The PD profile of REGN3918 was assessed utilizing a sheep red blood cell complement activity assay (CH50 assay) as well as serum concentrations of total C5. REGN3918 was administered as follows:Cohort 1: 1 mg/kg IV, single doseCohort 2a: 3 mg/kg IV, single doseCohort 2b: 300 mg SC, single doseCohort 3a: 10 mg/kg IV, single doseCohort 3b: 600 mg SC, single doseCohort 4: 30 mg/kg IV, single doseCohort 5: Loading dose of 15 mg/kg IV followed by 4 repeat SC doses of 400 mg administered once weekly for four weeks. RESULTS: REGN3918 was found to be well tolerated in single doses of up to 30 mg/kg IV and 600 mg SC. The multiple dose Cohort 5 has completed dosing in all subjects and is currently in safety follow-up. Thus far, there has been one SAE, salpingitis in a subject with an intra-uterine contraceptive device. The SAE occurred in a Cohort 5 subject after completion of dosing and has since resolved. REGN3918 exhibited dose-dependent increases in exposure in serum, with a trend toward prolonged serum concentrations at IV doses ≥10 mg/kg. Following SC administration, concentrations of REGN3918 in serum peaked at 4 to 8 days post dose and bioavailability was estimated as approximately 70%. REGN3918 exposure led to dose-dependent inhibition of CH50. In all 4 IV dosing cohorts, suppression of hemolysis was observed at 15 min post-injection. Complete suppression of hemolysis was achieved with ≥ 3mg/kg dosing. At 30 mg/kg, complete suppression of hemolysis was maintained for >6 weeks, consistent with observed prolonged REGN3918 concentrations following this dose. In the 2 SC cohorts, peak suppression of hemolysis was observed 3-7 days post dosing, again consistent with observed peak concentrations of REGN3918 in serum. In the multiple dose cohort 5, complete suppression of CH50 was observed over the four-week dosing period. CONCLUSIONS: REGN3918 was well tolerated and resulted in dose-dependent inhibition of hemolytic activity in normal healthy volunteers. A single IV infusion of R3918, at 30 mg/kg, blocked hemolytic activity completely for >6 weeks. Complete inhibition of hemolytic activity was maintained over a 4-week dosing period by a weekly SC regimen following an IV loading dose. Disclosures Weyne: Regeneron Pharmaceuticals: Employment, Equity Ownership. Ni:Regeneron Pharmaceuticals: Employment, Equity Ownership. DelGizzi:Regeneron Pharmaceuticals: Employment, Equity Ownership. Godin:Regeneron Pharmaceuticals: Employment. Morton:Regeneron Pharmaceuticals: Employment, Equity Ownership. Prasad:Regeneron Pharmaceuticals: Employment, Equity Ownership. Rankin:Regeneron Pharmaceuticals: Employment, Equity Ownership. Simek-Lemos:Regeneron Pharmaceuticals: Employment, Equity Ownership. Wang:Regeneron Pharmaceuticals: Employment, Equity Ownership. Rippley:Regeneron Pharmaceuticals: Employment, Equity Ownership. Harari:Regeneron Pharmaceuticals, Inc.: Employment.

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Pozelimab, a Human Antibody Against Complement Factor C5, Demonstrates Robust Inhibition of Alternative Complement Activity Both in Normal Human Serum and in Phase I Normal Healthy Volunteers

Blood ◽

10.1182/blood-2019-129865 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2278-2278 ◽

Cited By ~ 1

Author(s):

Kishor Devalaraja-Narashimha ◽

Yan G Ni ◽

Cong Huang ◽

Ming-Dauh Wang ◽

Umesh Chaudhari ◽

...

Keyword(s):

Phase I ◽

Healthy Volunteers ◽

Ex Vivo ◽

Alternative Pathway ◽

Classical Pathway ◽

Complement Factor ◽

Employment Equity ◽

Equity Ownership ◽

Dose Dependent ◽

Maximal Suppression

INTRODUCTION: Blockade of complement factor C5 has demonstrated benefit in paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, generalized myasthenia gravis and neuromyelitis optica. We have completed a Phase I study of pozelimab, a fully human anti-C5 IgG4, in healthy volunteers. Pozelimab was well tolerated and resulted in dose-dependent inhibition of hemolytic activity through the classical complement pathway in normal healthy volunteers. Complete inhibition of hemolytic activity was maintained over a 4-week dosing period by a weekly subcutaneous (SC) regimen following an intravenous (IV) loading dose (ASH2018 abstract). OBJECTIVE: To further characterize the impact of pozelimab on the alternative complement pathway activity, we investigated the effect of pozelimab on alternative pathway-mediated hemolysis using an AH50 assay in the completed first-in-human (FIH) study. In addition, we compared the effect of pozelimab in both alternative and classical pathway hemolysis assays with those of in-house eculizumab and in-house ravulizumab in pooled normal human serum (NHS) samples, ex vivo. METHODS: In total, 56 subjects were randomized to 4 sequential ascending IV single dose cohorts plus 2 sequential ascending SC single dose cohorts followed by 1 multiple dose cohort (consisting of an IV loading dose and weekly SC doses). Each cohort consisted of 8 subjects randomized to receive pozelimab or placebo (6 active: 2 placebo). Serum collected at multiple time-points was utilized to assess the effect of pozelimab on alternative pathway activity. For ex vivo spike experiments, pooled NHS was used to compare the hemolytic function of pozelimab, in-house produced eculizumab and in-house produced ravulizumab. Comparator antibodies were synthesized from published sequence. The alternative pathway (AP) and classical pathway (CP) hemolysis assays were performed based on lysis of rabbit red blood cells (RBCs) and sensitized sheep RBCs, respectively. Both assays measure the amount of hemoglobin released from red blood cells at 412 nm. RESULTS: In the FIH study, baseline AH50 was comparable across treatment groups with a mean of 110 U/mL (standard deviation = 19, n = 56). Pozelimab exposure led to dose-dependent inhibition of AH50. In all 4 IV dosing cohorts, peak suppression of hemolysis was observed at end of infusion (EOI). Maximal suppression of hemolysis was approximately −85% change from baseline. This was achieved with the 30 mg/kg IV group and the repeat dose 15 mg/kg IV + 400 mg SC QW group. In the 2 SC cohorts, peak suppression of hemolysis was observed 3-7 days post dosing, which was consistent with observed peak concentrations of pozelimab in serum. In an ex vivo spike study, pozelimab, in-house eculizumab and in-house ravulizumab were spiked into 10, 25 or 48% pooled NHS for AP, and 5, 10 or 25% for CP. The results from AP hemolysis assays showed that, for a given concentration of spiked antibody, the maximal suppression of hemolysis for all the antibodies decreased with increased percentage of serum (Figure). The maximal suppression of hemolysis was consistently higher (32-169%) for pozelimab compared with in-house eculizumab, and lower for in-house ravulizumab compared with pozelimab and in-house eculizumab at all serum percentages tested. The results from CP hemolysis assays showed that, although the maximal suppression of hemolysis was similar for all antibodies tested, in-house ravulizumab was required to be at least a log higher in concentration to achieve a similar effect as the other two anti-C5 antibodies. CONCLUSIONS: Ex vivo studies with pooled NHS demonstrate that pozelimab robustly blocks both CP and AP hemolysis. In-house ravulizumab appeared to be less potent compared with in-house eculizumab in both CP and AP hemolysis assays. The Phase I healthy volunteer study of pozelimab demonstrated dose-dependent and significant inhibition of alternative pathway hemolysis, with the maximal suppression of hemolysis approximately −85% change from baseline. Figure Disclosures Devalaraja-Narashimha: Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Ni:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Huang:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Wang:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Chaudhari:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Prasad:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Harari:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Rankin:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Morton:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. Weyne:Regeneron Pharmaceuticals, Inc.: Employment, Equity Ownership. OffLabel Disclosure: The drug is pozelimab and it is a fully human anti-C5 IgG4 being tested a Phase 1 study in healthy volunteers.

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

Blood ◽

10.1182/blood.v126.23.4548.4548 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4548-4548 ◽

Cited By ~ 1

Author(s):

Ann Barbier ◽

Bruce Silver ◽

Elizabeth Merica ◽

Marvin Cohen ◽

Charles Kung ◽

...

Keyword(s):

Blood Cell ◽

Pyruvate Kinase ◽

Red Blood Cell ◽

High Dose ◽

Phase 2 ◽

Employment Equity ◽

Phase 2 Trial ◽

Metabolic Defect ◽

Equity Ownership ◽

2,3 Dpg

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.

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Phenotype Does Not Always Equal Function: HDAC Inhibitors and UM171, but Not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-Negative Human Cells

Blood ◽

10.1182/blood.v130.suppl_1.659.659 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 659-659

Author(s):

Kevin A. Goncalves ◽

Megan D. Hoban ◽

Jennifer L. Proctor ◽

Hillary L. Adams ◽

Sharon L. Hyzy ◽

...

Keyword(s):

In Vitro Culture ◽

Small Molecule ◽

Hdac Inhibitors ◽

Employment Equity ◽

Nsg Mice ◽

Cd34 Cells ◽

Equity Ownership ◽

Human Hscs

Abstract Background. The ability to expand human hematopoietic stem cells (HSCs) has the potential to improve outcomes in HSC transplantation and increase the dose of gene-modified HSCs. While many approaches have been reported to expand HSCs, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods is incomplete. In the present study, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors, the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to expand cells that engraft NSG mice. Although all strategies increased the number of phenotypic HSC (CD34+CD90+CD45RA-) in vitro, SR1 was the most effective method to increase the number of NOD-SCID engrafting cells. Importantly, we found that HDAC inhibitors and UM171 upregulated phenotypic stem cell markers on downstream progenitors, suggesting that these compounds do not expand true HSCs. Methods. Small-molecules, SR1, HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid) and UM171 were titrated and then evaluated at their optimal concentrations in the presence of cytokines (TPO, SCF, FLT3L, and IL6) for the ability to expand human mobilized peripheral blood (mPB)-derived CD34+ cells ex vivo . Immunophenotype and cell numbers were assessed by flow cytometry following a 7-day expansion assay in 10-point dose-response (10 µM to 0.5 nM). HSC function was evaluated by enumeration of colony forming units in methylcellulose and a subset of the compounds were evaluated by transplanting expanded cells into sub-lethally irradiated NSG mice to assess engraftment potential in vivo . All cells expanded with compounds were compared to uncultured or vehicle-cultured cells. Results. Following 7 days of expansion, SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (>3-35-fold) resulted in an increase in CD34+CD90+CD45RA- number relative to cells cultured with cytokines alone; however, only SR1 (18-fold) and UM171 (8-fold) demonstrated enhanced engraftment in NSG mice. Interestingly, while HDAC inhibitors and UM171 gave the most robust increase in the number and frequency of CD34+CD90+CD45RA- cells during in vitro culture, these methods were inferior to SR1 at increasing NSG engrafting cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggested that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. We tested this hypothesis by sorting CD34+CD90-CD45RA+ cells and culturing these with the various compounds. These experiments confirmed that both HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- cells and may explain the disconnect between in vitro HSC phenotype and NSG engraftment in vivo . This was further confirmed by evaluation of colony forming unit frequency of CD34+CD90-CD45RA+ cells after culture with compounds. Conclusions. We have showed that AHR antagonism is optimal for expanding functional human HSCs using the NSG engraftment model. We also demonstrated that UM171 and HDAC inhibitors upregulate phenotypic HSC markers on downstream progenitors. This could explain the discrepancy between impressive in vitro phenotypic expansion and insufficient functional activity in the NSG mouse model. Therefore, these data suggest caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these approaches move into clinical trials in patients. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hoban: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Adams: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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Northstar-2: Updated Safety and Efficacy Analysis of Lentiglobin Gene Therapy in Patients with Transfusion-Dependent β-Thalassemia and Non-β0/β0 Genotypes

Blood ◽

10.1182/blood-2019-126046 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3543-3543 ◽

Cited By ~ 1

Author(s):

Alexis A. Thompson ◽

Mark C. Walters ◽

Janet L. Kwiatkowski ◽

Suradej Hongeng ◽

John B. Porter ◽

...

Keyword(s):

Gene Therapy ◽

Clinical Trials ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Adult Patients ◽

Employment Equity ◽

Advisory Committees ◽

Transfusion Independence ◽

Equity Ownership

Background Transfusion-dependent β-thalassemia (TDT) is treated with regular, lifelong red blood cell (RBC) transfusions and despite iron-chelating therapy, carries a risk of serious organ damage from iron overload and other complications. Transplantation with autologous CD34+ cells encoding a βA-T87Q-globin gene (LentiGlobin for β-thalassemia) is being evaluated in patients with TDT. Interim results are presented here from the ongoing, international, single-arm, phase 3 Northstar-2 study (HGB-207; NCT02906202) of LentiGlobin gene therapy in pediatric, adolescent, and adult patients with TDT (defined by receiving ≥100 mL/kg/yr of RBCs or ≥8 RBC transfusions/yr) and non-β0/β0 genotypes. Methods Patients undergo hematopoietic stem cell (HSC) mobilization with G-CSF and plerixafor. Following apheresis, CD34+ cells are transduced with BB305 lentiviral vector and infused into patients after pharmacokinetic-adjusted, single-agent busulfan myeloablation. The primary efficacy endpoint is transfusion independence (TI; weighted average hemoglobin [Hb] ≥9 g/dL without RBC transfusions for ≥12 months). HSC engraftment, βA-T87Q-globin expression, Hb levels, detection of replication competent lentivirus (RCL), and adverse events (AE) are also assessed. Patients are followed for 2 years and offered participation in a long-term follow-up study. Summary statistics are presented as median (min - max). Results Twenty patients were treated in Northstar-2 as of 13 December 2018 and have been followed for a median of 8.1 (0.5 - 22.2) months. At enrollment, median age was 16 (8 - 34) years; 5 patients were <12 years of age. Median drug product cell dose was 8.0 (5.0 - 19.9) x106 cells/kg and vector copy number was 3.2 (1.9 - 5.6) copies/diploid genome. Time to neutrophil and platelet engraftment in the 18/20 and 15/20 evaluable patients was 22.5 (13 - 32) and 45 (20 - 84) days, respectively. Non-hematologic grade ≥3 AEs in ≥3 patients after LentiGlobin infusion included stomatitis (n=12), febrile neutropenia (n=6), pyrexia (n=4), epistaxis (n=3), and veno-occlusive liver disease (n=3). One serious AE of grade 3 thrombocytopenia was considered possibly related to LentiGlobin. No patient died, had graft failure, or had detection of RCL. No insertional oncogenesis has been observed. Gene therapy-derived HbAT87Q stabilized approximately 6 months after infusion. In adolescent and adult patients treated with LentiGlobin, median HbAT87Q at Months 6, 12 and 18 was 9.5 (n=11), 9.2 (n=8), and 9.5 (n=3) g/dL, respectively. The median total Hb without transfusions at Months 6, 12, and 18 were 11.9 (n=11), 12.4 (n=8), 12.3 (n=2) g/dL, respectively. At Month 6, 91% (10/11) of patients had total Hb of >11 g/dL without transfusions. Five adolescent and adult patients were evaluable for the primary endpoint of transfusion independence, 4 (80%) of whom achieved TI. The median weighted average Hb during TI was 12.4 (11.5 - 12.6) g/dL which compared favorably to pre-transfusion nadir Hb levels before enrollment (median 9.1 g/dL [7.5 - 10.0 g/dL]). At time of analysis, the median duration of TI was 13.6 (12.0 - 18.2) months. One patient who did not achieve TI stopped transfusions for 11.4 months but resumed transfusions due to recurrent anemia. This patient had a 71.4% reduction in RBC transfusion volume from Month 6 to Month 18 compared to baseline. Marrow cellularity and myeloid:erythroid (M:E) ratios were evaluated in 8 adolescent and adult patients with ≥12 months follow-up to assess the effect of LentiGlobin treatment on dyserythropoiesis. Seven of 8 patients had improved marrow M:E ratios at Month 12 (0.63 - 1.90) compared with baseline (0.14 - 0.48). In patients who stopped transfusions, soluble transferrin receptor levels were reduced by a median of 72% (58% - 78%) at Month 12 (n=6). Updated outcomes in adolescents and adults and outcomes in pediatric patients will be reported. Summary In this update of the Northstar-2 study of LentiGlobin gene therapy in patients with TDT and non-β0/β0 genotypes, transfusion independence was observed in 4/5 evaluable adolescent and adults and 10/11 treated patients had total Hb of >11 g/dL without transfusion support 6 months after LentiGlobin infusion. HbAT87Q stabilized approximately 6 months after treatment and patients who stopped RBC transfusions had improved erythropoiesis. A safety profile consistent with busulfan conditioning was observed after LentiGlobin gene therapy. Disclosures Thompson: bluebird bio, Inc.: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Baxalta: Research Funding. Walters:TruCode: Consultancy; AllCells, Inc: Consultancy; Editas Medicine: Consultancy. Kwiatkowski:bluebird bio, Inc.: Consultancy, Research Funding; Terumo: Research Funding; Celgene: Consultancy; Agios: Consultancy; Imara: Consultancy; Apopharma: Research Funding; Novartis: Research Funding. Porter:Protagonism: Honoraria; Celgene: Consultancy, Honoraria; Bluebird bio: Consultancy, Honoraria; Agios: Consultancy, Honoraria; La Jolla: Honoraria; Vifor: Honoraria; Silence therapeutics: Honoraria. Thrasher:Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 4BIOCapital: Membership on an entity's Board of Directors or advisory committees. Thuret:BlueBird bio: Other: investigators for clinical trials, participation on scientific/medical advisory board; Celgene: Other: investigators for clinical trials, participation on scientific/medical advisory board; Novartis: Other: investigators for clinical trials, participation on scientific/medical advisory board; Apopharma: Consultancy. Elliot:bluebird bio, Inc.: Employment, Equity Ownership. Tao:bluebird bio, Inc.: Employment, Equity Ownership. Colvin:bluebird bio, Inc.: Employment, Equity Ownership. Locatelli:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Miltenyi: Honoraria.

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