Model Of Sickle Cell Disease Research Articles

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.

Oxycodone Produces Antinociception, but Worsens Functional Behaviors, in Humanized Sickle Cell Disease Mice

Introduction: Sickle Cell Disease (SCD) is a major cause of morbidity and mortality worldwide, and affects more than 100,000 people in the US. Acute, vaso-occlusive pain crises are the hallmark of the disease and the primary cause of hospitalization. Recurrent, acute pain episodes compound and result in chronic pain for many patients, though the mechanisms of this transition are poorly understood. Opioids remain the standard of care for chronic SCD pain, despite their adverse side effects (e.g., constipation, respiratory depression, abuse liability, dependence) and the fact that prolonged opioid treatment results in tolerance. Thus, a pressing need exists to identify effective non-opioid analgesics to reduce SCD chronic pain. Humanized mouse models of SCD, such as the Berkeley (BERK) model, provide a useful tool to investigate disease pathophysiology and evaluate novel therapeutic strategies. Though oxycodone is one of the most commonly prescribed analgesics for SCD chronic pain, it has yet to be evaluated in this SCD mouse model. Therefore, we assess whether oxycodone would ameliorate phenotypic hypernociception and behavioral deficits in the BERK mouse model of chronic pain. Methods: Male and female HbSS-BERK (sickle) and HbAA-BERK (control) mice were used as subjects. Stimulus-evoked nociceptive behaviors were assessed using the Von Frey and Hot Plate; and functional non-evoked behaviors were assessed using Grip Strength, Inverted Screen, and Nesting tests. Oxycodone (0, 1.25, 2.5, 5, and 10 mg/kg, s.c.) was administered one hour prior to testing. Data were analyzed as Student's t-test, one- and two-way ANOVAs followed by Tukey or Sidak post-hoc analysis when appropriate (p < 0.05 considered significant). Results: Oxycodone (2.5, 5, and 10 mg/kg) fully attenuated mechanical allodynia in HbSS-BERK mice; and produced significant antinociception in HbAA-BERK mice at the highest dose (10 mg/kg). Oxycodone (1.25 and 2.5 mg/kg) partially and fully attenuated thermal hyperalgesia in HbSS-BERK mice, respectively. 5 and 10 mg/kg of oxycodone in HbSS-BERK and 2.5, 5, and 10 mg/kg in HbAA-BERK mice produced significant antinociception. In the grip strength assay, oxycodone had no significant effect in HbSS-BERK mice, but worsened performance in HbAA-BERK mice. Conversely, oxycodone worsened inverted screen deficits in HbSS-BERK mice, with no significant effect in HbAA-BERK mice. Finally, oxycodone (2.5 and 5 mg/kg) worsened nest-building behavior regardless of genotype. Conclusion: Oxycodone, a gold standard analgesic for pain management, acutely reverses the hyperalgesic phenotype in BERK mice. However, oxycodone not only produces complete antinociception in those same assays, but also worsens functional deficits in the HbSS-BERK mice along with their controls. These findings demonstrate that oxycodone is suboptimal for restoring normal pain sensation as well as daily functioning.

A Phase 2a, Randomized, Open-Label Study to Evaluate Multiple Dosing Regimens of Subcutaneous ALXN1820 in Adult Patients with Sickle Cell Disease

Background:Sickle cell disease (SCD) is a group of inherited disorders that result in chronic hemolysis/anemia and vaso-occlusive crises (VOC). Current management options for SCD do not adequately address both anemia and VOC. Hematopoietic stem cell transplant is the only curative treatment but is associated with life-threatening complications. Complement activation may play an important role in the pathophysiology of SCD since intravascular hemolysis of sickle red blood cells (RBCs) leads to release of free hemoglobin triggering activation of the complement alternative pathway (CAP). Intravascular hemolysis and CAP activation promotes anemia and VOC in patients with SCD. Properdin is an endogenous activator of the CAP (Roumenina LT, et al. Am J Hematol. 2020;95(5):456-464; Vercellotti GM, et al. Am J Hematol. 2019;94(3):327-33). ALXN1820 is a novel, humanized, bispecific, VHH antibody that simultaneously binds human albumin and properdin. The antibody effectively and selectively inhibits CAP activation while binding to albumin, which confers an extended circulatory half-life to the molecule. Pretreatment with a mouse anti-properdin antibody significantly ameliorated signs of hemolysis and VOC in a mouse model of SCD. The safety, pharmacokinetics and pharmacodynamics of ALXN1820 are being evaluated in an ongoing study in healthy volunteers (NCT04631562). Study design, methods, and study population: This open-label Phase 2a study will evaluate the safety, tolerability, efficacy, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of multiple doses and dosing regimens of ALXN1820 administered subcutaneously to patients with SCD (see Figure for study schema). Key inclusion criteria are confirmed diagnosis of SCD (HbSS and HbSß0-thalassemia), hemoglobin levels 5.5-10g/dL at screening, 1-10 VOC episodes in the past 12 months, and vaccination requirements (meningococcal, Haemophilus influenzae type B (Hib) and Streptococcus pneumoniae). Concomitant treatment with stable dose of hydroxyurea is permitted during the study as applicable. Key exclusion criteria are receiving voxelotor or crizanlizumab within 60 days of providing informed consent and, prior treatment with complement inhibitors within 6 months of first study dose. Key endpoints of the study are shown in the Table. Safety analyses will be performed on the Safety Population and will be reported by each cohort and treatment arm. Efficacy analyses will include absolute and percentage change from baseline to the end of the Treatment Period (12 weeks) in complement biomarkers, hemoglobin, hemolysis markers, and hemopexin for Cohort 1 and Cohort 2. Conclusion: Complement activation may play an important role in the pathophysiology of SCD. Treatment with an anti-properdin antibody significantly ameliorated signs of hemolysis and VOC in a mouse model of SCD supporting the potential of ALXN1820 to treat SCD. Data from this Phase 2 study are anticipated to confirm the potential of ALXN1820 to treat SCD and guide the design of future studies in patients with SCD and other complement-mediated diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Sickle Cell Disease Patient Plasma Sensitizes iPSC-Derived Sensory Neurons from Sickle Cell Disease Patients

Individuals living with sickle cell disease (SCD) experience severe recurrent acute and chronic pain. The underlying neurobiology of the complex pain of SCD is not well understood. Limited and often ineffective treatment options and multifactorial healthcare disparities create an immediate need to develop non-opioid based SCD pain therapies. However, in order to develop novel therapies, we must better understand the neurobiological mechanisms underlying SCD pain. Although humanized mouse models of SCD do exist, growing evidence indicates there are striking differences in gene expression and functional responsiveness between human and mouse dorsal root ganglia (DRG) sensory neurons. Human DRG tissues are extremely limited in availability, postmortem tissues only provide static, endpoint molecular signatures, and obtaining DRGs from patients with SCD is untenable. These are all barriers to gaining mechanistic insight into pathogenic pain processes. Human induced pluripotent stem cell (iPSC)-derived sensory neurons (iSNs) have recently been used to model the altered function and expression of molecules in native DRG neurons in chronic pain disorders. These iPSC-derived SNs express canonical markers of human nociceptors and exhibit functional responses to noxious stimuli similar to those properties of native mammalian nociceptors. The objective of this study was to utilize iSNs differentiated from SCD patient iPSCs as well as age- and race-matched healthy controls (HCs) to characterize baseline receptor expression and function of SCD iSNs compared to HC iSNs. Our goal is to assess the intrinsic properties of SCD nociceptors that may contribute to the persistent sensitization of nociceptors in SCD patients with pain. To this end, both HC and SCD iSNs expressed transcripts for canonical markers of mature sensory neurons (NF200, CGRP), immune receptors (CCR1/2/3/5, CD4, CRLF2, CSF2Ra/b), and functional ion channels (HCN1, Nav1.7, P2X3/4, CACNA2D1, CHRNA9) (Table 1). Further, they expressed transcripts for ion channels relevant to SCD pain (Piezo1, Piezo2, TRPA1, TRPC5, TRPM3, TRPM8, TRPV1, TRPV4). We confirmed the expression of Peripherin, CGRP, TRPV1 at the protein level using immunocytochemistry. There were no significant differences in expression for any of these targets in iSNs derived from SCD patients compared to those from HCs. However, we did identify trends for higher endothelin 1 receptor type A (ETRa) and CC motif chemokine ligand 2 (CCL2) transcript production by SCD iSNs compared to HC iSNs (Table 1). Additionally, we observed robust responses with no significant differences in baseline function between HC and SCD iSNs when stimulated with known agonists of human SNs (KCl, Glutamate, ab-meATP, capsaicin) in traditional or high-throughput calcium imaging studies. To begin defining extrinsic mechanisms that may sensitize human SCD SNs, we tested the effects of SCD patient plasma (n=4 from SCD baseline [SS BL] and acute pain [SS IP]- paired samples, n=4 Black controls [HC]) on iSN hypersensitivity by exposing iSNs in vitro to human plasma diluted 1:10 in maintenance media for 30 minutes before analyzing calcium flux. Plasma samples from SCD patients experiencing acute pain (SS IP) were found to significantly increase SCD iSN response to capsaicin (p<0.05) compared to HC iSNs treated with SS IP plasma, while race-matched HC plasma or SCD patient plasma at baseline pain states (SS BL) did not induce this effect (Figure 1). Excitingly, these findings reproduce results identified in native DRG neurons from SCD mice and suggest that iSNs derived from patient iPSCs can be used in parallel with native SCD neurons from mouse models to investigate mechanisms underlying the chronic sensory neuron sensitization in SCD. They also identify potential intrinsic mechanisms of SCD pain which may extend beyond a blood-based disease pathology. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Apohemoglobin-Haptoglobin Complex Prevents Microvascular Dysfunction in Sickle Cell Mice: Potential Therapeutic Approach for Vaso-Occlusive Crisis

Background: Sickle cell disease (SCD) is associated with episodes of vaso-occlusive events (VOE) caused by the effects of hemoglobin S (HbS) polymerization in red blood cells (RBCs) and resultant hemolysis. In the acute setting, treatment for VOE is limited to supportive care such as hydration and aggressive pain management as no specific therapies can alter the course of acute VOE. The apohemoglobin-haptoglobin (apoHb-Hp) complex is a two-protein formulation that is capable of scavenging and neutralizing the toxic effects of cell-free hemoglobin (Hb) and heme released from the hemolysis of polymerized HbS RBCs. The primary objective of this study was to evaluate the microcirculatory effects of apoHb-Hp during hypoxia-induced VOE. Methods: The effects of apoHb-hp was studied in a mouse model of SCD using sickle cell transgenic mice (HbSS-Towne's mice, and compared to mice expressing normal human Hb (HbAA-Towne's mice). Each group consisted of 11 mice, they were initially kept in normal conditions (21% O2) for an hour followed by a hypoxic state (7% O2) for another hour and finally brought back to normal conditions (21% O2) for an hour before being sacrificed. ApoHb-hp (60 mg/mL) and Hp (85 mg/mL) were injected intravenously to yield equimolar total heme and Hb binding capacity between treatments prior to hypoxia exposure. During baseline, hypoxia and normoxia systemic parameters (mean arteriole pressure and heart rate), blood gases (pO2,A, pO2,v, sO2,A, sO2,v), hematological properties (total Hb concentration, hematocrit, and concentration of lactate) and microcirculatory hemodynamics (velocity, diameter, flow and functional capillary density) were measured. After the mice were sacrificed a count of present macrophages, neutrophils and lymphocytes were quantified in the pulmonary tissue. Results: There was no notable changes in blood gas concentrations with any of the treatments. ApoHb-Hp treatment improved microcirculatory hemodynamics of HbSS mice by preventing hypoxia-induced vasoconstriction and reduction of blood flow in arterioles <40µm (P=0.818, P<0.0001), arterioles >40 µm (P>0.9999, P<0.0001) and venules 20<x<60 µm (P=0.150, P=0.330) compared to HbAA mice. Furthermore, the functional capillary density saw favorable recovery to baseline when HbSS mice were treated with apoHb-Hp seeing a 36% drop initially, 50% drop after 24 hours, 35% drop after 48 hours, and 27% drop after 72 hours. For comparison, HbSS mice that didn't receive treatment saw a 50% drop initially, 77% drop after 24 hours, 80% drop after 48 hours, and 72% drop after 72 hours when compared to baseline. Conclusion: These results suggest a role for ApoHb-Hp complex in alleviating microvascular changes occurring during hypoxia mediated acute vaso-occlusion in SCD and merits further study for development as a potential therapeutic. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pathophysiological characterization of the Townes mouse model for sickle cell disease

A deeper pathophysiologic understanding of available mouse models of sickle cell disease (SCD), such as the Townes model, will help improve preclinical studies. We evaluated groups of Townes mice expressing either normal adult human hemoglobin (HbA), sickle cell trait (HbAS), or SCD (HbS), comparing younger versus older adults, and females versus males. We obtained hematologic parameters in steady-state and hypoxic conditions and evaluated metabolic markers and cytokines from serum. Kidney function was evaluated by measuring the urine protein/creatinine ratio and urine osmolality. In vivo studies included von Frey assay, non-invasive plethysmography, and echocardiography. Histopathological evaluations were performed in lung, liver, spleen, and kidney tissues. HbS mice displayed elevated hemolysis markers and white blood cell counts, with some increases more pronounced in older adults. After extended in vivo hypoxia, hemoglobin, platelet counts, and white blood cell counts decreased significantly in HbS mice, whereas they remained stable in HbA mice. Cytokine analyses showed increased TNF-alpha in HbS mice. Kidney function assays revealed worsened kidney function in HbS mice. The von Frey assay showed a lower threshold to response in the HbS mice than controls, with more noticeable differences in males. Echocardiography in HbS mice suggested left ventricular hypertrophy and dilatation. Plethysmography suggested obstructive lung disease and inflammatory changes in HbS mice. Histopathological studies showed vascular congestion, increased iron deposition, and disruption of normal tissue architecture in HbS mice. These data correlate with clinical manifestations in SCD patients and highlight analyses and groups to be included in preclinical therapeutic studies.

Amiodarone improves anemia in a murine model of sickle cell disease and is associated with increased erythrocyte bis(monoacylglycerol) phosphate

Sickle cell disease (SCD) is associated with altered plasma and erythrocyte lipid profiles. In a previous study, SCD mice with deficiency of proprotein convertase subtilisin/kexin type 9 (PCSK9) were observed to have more severe anemia and increased sickling compared to control SCD mice. Although PCSK9 affects circulating low density lipoprotein (LDL) by regulation of the LDL receptor, the effect of PCSK9 on anemia was independent of LDL receptor expression. In the current study, erythrocyte metabolomics were performed and revealed altered erythrocyte lipid species between SCD mice with and without PCSK9. Of particular interest, the late endosome-specific lipid bis(mono)acylglycerol phosphate (BMP) 44:12 was markedly decreased in erythrocytes from SCD mice deficient in PCSK9 mice relative to control SCD mice. Incubation of sickle erythrocytes with a neutralizing antibody to BMP increased erythrocyte sickling in vitro. In vitro treatment of SCD erythrocytes with amiodarone (1.5 μM) or medroxyprogesterone (6.75 μM), two pharmacologic compounds known to increase BMP, resulted in reduced erythrocyte sickling. Treatment of SCD mice with amiodarone (10 mg/kg) for 2 weeks resulted in increased BMP, improvement in anemia with reduced reticulocytosis, and decreased ex vivo sickling. In conclusion, severity of anemia in SCD is improved with amiodarone treatment, an effect which may be mediated through increased erythrocyte BMP.

Cannabidiol attenuates hyperalgesia in a mouse model of sickle cell disease

Cannabidiol attenuates hyperalgesia in a mouse model of sickle cell disease

Increased Susceptibility for Adverse Reactions to Ultrasound Enhancing Agents in Sickle Cell Disease

Pain-related adverse events (AEs) to ultrasound enhancing agents (UEAs) have been reported in patients with sickle cell disease (SCD). The aims of this study were to characterize the scope of these AEs in the SCD population and to investigate potential mechanisms on the basis of pathways involved in SCD vaso-occlusive crisis (VOC) and pain. The prevalence and classification of AEs were analyzed from two clinical trials in which high-dose Definity infusions were used in patients with SCD (n=55) or matched control subjects (n=43) to study muscle or myocardial microvascular perfusion. Because complement (C') activation can trigger VOC in SCD, C' activation and surface adhesion of C' proteins on lipid UEAs were studied invitro. C'-mediated UEA attachment to bone marrow immune cells was assessed using flow cytometry in a murine SCD model (Townes mice). Blood from patients receiving Definity was obtained to measure specific lysophospholipid metabolites of lipids in Definity thought to mediate SCD pain. Moderate or greater AEs, all of which were nociceptive (back or bone pain), occurred in one control subject and nine SCD subjects (2% vs 16%, P=.02). Patients with SCD who had AEs tended to have more severe manifestations of SCD. Three of the subjects with SCD had previously received Definity without complications. In patients with SCD, four AEs were classified as severe in intensity and as serious AEs on the basis of need for medical intervention. AEs were described to be similar to SCD-related pain, but there was no evidence for VOC, hemolysis, hypotension, or hypoxemia. At baseline, markers of C' activation were greater in patients with SCD than control subjects. However, after administration of lipid UEAs, SCD and control subjects were similar with regard to C' activation response, anaphylatoxin production, bone marrow microbubble retention, and production of lysophospholipids. There was a trend toward increased deposition of C3b and C3bi on lipid UEAs exposed to serum from patients with SCD. Patients with SCD are particularly susceptible to nociceptive AEs when given Definity at high doses. The mechanism for these AEs remains unclear but most are not related to the triggering of classic VOC.

Erythrocyte mitogen-activated protein kinases mediate hemolytic events under osmotic and oxidative stress and in hemolytic diseases

p38 MAPKs are key regulators of cellular adaptation to various stress stimuli, however, their role in mediating erythrocyte cell death and hemolysis is largely unknown. We hypothesized that activation of erythrocyte p38 MAPK is a common event in the stimulation of hemolysis, and that inhibition of p38 MAPK pathways could mitigate hemolysis in hemoglobinopathies. We exposed human erythrocytes to diamide-induced oxidative stress or to hypoosmotic shock in the presence or absence of p38 MAPK inhibitors (SCIO469, SB203580, CMPD1) and used immunoblotting to determine MAPK activity and to identify possible downstream effectors of p38 MAPK. We also evaluated the impact of p38 MAPK inhibitors on stress-induced hemolysis or hypoxia-induced sickling in erythrocytes from mouse models of sickle cell disease. We found that human erythrocytes express conventional MAPKs (MKK3, p38 MAPK, MAPKAPK2) and identified differential MAPK activation pathways in each stress condition. Specifically, p38 MAPK inhibition in diamide-treated erythrocytes was associated with decreased phosphorylation of Src tyrosine kinases and Band 3 protein. Conversely, hypoosmotic shock induced MAPKAPK2 and RSK2 phosphorylation, which was inhibited by SCIO469 or CMPD1. Relevant to hemoglobinopathies, sickle cell disease was associated with increased erythrocyte MKK3, p38 MAPK, and MAPKAPK2 expression and phosphorylation as compared with erythrocytes from healthy individuals. Furthermore, p38 MAPK inhibition was associated with decreased hemolysis in response to diamide treatments or osmotic shock, and with decreased erythrocyte sickling under experimental hypoxia. These findings provided insights into MAPK-mediated signaling pathways that regulate erythrocyte function and hemolysis in response to extracellular stressors or human diseases.

O-13: INHIBITION OF THE ENDOCANNABINOID REGULATING ENZYME MONOACYLGLYCEROL LIPASE AMELIORATES THE HYPER-NOCICEPTIVE PHENOTYPE IN THE BERKELEY MOUSE MODEL OF SICKLE CELL DISEASE

Purpose: Sickle Cell Disease (SCD) is a major cause of morbidity and mortality worldwide, and affects more than 100,000 people in the US. Recurrent episodes of acute, vaso-occlusive pain crisis, a hallmark of the disease, lead to chronic pain for many patients, though the mechanisms of this transition are poorly understood. Although opioids remain the standard of care to treat SCD chronic pain, their myriad adverse side effects (e.g., constipation, respiratory depression, abuse liability, dependence) as well as the fact that SCD chronic pain requires prolonged opioid treatment that results in tolerance, severely limit their therapeutic utility. Thus, a pressing need exists to identify effective non-opioid analgesic strategies to reduce SCD chronic pain. Humanized mouse models of SCD, such as the Berkeley (BERK) model, provide a useful tool to investigate disease pathophysiology and evaluate novel therapeutic strategies. Dorsal Root Ganglion (DRG) neurons are peripheral sensory neurons essential for the transmission of nociceptive afferents to the CNS. In rodent models of neuropathic pain, harvested DRG neurons show hyperexcitability. Inhibitors of the major degradative enzyme of 2-arachidonoylglycerol, monoacylglycerol lipase (MAGL), reduce nociceptive behavior in neuropathic and inflammatory preclinical models of pain through cannabinoid receptor-dependent and -independent mechanisms. As MAGL inhibitors have yet to be tested in BERK mice, here we test whether this approach will ameliorate the hyper-nociceptive phenotype of HbSS-BERK mice. Materials and methods: Male and female HbSS-BERK (sickle) and HbAA-BERK (control) mice were used as subjects for these experiments. Nociceptive behaviors were assessed using the von Frey and Hot Plate tests. Motor-functional behavior was assessed using the Grip Strength and Nesting assays. Neuronal hyperexcitability was assessed using whole cell patch clamp electrophysiology of L4-S1 DRG neurons. Data were analyzed as two- and three-way ANOVAs followed by Tukey post-hoc analysis when appropriate (p < 0.05 considered significant). Results: HbSS-BERK mice displayed profound mechanical, thermal and deep tissue/musculoskeletal hypersensitivity. HbSS-BERK mice also exhibited deficits in nest-building behavior. Patch clamp studies revealed that DRG neurons harvested from HbSS-BERK mice displayed extremely hyperexcitability. MJN-110 dose-dependently reduced mechanical allodynia and thermal hyperalgesia, and ameliorated deficits in grip strength in HbSS-BERK mice. Importantly, seven days of daily injections of MJN-110 (5 mg/kg) continued to ameliorate the hyper-nociceptive phenotype of HbSS-BERK mice, which did not undergo tolerance. Moreover, DRG harvested from the MJN-treated HbSS-BERK mice showed similar DRG neuronal activity as seen in control mice. Conclusion: These findings validate that HbSS-BERK mice show hypersensitive responses to mechanical and heat stimuli, and exhibit motor-functional deficits in grip strength and nest building behavior. Additionally, this work demonstrates hyperexcitability of sensory neurons from the lower lumbar region of the spine of HbSS-BERK mice. Finally, the observations that MJN110 reduces these hyper-nociceptive behaviors and ameliorates neuronal hyperexcitability in HbSS-BERK mice suggest that MAGL inhibition represents a viable strategy to reduce chronic pain related to sickle cell disease. The authors do not declare any conflict of interest

Endothelial VWF is critical for the pathogenesis of vaso-occlusive episode in a mouse model of sickle cell disease

Vaso-occlusive episode (VOE) is a common and critical complication of sickle cell disease (SCD). Its pathogenesis is incompletely understood. von Willebrand factor (VWF), a multimeric plasma hemostatic protein synthesized and secreted by endothelial cells and platelets, is increased during a VOE. However, whether and how VWF contributes to the pathogenesis of VOE is not fully understood. In this study, we found increased VWF levels during tumor necrosis factor (TNF)-induced VOE in a humanized mouse model of SCD. Deletion of endothelial VWF decreased hemolysis, vascular occlusion, and organ damage caused by TNF-induced VOE in SCD mice. Moreover, administering ADAMTS13, the VWF-cleaving plasma protease, reduced plasma VWF levels, decreased inflammation and vaso-occlusion, and alleviated organ damage during VOE. These data suggest that promoting VWF cleavage via ADAMTS13 may be an effective treatment for reducing hemolysis, inflammation, and vaso-occlusion during VOE.

Evaluation of the mechanisms of heme-induced tissue factor activation: Contribution of innate immune pathways.

Hemolytic diseases such as Sickle Cell Disease (SCD) are characterized by a natural propensity for both arterial and venous thrombosis. The ability of heme to induce tissue factor (TF) activation has been shown both in animal models of SCD, and in human endothelial cells and monocytes. Moreover, it was recently demonstrated that heme can induce coagulation activation in the whole blood of healthy volunteers in a TF-dependent fashion. Herein, we aim to further explore the cellular mechanisms by which heme induces TF-coagulation activation, using human mononuclear cells, which have been shown to be relevant to in vivo hemostasis. TF mRNA expression was evaluated by qPCR and TF procoagulant activity was evaluated using a 2-stage assay based on the generation of activated factor X (FXa). Heme was capable of inducing both TF expression and activation in a TLR4-dependent pathway. This activity was further amplified after TNF-α-priming. Our results provide additional details on the mechanisms by which heme is involved in the pathogenesis of hypercoagulability in hemolytic diseases.

P1560: IMR-261, A NOVEL, ORAL NRF2 ACTIVATOR, REDUCES IRON OVERLOAD IN AN EXPERIMENTAL MOUSE MODEL BY ACTIVATING BMP6-MEDIATED HEPCIDIN SYNTHESIS

Background: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates several cellular defense mechanisms, including the oxidative stress response. Recent studies elucidated a protective role for Nrf2 under conditions of iron overload. Accumulation of toxic iron activates Nrf2, which activates Bmp6-mediated synthesis of hepcidin, thereby inhibiting ferroportin and improving iron homeostasis (Lim et al, Nat Metab 2019). IMR-261 is a novel, oral Nrf2 activator which has been tested in Phase 2 clinical trials (previously as CXA-10). Our previous work in mouse models of sickle cell disease and b-thalassemia showed that activation of Nrf2 by IMR-261 increases fetal hemoglobin (HbF) and hemoglobin (Hb), decreases markers of hemolysis and adhesion, reduces VOCs and improves ineffective erythropoiesis. Aims: The objective of this study was to evaluate the ability of IMR-261 to activate Bmp6-mediated synthesis of hepcidin (encoded by the Hamp gene), regulate iron stores and modulate transferrin saturation (TSAT) in a mouse model of iron overload. Methods: b-thalasssemic mice (Hbbth1/th1) were treated with IMR-261 at 37.5 or 50 mg/kg BID for 4 weeks. To assess activation of the Bmp6-hepcidin pathway, hepatic mRNA and plasma protein levels were measured. Iron concentrations in serum and tissue (liver, kidney and spleen) as well as markers of iron overload were also evaluated, and standard hematologic assessments were performed. Results: IMR-261 showed a significant, dose-dependent increase in hepatic mRNA levels of Bmp6 and Hamp (~3-fold increase; high dose vs vehicle) as well as Bmp6 target genes, Smad7 and Id1. A corresponding significant increase in Bmp6 and hepcidin protein levels in plasma was also observed. IMR-261 significantly decreased iron in serum by 32% (high dose vs vehicle) and in all tissues tested (34% in liver, 39% in spleen and 24% in kidney; high dose vs vehicle). Mice treated with IMR-261 also showed a significant decrease in TSAT and ferritin levels (up to 30% and 27%, respectively), as well as a reduction in GDF15 and erythropoietin (up to 72% and 48%, respectively) relative to vehicle. IMR-261 improved hematologic markers including a 30% decrease in reticulocytes and significant increases in RBCs, hemoglobin and hematocrit (consistent with our previous data). Image:Summary/Conclusion: Pharmacologic activation of Nrf2 by IMR-261 in a mouse model of iron overload activates hepcidin synthesis (likely via the Bmp6 signaling pathway) and decreases iron in serum, liver, spleen and kidney. IMR-261 also showed significant reductions in TSAT, ferritin, GDF15 and erythropoietin, which further support its role in iron homeostasis. Induction of hepcidin synthesis via Nrf2 activation is a novel therapeutic approach, and these results support further clinical development of IMR-261 as a treatment for conditions associated with iron dysregulation, including hereditary hemochromatosis.

S267: PROPERDIN-BLOCKING ANTIBODIES ATTENUATE COMPLEMENT ALTERNATIVE PATHWAY ACTIVATION TRIGGERED BY CELL-FREE HEME IN SICKLE CELL DISEASE MODELS

Background: Complement activation may play an important role in sickle cell disease (SCD) pathophysiology. Heme has been identified as a trigger of complement activation in SCD patients and is present in excess during severe vaso-occlusive crises. Heme is also a DAMP (damage associated molecular pattern) and induces thrombo-inflammation through tissue factor expression. ALXN1820 is a bi-specific VHH nanobody that simultaneously binds human albumin and properdin, thereby effectively and selectively inhibiting alternative pathway (AP) activation and formation of C3 and C5 convertases. The safety of ALXN1820 has been assessed in non-human primates and is currently being tested in a phase 1 study in healthy participants. Aims: 1) To assess if complement activation and heme are functionally linked; 2) To investigate the therapeutic potential of ALXN1820 in in vitro SCD models and in in vivo SCD models using a mouse properdin-blocking monoclonal antibody (mAb), 14E1. Methods:In vitro, complement inactivated C3b (iC3b) and C5b-9 deposition on red blood cells (RBCs) from patients with SCD and complement iC3b and C5b-9 deposition on the endothelial cell line HMEC-1 were assessed by flow cytometry after exposure to heme +/- ALXN1820. In vivo, in Townes SCD mice, vaso-occlusion and hyper-hemolysis were induced by intravenous heme injection (50 µmol/kg) or hypoxia-reoxygenation (8% O2 for 3h then 21% O2 for 1h) in the presence and absence of 14E1 (40 mg/kg), a mouse-specific properdin inhibitor. Vaso-occlusion was measured by staining RBCs with immunofluorescence-labeled Ter-119 antibodies on lung and liver sections. C3b and C5b-9 deposition on SCD RBCs was assessed by flow cytometry. Markers of hemolysis (bilirubin, lactate dehydrogenase, free hemoglobin, free heme) were quantified in plasma using commercial assays. Results: In RBCs from patients with SCD, cell-free heme triggered marked iC3b and C5b-9 deposition (Table). Deposition was blocked in the presence of ALXN1820 by >95% for iC3b and by >85% for C5b-9. HMEC-1 cells exposed to heme showed marked deposition of iC3b and C5b-9. In the presence of ALXN1820, deposition was blocked by >70% for iC3b and >85% for C5b-9. Pretreatment of mice with 14E1 markedly ameliorated vaso-occlusion and reduced both C3b and C5b-9 deposition on RBCs, and hemolysis biomarkers. Image:Summary/Conclusion: These data strengthen the hypothesis that in SCD, cell-free heme is a potent trigger of complement AP activation, which can be blocked by targeting properdin. Investigating the efficacy and safety of anti-properdin ALXN1820 in patients with SCD is warranted as a novel approach to treatment of acute and chronic SCD complications.

S269: MACROPHAGE FUNCTIONAL AND METABOLIC REWIRING THROUGH PPAR/PGC1 MODULATION IMPROVES HEME/IRON-DRIVEN DEFECTIVE EFFEROCYTOSIS AND PROMOTES TISSUE DAMAGE RESOLUTION IN SICKLE CELL DISEASE

Background: Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory status, which is contributed by pro-inflammatory macrophages. Macrophages are highly plastic immune cells which integrate and respond to a variety of signals they are exposed to in the surrounding microenvironment. One of these stimuli is represented by heme, which is released from oxidized hemoglobin upon hemolysis and acts as damage-associated molecular pattern to stimulate macrophage pro-inflammatory phenotypic switching through TLR4 signaling activation and ROS production. Heme-induced M1 pro-inflammatory activation program in macrophages promotes sterile inflammation and aggravates hepatic fibrosis in SCD, contributing to organ damage typically associated with this disease. Aims: While previous studies addressed heme ability to induce inflammatory cytokine production in macrophages, how heme alters cell functional properties and whether this exacerbates tissue injury remain unexplored. Macrophage functions as immune cell recruitment ability and apoptotic cell clearance are relevant in the context of SCD, where tissue damage and cell apoptosis occur frequently due to vaso-occlusive episodes, hypoxia and ischemic injury. Methods: Here we analyzed macrophage response to apoptotic stimuli in vivo, in mouse models of heme overload and SCD, as well as in vitro, in bone-marrow-derived macrophages, to unveil the impact of hemolysis on macrophage functional properties. Results: Our results demonstrate that, in addition to inflammatory activation, heme strongly alters macrophage functional response to apoptotic cell damage by exacerbating their immune cell recruitment ability and impairing their efferocytic capacity. We show that exposure to heme and iron excess drives defective efferocytosis of apoptotic neutrophils in vitro, in cultured bone-marrow-derived macrophages, and in vivo, in mouse model of heme overload. This is fully recapitulated in SCD mice, where limited efferocytosis contributes to impaired apoptotic cell clearance and exacerbated tissue damage. Mechanistically, altered efferocytosis depends on heme-driven activation of TLR4 signaling pathway and the suppression of the transcription factor PPARγ and its coactivator PGC1α. These changes lead to reduced expression of efferocytic receptors and impaired mitochondrial biogenesis as well as mitochondrial dysfunction, and is associated with metabolic skewing. This results in limited recognition and engulfment of apoptotic cells and decreased shift to aerobic mitochondrial fatty acid β-oxidation and anti-inflammatory IL-4/10 release, with consequent inhibition of continual efferocytosis, inflammation resolution and tissue repair. We further demonstrate that impaired phagocytic capacity and tissue damage are improved by hemopexin-mediated heme scavenging and anti-inflammatory IL-4 treatment in SCD mice through phenotypic and functional macrophage rewiring. Interestingly, defective efferocytosis is reproduced in vitro by macrophage exposure to SCD patients’ plasma and rescued by hemoglobin/heme scavenging via haptoglobin and hemopexin and PPARγ/PGC1α modulation via PPARγ agonist or IL-4. Summary/Conclusion: Our data indicate that the therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PPARγ/PGC1α modulation promotes the resolution of inflammation and ameliorates tissue damage associated with SCD pathophysiology. Thus, macrophage functional rewiring offers potentially valuable therapeutic strategies for SCD patients to improve tissue damage resolution upon vaso-occlusive crisis and ischemic events.

P1483: EPELEUTON, A NOVEL SYNTHETIC SECOND GENERATION W-3 FATTY ACID, PROTECTS HUMANIZED SICKLE CELL MICE AGAINST HYPOXIA/REOXYGENATION ORGAN DAMAGE

Background: Sickle cell disease (SCD) is a hereditary red cell disorder with high mortality and morbidity. Although crizanlizumab, a P-selectin inhibitor, and voxelotor, an oral anti-sickling agent, have been recently approved for clinical management of SCD, therapeutic options to limit acute events and to reduce disease progression are still required. An attempt to target inflammatory vasculopathy and to modulate inflammatory response has been made based on evidence in other diseases such as in cardiovascular disease looking to administration of ω-3 fatty acids. In humanized SCD mice, we previously showed that polyunsaturated fatty acid supplementation protects against acute sickle cell-related lung and liver damages during hypoxia/reoxygenation (H/R) induced vaso-occlusive crisis (VOCs) (Kalish B et al. Haematologica 2016). This agrees with the observation that the resolution process actively controlled by specialized pro-resolving lipid mediators is defective in SCD (Matte A et al, Blood 2019). Epeleuton (15-HEPE, 15 hydroxy eicosapentaenoic acid) is a novel synthetic second generation w-3 fatty acid which (i) has a favorable clinical safety profile, (ii) modulates systemic inflammation and triglyceride synthesis in patients with cardiometabolic disease (Climax J et al J Am Heart As 2020) and (ii) beneficially affects red cell indices in long-term studies in rats. Aims: To evaluate the impact of epeleuton on acute organ damage in a humanized mouse model of SCD. Methods: 3-4 month old male and female mice healthy control (Hbatm1(HBA)Tow Hbbtm3(HBG1,HBB)Tow) and Tim Townes sickle mice (SCD, Hba tm1(HBA)Tow Hbb tm2(HBG1,HBB*)Tow) were used. Animals were treated for 6 weeks with epeleuton 1,000 mg/kg once daily or vehicle by gavage. Mice underwent 10 hours of hypoxia (8% oxygen), followed by 3 hours of reoxygenation (21% oxygen) to mimic acute VOCs (Matte A et al, Blood 2019). Hematologic parameters and molecular analysis of target organs for SCD such as lung were carried out. Results: In SCD mice exposed to H/R stress, we found that epeleuton (i) reduces H/R induced hemolysis; (ii) normalizes tyrosine-phosphorylation profile of red cell membrane proteins; and (iii) decreases neutrophil count. In lungs of SCD mice exposed to H/R stress, we observed that epeleuton prevents the H/R induced activation of inflammatory and redox related transcription factors NF-kB, p65 and Nrf2 local inflammatory response compared with vehicle-treated animals. Epeleuton treated SCD mice exposed to H/R stress showed down-regulation of VCAM-1 and E-selectin, markers of inflammatory vasculopathy as well as reduction of lung protein oxidation and expression of anti-oxidant systems such as heme-oxygenase-1 (HO-1) or peroxiredoxin-2. Similar changes were also observed in livers of epeleuton-treated SCD mice exposed to H/R stress compared with vehicle-treated SCD animals. Of note, epeleuton prevented the H/R induced up-regulation of both VCAM-1 and ICAM-1, suggesting a direct impact of treatment on vascular activation and inflammation. Summary/Conclusion: In humanized SCD mice exposed to H/R stress to mimic acute VOCs, epeleuton acts as a multimodal agent targeting red cells, hemolysis, inflammatory response, and vascular dysfunction. Our data provide the rationale for epeleuton as a potential novel therapeutic option for clinical management of patients with SCD.

The oral ferroportin inhibitor vamifeport improves hemodynamics in a mouse model of sickle cell disease

AbstractSickle cell disease (SCD) is an inherited hemolytic anemia caused by a single point mutation in the β-globin gene of hemoglobin that leads to synthesis of sickle hemoglobin (HbS) in red blood cells (RBCs). HbS polymerizes in hypoxic conditions, leading to intravascular hemolysis, release of free hemoglobin and heme, and increased adhesion of blood cells to the endothelial vasculature, which causes painful vaso-occlusion and organ damage. HbS polymerization kinetics are strongly dependent on the intracellular HbS concentration; a relatively small reduction in cellular HbS concentration may prevent HbS polymerization and its sequelae. We hypothesized that iron restriction via blocking ferroportin, the unique iron transporter in mammals, might reduce HbS concentration in RBCs, thereby decreasing hemolysis, improving blood flow, and preventing vaso-occlusive events. Indeed, vamifeport (also known as VIT-2763), a clinical-stage oral ferroportin inhibitor, reduced hemolysis markers in the Townes model of SCD. The RBC indices of vamifeport-treated male and female Townes mice exhibited changes attributable to iron-restricted erythropoiesis: decreased corpuscular hemoglobin concentration mean and mean corpuscular volume, as well as increased hypochromic and microcytic RBC fractions. Furthermore, vamifeport reduced plasma soluble VCAM-1 concentrations, which suggests lowered vascular inflammation. Accordingly, intravital video microscopy of fluorescently labeled blood cells in the microvasculature of Townes mice treated with vamifeport revealed diminished adhesion to the endothelium and improved hemodynamics. These preclinical data provide a strong proof-of-concept for vamifeport in the Townes model of SCD and support further development of this compound as a potential novel therapy in SCD.

Limitations of mouse models for sickle cell disease conferred by their human globin transgene configurations.

ABSTRACTWe characterized the human β-like globin transgenes in two mouse models of sickle cell disease (SCD) and tested a genome-editing strategy to induce red blood cell fetal hemoglobin (HbF; α2γ2). Berkeley SCD mice contain four to 22 randomly arranged, fragmented copies of three human transgenes (HBA1, HBG2-HBG1-HBD-HBBS and a mini-locus control region) integrated into a single site of mouse chromosome 1. Cas9 disruption of the BCL11A repressor binding motif in the γ-globin gene (HBG1 and HBG2; HBG) promoters of Berkeley mouse hematopoietic stem cells (HSCs) caused extensive death from multiple double-strand DNA breaks. Long-range sequencing of Townes SCD mice verified that the endogenous Hbb genes were replaced by single-copy segments of human HBG1 and HBBS including proximal but not some distal gene-regulatory elements. Townes mouse HSCs were viable after Cas9 disruption of the HBG1 BCL11A binding motif but failed to induce HbF to therapeutic levels, contrasting with human HSCs. Our findings provide practical information on the genomic structures of two common mouse SCD models, illustrate their limitations for analyzing therapies to induce HbF and confirm the importance of distal DNA elements in human globin regulation. This article has an associated First Person interview with the first author of the paper.

Testosterone Deficiency in Sickle Cell Disease: Recognition and Remediation.

Hypogonadism is common in men with sickle cell disease (SCD) with prevalence rates as high as 25%. Testicular failure (primary hypogonadism) is established as the principal cause for this hormonal abnormality, although secondary hypogonadism and compensated hypogonadism have also been observed. The underlying mechanism for primary hypogonadism was elucidated in a mouse model of SCD, and involves increased NADPH oxidase-derived oxidative stress in the testis, which reduces protein expression of a steroidogenic acute regulatory protein and cholesterol transport to the mitochondria in Leydig cells. In all men including those with SCD, hypogonadism affects physical growth and development, cognition and mental health, sexual function, as well as fertility. However, it is not understood whether declines in physical, psychological, and social domains of health in SCD patients are related to low testosterone, or are consequences of other abnormalities of SCD. Priapism is one of only a few complications of SCD that has been studied in the context of hypogonadism. In this pathologic condition of prolonged penile erection in the absence of sexual excitement or stimulation, hypogonadism exacerbates already impaired endothelial nitric oxide synthase/cGMP/phosphodiesterase-5 molecular signaling in the penis. While exogenous testosterone alleviates priapism, it disadvantageously decreases intratesticular testosterone production. In contrast to treatment with exogenous testosterone, a novel approach is to target the mechanisms of testosterone deficiency in the SCD testis to drive endogenous testosterone production, which potentially decreases further oxidative stress and damage in the testis, and preserves sperm quality. Stimulation of translocator protein within the transduceosome of the testis of SCD mice reverses both hypogonadism and priapism, without affecting intratesticular testosterone production and consequently fertility. Ongoing research is needed to define and develop therapies that restore endogenous testosterone production in a physiologic, mechanism-specific fashion without affecting fertility in SCD men.