Global Blood Therapeutics Research Articles

Population Pharmacokinetic Modeling of Different Formulations of Inclacumab Using Phase 1 Data in Healthy Participants

Introduction: P-selectin is a cell adhesion molecule that contributes to vaso-occlusive crises (VOCs) in sickle cell disease (SCD). Inclacumab is a recombinant, fully human, immunoglobulin G4 monoclonal antibody that selectively binds to P-selectin to prevent binding with P-selectin glycoprotein ligand-1. Inclacumab was previously evaluated by Roche in clinical trials with healthy participants and patients with cardiovascular disease and is now being developed as a potential treatment to reduce the frequency of VOCs in individuals with SCD. Changes in the manufacturing of inclacumab have resulted in 3 formulations/drug materials: Roche v0.1, Roche v0.2, and Global Blood Therapeutics (GBT). GBT-manufactured inclacumab utilizes a similar formulation and the same cell line as Roche v0.2 (Roche v0.1 utilizes a different cell line), and analytical and nonclinical comparability between Roche v0.2 and GBT inclacumab have been demonstrated (Mihaila et al. Expert Opin Biol Ther 2022). Here we report results from a population pharmacokinetic (PopPK) model developed to compare the pharmacokinetics (PK) of different formulations using data from 4 phase 1 clinical studies in healthy participants administered Roche v0.1, Roche v0.2, or GBT inclacumab. The objectives were to develop a PopPK model to describe inclacumab PK in healthy adult participants and to quantify potential differences in inclacumab PK associated with different formulations/drug materials. Methods: A PopPK model was developed using single- and multiple-dose PK data from healthy participants who received intravenous (IV) inclacumab across 4 studies (BP21112 [Roche v0.1, single ascending dose (SAD)], BP21617 [Roche v0.1, multiple ascending dose], BP28134 [Roche v0.2, SAD], and GBT2104-111 [GBT, SAD]). Evaluable participants had ≥1 measurable postdose concentration with an associated sample time, relevant dosing information prior to each measurable concentration, and no protocol violation that could confound PK analyses. A nonlinear mixed effects model was fit to the inclacumab plasma concentration-time data using first-order conditional estimation with eta-epsilon interaction in NONMEM ® (v7.3). Model selection was based on the plausibility of parameter estimates, goodness-of-fit plots, and the objective function value. The final model was evaluated using a prediction-corrected visual predictive check (pcVPC). Results: The PopPK dataset comprised 145 healthy participants, of which 2622 inclacumab plasma concentrations (84.9%) from 143 participants were evaluable (Roche v0.1: n=68 [47.6%]; Roche v0.2: n=60 [42.0%]; GBT: n=15 [10.5%]). Median (range) age and body weight were 42 (21-63) years and 73.2 (45.8-109) kg, respectively. Nominal doses ranged from 0.03 to 40 mg/kg IV; 23.8% of participants received 3 mg/kg and 29.4% received 20 mg/kg. A 3-compartment disposition model with parallel linear clearance (CL=0.125 L/d) and nonlinear (concentration-dependent; V max=1.19 mg/d; K m=0.784 μg/mL) clearance from the central compartment described the plasma concentration-time data. The effect of drug material was incorporated a priori as relative bioavailability term F in the base model using exploratory analyses of observed exposures. Dosing of Roche v0.1 and Roche v0.2 inclacumab resulted in 23% and 2% higher exposure than GBT inclacumab, respectively. Between-subject variability was estimated for linear clearance, nonlinear clearance, and all volume parameters, and residual error was described using a proportional model (Table). Of the tested covariates, baseline body weight was a statistically significant covariate effect and was included on all linear clearance and volume terms. Model qualification by pcVPC showed good predictive performance of the final model. Conclusions: The PopPK of IV inclacumab in healthy participants was described by a 3-compartment disposition model with parallel linear and nonlinear clearances. Estimated relative exposure of Roche v0.2 and GBT-manufactured inclacumab was similar, indicating comparability in drug exposure. Understanding the relationship between exposure and dose for different formulations facilitates more accurate assessment of safety data across multiple clinical studies. These data provide support for the ongoing phase 3 clinical development in SCD using GBT-manufactured inclacumab without further clinical comparability testing.

Red Blood Cell Deformability and Morphological Dynamic Sickling As a Metric to Monitor Pharmacodynamic Response to Voxelotor

The recent introduction of new therapies, such as hemoglobin (Hb) oxygen affinity modifiers, fetal Hb (HbF) inducers, and gene therapy (replacement of HbS with non-sickling Hb), that affect the Hb polymerization rate for sickle cell disease (SCD), highlight the need for new metrics to assess changes in sickling characteristics as therapeutic response. Primary outcomes in clinical trials often focus on indirect clinical improvement i.e. decrease in vaso-occlusive events (VOC), Hb increase, fatigue and pain score. There is currently a lack of direct biomarkers to assess the pharmacodynamic impact of these therapies. Two methods have been developed to assess downstream Hb polymerization, LoRRca (oxygenscan), a commercial oxygen gradient ektacytometry that measures deformability as a function of oxygen tension (pO 2), and dynamic sickling assay (DSA), an enzymatic oxygen scavenging system that directly assesses morphological sickling. In the present study, we investigate the relationship between red blood cell (RBC) deformability and kinetics of sickling morphology after a treatment with different doses of voxelotor (GBT440, Global Blood Therapeutics), the only FDA approved Hb oxygen modifier. Blood samples from eight SCD patients were collected following the IRB #041718MP2E & IRB #Pro00066220 protocols. RBCs were incubated with voxelotor at five different doses based on sample hematocrit (Hct): 25% (0.32 ± 0.06 mM), 50% (0.65 ± 0.11 mM), 75% (0.97 ± 0.17 mM), 100% (1.29 ± 0.23 mM) and 125% (1.62 ± 0.28 mM), or with an equal volume of DMSO-containing buffer (vehicle). Oxygenscan is performed (LoRRca, RR Mechatronics, The Netherlands) as per manufacturer's instructions. The following parameters are calculated: i) elongation index (EImin), measured at minimum RBC deformability, ii) point of sickling (PoS), the pO 2 at which RBC deformability decreases below 5% of EImax during deoxygenation and iii) ΔEI, the difference between EImin and EImax. DSA uses an enzymatic reaction that allows for tight control of the rate and depth of hypoxia, resulting in RBC morphological changes observed through time-lapse photography. Sickling profiles were constructed to represent: i) morphological points of sickling at 50% of induced fraction (mPoS i@50%), ii) rate of sickling at 50% of induced fraction (rate i@50%) and iii) area under the curve at 15 minutes (AUC 15). Paired t-test analysis of the LoRRca elongation curves show a significant dose response in Elmin (Vehicle: 0.16 ± 0.16; 25%: 0.20 ± 0.16, p=0.0033; 50%: 0.23 ± 0.15, p=0.0001; 75%: 0.27 ± 0.15, p=0.0004; 100%: 0.30 ± 0.14, p=0.0009; 125%: 0.35 ± 0.13, p=0.0015), ΔEl (Vehicle: 0.26 ± 0.11; 25%: 0.22 ± 0.11, p=0.0016; 50%: 0.18 ± 0.11, p=0.0003; 75%: 0.15 ± 0.08, p=0.0002; 100%: 0.11 ± 0.06, p=0.0004; 125%: 0.09 ± 0.07, p=0.0011) and PoS (Figure 1; Vehicle: 35 ± 11 mmHg; 25%: 32 ± 6, p=0.0092; 50%: 28 ± 7, p=0.0038; 75%: 25 ± 6, p<0.0001; 100%: 17 ± 5, p=0.0007; 125%: 14 ± 4, p=0.0015). These findings indicate improvements in RBC deformability under hypoxic conditions when the drug is present. Furthermore, paired t-test of DSA data showed a significant dose response with AUC 15 (Vehicle: 958 ± 104; 25%: 909 ± 105, p=0.0061; 50%: 845 ± 146, p=0.0004; 75%: 768 ± 194, p=0.0014; 100%: 672 ± 179, p<0.0001; 125%: 623 ± 192, p=0.0002), rate i@50% (Vehicle: 0.87 ± 0.26; 25%: 0.77 ± 0.30, p=0.1236; 50%: 0.61 ± 0.29, p=0.0003; 75%: 0.33 ± 0.13, p<0.0001; 100%: 0.28 ± 0.10, p<0.0001; 125%: 0.25 ± 0.09, p=0.0004) and mPoS i@50% (Figure 2; Vehicle: 4.4 ± 0.7 minutes; 25%: 4.7 ± 0.9, p=0.0260; 50%: 5.0 ± 1.2, p=0.0407; 75%: 5.9 ± 1.7, p=0.0082; 100%: 7.0 ± 1.6, p=0.0002; 125%: 7.8 ± 1.8, p=0.0004). These results indicate a delay in morphological sickling due to drug treatment. Based on the results of our study there is a dose-dependent decrease in RBC deformability and in the kinetics of morphologic sickling based of DSA following voxelotor treatment. These two methods have demonstrated a comparable ability to assess downstream therapeutic effects on delaying/inhibiting HbS polymerization. The combination of these two unique properties, RBC deformability and morphology change, can possibly provide a comprehensive approach to describe the cellular level of phenotypes, which may predict SCD severity. With further development these complementary assays have the potential to become important tools for drug development and monitoring response to SCD therapies.

Inclacumab Reduce Preexisting Red Blood Cell Adhesion to Activated Endothelial Cells: In-Vitro Assessment of the Microfluidic Platform Endothelium-on-a-Chip

Background: In 2019 FDA approved the use of an antibody anti P-selectin (Crizanlizumab) for the treatment of sickle cell disease (SCD) to reduce the vaso occlusive crisis (VOCs). New anti-adhesive treatments are being developed recently. Global Blood Therapeutics, Inc., acquired by Pfizer Inc. in October 2022, acquired Inclacumab from Roche and started two clinical trials now in phase 3 to evaluate the capacity of this humanized IgG4 monoclonal antibody anti P-selectin to reduce the VOCs in patients with SCD. In our recent work presented at EHA 23 in Frankfurt Germany, we showed that Inclacumab prevents the adhesion of RBCs to chronically and acutely heme activated human umbilical vein endothelial cells (HUVECs) with a statistically significant higher effect in comparison to Crizanlizumab. In this work we used a similar approach to test the capacity of Inclacumab in the reduction of preexisting adhesion events on acutely heme activated HUVECs. Methods:Whole blood EDTA samples from six SCD subjects all with HbSS genotypes were collected at University Hospital Cleveland Medical Center, Cleveland, OH, USA. To standardize the samples, we isolated RBCs by centrifugation and subsequential washes in Phospahte buffer s of the whole blood and resuspended in basal cell culture medium (EBM; Lonza, Morristown, NJ, USA) at a hematocrit of 20% with 10 mM of HEPES. HUVECs (Lonza, Morristown, NJ, USA) were introduced and maintained within the endothelialized microfluidic channels at physiological flow for at least 48-72 hours prior to experiments. We performed an acute short-term activation, where the blood samples were supplemented with 40 µM heme +/- 100 µg/ml injected through the microfluidic channels for 15 minutes, after the adhesion took place we washed the non-adherent cells with Inclacumab and Crizanlizumab and we compared with untreated washing buffer (both compounds were provided by Global Blood Therapeutics, Inc., a wholly owned subsidiary of Pfizer Inc. South San Francisco, CA, USA) A phase-contrast images of the remaining RBCs were acquired with an inverted microscope (DMi8 Leica Microsystems Inc. Deerfield, IL, USA) and quantified based on previous published methods (Figure 1A). Paired t-test was used to calculate statistical significance. Results: Acute short-term heme activation induces high level of RBCs adhesion. Both compounds Inclacumab and Crizanlizumab showed a reduction of the number of adherent RBCs to the activated heme, but only Inclacumab results statistically a significant higher reduction compared to untreated RBCs (Figure 1B). Conclusions: Inclacumab is able not only to prevent the adhesion of RBCs to acutely and chronically activated HUVECs but is able to remove the preexisting adhesion and results in significant higher reduction compared to untreated. RBCs adhesion assay on Endothelium-on-a-chip is a novel and efficient way to study the efficacy of the new antiadhesive drug and could be used to monitor patient response to new therapies in SCD. Figure1 : Effect of Inclacumab vs Crizanlizumab on removal of SCD RBC adhesion to heme activated HUVECs. A) Schematic of adhesion experiment and inverted microscope contrast of phase images 10X of adherent RBCs at acutely heme activated HUVECs. B) Inclacumab has statistically significant effect on remove adherent RBCs to acutely heme activated HUVECs (p=0.009, N=7) For all experiments, the heme concentrations were 40 µM and Inclacumab and Crizanlizumab were 100 µg/ml. Scale bare 20 µm. P-values were based on paired t-test. Error bars represent the standard error of the mean (SEM)

Microvascular Cerebral Blood Flow Monitoring Detects Response to Intrathecal Nicardipine Treatment for Vasospasm (S4.005)

<h3>Objective:</h3> Determining the effect of intrathecal nicardipine on microvascular cerebral blood flow <h3>Background:</h3> A common complication of subarachnoid hemorrhage is cerebral vasospasm, which is associated with delayed cerebral ischemia (DCI), a predictor of poor functional outcome. Intrathecal administration of nicardipine (ITn) upon detection of vasospasm holds promise as a treatment that reduces the incidence of secondary stroke. Herein, we utilize a novel non-invasive optical measurement of regional microvascular perfusion to determine the effect of ITn on microvascular cerebral blood flow (CBF). <h3>Design/Methods:</h3> In this observational study, we prospectively studied 20 patients with non-traumatic subarachnoid hemorrhage who were treated with ITn as part of usual care for evolving cerebral vasospasm. Regional microvascular CBF in the frontal cortex was measured with diffuse correlation spectroscopy for up to 90 min after the first treatment dose, and for a subset of patients, on days 2 and 3 of treatment as well. Outcome was assessed as the presence of DCI on imaging. <h3>Results:</h3> CBF increased significantly over the 90 min of monitoring after the first and subsequent doses (p&lt;0.001) in the absence of systemic hemodynamic changes. In general, the CBF response was heterogeneous across subjects. Using data from the first dose, a latent class mixture model was used to classify 19/20 patients into two distinct classes of cerebral blood flow response: patients in class 1 (n=6) showed a decrease or no change in CBF, while patients in class 2 (n=13) showed a pronounced increase in CBF in response to nicardipine. The incidence of DCI was 5/6 in class 1 and 0/13 in class 2 (p&lt;0.001). Similar significant differences in CBF response in patients with and without DCI were also observed on days 2 and 3 (both p&lt;0.001). <h3>Conclusions:</h3> Microvascular CBF monitoring is feasible in critically ill subarachnoid hemorrhage patients. CBF response may provide a valuable biomarker for treatment responsiveness. <b>Disclosure:</b> Dr. Sathialingam has nothing to disclose. Mr. Cowdrick has nothing to disclose. Ms. Urner has nothing to disclose. Ms. Liew has nothing to disclose. Dr. Sigman has nothing to disclose. Dr. Akbik has nothing to disclose. Dr. Samuels has received publishing royalties from a publication relating to health care. Dr. Kandiah has nothing to disclose. Dr. Buckley has received research support from Global Blood Therapeutics. Dr. Buckley has received research support from NIH. Ofer Sadan has received intellectual property interests from a discovery or technology relating to health care.

Sleep quality associated with neuroimaging measures of CSF flow in individuals with Parkinson’s disease (N5.001)

<h3>Objective:</h3> To investigate the relationship between sleep quality and cerebrospinal fluid (CSF) flow in individuals with Parkinson’s disease (PD). <h3>Background:</h3> Previous studies have emphasized the role of CSF circulation in the clearance of brain waste products, with recent findings demonstrating increased CSF flow during sleep. CSF circulation impairment could play a major role in neurodegenerative proteinopathies such as PD, a commonly encountered feature of which is disrupted sleep. Here, we assessed the relationship between patient-reported sleep quality and imaging assays of CSF flow. <h3>Design/Methods:</h3> Imaging and clinical assessments were completed in 23 PD participants aged 56 to 79 (M_age=66.70; 16 males). The Montreal Cognitive Assessment (MoCA) and Unified Parkinson’s Disease Rating Scale (UPDRS) were administered; subjective sleep measurements were recorded using the Sleep Disturbance (SD) and Sleep-Related Impairment (SRI) items from the Patient-Reported Outcomes Measurement Information System (PROMIS) scale. MRI phase contrast sequences quantified CSF flux (mL/min) via the cerebral aqueduct. Net CSF flow was defined as the difference between caudal and cranial flow, whereas absolute flow was calculated as the sum of total caudal and cranial flow per cardiac cycle. <h3>Results:</h3> Participants had mean MoCA scores of 24.17 (range 13–30) and UPDRS scores of 30.70 (7–59). PROMIS scores for SD and SRI were 20.14 (9–32) and 15.00 (8–24), respectively. Mean CSF measurements were 0.25 mL/min (−0.29–0.88) for net and 8.11 mL/min (3.55–16.34) for absolute flow. Higher SRI scores correlated with greater net CSF flow while awake (r=0.531, p=0.023). Additionally, SRI and age explained 73.4% of variation in absolute CSF flow in a linear regression model (F(3,15)=24.5, p&lt;0.0001). <h3>Conclusions:</h3> Self-reported sleep impairment is associated with increased CSF flow in awake individuals with PD. This study is the first to link patient-reported measures of sleep with alterations to CSF flow. These data support novel outcomes for clinical studies that improve sleep. <b>Disclosure:</b> Mr. Ponzo has nothing to disclose. Dr. Hett has nothing to disclose. Mr. Eisma has nothing to disclose. Mr. Elenberger has nothing to disclose. Mr. Song has nothing to disclose. An immediate family member of Dr. McKnight has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Deciphera. The institution of Dr. McKnight has received research support from RSNA. Dr. Considine has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for MDisrupt. Dr. Considine has received personal compensation in the range of $10,000-$49,999 for serving as an Expert Witness for Park Dietz &amp; Associates. The institution of Dr. Considine has received research support from NIH. The institution of Dr. Considine has received research support from DoD. The institution of Dr. Considine has received research support from Acadia Pharmaceuticals. Manus Donahue, 13717 has received personal compensation for serving as an employee of Alterity. Manus Donahue, 13717 has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Alterity. Manus Donahue, 13717 has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Pfizer. Manus Donahue, 13717 has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Global Blood Therapeutics. Manus Donahue, 13717 has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Pfizer. The institution of Manus Donahue, 13717 has received research support from National Institutes of Health. Dr. Claassen has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Teva Neuroscience. Dr. Claassen has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Spark . The institution of Dr. Claassen has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for Alterity. Dr. Claassen has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Teva Neuroscience. Dr. Claassen has received personal compensation in the range of $500-$4,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for HD Insights. The institution of Dr. Claassen has received research support from NIH. The institution of Dr. Claassen has received research support from CHDI. The institution of Dr. Claassen has received research support from HDSA. The institution of Dr. Claassen has received research support from Department of Defense. The institution of Dr. Claassen has received research support from Griffin Family Foundation. The institution of Dr. Claassen has received research support from Neurocrine. The institution of Dr. Claassen has received research support from Vaccinex. The institution of Dr. Claassen has received research support from AbbVie. The institution of Dr. Claassen has received research support from CHDI. The institution of Dr. Claassen has received research support from Genentech/ Roche. The institution of Dr. Claassen has received research support from Prilenia. The institution of Dr. Claassen has received research support from Neurocrine/ HSG.

5572697 TRIALS IN PROGRESS: THE THRIVE STUDIES EVALUATING THE EFFICACY, SAFETY, AND LONG-TERM TREATMENT WITH INCLACUMAB, A P-SELECTIN INHIBITOR, IN PATIENTS WITH SICKLE CELL DISEASE

Background: Sickle cell disease (SCD) is an autosomal recessive red blood cell (RBC) disorder caused by a single point mutation in the β-globin gene, resulting in the production of hemoglobin S. Hemoglobin S polymerization within deoxygenated RBCs contributes to hemolysis, anemia, vaso-occlusion, and end-organ damage. Additionally, abnormal heterocellular adhesive interactions between RBCs and endothelial cells, leukocytes, and platelets play a central role in triggering painful vaso-occlusive crises (VOCs) in patients with SCD. Inclacumab is a recombinant, fully human monoclonal antibody directed against human P-selectin, a cell adhesion molecule produced by endothelial cells and platelets. Inclacumab interferes with the adhesion of sickle RBCs, platelets, and leukocytes to endothelium by binding P-selectin and preventing P-selectin binding to its ligands. This is the putative mechanism by which inclacumab may potentially reduce the incidence of VOCs. Aims: We aim to conduct two global, multicenter, phase 3 studies and an open-label extension (OLE) study to evaluate the safety and efficacy of inclacumab in individuals with SCD. Methods: THRIVE-131 (NCT04935879) is a randomized, double-blind, placebo-controlled, multicenter trial in which ~240 participants aged ≥12 years experiencing 2 to 10 VOCs in the previous 12 months will be randomized 1:1 to receive intravenous (IV) inclacumab or placebo every 12 weeks for 48 weeks (Figure). The primary endpoint is the rate of VOCs during the 48-week treatment period. THRIVE-132 (NCT04927247) is a randomized, double-blind, placebo-controlled, multicenter trial in which ~280 participants aged ≥12 years experiencing 2 to 10 VOCs in the previous 12 months will be randomized 1:1 to receive a single dose of IV inclacumab or placebo within 5 days of resolution of an index VOC that required admission to a healthcare facility and treatment with parenteral pain medication. The primary endpoint is the proportion of participants with readmission for a VOC within 90 days of randomization. After completion of their participation in THRIVE-131 or THRIVE-132, participants may enroll in THRIVE-133 OLE, in which the long-term safety of inclacumab will be investigated. In THRIVE-133 OLE, participants will receive IV inclacumab every 12 weeks as long as the clinical benefit of treatment outweighs the risk and until access to inclacumab from an alternative source (eg, through commercialization or a managed-access program) becomes available. Results: Recruiting for both THRIVE-131 and THRIVE-132 began in October 2021 and is ongoing. Summary – Conclusion: The THRIVE-131 and THRIVE-132 phase 3 studies will examine the efficacy of inclacumab in reducing the frequency of VOCs and readmissions due to VOCs, and the THRIVE-133 OLE study will examine the long-term safety of inclacumab in individuals with SCD. Funding: These studies are supported by Global Blood Therapeutics. Figure: Designs of the THRIVE Studies

5610417 IN-VITRO REAL-TIME OBSERVATION OF MODERATE SICKLING KINETICS IN SICKLE ERYTHROCYTES TREATED BY VOXELOTOR UNDER MONOTONIC AND CYCLIC HYPOXIA

Background: Hypoxia-induced sickle hemoglobin (HbS) polymerization in sickle red blood cells (sRBCs) is the root cause of the symptoms and complications in sickle cell disease (SCD). In microcirculation, timing is critical for sRBCs to pass through narrow capillaries before they become sickled under hypoxia and rigid enough to get trapped. Hence, the delay time in cell sickling due to HbS polymerization is likely a key parameter predictive of the SCD severity. Recent studies have also shown the delay time is shortened due to repeated hypoxia cycles. Voxelotor is a known hemoglobin oxygen-affinity modulator, which has been proven to effectively inhibit HbS polymerization and favorably modify SCD outcomes. However, there has been no direct in-situ observation of the dose-dependent anti-sickling effect of voxelotor treatment under monotonic hypoxia or multiple hypoxia cycles. Aims: 1. We will test the anti-sickling effect of voxelotor on sRBCs by measuring their dose-dependent hypoxia-induced sickling kinetics using our microfluidics assay. 2. We will further test the efficacy of voxelotor on the cumulative impairment to sRBCs from repetitive sickling cycles. Methods: Blood samples from patients with homozygous SCD were studied. Sample specimens used for this study are restricted to patients that were not in crisis and patients with transfusions 30 days or longer prior to blood draw. Details about the microfluidic device and related methods can be found elsewhere [1-3]. Single deoxygenation-reoxygenation cycle or multiple deoxygenation-reoxygenation cycles is created by switching between two gas supplies with 2% and 20% O2 at predetermined time intervals [3]. Results: Figure 1A-B shows significant decrease in sickled fraction with the increase of voxelotor modification percentage from 0% to 30%, 60% and 100%, which are consistent among patients taking hydroxyurea (HU ON) or not taking hydroxyurea (HU OFF). Secondly, we have investigated the efficacy of voxelotor (30% modification) on sickling kinetics progression after 20 repeated deoxygenation-reoxygenation cycles (each cycle includes 60s of deoxygenation and 30s of oxygenation). From the measurements from 4 patient samples, we have confirmed that the sickling kinetics of sickle cells (vehicle, 0% modification) become progressively faster with subsequent deoxygenation-oxygenation cycles, which is quantified by the reduced delay time and higher sickled fraction versus deoxygenation time. Figure 1C-D shows that the treatment of voxelotor (30% modification) has significant efficacy in mitigating the progressively accelerated sickling of sRBCs due to repeated deoxygenation-oxygenation cycles in vitro. The sickled fraction measured at t = 20s in the 20th cycle decreases from 53%±9% for vehicle samples (0% modification) to 21%±11% for voxelotor treated samples (30% modification) (p < 0.05). Similarly, the sickled fraction measured at t = 60s in the 20th cycle decreases from 64%±11% (0% modification) to 30%±16% (30% modification) (p < 0.05).Summary-Conclusion: Voxelotor significantly slows down the rate of sickling and reduces the sickled fraction of sRBCs under monotonic hypoxia with dose-dependency. Furthermore, voxelotor is effective for mitigating the cumulative impairment to sRBCs from repetitive sickling events. Funding statement: This study has been supported by Global Blood Therapeutics Inc. Y.Q. and M.D. acknowledge partial support from NIH R01HL158102.

Health Equity in Sickle Cell Research and Access to Therapy

Health Equity in Sickle Cell Research and Access to Therapy

Diversity, equity, and inclusion: Moving from ambition to action.

Natasha M. Archer received clinical trial fees from Global Blood Therapeutics for a clinical trial outside the scope of this submitted work.

Investigating Gene Expression Signatures and Cell-Type Composition Changes in Sickle Cell Disease Using Whole Blood Transcriptomics

Background: Sickle cell disease (SCD) is a devastating Mendelian disease affecting millions of people worldwide. Although SCD is caused by a single-point mutation (Glu6Val) in the β-globin chain of hemoglobin, the clinical manifestations are heterogeneous and systemic, impacting the functions of multiple organs and leading to early mortality. Predicting disease severity and identifying the molecular mechanisms underlying disease pathology and progression remain a challenge. Objective: The goal of this work was to characterize the clinical heterogeneity associated with SCD using transcriptomics. Methods: We integrated publicly available bulk and single-cell RNA sequencing (scRNA-seq) data sets that included samples from pediatric to young adult patients and contained clinical phenotypes to identify molecular signatures of SCD severity and heterogeneity (Table). Our integrative transcriptomics analyses included the following: Identify cell-type clusters and derive novel progenitor signatures from an integrative analysis of the 2 independent scRNA-seq data sets capturing CD34+-enriched or unselected bone marrow (BM) cells from SCD and control samples.Calculate the relative proportions of cell types in whole blood by deconvolution using both the scRNA-seq-derived novel cell-type signatures (dtangle) and publicly available immune cell-type signatures (xCell, EPIC, quanTIseq).Identify signatures (genes, pathways, cell types) associated with disease severity and hemoglobin genotypes using various bioinformatics approaches, including differential gene expression and single-sample Gene Set Enrichment Analysis, among others, and examine relationships of enriched cell types and pathways (queried from MSigDB) in discovery followed by confirmation in validation data sets. Results: Cell-type clusters and gene signatures were generated by combining and integrating scRNA-seq data sets from BM SCD and control samples (Table). Using our fine-mapped cell-type clusters, we found an erythroid progenitor subclass to be depleted, whereas a lymphoid progenitor subclass expanded in SCD BM samples, increasing granularity of the cell types reported by Hua et al. Cell-type deconvolution of bulk transcriptome data using the combined scRNA-seq-derived signatures revealed enriched monocyte relative proportions in acute crisis compared with steady-state SCD samples (β=0.01, P=1.13×10−7) and enriched erythroblast relative proportions in SCD compared with control samples (β=0.02, P=1.10×10−12). Deconvolution using publicly available immune cell signatures showed significant enrichment of neutrophil and macrophage signatures in acute SCD samples (Padj<0.1). Analysis of bulk RNA-seq data indicated a set of highly correlated genes differentially expressed in steady-state SCD and associated with varied pathways, including red blood cell (RBC) morphology, hematopoiesis, anemia, and cholelithiasis. Higher pathway scores were seen in severe (HbSS) genotype samples than in milder (HbSC) genotype samples, consistent with known disease pathology and severity (Figure). Building on results from integrative analyses, we found that interferon and inflammatory response genes were enriched in acute SCD in both discovery and validation data sets. We found significant correlations between monocyte relative proportions and interferon signaling pathway scores, and between erythroblast relative proportions and the pathway scores affecting RBC production, morphology, and pathology and potential SCD comorbidities such as cholelithiasis (Figure). Conclusions: Our integrative scRNA-seq analysis led to robust cell cluster identification to improve deconvolution analysis of bulk transcriptomics data. We found elevated interferon and inflammatory pathway activity in acute SCD samples, consistent with findings in the literature (PMIDs: 34166491, 32764656). Furthermore, we found enriched neutrophils and monocyte signatures in acute SCD, suggesting their role in mediating the innate immune response. These findings provide motivation to characterize the role of neutrophils and monocytes in acute SCD crises. Our integrative transcriptomics analyses showed defective RBC production and maturation in SCD. Using the signatures from this work, we aim to elucidate the heterogeneity within SCD pathophysiology. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Trials in Progress: Two Randomized, Double-Blind, Multicenter, Placebo-Controlled Trials Investigating the Efficacy of Voxelotor on Cerebral Blood Flow and Neurocognitive Function in Patients with Sickle Cell Disease

Background: Sickle cell disease (SCD) is an inherited blood disorder caused by a point mutation in the beta chain of hemoglobin (Hb) that results in the formation of sickle hemoglobin, HbS. Polymerization of HbS under conditions of deoxygenation causes red blood cell sickling that can lead to hemolytic anemia and vascular occlusion with subsequent end-organ damage, infarction, and cognitive dysfunction. In patients with SCD, cerebral blood flow (CBF) is elevated throughout the brain, with silent cerebral infarcts (SCIs) occurring mainly in brain regions with the lowest CBF. SCIs, overt strokes, and cognitive impairment are some of the most common complications of SCD, all of which have a substantial impact on the education, employment, and quality of life of patients with SCD. Cognitive impairment in patients with SCD can also manifest in those without SCI or overt stroke, suggesting a potential role of chronic anemia and cerebral hemodynamics in neurocognitive dysfunction. Despite progress in the development of therapies for SCD, little is known about their effect on cognitive function and cerebral hemodynamics. Voxelotor is a HbS polymerization inhibitor approved in the United States for the treatment of SCD in patients aged ≥4 years and in the United Kingdom, European Union, and the United Arab Emirates for patients aged ≥12 years. Objective: To understand the impact of voxelotor on brain function in patients with SCD, 2 post-approval studies of voxelotor are currently recruiting and are described here. Methods: The Neurocognitive Function study (NCT05228834) is a phase 3b, randomized, double-blind, placebo-controlled, multicenter trial investigating the effect of once-daily oral voxelotor (Figure) on neurocognitive function in children and adolescents with SCD. The Cerebral Hemodynamics study (NCT05228821) is a phase 4, randomized, double-blind, placebo-controlled, multicenter trial investigating the impact of once-daily oral voxelotor (Figure) on CBF in adolescents and adults with SCD. An overview of the study population, study design, and key endpoints for both studies is provided in the Table. Results: The primary objective of the Neurocognitive Function study is to assess the effect of voxelotor compared with placebo on the neurocognitive function in children and adolescents with SCD (aged 8 to <18 years), which will be evaluated by measuring the change from baseline at week 12 in the executive abilities composite score, as assessed by the National Institutes of Health Toolbox Cognition Module. The primary objective of the Cerebral Hemodynamics study is to assess the efficacy of voxelotor compared with placebo on CBF in adolescents and adults (aged 12-30 years) with homozygous hemoglobin S (HbSS) or sickle beta zero thalassemia (HbSβ0), which will be evaluated by assessing the change from baseline in CBF measured by magnetic resonance imaging at week 12. Conclusions: Treatments that improve neurocognitive dysfunction represent an important unmet need in the management of SCD. Results from the Neurocognitive Function study and the Cerebral Hemodynamics study (the latter will enroll adult patients) will provide important information on the impact that inhibition of HbS polymerization may have on cognitive function and cerebral hemodynamics by improving anemia and hemolysis in patients with SCD. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Insights into Vaso-Occlusive Crises-Related Readmissions in Patients with SCD across Geographic Regions

Background: Sickle cell disease (SCD) is an inherited red blood cell disorder characterized by chronic anemia and hemolysis, inflammation, and vaso-occlusion that manifests clinically as recurrent, painful vaso-occlusive crises (VOCs) and widespread organ damage. VOCs are the main reason for admission to a healthcare facility (eg, hospital, emergency department [ED], observation unit, or day care). Frequent admission for VOCs is an indicator of severe disease and is associated with early mortality. In the United States, 33% of patients admitted to a healthcare facility for VOCs are readmitted within 30 days, and approximately 50% of patients are readmitted within 90 days. These high readmission rates place an enormous burden on patients, caregivers, and the healthcare system. US readmission rates may not represent 90-day readmission rates among individuals with SCD outside of the United States due to variability in healthcare settings, practices, access, and other potentially confounding factors. Objective: To explore regional differences in 90-day readmission rates for VOCs, this retrospective chart review was conducted in a sample of patients with SCD at selected sites across geographic regions. Methods: Medical records from patients with SCD (all genotypes), aged ≥12 years, who were admitted to the ED or hospital for management of a VOC during a 2-year period were evaluated for this retrospective chart review. The evaluation took place at 8 sites on 5 continents (North America, Africa, Asia, Europe, and South America). Eligible patients had a history of painful VOC episodes, including complicated VOCs of acute chest syndrome, hepatic sequestration, splenic sequestration, and priapism, requiring admission to a medical facility (hospital, ED, observation unit, or day care) for treatment. For each patient, dates of admission for a VOC requiring treatment with parenteral (intravenous or intramuscular) pain medications (eg, nonsteroidal anti-inflammatory drugs, narcotics) in the selected time were collected. Patients with SCD who had 2 to 10 VOCs within year 1 were eligible for inclusion in the chart analysis. The first admission for VOC during the second year of the study was designated as the index VOC. A subsequent VOC occurring within 90 days of the index VOC was used to determine the 90-day readmission rate. Results: The study included a review of 500 patient charts across 8 geographic regions in 6 countries: Brazil, Egypt, Italy, Lebanon, Nigeria, and the United States (Figure 1). Out of the 500 patient charts reviewed, 290 (58%) were eligible for this study. Of the eligible patients with SCD who were admitted for a VOC, those who were readmitted within 90 days were included for analysis. The proportion of patients readmitted for a VOC within 90 days ranged from 30% to 51% (Figure 2). Conclusions: Recurrent and prolonged VOC-related hospitalizations substantially disrupt the daily lives and emotional well-being of patients with SCD and are associated with increased risk of mortality. VOCs also remain the main cause for inpatient admissions, and there is an urgent need to reduce the burden of VOC-related hospitalizations. This study provides insights into 90-day readmission rates for VOCs in patients with SCD across different geographic regions. Rates of readmission due to VOCs were generally similar across the sites and consistent with rates observed in the United States. The 90-day readmission rates observed in this study suggest that similar factors may influence VOC hospitalization rates. Identification of these factors is critical for formulating interventions to reduce readmissions and the associated social and economic burden, and for facilitating efficient allocation of resources. Future analyses will include the assessment of site and practice-specific factors contributing to these results to inform physicians treating patients with SCD on potential ways to alleviate this burden on patients, caregivers, and the medical community. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Leg Ulcer Prevalence and Outcomes in a Real-World, Multicenter, Retrospective Study of Patients Treated with Voxelotor

Background: Sickle cell disease (SCD) is an inherited disorder driven by the polymerization of sickle hemoglobin in the deoxygenated state, leading to chronic hemolytic anemia. Leg ulcers are a severe, chronic, and often recurrent complication of SCD, occurring in about 14% to 18% of patients in the US and more commonly in men and those with severe SCD genotypes. Leg ulcers are painful and decrease physical function, increase pain interference, and cause psychosocial impacts. Symptoms and complications associated with leg ulcers include chronic hemolysis, edema, impaired endothelial function, and venous incompetence and vaso-occlusion. Leg ulcers in SCD are commonly treated with standard wound care, including mechanical/autolytic detersion and modern dressings. Studies on local and systemic treatments have been inconclusive. Voxelotor is a sickle hemoglobin polymerization inhibitor approved in the US for the treatment of SCD in patients aged ≥4 years and in the UK, EU, and UAE for patients aged ≥12 years. Phase 3 clinical trials have shown that voxelotor increases hemoglobin and reduces markers of hemolysis with an overall favorable safety profile. The Retrospective Real World Oxbryta Data Collection and Analysis Study (RETRO) aimed to characterize real-world safety and effectiveness of voxelotor in patients aged ≥12 years. RETRO expands upon our understanding of voxelotor, providing valuable insights and driving future studies that will investigate its descriptive results. Objective: To examine leg ulcer prevalence and outcomes before and during voxelotor treatment in the RETRO study. Methods: RETRO is a post-marketing study designed to collect and characterize real-world, retrospective laboratory and clinical data from patients aged ≥12 years with SCD (all genotypes) treated with voxelotor as part of their usual care. The study was conducted in 9 US sites. Data from patients treated with voxelotor for ≥2 consecutive weeks and with available medical records 1 year before and up to 1 year after their first voxelotor dose were included. Results: A total of 216 patients with a mean (SD) age of 33.5 (14.21) years were included. Overall, 8.8% (19/216) of patients had an active or previous leg ulcer any time during the 1-year period before voxelotor initiation and up to 1-year post-treatment. Of these patients, 57.9% (11/19) reported 1 leg ulcer, and 42.1% (8/19) reported ≥2 leg ulcers; the mean (SD) age of these 19 patients was 40.5 (10.9) years, 36.8% (7/19) were female, and 79.0% (15/19) were Black or African American. Most patients (84.2% [16/19]) had the HbSS genotype, and 57.9% (11/19) were taking hydroxyurea. Five leg ulcers in 4 patients resolved before voxelotor initiation. A total of 23 leg ulcers were ongoing at treatment initiation, meaning the ulcer either occurred before the first dose of voxelotor or occurred after treatment initiation. A total of 14/23 (60.9%) leg ulcers occurred before voxelotor initiation, with half of them (7/14) resolving after voxelotor initiation. The other 9 leg ulcers (39.1% [9/23]) occurred after voxelotor initiation; of these, 5 (55.6%) resolved during voxelotor treatment. Further descriptive analyses of leg ulcer events and resolutions are ongoing. Conclusion: RETRO is the largest multicenter, retrospective study on voxelotor to date. Data reported here describe the prevalence and incidence of leg ulcers in this cohort and the reported outcomes before and during voxelotor treatment. The limitations of this retrospective study include small sample sizes, the reliance on data captured in medical records, and the lack of a control group. To address this gap in knowledge, the RESOLVE study to investigate the impact of voxelotor on leg ulcers is currently underway (PACTR202102669041711). Funding: Global Blood Therapeutics.

Increased Hemoglobin Affinity for Oxygen with GBT021601 Reduces Pulmonary Hypertension and Lung Vascular Inflammation in a Murine Model of Sickle Cell Disease

Background: Sickle cell disease (SCD) is caused by a single nucleotide change in the β-globin gene coding sequence that results in the production of hemoglobin S (HbS). Under conditions of red blood cell (RBC) deoxygenation, HbS polymerizes and leads to RBC sickling, resulting in hemolysis, anemia, diminished microvascular blood flow, vascular endothelial cell activation, vaso-occlusion, and ischemia. Despite improvements in treatment options, SCD remains a debilitating condition, and most patients experience progressive end-organ damage with associated complications and repeated hospitalizations. Pulmonary hypertension (PH), which is associated with increased mortality, is a common complication in patients with SCD, with nearly 10% of the SCD population reporting PH. PH in patients with SCD is multifactorial and may be caused by hemolysis, nitric oxide scavenging, and/or tissue hypoxia. Pharmacological increase of the concentration of oxygenated HbS (oxyHbS) in RBCs by voxelotor, a first-generation HbS polymerization inhibitor, reduces RBC sickling and preserves oxygen (O2) delivery by reducing anemia and improving blood rheology. Limited data are currently available on whether inhibiting HbS polymerization by increasing oxyHbS concentration may improve end-organ damage and associated complications, including PH. Objective: To examine the effects of GBT021601, a small-molecule, investigational, second-generation HbS polymerization inhibitor, on hemoglobin (Hb)-O2 affinity, PH, right ventricular dysfunction, and lung inflammation in a murine model of SCD. Methods: Townes HbSS mice treated with GBT021601 (75 mg/kg orally once daily for 14 days) were exposed to acute hypoxia (8% O2) for 1 hour and reoxygenated for 72 hours. Untreated HbAA Townes mice were used as controls. Lung inflammation was assessed in terms of accumulation of inflammatory cells, activation of nuclear factor kappa B subunit 1, and expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1. To evaluate the impact of GBT021601 treatment on Hb-O2 affinity and anemia, p50 (partial O2 pressure at which Hb is 50% saturated with O2) and hematocrit levels were measured in SCD mice. In vivo pulmonary hemodynamic measurements included pulmonary artery pressure and right atrial pressure, whereas right ventricular hypertrophy was assessed using the Fulton index, which is the ratio of right ventricular weight to the weight of the left ventricle plus septum. Results: The total number of inflammatory cells and the specific numbers of lymphocytes, macrophages, and neutrophils were lower in untreated HbAA Townes mice and in HbSS Townes mice treated with GBT021601 compared with vehicle-dosed HbSS mice (Figure, panel A). Chronic treatment with GBT021601 increased Hb-O2 affinity, reducing the p50 in SCD mice from 32 mm Hg to 14 mm Hg. The treatment significantly reduced anemia, increasing hematocrit from 27.2% in the vehicle-dosed HbSS mice to 38.5% in the mice treated with GBT021601. Chronic GBT021601 treatment in HbSS mice also reduced pulmonary artery and right atrial pressure compared with those in untreated HbSS mice. Additionally, GBT021601 reduced right ventricular hypertrophy in HbSS mice, as evidenced by a reduction in the Fulton ratio (Figure, panel B). Conclusions: GBT021601 reduced PH, right ventricular dysfunction, and lung inflammation in a murine model of SCD. Our findings suggest that reducing RBC sickling, anemia, and hemolysis by increasing HbS affinity for O2 plays a critical role in modulating the immune system in PH and lung vasculature. These findings highlight the potential therapeutic efficacy of GBT021601 based on its ability to increase HbS affinity for O2 and prevent HbS polymerization and RBC sickling to reduce PH in SCD. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

GBT021601 Increases Hemoglobin and Improves Red Blood Cell Deformability without Rebound Hyperviscosity upon Drug Clearance in a Sickle Mouse Model

Background: Whole-blood viscosity and red cell deformability are markedly abnormal in sickle cell disease (SCD) due to sickling of red blood cells (RBCs). Increased whole-blood viscosity is a risk factor for vaso-occlusive crisis (VOC), avascular necrosis, and proliferative retinopathy. Blood viscosity is strongly impacted by hemoglobin (Hb) levels and RBC deformability. GBT021601 is a potent next-generation sickle hemoglobin (HbS) polymerization inhibitor, with anti-sickling properties, that increases the oxygen affinity of Hb. In preclinical studies, treatment with GBT021601 achieved higher levels of Hb than the US Food and Drug Administration-approved first-generation compound voxelotor, raising concern that this rise in Hb would substantially increase whole-blood viscosity and impose a VOC risk after treatment cessation. Objective: To investigate a potential rebound hyperviscosity effect from GBT021601 cessation in a murine model of SCD by determining the effect on whole-blood viscosity and on RBC deformability under a range of oxygen concentrations using oxygen gradient ektacytometry. Methods: Nine-week-old Townes HbSS mice were fed chows containing 0.2% GBT021601 for 5 weeks, then transitioned to control chow. Whole blood was collected in an EDTA tube. All tests were conducted within 4 hours of collection. Using an Element HT5 hematology analyzer (Heska), complete blood count was obtained from mice while on GBT021601 (day 0) and then on days 1, 3, and 5 after transition to control chow. Blood viscosity was measured with a cone/plate viscometer (AMETEK Brookfield) at 37 °C at shear rates of 45 s-1 and 225 s-1. Hematocrit to viscosity ratio (HVR) was calculated to determine RBC oxygen-carrying capacity. RBC deformability was measured by oxygen gradient ektacytometry (Lorrca, RR Mechatronics). Results: Treatment with GBT021601 significantly increased Hb from 8.0 to 12.9 g/dL (P<0.0001) without increasing HVR. Compared with previous studies with the voxelotor analog GBT1118, Hb dropped off more slowly with GBT021601 treatment; this allowed for the ideal assessment of whole-blood viscosity and HVR when the drug had cleared, and HbS was no longer modified, despite elevated Hb levels. HVR at both 45 s-1 and 225 s-1 showed no significant change with drug clearance. GBT021601 treatment also improved RBC deformability, as indicated by a higher elongation index (EI) under both normoxic (EImax, P<0.001) and hypoxic (EImin, P<0.0001) conditions. Point of sickling (PoS) was also reduced (P<0.0001), indicating that treated RBCs tolerated lower oxygen concentrations before sickling. When the drug was withdrawn, PoS and EImin returned to baseline levels, whereas EImax remained elevated until day 5 off treatment. Hb also remained elevated until day 5 off treatment, despite the return to baseline levels of the EImin, PoS, and p50 (partial oxygen pressure at which Hb is 50% saturated with oxygen), indicating drug clearance. Conclusions: In response to GBT021601, sickle cell mice showed a robust increase in Hb, comparable to that of a HbAS mouse, as well as a marked improvement in PoS and RBC deformability. When normalized to HVR, there was no significant difference in blood viscosity during treatment and after cessation of treatment. This finding suggests that oxygen-carrying capacity is not impaired and provides no indication for rebound hyperviscosity with drug clearance. Moreover, the observation that EImax remained elevated until 5 days after drug cessation suggests that some residual rheological improvement occurred in the RBC population that did not dissipate when the drug was cleared. In vitro addition of GBT021601 (and other related compounds) to blood rapidly increases the EImin and reduces the PoS of RBCs but does not alter the EImax. In contrast, an increase in the EImax of RBCs has been consistently observed with GBT021601 treatment in vivo, including in early clinical studies in patients with SCD. The precise mechanism by which this benefit is obtained requires further study. Funding: Global Blood Therapeutics.

GBT021601 Prevents Microvascular Dysfunction and Improves Hypoxia-Reoxygenation-Induced Vaso-Occlusion in a Murine Model of Sickle Cell Disease

Background: In sickle cell disease (SCD), a single nucleotide change in the β-globin gene coding sequence results in the production of hemoglobin S (HbS), which polymerizes when deoxygenated and leads to red blood cell (RBC) sickling, hemolysis, anemia, diminished microvascular blood flow, vascular endothelial cell activation, vaso-occlusion, and ischemia. Pharmacological increase of the oxygenated HbS concentration in RBCs has been shown to be a well-tolerated and effective approach to inhibiting HbS polymerization, reducing RBC sickling, and improving oxygen (O2) delivery by reducing anemia and improving blood rheology. GBT021601, a potent second-generation small-molecule HbS polymerization inhibitor, is currently under investigation and has previously been shown to increase hemoglobin (Hb), improve hemolysis, and improve RBC health in a murine model. However, little is known regarding its impact on vaso-occlusion and microvascular dysfunction. Objective: To examine the effects of GBT021601 on SCD-induced microvascular dysfunction, capillary perfusion and density, Hb-O2 oxygen affinity, and Hb and hematocrit levels in a murine model of SCD. Methods: Capillary perfusion was measured by functional capillary density (FCD, a marker of capillary perfusion defined as the number of capillaries with RBCs in transit) in a murine model of SCD with an implanted dorsal window chamber (Figure). HbSS Townes mice were treated with GBT021601 (75 mg/kg) by oral gavage daily for 14 days and were compared with untreated HbSS and HbAA Townes mice. Results: Chronic treatment of HbSS mice with GBT021601 led to increased Hb-O2 affinity, evidenced by a reduction in blood p50 (partial O2 pressure at which Hb is 50% saturated with O2) from 32 mm Hg to 14 mm Hg. GBT021601 significantly reduced anemia by increasing total Hb and hematocrit from 8.3 g/dL and 26% in untreated HbSS mice to 11.2 g/dL and 37% in GBT021601-treated HbSS mice, respectively (P<0.01). Moreover, GBT021601 treatment prevented microvascular dysfunction (reduced RBC velocities and blood flow) in the arterioles and venules of HbSS mice. Increased capillary density is indicative of greater oxygen transport. Challenging HbSS mice with hypoxia (8% O2 for 1 hour) followed by reoxygenation (21% O2) induced vaso-occlusion, causing reduced microvascular blood flow and FCD in untreated mice. However, in GBT021601-treated HbSS mice, microvascular blood flow and FCD were significantly higher than those in untreated HbSS mice. Unlike in untreated HbSS mice, microvascular blood flow and FCD returned to baseline values after 72 hours of reoxygenation in treated HbSS mice. These results suggest that by increasing HbS-O2 affinity and thereby inhibiting HbS polymerization, GBT021601 may attenuate hypoxia-induced vaso-occlusion and impaired capillary perfusion. Furthermore, GBT021601 treatment in HbSS mice reduced both circulating adhesion markers and leukocyte-endothelium interactions in venules after hypoxia and reoxygenation. Conclusions: GBT021601 increased microvascular perfusion, reduced vaso-occlusion, and improved the hypoxia-induced reduction in capillary perfusion in HbSS mice, suggesting that chronic GBT021601 treatment prevents SCD-induced microvascular dysfunction and mitigates the rebound vaso-occlusion induced by hypoxia and reoxygenation in a murine model of SCD. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Active Study: Impact of Voxelotor on Sleep Quality, Physical Activity, and Patient-Reported Outcomes

Background: Sickle cell disease (SCD) is an inherited disorder in which sickle hemoglobin (HbS) polymerization causes red blood cell sickling, resulting in complications such as chronic hemolytic anemia, vaso-occlusion, end-organ damage, and decreased quality of life. Voxelotor is a first-in-class HbS polymerization inhibitor approved in the US for the treatment of SCD in patients aged ≥4 years. The pivotal phase 3 HOPE and phase 2a HOPE-KIDS 1 trials demonstrated the safety and efficacy of voxelotor in increasing hemoglobin (Hb) and decreasing hemolytic markers. Objective: Here we report Hb response, patient-reported quality-of-life (QOL) outcomes, and safety data from the ongoing ActIVe study (NCT04400487), a phase 4, multicenter, open-label study evaluating the effect of voxelotor on physical activity, sleep quality, and overall well-being in patients with SCD. Methods: Patients aged 12 to 55 years with SCD received voxelotor 1500 mg/day for up to 24 weeks across 10 US sites. QOL assessments were completed during baseline (BL) run-in before the start of voxelotor. Hb levels were measured via laboratory assessments at BL and at weeks 2, 12, and 24. The proportion of patients with any increase in Hb from BL was noted. Patient-reported outcomes were evaluated using the Patient Global Impression of Change (PGI-C) scale, the National Institutes of Health Patient-Reported Outcome Measurement Information System-43 (PROMIS-43) for adults (>17 years), PROMIS-37 for adolescents (≤17 years), and a 10-point numeric rating scale (NRS) for pain intensity. A minimum response deemed meaningful was interpreted at the group level as a ≥5-point change from BL (CFB) in PROMIS domains and a ≥2-point change on the NRS. Clinicians' perceptions of the impacts of voxelotor on patients' overall health status were captured using the Clinical Global Impression of Change (CGI-C). All treatment-emergent adverse events (TEAEs) were recorded from the time of consent/assent to 28 days after the last dose of voxelotor. Results: A total of 25 patients were enrolled, with a median (range) age of 20.0 (12-51) years; 64.0% of patients were female and 92.0% were Black or African American. Two (8.0%) patients permanently discontinued voxelotor due to non-compliance and loss to follow-up. A total of 14 of 18 (78%) patients had an increase in Hb at week 24; the mean (SD) increase was 0.53 (0.839) g/dL. Clinicians reported an improvement in health status among 84.6% (11/13) of patients at week 24: 7.7% (1/13) were "very much improved," 46.2% (6/13) were "much improved," and 30.8% (4/13) were minimally improved; 2 patients had no change. At week 24, 92.9% (13/14) of patients self-reported improved health status: 21.4% (3/14) were "very much improved," 42.9% (6/14) were "much improved," and 28.6% (4/14) were minimally improved; 1 patient reported minimally worse health status. At week 24, 14 patients (9 adults, 5 adolescents) completed PROMIS measures; improvements in the mean CFB were reported across several PROMIS health domains. In adults, a meaningful CFB was found for both fatigue and pain interference. Some domains reached near-meaningful improvements, including anxiety, sleep disturbance, and ability to participate (in social roles/other activities), and others improved more modestly (Table 1). While some domains improved among adolescents, none reached a meaningful level (Table 2). A total of 18 (72.0%) patients had ≥1 non-SCD-related TEAE, and 3 (12.0%) patients had ≥1 grade 3 non-SCD-related TEAE. No non-SCD TEAEs of grade 4 or higher were reported. The most common non-SCD-related TEAEs (>10%) were diarrhea (32.0%), headache (20.0%), nausea (20.0%), and vomiting (12.0%). Conclusions: Over three-quarters of voxelotor-treated patients achieved an increase in Hb at week 24, and most patients and clinicians reported improvement in patients' overall health status. The PROMIS data in voxelotor-treated adults provided insights into improvements in general well-being, such as fatigue, pain interference, and ability to participate; most notable were fatigue and pain interference, both of which reached a CFB threshold considered meaningful. The safety profile reported in ActIVe is consistent with previous studies of voxelotor treatment. Overall, results suggest that voxelotor-treated patients may experience improvements in QOL, including in their physical and emotional well-being. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effect of Voxelotor on Red Blood Cell Adhesion Under Normoxia Using an Endothelialized Microfluidic System

Introduction: Sickle cell disease (SCD) is characterized by the polymerization of hemoglobin S (HbS) that results in sickle red blood cell (sRBC) formation under deoxygenation. Subsequent alterations in the rheological properties of sRBC lead to abnormal deformability, adhesion, and ultimately vasculopathy. Vascular occlusion leads to repeated cycles of organ ischemia and pain. Voxelotor (Oxbryta, Global Blood Therapeutics, South San Francisco, CA, USA) is an FDA-approved medication that reversibly binds to sickle hemoglobin in a dose-dependent fashion to increase its oxygen affinity and reduce sickling and polymerization. Previous studies using voxelotor under hypoxic conditions have shown an improvement in blood flow (Azul & Wood, 2020) and sRBC deformability (Dufu et al., 2018). As previously published, endothelialized microfluidic devices have been used to quantify sRBC adhesion (Kucukal et al., 2021). This experimental study is the first in-vitro assessment of the effect of voxelotor on adhesion under normoxic conditions using an endothelialized microfluidic system. Methods: Human umbilical vein endothelial cells (HUVECs, Lonza, Morristown, USA) were cultured within a microfluidic system for at least 72 hours prior to experiments. HUVECs underwent four-hour activation with tumor necrosis factor alpha (TNFα) at a concentration of 20ng/mL. Under an IRB-approved study, whole blood samples from 12 patients with HbSS SCD were collected in EDTA tubes and red blood cells were isolated via centrifugation. RBCs were then resuspended in basal media (Lonza) with HEPES for pH-adjustment and treated with and without voxelotor (Selleckchem, Radnor, PA, USA) to a final hematocrit of 20% and concentration of 600μM. After one-hour in the incu-shaker, the samples were injected through the activated channels under normoxia at a rate of 2μL/min. Non-adherent cells were then removed by flushing the channels with additional media with HEPES. Adhered cells were counted via Adobe Photoshop Software. Statistical significance was calculated via paired t-test. Results: RBC adhesion ranged between 580 and 15,492 cells in untreated, activated samples and between 516 and 14,216 cells in voxelotor-treated, activated samples (Figure 1). Voxelotor treatment resulted in decreased sRBC adhesion in all but two of twelve samples and the percentage of adhesion reduction ranged between 7 and 70 percent (Figure 2, p=0.2). No conclusions were made when comparing patient's laboratory values (white blood cell, hemoglobin, platelets, absolute neutrophil count, absolute reticulocyte count, lactate dehydrogenase) to percent of adhesion reduction. Conclusion: Our results show that voxelotor does result in adhesion reduction under normoxia although, overall, the drug did not significantly decrease RBC adhesion. We theorize that by improving oxygen affinity and decreasing polymerization, voxelotor may limit the downstream effects of polymerization, specifically adhesion and vaso-occlusion. One major limitation is that RBCs were not deoxygenated and as mentioned above, voxelotor has improved RBC rheology under hypoxia. Therefore, future experiments will assess the effect of voxelotor on adhesion under hypoxic conditions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Analytical comparability demonstrated for an IgG4 molecule, inclacumab, following transfer of manufacturing responsibility from Roche to Global Blood Therapeutics

ABSTRACT Background Inclacumab is a recombinant, fully human, immunoglobulin IgG4 monoclonal antibody that selectively binds to P-selectin. Initially discovered and developed by Roche through phase 2 clinical studies in peripheral arterial disease and coronary artery disease, inclacumab has been in-licensed by Global Blood Therapeutics (GBT) as a potential treatment to reduce the frequency of vaso-occlusive crises in individuals with sickle cell disease. Research design and methods GBT sought to demonstrate the analytical comparability between material produced by Roche and material produced by GBT to ensure that no meaningful differences in identity, safety, purity, potency, or bioavailability exist between the GBT and Roche lots. Results Inclacumab samples produced by GBT were found to be comparable to the Roche v0.2 inclacumab samples based on (1) comparable primary and higher-order structures; (2) comparable purity profiles; (3) comparable potency, in vitro functional activities, and in vivo plasma exposures and pharmacokinetic profiles; and (4) comparable degradation patterns and kinetics under forced degradation conditions. Conclusions Based on the design of this comparability study and the results obtained, the US Food and Drug Administration approved the changes to the manufacturing process and gave clearance for GBT to proceed with phase 3 clinical trials.

TRIALS IN PROGRESS: THE THRIVE STUDIES EVALUATING THE EFFICACY, SAFETY, AND LONG-TERM TREATMENT WITH INCLACUMAB, A P-SELECTIN INHIBITOR, IN PATIENTS WITH SICKLE CELL DISEASE

Sickle cell disease (SCD) is an autosomal recessive red blood cell (RBC) disorder caused by a single point mutation in the β-globin gene, resulting in the production of hemoglobin S. Hemoglobin S polymerization within deoxygenated RBCs contributes to hemolysis, vaso-occlusion, and end-organ damage. Additionally, abnormal heterocellular adhesive interactions between RBCs and endothelial cells, leukocytes, and platelets play a central role in triggering painful vaso-occlusive crises (VOCs) in patients with SCD. Inclacumab is a recombinant, fully human, monoclonal antibody directed against human P-selectin, a cell adhesion molecule produced by endothelial cells and platelets. Inclacumab interferes with the adhesion of sickle RBCs, platelets, and leukocytes to the endothelium by binding P-selectin and preventing P-selectin from binding to its ligands. This is the putative mechanism by which inclacumab may potentially reduce the incidence of VOCs. We aim to conduct 2 global, multicenter, phase 3 studies and an open-label extension (OLE) study to evaluate the safety and efficacy of inclacumab in individuals with SCD. The THRIVE studies (THRIVE-131, THRIVE-132, THRIVE OLE) are being conducted in approximately 75 sites worldwide, including 13 sites in Brazil. Of the Brazilian sites, 2 are currently activated. THRIVE-131 (NCT04935879) is a randomized, double-blind, placebo-controlled, multicenter trial in which 240 participants aged ≥12 years experiencing 2 to 10 VOCs in the previous 12 months will be randomized 1:1 to receive intravenous (IV) inclacumab or placebo every 12 weeks for 48 weeks. The primary endpoint is the rate of VOCs during the 48-week treatment period. THRIVE-132 (NCT04927247) is a randomized, double-blind, placebo-controlled, multicenter trial in which 280 participants aged ≥12 years experiencing 2 to 10 VOCs in the previous 12 months will be randomized 1:1 to receive a single dose of IV inclacumab or placebo within 5 days of resolution of an index VOC that required admission to a healthcare facility and treatment with parenteral pain medication. The primary endpoint is the proportion of patients with readmission for a VOC within 90 days of randomization. After completion of their participation in THRIVE-131 or THRIVE-132, participants may enroll in THRIVE-133 OLE in which the long-term safety of inclacumab will be investigated. In THRIVE-133 OLE, participants will receive IV inclacumab every 12 weeks as long as the clinical benefit of treatment outweighs the risk and until access to inclacumab from an alternative source (eg, through commercialization or a managed-access program) becomes available. Recruiting for both THRIVE-131 and THRIVE-132 began in October 2021 and is ongoing. The THRIVE-131 and THRIVE-132 phase 3 studies will examine the efficacy of inclacumab in reducing the frequency of VOCs and readmissions due to VOCs, and the THRIVE-133 OLE study will examine the long-term safety of inclacumab in individuals with SCD. The phase 3 THRIVE studies are expected to provide valuable data on the safety and efficacy of inclacumab as a potential therapy for VOCs associated with SCD. These studies are supported by Global Blood Therapeutics.