Phase 1 Multiple Ascending Dose Study of Safety, Tolerability, and Pharmacokinetics/Pharmacodynamics of Mitapivat (AG-348) in Subjects with Sickle Cell Disease

Background. Sickle cell disease (SCD) is a devastating disorder initiated by polymerization of the deoxy-hemoglobin S (HbS) to form fibers that distort (sickle) erythrocytes. Increased intracellular 2,3- diphosphoglycerate (2,3-DPG) stabilizes fibers and promotes sickling, while decreased intracellular adenosine triphosphate (ATP) levels can lead to hemolysis. Mitapivat (AG-348) is an oral, small molecule, allosteric activator capable of activating both mutant and wild type red cell pyruvate kinase (PKR), thus decreasing 2,3-DPG and increasing ATP levels in red cells and potentially acting as an anti-sickling agent. The safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of multiple ascending doses of mitapivat were assessed in subjects with SCD in a Phase I study. Methods. Subjects aged ≥ 18 years with a confirmed diagnosis of SCD (HbSS), adequate organ function, baseline Hb ≥ 7 g/dL, and no transfusions or erythropoietin therapy in the prior 3 months were eligible. Concomitant stable hydroxyurea (HU) and/or L-Glutamine therapy was permitted on study. Subjects received either 3 or 4 ascending dose levels of mitapivat (5 mg BID, 20 mg BID, 50 mg BID, 100 mg BID) for 2 weeks' duration each, followed by a 12-15-day drug taper. The primary endpoint was safety and tolerability as assessed by frequency and severity of adverse events (AEs) and laboratory parameters. Secondary endpoints included changes in hematological parameters, 2,3-DPG and ATP levels, and markers of Hb S polymerization (p50 and t50). Results. Nine subjects have been enrolled as of July 2020. One patient discontinued treatment early and was lost to follow-up. Results pertain to the 8 subjects who completed study: mean age was 43.6 years (range 34-55 years), 5 were male, and 7 were stabilized on HU. The initial 6 subjects escalated to a maximum dose of 50 mg BID, and the subsequent 2 subjects escalated up to a 100 mg BID dose following a protocol amendment. All 8 subjects reported AEs; the most common treatment related AEs were hypertension (n=3, Grades 1-3), insomnia (n=3, Grade 2), and asymptomatic heart rate increase on vital signs (n=3, Grade 1). 5 serious AEs (SAEs) were reported in 4 subjects, with only 1 SAE possibly attributable to study drug - a vaso-occlusive crisis (VOC) during drug taper, after which the protocol was amended to increase the length of taper from 9 to 12 days for the 50 mg BID dose and 15 days for the 100 mg BID dose. No VOCs have occurred during core period or on the prolonged drug taper. The SAEs not attributable to study drug included a VOC at the end of study, VOC-related transaminitis (Grade 2), a non-VOC-related pain episode, and a pre-existing pulmonary embolism discovered shortly after study drug initiation. Table 1 details the PD and clinical laboratory measures. Mean 2,3-DPG levels decreased and ATP levels increased in a dose-dependent manner. ATP levels remained elevated immediately following drug taper in 5/8 subjects. Both ATP and 2,3-DPG levels returned to near baseline by 4 weeks after drug taper. Decreases in p50 and increases in t50 at various dose levels support an anti-sickling effect of mitapivat in a subset of subjects, though responses were highly variable. Mitapivat increased Hb levels in a dose-dependent manner with a concomitant decrease in hemolytic markers. Mean change in Hb from baseline for all 8 subjects at the 50 mg BID dose level was 1.2 g/dL (range -0.3-2.7 g/dL), followed by near return to mean Hb baseline after drug taper. Hemolytic markers similarly returned to near baseline after drug taper. 3/8 and 5/8 subjects achieved a Hb increase of ≥ 1 g/dL at the 20 and 50 mg BID dose levels, respectively. A small dose-dependent increase in mean corpuscular volume (MCV) was also observed, suggesting a potential alternative mechanism of mitapivat, acting through the ATP-dependent Gardos channel to increase red cell hydration, decrease hemolysis, and improve red cell survival in SCD. Conclusion. Mitapivat demonstrated an acceptable safety profile across the tested dose levels in 8 subjects with SCD. Analyses of data show promising evidence of efficacy in terms of Hb increase from baseline with concomitant decreases in hemolytic markers. The accompanying changes in metabolites and sickling studies are consistent with the proposed mechanism of the drug. The study is ongoing with a planned sample size of 15 subjects completing 6-8 weeks of treatment. Additional data including PK will be presented. Disclosures Iyer: Novartis: Current equity holder in publicly-traded company; Agios Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Mangus:Agios Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Kung:Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Dang:Agios Pharmaceuticals Inc.: Current Employment, Current equity holder in publicly-traded company. Kosinski:Agios Pharmaceuticals Inc: Current Employment, Current equity holder in publicly-traded company. Hawkins:Infinity Pharmaceuticals: Current equity holder in publicly-traded company; Bristol-Myers Squibb: Current equity holder in publicly-traded company; Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company; Jazz Pharmaceuticals: Current equity holder in publicly-traded company.

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Mitapivat (AG-348) Demonstrates Safety, Tolerability, and Improvements in Anemia, Hemolysis, Oxygen Affinity, and Hemoglobin S Polymerization Kinetics in Adults with Sickle Cell Disease: A Phase 1 Dose Escalation Study

Blood ◽

10.1182/blood-2021-145398 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 10-10

Author(s):

Julia Z. Xu ◽

Anna Conrey ◽

Ingrid Frey ◽

Eveline Gwaabe ◽

Laurel A Menapace ◽

...

Keyword(s):

Dose Level ◽

Stock Options ◽

Phase 1 ◽

Oxygen Affinity ◽

Red Cell ◽

Publicly Traded ◽

Atp Levels ◽

Dose Levels ◽

2,3 Dpg ◽

Privately Held

Abstract Background. Hemoglobin S (HbS) polymerization causes red cell sickling, hemolysis, and vaso-occlusion, key pathological features of sickle cell disease (SCD). Mitapivat (AG-348) has potential as an oral anti-sickling agent in SCD via increasing glycolytic activity, which reduces intracellular levels of 2,3-diphosphoglycerate (2,3-DPG) in parallel with increasing adenosine triphosphate (ATP). Reducing 2,3-DPG decreases HbS polymerization, while increasing ATP improves red cell membrane integrity. Here, we report the complete results of our single-center Phase 1 study of multiple ascending doses of mitapivat in subjects with SCD. Methods. We enrolled adult subjects (age ≥ 18 years) with confirmed SCD (HbSS) and baseline Hb ≥ 7 g/dL; with no recent transfusions, erythropoietin therapy, or changes in SCD-specific therapies including hydroxyurea (HU) and L-glutamine. Subjects received either 3 or 4 ascending dose levels of mitapivat (5 mg BID, 20 mg BID, 50 mg BID, 100 mg BID) for 2 weeks' duration each, followed by a 12-15 day drug taper. Safety and tolerability were assessed by frequency and severity of adverse events (AEs) and changes in hemoglobin (Hb) level and hemolytic markers. For each dose level, pharmacokinetics (PK), pharmacodynamics (PD; 2,3-DPG and ATP levels), and markers of oxygen (O 2) affinity (p50) and HbS polymerization (t50) were assessed pre-dose, post-dose, at end of taper, and at end of study. p50 is the partial pressure of O 2 at which 50% of the hemes in the Hb molecule have O 2 bound; t50 is the time at which 50% of erythrocytes are sickled in response to gradual deoxygenation with nitrogen to a final O 2 partial pressure of 38 torr. Results. Out of 17 subjects enrolled, 16 escalated to 50 mg BID. One subject, withdrawn 3 days after starting the study for a pre-existing pulmonary embolus, was not evaluable for response. After a protocol amendment, 9/10 eligible subjects completed the 100 mg BID dose level; 1 subject self-discontinued treatment after completing 3 dose levels. Mean age of the 17 subjects was 39 years (range 23-55 years); 11 were male, and 12 were on HU. Mitapivat was well tolerated; the most commonly reported drug-related AEs were insomnia (n=6 subjects, Grades 1-2), arthralgia (n=3, Grades 1-2), and hypertension (n=3, Grades 1-3). Six serious AEs (SAEs) were reported in 6/17 subjects, including 4 vaso-occlusive crises (VOCs), 1 non-VOC-related pain, and 1 pre-existing pulmonary embolism; 2/6 SAEs were deemed possibly drug-related. Of the 4 VOCs, 2 occurred during drug taper and were possibly drug-related, and 2 occurred during the 28-day safety follow up post-treatment in the setting of known VOC triggers. In 16 evaluable subjects, a dose-dependent decrease in mean 2,3-DPG levels and increase in mean ATP levels were consistently observed, followed by a return to near baseline by end of study (Figure 1A-B). There was a mean decrease in p50 and increase in t50 (Figure 1D-E), indicating increased oxygen affinity and slower sickling, respectively. The mean Hb increase at the 50 mg BID dose level was 1.2 g/dL (range -0.3-2.9 g/dL; Figure 1C). Over half (9/16, 56.3%) of subjects achieved a Hb response, defined as a ≥ 1 g/dL increase in Hb at any dose level compared to baseline. Subjects also experienced a mean reduction in the hemolytic markers of lactate dehydrogenase, total serum bilirubin, absolute reticulocyte count, and aspartate aminotransferase during the dose escalation period (Figure 1F-I), though responses were variable. Mean corpuscular volume (MCV) and HbF levels remained relatively stable throughout the study, supporting the notion that hydroxyurea exposure remained stable throughout the treatment period. Conclusion. During a 6-8 week treatment period, mitapivat demonstrated an acceptable safety and tolerability profile at multiple ascending dose levels in subjects with SCD. Mitapivat improved anemia, reduced markers of hemolysis, decreased 2,3-DPG and increased ATP levels, improved oxygen affinity, and decreased sickling rate, signaling its potential to improve clinically meaningful outcomes in SCD. Long-term disease modifying effects of mitapivat treatment in SCD are being evaluated in an ongoing extension study (ClinicalTrials.gov NCT04610866). Figure 1 Figure 1. Disclosures Iyer: Novartis: Current equity holder in publicly-traded company; Agios Pharmaceuticals: Current Employment, Current holder of stock options in a privately-held company. Mangus: Agios Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company; Bristol-Myers Squibb: Current equity holder in publicly-traded company. Kung: Agios Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Dang: Agios Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Kosinski: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Hawkins: Bristol-Myers Squibb: Current equity holder in publicly-traded company; Agios: Current equity holder in publicly-traded company.

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Novel Structurally-Modified Allosteric Effectors of Hemoglobin Exhibit Superior Antisickling Properties

Blood ◽

10.1182/blood.v124.21.218.218 ◽

2014 ◽

Vol 124 (21) ◽

pp. 218-218

Author(s):

Osheiza Abdulmalik ◽

Tanvi Deshpande ◽

Mohini Ghatge ◽

Yan Zhang ◽

Jurgen Venitz ◽

...

Keyword(s):

Sickle Cell Disease ◽

Phase I ◽

Sickle Cell ◽

Clinical Studies ◽

Ex Vivo ◽

Dependent Manner ◽

Cell Disease ◽

Hb S ◽

Dose Dependent

Abstract Sickle cell disease (SCD) continues to cause significant morbidity, mortality and healthcare disparities. Despite considerable progress in understanding the underlying pathophysiology and investigating various therapeutic strategies, novel pharmacologic approaches to ameliorate SCD continue to hold immense potential and promise, especially for patients in developing countries. Our group and others have recently renewed and refocused attention to candidate drugs that directly bind to hemoglobin (Hb) and increase oxygen (O2) affinity, preventing the fundamental pathophysiology of the disease, i.e., sickle Hb (Hb S) polymerization and red blood cell (RBC) sickling. While several candidate drugs have shown biological activity in-vitro, ex-vivo and in animal studies, their ultimate success in clinical studies was hampered by toxicity concerns and/or low oral bioavailability. Recent promising reports from a phase I/II study on 5-HMF renews optimism for this therapeutic approach. We reasoned that modifications of vanillin--a previously reported antisickling agent and food constituent without known toxicities--to enhance its efficacy, would represent a feasible approach in rationally developing clinically useful candidate drugs. Consequently, we designed and synthesized two classes of compounds: INN and TD series. The former are pyridyl derivatives of vanillin, rationalized to stereospecifically inhibit deoxy-Hb S polymer formation while increasing the fraction of the soluble oxy-Hb S in regions of low O2 tension. The TD compounds represent further modification of corresponding INN compounds (with a methoxyl group on the pyridine ring), rationalized to exhibit similar dual antisickling effects, but with enhanced direct polymer destabilization properties. We subjected a prototypical compound from each class (INN-270 and TD-7) to our battery of exploratory in-vitro assays, specifically: 1) rates of Hb S binding/modification, 2) corresponding change in O2 affinity, 3) direct inhibition of Hb S polymerization, and 4) inhibition of RBC sickling under hypoxia. We incubated 0.5, 1, or 2 mM of either INN-270 or TD-7 with RBCs from patients with homozygous SCD, under hypoxia (4% O2/96% N2 gas mixture) in a shaker-incubator at 37 ˚C for 3 h. Assays were conducted in at least three replicates utilizing different samples on different days. At the conclusion of each assay, aliquot samples (~ 10 μl each) were drawn into a fixing solution under hypoxia to preserve RBC morphology for analyses. Residual RBC suspensions were washed, hemolyzed, and subjected to: cation-exchange HPLC (to determine Hb modification); P50 analyses to establish change in O2 affinity; and temperature-dependent delay time studies to establish a delay in Hb S polymerization. Our results show that both compounds permeated RBC membranes without causing hemolysis, bound to and modified intracellular Hb at high levels in a dose dependent manner, increased O2 affinity significantly, and inhibited sickling of RBCs under hypoxia. TD-7 modified Hb S in a dose-dependent manner (to 92.3 ± 5.2 %, n=4 at 2 mM), shifted O2 equilibrium to the left (Δp50 = 45.6 ± 8.2 %, n=3 at 2 mM), and inhibited RBC sickling (by 95 -100 %, n=4). Preliminary delay time analyses also showed that at 2 mM, TD-7 increased the Hb S polymerization times from 18.1 ± 1.0 min to 24.5 ± 0.5 min. INN-270 showed a similar profile, however with a lower efficacy (at 2 mM) for Hb S modification (to ~ 75 %), Δp50 of 40.3 %, sickling inhibition by ~ 70 %, and increased delay times from 15.6 ± 0.5 min to 19.7 ± 1.0 min. We have elucidated the dual antisickling mechanism of action of INN-270 and TD-7 by X-ray crystallography. Two molecules of each compound bind to Hb via Schiff-base, and a series of hydrogen-bond/hydrophobic interactions that favor a high-O2-affinity Hb state. Importantly, the methoxyl group on the pyridine ring of TD-7 forms hydrogen-bond interactions with the surface-located αF-helix, resulting in a conformational change, possibly explaining the improved potency. Based on our results, both TD7 and INN 270 exhibited greater than a 40- and 3-fold superiority in efficacy compared to vanillin and 5-HMF, respectively. We conclude that our findings justify a prospective, structure-based approach to designing novel antisickling agents with enhanced potency. In-vitro/ex-vivo murine and human PK/PD studies are currently ongoing to help guide planned in-vivo PK/PD studies in mice. Disclosures Venitz: Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: Consultancy. Safo:Baxter and AesRx companies have licensed our patented antisickling compounds. Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: #7160910; #7119208 Patents & Royalties, Consultancy, Research Funding.

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Effects of NO-Donors on Thrombus Formation and Microcirculation in Cerebral Vessels of the Rat

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650532 ◽

1996 ◽

Vol 76 (01) ◽

pp. 111-117 ◽

Cited By ~ 10

Author(s):

Yasuto Sasaki ◽

Junji Seki ◽

John C Giddings ◽

Junichiro Yamamoto

Keyword(s):

Blood Flow ◽

Thrombus Formation ◽

Dependent Manner ◽

Red Cell ◽

Cell Velocity ◽

Red Cell Velocity ◽

The Mean ◽

Wall Shear ◽

Dose Dependent

SummarySodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), are known to liberate nitric oxide (NO). In this study the effects of SNP and SIN-1 on thrombus formation in rat cerebral arterioles and venules in vivo were assessed using a helium-neon (He-Ne) laser. SNP infused at doses from 10 Μg/kg/h significantly inhibited thrombus formation in a dose dependent manner. This inhibition of thrombus formation was suppressed by methylene blue. SIN-1 at a dose of 100 Μg/kg/h also demonstrated a significant antithrombotic effect. Moreover, treatment with SNP increased vessel diameter in a dose dependent manner and enhanced the mean red cell velocity measured with a fiber-optic laser-Doppler anemometer microscope (FLDAM). Blood flow, calculated from the mean red cell velocity and vessel diameters was increased significantly during infusion. In contrast, mean wall shear rates in the arterioles and venules were not changed by SNP infusion. The results indicated that SNP and SIN-1 possessed potent antithrombotic activities, whilst SNP increased cerebral blood flow without changing wall shear rate. The findings suggest that the NO released by SNP and SIN-1 may be beneficial for the treatment and protection of cerebral infarction

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Increased Procoagulant Activity of Red Blood Cells from Patients with Homozygous Sickle Cell Disease and β-Thalassemia

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650577 ◽

1996 ◽

Vol 76 (03) ◽

pp. 322-327 ◽

Cited By ~ 73

Author(s):

Dominique Helley ◽

Amiram Eldor ◽

Robert Girot ◽

Rolande Ducrocq ◽

Marie-Claude Guillin ◽

...

Keyword(s):

Sickle Cell Disease ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Annexin V ◽

Mean Value ◽

Procoagulant Activity ◽

Dependent Manner ◽

Cell Disease ◽

Homozygous Sickle Cell Disease

SummaryIt has recently been proved that, in vitro, red blood cells (RBCs) from patients with homozygous β-thalassemia behave as procoagulant cells. The procoagulant activity of β-thalassemia RBCs might be the result of an increased exposure of procoagulant phospholipids (i. e. phosphatidylserine) in the outer leaflet of the membrane. In order to test this hypothesis, we compared the catalytic properties of RBCs of patients with β-thalassemia and homozygous sickle cell disease (SS-RBCs) with that of controls. The catalytic parameters (Km, kcat) of prothrombin activation by factor Xa were determined both in the absence and in the presence of RBCs. The turn-over number (kcat) of the reaction was not modified by normal, SS- or (3-thalassemia RBCs. The Km was lower in the presence of normal RBCs (mean value: 9.1 µM) than in the absence of cells (26 µM). The Km measured in the presence of either SS-RBCs (mean value: 1.6 µM) or β-thalassemia RBCs (mean value: 1.5 pM) was significantly lower compared to normal RBCs (p <0.001). No significant difference was observed between SS-RBCs and p-thalassemia RBCs. Annexin V, a protein with high affinity and specificity for anionic phospholipids, inhibited the procoagulant activity of both SS-RBCs and (3-thalassemia RBCs, in a dose-dependent manner. More than 95% inhibition was achieved at nanomolar concentrations of annexin V. These results indicate that the procoagulant activity of both β-thalassemia RBCs and SS-RBCs may be fully ascribed to an abnormal exposure of phosphatidylserine at the outer surface of the red cells.

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PHYTOCHEMICAL AND BIOLOGICAL STUDIES OF ROTULA AQUATICA LOUR. ROOTS

INDIAN DRUGS ◽

10.53879/id.50.11.p0034 ◽

2013 ◽

Vol 50 (11) ◽

pp. 34-38

Author(s):

T. Shyam ◽

◽

S Ganapaty

Keyword(s):

Body Weight ◽

Spectroscopic Data ◽

Methanolic Extract ◽

Hepatoprotective Activity ◽

Dependent Manner ◽

Biological Studies ◽

Bacteria And Fungi ◽

Dose Levels ◽

Dose Dependent ◽

Chemical Evidence

Four compounds viz α-amyrin, β- amyrin, bauerenol and ellagic acid were isolated from the methanolic extract of Rotula aquatica roots. The structures of these compounds were elucidated on the basis of spectroscopic data analysis and chemical evidence. The extract was evaluated for hepatoprotective activity against carbon tetrachloride induced hepatotoxic model at a dose levels of 200,400 and 800 mg/ kg body weight and compared with that of the standard silymarin (25mg/kg body weight). It showed good hepatoprotective activity in a dose dependent manner. The extract was also screened for antimicrobial activity against various types of organisms like bacteria and fungi.

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MO258SAFETY, TOLERABILITY, PHARMACOKINETICS AND PHARMACODYNAMICS OF VIS649, AN APRIL-NEUTRALIZING IGG2 MONOCLONAL ANTIBODY, IN HEALTHY VOLUNTEERS: PHASE 1, RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED, SINGLE ASCENDING DOSE STUDY

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab104.0016 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Yusuke Suzuki ◽

Mohit Mathur ◽

Jonathan Barratt ◽

Frank Engler ◽

Jill Yarbrough ◽

...

Keyword(s):

Phase 1 ◽

Study Drug ◽

Dependent Manner ◽

Double Blind ◽

Serum Iga ◽

Dose Study ◽

Dose Response Effect ◽

Japanese Descent ◽

Dose Dependent ◽

Single Ascending Dose Study

Abstract Background and Aims Immunoglobulin A (IgA) nephropathy (IgAN) is a glomerulonephritis characterized by the presence of circulating and glomerular immune complexes containing galactose-deficient (Gd) IgA1. A proliferation-inducing ligand (APRIL), a member of the tumor necrosis factor superfamily of ligands, is thought to play a key role in the pathogenesis of IgAN by virtue of its role in class-switching to IgA production. VIS649, a humanized immunoglobulin G (IgG2) monoclonal antibody that binds to and blocks the biological actions of APRIL, is in clinical development as a potential treatment for IgAN. The primary objective of this first-in-human study was to evaluate the safety and tolerability of VIS649 in healthy volunteers. Secondary objectives included characterization of the pharmacokinetics (PK) and pharmacodynamics (PD) of VIS649. Method This was a Phase 1, randomized, double-blind, placebo-controlled, single ascending dose study of VIS649 in healthy adult male and female volunteers (ClinicalTrials.gov identifier: NCT03719443). The study was conducted in sequential dosing cohorts. The first four cohorts (0.5, 2.0, 6.0, and 12.0 mg/kg, respectively) each enrolled 9 participants (4 of Japanese descent and 5 of non-Japanese descent) who were randomized to VIS649 or placebo in a ratio of 7:2. In addition, a fifth cohort enrolled 15 adults randomized to receive VIS649 6.0 mg/kg or placebo (10:5), followed by tetanus/diphtheria vaccine challenge after 28 days (TENIVAC®, Sanofi Pasteur Limited; the effect of APRIL inhibition on vaccine response is described in a companion abstract). Participants received intravenous administration of study drug on Day 1, were discharged from the institution on Day 2, and were followed for 16–24 weeks on an outpatient basis. Standard safety assessments and blood sampling for PK and PD were performed at regular intervals. Results 51 participants were randomized and dosed with study drug, of whom 47 (92.2%) completed the study. VIS649 was well tolerated, with no serious adverse events (AEs) or AEs that led to study discontinuation. Most treatment-emergent AEs (TEAEs) were mild; the incidence and severity of TEAEs were not dose dependent. One participant in the 2.0 mg/kg group experienced a severe TEAE of syncope following phlebotomy that the investigator considered unlikely to be related to study drug. There was no clinically relevant effect of treatment on laboratory tests, vital signs, electrocardiogram parameters, or physical examinations. VIS649 had non-linear PK: half-life (t½) increased with dose, while drug exposure (AUC) increased in a greater than dose proportional manner. Serum IgA, Gd-IgA1, IgG, and IgM were reversibly suppressed in a dose-dependent manner following VIS649 administration. The maximum mean percentage reductions from baseline occurred at Week 12 for the 12.0 mg/kg dose: IgA, -57.2% (Figure); Gd-IgA1, -71.6% (Figure); IgG, -33.6%; and IgM, -67.2%. These reductions were reversible and showed a dose-response effect with respect to time-to-recovery. Mean free (non-VIS649 bound) serum APRIL levels decreased to the lower limit of quantification (50 pg/mL) for all VIS649 doses at Week 1, and also showed a dose-response effect with respect to time-to-recovery. No depletions in circulating lymphocyte populations were observed. There were no significant PK or PD differences between Japanese and non-Japanese participants. Conclusion A single dose of VIS649, up to 12.0 mg/kg, was safe and well tolerated in healthy adults and was able to suppress free serum APRIL to the lower level of quantification. Serum Gd-IgA1 decreased in parallel with total serum IgA and recovered in a dose-dependent manner following reappearance of free APRIL in serum. These data support the further clinical development of VIS649 as a potential treatment for IgAN.

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PHARMACOLOGICAL SCREENING OF ANTI-OBESITY POTENTIAL OF ACORUS CALAMUS LINN. IN HIGH FAT CAFETERIA DIET FED OBESE RATS

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2017.v10i4.16893 ◽

2017 ◽

Vol 10 (4) ◽

pp. 384 ◽

Cited By ~ 2

Author(s):

Athesh K ◽

Joshi G

Keyword(s):

Body Weight ◽

High Fat Diet ◽

Serum Glucose ◽

In Vivo Studies ◽

Acorus Calamus ◽

Dependent Manner ◽

Fat Pad ◽

High Fat ◽

Dose Levels ◽

Dose Dependent

Objective: To study the anti-obesity potential of aqueous rhizome extract of Acoruscalamus Linn. (AREAC)in high fat diet fed obese rats.Methods: Adult strain male Wistar rats used in this study were fed with High Fat Diet (HFD) for 60 days. For the treatment groups,AREAC was administered in a dose levels of100, 200 and 300 mg/kgbw, orally once a day along with HFD. Rats fed with normal pellet chow were served as normal control. The effect of AREAC on physical parameterssuch as body weight, organ weight, fat pad weights and various biochemical parameterslike serum glucose, insulin, leptin,lipid profile, liver markers, kidney markers and oxidative stress markers were analysed.In-vitro pancreatic lipase inhibition assay of AREAC was also studied.Results: Data of in-vivo studies revealedsignificant (p<0.05) reduction in percentage body weight gain, organ weights, fat pad weights and levels of serum glucose, insulin and leptin after treatment with AREAC in a dose dependent manner. Also, administration of AREAC significantly inhibited the increases in the concentrations of triglycerides, total cholesterol, LDL-cholesterol, VLDL-cholesterol, free-fatty acid and phospholipids in a dose dependent manner whereas, the level of HDL-cholesterol was found to be elevated on treatment. Moreover, on treatment with test drug,the elevated levels of serum liver and kidney markerssuch as AST, ALT, ALP, urea, creatinine were also brought back to near normalcy. Antioxidant status was found to be enhanced in liver tissues after treatment.In-vitro studies showed significant inhibition in the activity of pancreatic lipaseby AREAC.Conclusion: The data of the results obtained clearly depicted that AREAC was found to have pronounced anti-obesity activity particularly at the dose levels of 300 mg/kg bw.Key Words: Obesity, High Fat Diet, Leptin, AcoruscalamusLinn., Orlistat.

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Reconstruction of Sickle Cell Disease with Circulating Sickling Red Blood Cells in Novel Humanized Cytokines and Liver Mistrg Mice

Blood ◽

10.1182/blood-2020-141603 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 29-30

Author(s):

Yuanbin Song ◽

Rana Gbyli ◽

Liang Shan ◽

Wei Liu ◽

Yimeng Gao ◽

...

Keyword(s):

Sickle Cell Disease ◽

Mouse Model ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Red Cell ◽

Cell Disease ◽

Ineffective Erythropoiesis ◽

Cd34 Cells

In vivo models of human erythropoiesis with generation of circulating mature human red blood cells (huRBC) have remained elusive, limiting studies of primary human red cell disorders. In our prior study, we have generated the first combined cytokine-liver humanized immunodeficient mouse model (huHepMISTRG-Fah) with fully mature, circulating huRBC when engrafted with human CD34+ hematopoietic stem and progenitor cells (HSPCs)1. Here we present for the first time a humanized mouse model of human sickle cell disease (SCD) which replicates the hallmark pathophysiologic finding of vaso-occlusion in mice engrafted with primary patient-derived SCD HSPCs. SCD is an inherited blood disorder caused by a single point mutation in the beta-globin gene. Murine models of SCD exclusively express human globins in mouse red blood cells in the background of murine globin knockouts2 which exclusively contain murine erythropoiesis and red cells and thus fail to capture the heterogeneity encountered in patients. To determine whether enhanced erythropoiesis and most importantly circulating huRBC in engrafted huHepMISTRG-Fah mice would be sufficient to replicate the pathophysiology of SCD, we engrafted it with adult SCD BM CD34+ cells as well as age-matched control BM CD34+ cells. Overall huCD45+ and erythroid engraftment in BM (Fig. a, b) and PB (Fig. c, d) were similar between control or SCD. Using multispectral imaging flow cytometry, we observed sickling huRBCs (7-11 sickling huRBCs/ 100 huRBCs) in the PB of SCD (Fig. e) but not in control CD34+ (Fig. f) engrafted mice. To determine whether circulating huRBC would result in vaso-occlusion and associated findings in SCD engrafted huHepMISTRG-Fah mice, we evaluated histological sections of lung, liver, spleen, and kidney from control and SCD CD34+ engrafted mice. SCD CD34+ engrafted mice lungs showed an increase in alveolar macrophages (arrowheads) associated with alveolar hemorrhage and thrombosis (arrows) but not observed control engrafted mice (Fig. g). Spleens of SCD engrafted mice showed erythroid precursor expansion, sickled erythrocytes in the sinusoids (arrowheads), and vascular occlusion and thrombosis (arrows) (Fig. h). Liver architecture was disrupted in SCD engrafted mice with RBCs in sinusoids and microvascular thromboses (Fig. i). Congestion of capillary loops and peritubular capillaries and glomeruli engorged with sickled RBCs was evident in kidneys (Fig. j) of SCD but not control CD34+ engrafted mice. SCD is characterized by ineffective erythropoiesis due to structural abnormalities in erythroid precursors3. As a functional structural unit, erythroblastic islands (EBIs) represent a specialized niche for erythropoiesis, where a central macrophage is surrounded by developing erythroblasts of varying differentiation states4. In our study, both SCD (Fig. k) and control (Fig. l) CD34+ engrafted mice exhibited EBIs with huCD169+ huCD14+ central macrophages surrounded by varying stages of huCD235a+ erythroid progenitors, including enucleated huRBCs (arrows). This implies that huHepMISTRG-Fah mice have the capability to generate human EBIs in vivo and thus represent a valuable tool to not only study the effects of mature RBC but also to elucidate mechanisms of ineffective erythropoiesis in SCD and other red cell disorders. In conclusion, we successfully engrafted adult SCD patient BM derived CD34+ cells in huHepMISTRG-Fah mice and detected circulating, sickling huRBCs in the mouse PB. We observed pathological changes in the lung, spleen, liver and kidney, which are comparable to what is seen in the established SCD mouse models and in patients. In addition, huHepMISTRG-Fah mice offer the opportunity to study the role of the central macrophage in human erythropoiesis in health and disease in an immunologically advantageous context. This novel mouse model could therefore serve to open novel avenues for therapeutic advances in SCD. Reference 1. Song Y, Shan L, Gybli R, et. al. In Vivo reconstruction of Human Erythropoiesis with Circulating Mature Human RBCs in Humanized Liver Mistrg Mice. Blood. 2019;134:338. 2. Ryan TM, Ciavatta DJ, Townes TM. Knockout-transgenic mouse model of sickle cell disease. Science. 1997;278(5339):873-876. 3. Blouin MJ, De Paepe ME, Trudel M. Altered hematopoiesis in murine sickle cell disease. Blood. 1999;94(4):1451-1459. 4. Manwani D, Bieker JJ. The erythroblastic island. Curr Top Dev Biol. 2008;82:23-53. Disclosures Xu: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Flavell:Zai labs: Consultancy; GSK: Consultancy.

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Alkaline Phosphatase Is Associated with Red Cell Alloimmunization in the Pulmonary Hypertension and Hypoxic Response (PUSH) Sickle Cell Disease Cohort

Blood ◽

10.1182/blood-2020-143036 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 20-20

Author(s):

Victoria Brooks ◽

Oluwalonimi Adebowale ◽

Victor R. Gordeuk ◽

Sergei Nekhai ◽

James G. Taylor

Keyword(s):

Risk Factors ◽

Alkaline Phosphatase ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Research Funding ◽

Severe Disease ◽

Case Control ◽

Red Cell ◽

Cell Disease ◽

History Of

Background: Blood transfusion is a common therapy for sickle cell disease (SCD). Although, highly effective, a major limitation is development of alloantibodies to minor blood group antigens on donor red cells. Alloimmunization has a prevalence of 2-5% for transfusions in the general population, but it is significantly higher in SCD. Risk factors for alloimmunization have been poorly characterized, although number of lifetime transfusions is an important risk factor. Alloimmunization has been clinically observed in children with a prevalence of about 7%. With development of each antibody, blood donor matching becomes increasingly difficult and expensive with an increased risk for transfusion reactions and diminished availability of compatible red cell units for treatment of SCD. The ability to identify risk factors for developing alloantibodies would be beneficial for clinicians. To identify markers for alloimmunization in SCD, we have analyzed children and adults who developed this complication. Methods: We analyzed The Pulmonary Hypertension and Hypoxic Response in Sickle Cell Disease (PUSH) study, which enrolled n=468 pediatric and n=59 adult SCD subjects. In both children and adults, alloimmunization cases were defined as a history of at least 1 alloantibody. Controls in both cohorts were defined as subjects with no history of alloantibodies and receipt of more than 10 lifetime red cell transfusions. All others within the study who did not meet these criteria were assigned to a third comparison group. To identify differences between cases, controls and all others, we performed univariate analyses (using ANOVA or Kruskal Wallace where appropriate) for clinical parameters and laboratories. Case control comparisons were also performed for selected variables and plasma levels for 11 cytokines. Results were further analyzed using regression modeling. Results: The overall prevalence of alloimmunization was 7.3% among children (34/468 subjects; median age 12, range 3-20 years) compared to 28.8% in adults (17/59 subjects; median age 37, range 18-73 years). When only considering those with >10 lifetime transfusions, the prevalence was considerably higher at 29.3% and 54.8% in children and adults, respectively. At the same time, 8 pediatric (23.5%) and 5 adult (29.4%) alloimmunization cases had received fewer than 10 transfusions. In a 3-way pediatric cohort comparison (cases, controls and all others), risk factors associated with alloimmunization included SS genotype, older age and markers of more severe disease (higher ferritin, WBCs, platelets and total bilirubin). Comparison of cases to controls showed alkaline phosphatase (P=0.05) was significantly lower in cases, whereas AST (P=0.02) was significantly higher even with adjustment for age. Levels of plasma cytokines MCP-1 (P=0.01) and IFNgamma (P=0.08) were lower in cases from a subset of the pediatric cohort. In adults, only 4/59 (6.8%) subjects had never received a lifetime transfusion (all non-SS). In the adult 3-way comparisons, only SS genotype and higher ferritin were associated with alloimmunization. The adult case control analysis showed higher absolute monocyte count (P=0.02), absolute eosinophil count (P=0.04) and absolute basophil count (P=0.008) in association with alloimmunization cases. In addition, alkaline phosphatase was again significantly lower among cases (P=0.02) as seen in the pediatric cohort. There were no significant differences in cytokine levels among adults. Conclusions: When considering only transfused SCD patients, the prevalence of alloimmunization is higher than 30%. As seen in prior studies, higher lifetime red cell transfusions are an important risk factor especially among adults where most patients have received transfusions. Children who develop alloantibodies appear to have laboratory markers of more severe disease, but this is not observed in adults. A novel association observed across both pediatric and adult subjects is a significantly lower serum alkaline phosphatase in those with alloantibodies. The results of this study suggest a need for improved tracking of red cell transfusion therapy in the US for SCD patients due to a high prevalence of alloimmunization. Further study is also needed to elucidate the significance of the alkaline phosphatase association. Disclosures Gordeuk: CSL Behring: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; Ironwood: Research Funding; Imara: Research Funding.

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Sickle Cell Disease Model Mice Lacking 2,3-Dpg Show Reduced RBC Sickling and Improvements in Markers of Hemolytic Anemia

Blood ◽

10.1182/blood-2020-142052 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-28

Author(s):

Kelly M. Knee ◽

Amey Barakat ◽

Lindsay Tomlinson ◽

Lila Ramaiah ◽

Zane Wenzel ◽

...

Keyword(s):

Sickle Cell Disease ◽

Blood Cell ◽

Hemolytic Anemia ◽

Sickle Cell ◽

Red Blood Cell ◽

Organ Damage ◽

Complete Elimination ◽

Cell Disease ◽

The Impact ◽

2,3 Dpg

Sickle cell disease (SCD) is a severe genetic disorder caused by a mutation in hemoglobin (b6Glu-Val), which allows the mutant hemoglobin to assemble into long polymers when deoxygenated. Over time, these polymers build up and deform red blood cells, leading to hemolytic anemia, vaso-occlusion, and end organ damage. A number of recent therapies for SCD have focused on modulating the mutant hemoglobin directly, however, reduction or elimination of 2,3-DPG to reduce Hb S polymerization and RBC sickling has recently been proposed as a therapeutic strategy for SCD. Current clinical studies focus on activation of pyruvate kinase to reduce 2,3-DPG, however, direct targeting of the enzyme which produces 2,3-DPG; Bisphosphoglycerate Mutase (BPGM) may also be possible. In this study we evaluate the impact of elimination of 2,3-DPG on SCD pathology by complete knockout of BPGM in Townes model mice. Animals with complete knockout of BPGM (BPGM -/-) have no detectable 2,3-DPG, while animals that are heterozygous for BPGM (BPGM -/+) have 2,3-DPG levels comparable to Townes mice. Western Blot analysis confirms that BPGM -/- animals completely lack BPGM, while BPGM -/+ animals have BPGM levels that are nearly equivalent to Townes mice. As expected from the lack of 2,3-DPG, BPGM -/- animals have increased oxygen affinity, observed as a 39% decrease in p50 relative to Townes mice. Complete elimination of 2,3-DPG has significant effects on markers of hemolytic anemia in BPGM -/- mice. Mice lacking 2,3-DPG have a 60% increase in hemoglobin (3.7 g/dL), a 53% increase in red blood cell count, and a 29% increase in hematocrit relative to Townes mice. The BPGM -/- mice also have a 57% decrease in reticulocytes, and a 61% decrease in spleen weight relative to Townes animals, consistent with decreased extramedullary hematopoiesis. Consistent with the reduction in hemolysis, BPGM -/- animals had a 59% reduction in red blood cell sickling under robust hypoxic conditions. BPGM -/+ animals had hemoglobin, RBC, and hematocrit levels that were similar to Townes animals, and a similar degree of RBC sickling to Townes mice. Liver phenotype was similar across all variants, with areas of random necrosis observed in BPGM -/-, BPGM -/+ and Townes mice. Higher percentages of microcytic and/or hyperchromic RBCs were observed in BPGM -/- animals relative to BPGM -/+ or Townes animals. These results suggest that modulation of 2,3-DPG has a positive effect on RBC sickling and hemolytic anemia, which may have therapeutic benefits for SCD patients. However, the lack of improvement in organ damage suggests that modulation of 2,3-DPG alone may not be sufficient for complete elimination of SCD phenotypes, and further investigation of this therapeutic avenue may be necessary. Disclosures No relevant conflicts of interest to declare.

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