scholarly journals A Novel Non-Covalent Modulator of Hemoglobin Improves Anemia and Reduces Sickling in a Mouse Model of Sickle Cell Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 207-207
Author(s):  
Kelly M. Knee ◽  
Reema Jasuja ◽  
Amey Barakat ◽  
Dharani Rao ◽  
Zane Wenzel ◽  
...  
Keyword(s):  
Schiff Base ◽  
Sickle Cell Disease ◽  
Hemolytic Anemia ◽  
Oxygen Affinity ◽  
Treated Group ◽  
Cell Disease ◽  
Hypoxic Conditions ◽  
Hb S ◽  
Reactive Aldehyde

Sickle cell disease (SCD) is a severe genetic disorder caused by a single point mutation on the β-chain of adult hemoglobin (Hb A), β6 Glu→Val (Hb S). In the deoxygenated state Hb S polymerizes, leading to RBC sickling and precipitating all downstream consequences, including vaso-occlusion (pain crisis), hemolytic anemia, and stroke. Over time, these features cause significant organ damage and eventual organ failure, dramatically impacting both quality of life and expected lifespan. Numerous small molecules which covalently bind to Hb S have been evaluated clinically, however, the molecules that have demonstrated clinical efficacy all carry a reactive aldehyde group. The reactive aldehyde, a moiety that has the potential to react with any free amine, forms a covalent Schiff base with the N-terminal amine of the α1-Val. At least one member of this class of molecules, Tucaresol, showed a significant safety signal attributed to off-target Schiff base formation. An early investigation of covalent hemoglobin modification, extracorporeal carbamylation, both improved anemia and decreased the frequency of vaso-occlusive events by 80%, when there was a sufficiently high level of modification (30-50%). These results suggest that a molecule that binds Hb S and stabilizes the oxygenated state can impact both hemolytic anemia and vaso-occlusive crisis, if the molecule can achieve the necessary degree of hemoglobin modification. PFE-001 is a non-covalent molecule which binds selectively to Hb S and stabilizes the oxygenated state. Biochemical and biophysical studies show that PFE-001 binds specifically to Hb with double digit nanomolar potency and exhibits strong in vivo partitioning into RBCs. In a two-week multiple dose study using Townes SCD model animals (200 mg/kg, twice daily), PFE-001 significantly improved markers of hemolytic anemia, increased oxygen affinity, and reduced RBC sickling. Following 15 days of treatment blood drawn from PFE-001 treated animals and exposed to intense hypoxic conditions (4% O2, 4 hr) showed a 37.8% reduction in sickling compared to vehicle treated mice. Oxygen affinity was increased, demonstrated by a 53.7% reduction in p50 and an 84.4% reduction in p20 in the PFE-001 treated group. Hemoglobin levels in mice treated with PFE-001 increased by 42%, a mean increase of 5 g/dL. Hematocrit in the PFE-001 treated group increased to 42%, in contrast to 29% in the vehicle group. Reticulocyte percentages were reduced from 53% in vehicle treated animals to 24% in PFE-001 treated animals. In addition to the significant impact PFE-001 had on hemolytic anemia, a 10% reduction in sVCAM-1 levels in the PFE-001 treated group indicates a small but statistically significant improvement in vasculopathy following 15 days of treatment. This improvement in vasculopathy suggests that PFE-001 has the potential to address vaso-occlusive crisis in addition to anemia. In total, the in vitro and in vivo data suggest that PFE-001 is a potent, selective, and effective inhibitor of Hb S polymerization and RBC sickling. PFE-001 can reduce hemolytic anemia, improve vasculopathy, increase oxygen affinity, and reduce RBC sickling under hypoxic conditions. Plans for advancement of PFE-001 to clinical trials are in progress. Disclosures Knee: Pfizer Inc: Employment. Jasuja:Pfizer Inc.: Employment. Barakat:Pfizer Inc.: Employment. Rao:Pfizer Inc.: Employment. Wenzel:Pfizer Inc.: Employment. Sahasrabudhe:Pfizer Inc.: Employment. Narula:Pfizer Inc.: Employment. Jasti:Pfizer Inc.: Employment. Chang:Pfizer Inc.: Employment. Beaumont:Pfizer Inc.: Employment. Piotrowski:Pfizer Inc.: Employment. Janz:Pfizer Inc.: Employment.

scholarly journals GBT440 reverses sickling of sickled red blood cells under hypoxic conditions in vitro

2018 ◽  
Vol 10 (2) ◽  
Author(s):  
Kobina Dufu ◽  
Donna Oksenberg
Keyword(s):  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Oxygen Affinity ◽  
Microvascular Blood Flow ◽  
Cell Disease ◽  
Hypoxic Conditions

Sickle cell disease is characterized by hemolytic anemia, vasoocclusion and early mortality. Polymerization of hemoglobin S followed by red blood cell sickling and subsequent vascular injury are key events in the pathogenesis of sickle cell disease. Sickled red blood cells are major contributors to the abnormal blood rheology, poor microvascular blood flow and endothelial injury in sickle cell disease. Therefore, an agent that can prevent and or reverse sickling of red blood cells, may provide therapeutic benefit for the treatment of sickle cell disease. We report here that GBT440, an anti-polymerization agent being developed for the chronic treatment of sickle cell disease, increases hemoglobin oxygen affinity and reverses in vitro sickling of previously sickled red blood cells under hypoxic conditions. Our results suggest that besides preventing sickling of red blood cells, GBT440 may mitigate vasoocclusion and microvascular dysfunction by reversing sickling of circulating sickled red blood cells in vivo.

scholarly journals Biologic Complexity in Sickle Cell Disease: Implications for Developing Targeted Therapeutics

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Beatrice E. Gee
Keyword(s):  
Clinical Trials ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Vascular Occlusion ◽  
Current Therapy ◽  
Cell Disease ◽  
Primary Defect ◽  
Relative Contribution ◽  
Hb S

Current therapy for sickle cell disease (SCD) is limited to supportive treatment of complications, red blood cell transfusions, hydroxyurea, and stem cell transplantation. Difficulty in the translation of mechanistically based therapies may be the result of a reductionist approach focused on individual pathways, without having demonstrated their relative contribution to SCD complications. Many pathophysiologic processes in SCD are likely to interact simultaneously to contribute to acute vaso-occlusion or chronic vasculopathy. Applying concepts of systems biology and network medicine, models were developed to show relationships between the primary defect of sickle hemoglobin (Hb S) polymerization and the outcomes of acute pain and chronic vasculopathy. Pathophysiologic processes such as inflammation and oxidative stress are downstream by-products of Hb S polymerization, transduced through secondary pathways of hemolysis and vaso-occlusion. Pain, a common clinical trials endpoint, is also complex and may be influenced by factors outside of sickle cell polymerization and vascular occlusion. Future sickle cell research needs to better address the biologic complexity of both sickle cell disease and pain. The relevance of individual pathways to important sickle cell outcomes needs to be demonstratedin vivobefore investing in expensive and labor-intensive clinical trials.

scholarly journals Sickle cell disease of transgenic SAD mice

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 3189-3197 ◽  
Author(s):  
M Trudel ◽  
ME De Paepe ◽  
N Chretien ◽  
N Saadane ◽  
J Jacmain ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Acute Hypoxia ◽  
Oxygen Affinity ◽  
Premature Death ◽  
Cellular Level ◽  
Cell Disease ◽  
Severe Renal Disease

Erythrocyte sickling on deoxygenation in vitro occurs in transgenic SAD mice, hemizygous for a modified human sickle hemoglobin, HbSAD [alpha 2 beta 2S(beta 6val)Antilles (beta 23 lle)D- Punjab (beta 121Gln)] (SAD- 1, 19% HbSAD; beta-thal/SAD-1, 26% HbSAD). The present study examines the cellular defects in vivo and pathologic changes observed in SAD-1 mice at atmospheric oxygenation as well as the effect of acute hypoxia. The transgenic mice showed generalized congestion and microvascular occlusions, occasionally with thrombosis and infarctions of lung, kidneys, penis, and myocardium. The most prevalent chronic organ lesions were congestive splenomegaly (83% of animals) and renal glomerulopathy, which affected 75% of animals by 10 months of age. Further, SAD mice have a mean lifespan that was reduced by 40% when compared with nontransgenic littermates. Premature death of SAD mice was associated with acute vasoocclusive events or severe renal disease. SAD mice developed lethal vasoocclusive processes when exposed to reduced pO2 conditions, whereas control mice survived normally. The sensitivity to hypoxia appears to depend on the cellular level of HbSAD, because death occurred at pO2 of 42 mmHg for SAD mice and 49 mmHg for beta-thal/SAD. Administration of an antisickling agent that increases oxygen affinity (BW12C79) protected SAD and beta-thal/SAD mice from the lethal hypoxic stress. In conclusion, the transgenic SAD and beta-thal/SAD mice developed a pathophysiology that strongly resembles human sickle cell disease. Moreover, this animal model allows studies on the effect of antisickling agents.

scholarly journals Treatment of sickle cell disease by increasing oxygen affinity of hemoglobin

Blood ◽  
2021 ◽  
Author(s):  
Eric R Henry ◽  
Belhu Metaferia ◽  
Quan Li ◽  
Julia Harper ◽  
Robert B Best ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Oxygen Delivery ◽  
Cell Damage ◽  
Oxygen Affinity ◽  
Red Cell ◽  
Cell Disease ◽  
Oxygen Dissociation ◽  
Dissociation Curves

The issue of treating sickle cell disease with drugs that increase hemoglobin oxygen affinity has come to the fore with the FDA approval in 2019 of voxelotor, the only anti-sickling drug approved since hydroxyurea in 1998. Voxelotor reduces sickling by increasing the concentration of the non-polymerizing, high oxygen affinity R (oxy) conformation of HbS. Treatment of sickle cell patients with voxelotor increases Hb levels and decreases indicators of hemolysis, but with no indication as yet that it reduces the frequency of pain episodes. Here we use the allosteric model of Monod, Wyman, and Changeux to simulate whole blood oxygen dissociation curves and red cell sickling in the absence and presence of voxelotor under the in vivo conditions of rapid oxygen pressure decreases. Our modeling agrees with experiments using a new robust assay, which shows the very large, expected decrease in sickling from the drug. The modeling indicates, however, that the increase in oxygen delivery from reduced sickling is largely offset by the increase in oxygen affinity. The net result is that the drug increases overall oxygen delivery only at the very lowest oxygen pressures. Reduction of sickling does, however, mitigate against red cell damage and explains the observed decrease in hemolysis. More importantly, our modeling of in vivo oxygen dissociation, sickling, and oxygen delivery suggests that drugs that increase fetal hemoglobin or decrease MCHC, should be more therapeutically effective than drugs that increase oxygen affinity.

Pharmacological modification of oxygen affinity improves deformability of deoxygenated sickle erythrocytes: a possible therapeutic approach to sickle cell disease

1989 ◽  
Vol 76 (4) ◽  
pp. 357-362 ◽  
Author(s):  
A. J. Keidan ◽  
M. C. Sowter ◽  
C. S. Johnson ◽  
S. S. Marwah ◽  
J. Stuart
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Oxygen Affinity ◽  
Saturation Curve ◽  
Cell Disease ◽  
Filtration Method ◽  
Sickle Cells ◽  
Sickle Erythrocytes ◽  
Haemoglobin S

1. The formation of polymerized haemoglobin S in sickle cells is critically dependent on the concentration of deoxygenated haemoglobin so that compounds which increase the oxygen affinity of haemoglobin S are potential anti-sickling agents. 2. BW12C [5-(2-formyl-3-hydroxyphenoxy)pentanoic acid] and BWA589C [4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid] are aromatic benzaldehydes that cause a dose-dependent left-shift of the oxygen saturation curve of haemoglobin S by stabilization of its oxy-(R)-conformation. 3. A 5 μm pore filtration method, which is highly sensitive to polymerization of haemoglobin S, was used to demonstrate a significant improvement in the deformability of deoxygenated sickle erythrocytes at concentrations (0.75–1.5 mmol/l) of BW12C and BWA589C that are achievable in vivo. Both compounds may therefore be of value for the treatment of sickle cell disease. 4. Filtration of sickle cells through pores of 5μm diameter is a sensitive technique for evaluating the rheological effects of potential anti-sickling compounds.

scholarly journals Hypoxia Enhanced Red Cell Adhesion in Vitro May Identify Patients at Risk for Vasculopathy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3672-3672
Author(s):  
Charlotte Yuan ◽  
Erina Quinn ◽  
Sargam Kapoor ◽  
Myeongseop Kim ◽  
Erdem Kucukal ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Research Funding ◽  
Cell Disease ◽  
Hypoxic Conditions ◽  
Significant Difference ◽  
History Of ◽  
Male Subjects

Abstract Background: Priapism is a serious complication associated with Sickle Cell Disease (SCD) that may be a manifestation of underlying vasculopathy. The Centers for the Study of Complex Diseases of Childhood (CSCCD), comprising independent Comprehensive Sickle Cell Centers, demonstrated an association of priapism with hemolysis.1 Previously, we identified two groups of people with SCD based on red blood cell (RBC) adhesion under hypoxic conditions: those patients whose RBCs showed hypoxia-enhanced adhesion (HEA) and those whose did not (non-HEA).2 Patients with HEA had evidence for more hemolysis in vivo. Here, we aimed to examine (1) the association of HEA with hypoxia in vivo, and (2) RBC adhesion in normoxic and hypoxic conditions in male patients with or without a history of priapism. Methods: This retrospective study was conducted at the Adult Sickle Cell Disease Clinic at the University Hospitals Seidman Cancer Center, in Cleveland, OH between 2015 to 2018. Blood samples were obtained from 26 male subjects (29 samples, 25 HbSS and 1 HbSS HPFH). Adhesion experiments were performed as previously reported by passing surplus whole blood through LN-immobilized microchannels at physiological conditions under both normoxic and hypoxic conditions.2,3 Adherent RBCs were then quantified with microscope after a wash step. The median value was used for data analyses from multiple samples obtained from an individual. Chart review was conducted to examine results of hypoxia testing obtained in vivo as part of routine clinical care. Results: Male subjects with HbSS and a history of priapism had higher HEA in comparison to subjects without a history of priapism (3268 ± 5647 vs. 122 ± 1218, p=0.016). However, there was no significant difference between RBC adhesion of the two groups under normoxic conditions (529 ± 1528 vs. 402 ± 280). More male subjects with priapism had hypoxia in vivo (10 out of 14) than subjects without priapism (5 out of 12). Compared to male subjects with a history of priapism, those without a history of priapism had lower lactate dehydrogenase levels (474 ± 267 vs. 290 ± 215, p=0.008). Conclusions: Our data showed that subjects with a history of priapism had a higher HEA and tended to have more evidence for hypoxia in vivo than did subjects without a history of priapism. Further, male subjects with hypoxia in vivo had more HEA than did those without hypoxia in vivo (not shown). Hypoxia in vivo may cause increased RBC damage (reflected by HEA), hemolysis, nitric oxide depletion, and consequent vasculopathy, resulting in priapism. Hypoxia may be treatable, when identified in subjects with a history priapism in vivo or possibly with HEA in vitro. This could plausibly modify disease severity in some cases. References: Nolan VG, Wyszynski DF, Farrer LA, Steinberg MH. Blood. 20015 Nov;106(9):3264-7. doi: 10.1182/blood-2005-04-1594 Kim M, Alapan Y, Adhikari A, Little JA, Gurkan Microcirculation. 2017 Jul;24(5). doi: 10.1111/micc.12374. Alapan Y, Kim C, Adhikari A, Gray KE, Gurkan-Cavusoglu E, Little JA, Gurkan Transl Res. 2016 Jul;173:74-91.e8. doi: 10.1016/j.trsl.2016.03.008. Epub 2016 Mar 19. Disclosures Little: NHLBI: Research Funding; PCORI: Research Funding; Hemex: Patents & Royalties: Patent, no honoraria; Doris Duke Charitable Foundations: Research Funding.

scholarly journals Rational modification of vanillin derivatives to stereospecifically destabilize sickle hemoglobin polymer formation

2018 ◽  
Vol 74 (10) ◽  
pp. 956-964 ◽  
Author(s):  
Tanvi M. Deshpande ◽  
Piyusha P. Pagare ◽  
Mohini S. Ghatge ◽  
Qiukan Chen ◽  
Faik N. Musayev ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Biological Effects ◽  
Oxygen Affinity ◽  
Cell Disease ◽  
Sickle Hemoglobin ◽  
Polymer Formation ◽  
Promising Therapeutic Approach ◽  
Hb S

Increasing the affinity of hemoglobin for oxygen represents a feasible and promising therapeutic approach for sickle cell disease by mitigating the primary pathophysiological event,i.e.the hypoxia-induced polymerization of sickle hemoglobin (Hb S) and the concomitant erythrocyte sickling. Investigations on a novel synthetic antisickling agent, SAJ-310, with improved and sustained antisickling activity have previously been reported. To further enhance the biological effects of SAJ-310, a structure-based approach was employed to modify this compound to specifically inhibit Hb S polymer formation through interactions which perturb the Hb S polymer-stabilizing αF-helix, in addition to primarily increasing the oxygen affinity of hemoglobin. Three compounds, TD-7, TD-8 and TD-9, were synthesized and studied for their interactions with hemoglobin at the atomic level, as well as their functional and antisickling activitiesin vitro. X-ray crystallographic studies with liganded hemoglobin in complex with TD-7 showed the predicted mode of binding, although the interaction with the αF-helix was not as strong as expected. These findings provide important insights and guidance towards the development of molecules that would be expected to bind and make stronger interactions with the αF-helix, resulting in more efficacious novel therapeutics for sickle cell disease.

scholarly journals Improving the Solubility and Oral Bioavailability of a Novel Aromatic Aldehyde Antisickling Agent (PP10) for the Treatment of Sickle Cell Disease

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1148
Author(s):  
Tarek A. Ahmed ◽  
Khalid M. El-Say ◽  
Fathy I. Abd-Allah ◽  
Abdelsattar M. Omar ◽  
Moustafa E. El-Araby ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Binary System ◽  
Aromatic Aldehyde ◽  
Sickle Cell ◽  
Oxygen Affinity ◽  
Oral Drug ◽  
Cell Disease ◽  
Sprague Dawley

Background: Aromatic aldehydes, with their ability to increase the oxygen affinity of sickle hemoglobin, have become important therapeutic agents for sickle cell disease (SCD). One such compound, voxelotor, was recently approved for SCD treatment. Methyl 6-((2-formyl-3-hydroxyphenoxy)methyl) picolinate (PP10) is another promising aromatic aldehyde, recently reported by our group. Like voxelotor, PP10 exhibits O2-dependent antisickling activity, but, unlike voxelotor, PP10 shows unique O2-independent antisickling effect. PP10, however, has limited solubility. This study therefore aimed to develop oral and parenteral formulations to improve PP10 solubility and bioavailability. Methods: Oral drug tablets with 2-hydroxypropyl beta cyclodextrin (HP-β-CD), polyvinylpyrrolidone, or Eudragit L100-55 PP10-binary system, and an intravenous (IV) formulation with d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) or HP-β-CD, were developed. The pharmacokinetic behavior of the formulations was studied in Sprague-Dawley rats. PP10, a methylester, and its acid metabolite were also studied in vitro with sickle whole blood to determine their effect on Hb modification, Hb oxygen affinity, and sickle red blood cell inhibition. Results: Aqueous solubility of PP10 was enhanced ~5 times with the HP-β-CD binary system, while the TPGS aqueous micelle formulation was superior, with a drug concentration of 0.502 ± 0.01 mg/mL and a particle size of 26 ± 3 nm. The oral tablets showed relative and absolute bioavailabilities of 173.4% and 106.34%, respectively. The acid form of PP10 appeared to dominate in vivo, although both PP10 forms demonstrated pharmacologic effect. Conclusion: Oral and IV formulations of PP10 were successfully developed using HP-β-CD binary system and TPGS aqueous micelles, respectively, resulting in significantly improved solubility and bioavailability.

scholarly journals MetAP2 Inhibition Modifies Hemoglobin S (HbS) to Delay Polymerization and Improve Blood Flow in Sickle Cell Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2260-2260
Author(s):  
Melanie Demers ◽  
Sarah Sturtevant ◽  
Kevin Guertin ◽  
Dipti Gupta ◽  
Kunal Desai ◽  
...  
Keyword(s):  
Blood Flow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Whole Blood ◽  
Research Funding ◽  
Oxygen Affinity ◽  
Enzyme Kinetic ◽  
Erythroid Cells ◽  
Cell Disease

Dilution of HbS with non-sickling hemoglobin or hemoglobin with increased oxygen affinity is clinically beneficial in sickle cell disease. Aldehydes, including 5-HMF, tucaresol or GBT440, modify the N-terminus of HbS by reversible covalent imine formation generating modified forms of HbS that resist polymerization under low oxygen concentrations. In contrast to reversible imine formation by aldehydes, we hypothesize that stable modification of HbS will result from N-terminal retention of the initiator methionine (iMet) and subsequent N-terminal acetylation of the iMet (acetyl-iMet). MetAP2 is the methionine aminopeptidase able to cleave iMet from Val1 on α-globin and βS-globin as the unfolded N-terminal peptides emerge from the ribosome. Enzyme kinetic studies with pure MetAP2 and N-terminal octapeptides showed that βS-globin peptide is a 5-fold better substrate than α-globin peptide. Lentiviral shRNA knock-down of MetAP2 in differentiating erythroid HUDEP cells in vitro confirmed that α-globin is more extensively modified than βS-globin, consistent with the enzyme kinetic data. Selective MetAP2 inhibitors used to treat cultured human erythroid cells (HUDEP and PBMC derived CD34+) and Townes SCD mice in vivo confirmed that both α-globin and βS-globin domains of HbS are extensively modified by N-terminal iMet and acetyl-iMet. N-terminal retention of iMet and subsequent acetylation creates a mixture of modified HbS tetramers with combined modifications on both globins. Cation exchange chromatography separated nine different modified HbS variants from unmodified HbS as identified by LCMS. Purified samples of HbS modified by N-terminal iMet and acetyl-iMet had increased oxygen affinity as measured by decreased P50. Modified HbS containing the acetyl-iMet-βS-globin were found to have delayed polymerization under complete hypoxia (sodium metabisulfite triggered hypoxia in 1.8 M phosphate). Two modified HbS variants were further purified for X-ray crystallography studies (βS-globin / iMet-α-globin and acetyl-iMet-βS-globin / iMet-α-globin). Oxyhemoglobin structures of both modified HbS variants were in the R2-state previously described in structures of aldehyde modified HbS. This R2-state stabilizes the oxygenated R-state of HbS from conversion to the deoxygenated T-state that initiates HbS polymerization in sickle RBC. Treatment by selective irreversible covalent or reversible MetAP2 inhibitors resulted in high levels of HbS modification (>75%) in cultured erythroid cells (HUDEP and CD34+ cells). Dose dependent modification of HbS was observed in Townes sickle cell mouse blood RBC in vivo with total modification of HbS approaching 50%. In whole blood ex vivo studies, modification of HbS decreased RBC sickling under hypoxia (4% O2) and significantly increased the affinity of RBC for oxygen (decreased P50). Blood samples from MetAP2 inhibitor treated mice were analyzed for single-cell O2 saturation of the RBC and for the fractional flow velocity drop in whole blood rheology under decreasing partial oxygen pressures. In blood from vehicle treated sickle mice, a low-saturation peak of deoxy-HbS was observed in 7.8% O2, in contrast to blood from MetAP2 inhibitor-treated mice where the low-saturation peak was only observed in 6.4% O2. Similarly, in an assay of O2 dependent blood flow rheology, the half-maximum fractional velocity drop occurred at 5% O2 in control blood decreasing to 2% O2 in MetAP2 inhibitor treated blood. Our studies show that MetAP2 inhibition results in retention of iMet on βS-globin and α-globin and allows further acetylation of the retained iMet to create a mixture of N-terminal modified HbS tetramers. These modified HbS variants resist polymerization and RBC sickling under conditions of low O2 by delaying HbS polymerization and increasing O2 affinity. Our data suggests that MetAP2 may warrant further study as a potential therapeutic target for sickle cell disease. Disclosures Demers: Sanofi: Employment. Sturtevant:Sanofi: Employment. Guertin:Sanofi: Employment. Gupta:Sanofi: Employment. Desai:Sanofi: Employment. Vieira:Sanofi: Employment. Hicks:Sanofi: Employment. Ismail:Sanofi: Employment. Safo:Sanofi: Consultancy, Research Funding; Virginia Commonwealth University: Patents & Royalties. Wood:Sanofi: Consultancy, Research Funding. Higgins:Sanofi: Consultancy, Research Funding. Light:Sanofi: Employment.

IHP-Loaded Red Blood Cells as Preventive Therapy In Sickle Cell Disease

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1625-1625
Author(s):  
Vanessa Bourgeaux ◽  
Yannick Campion ◽  
Emeline Aufradet ◽  
Cyril Martin ◽  
Yann Godfrin
Keyword(s):  
Sickle Cell Disease ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Blood Cells ◽  
Cell Disease ◽  
Transfusion Therapy ◽  
Platelet Counts ◽  
Hypoxic Conditions ◽  
Hypotonic Lysis

Abstract Abstract 1625 Sickle cell disease (SCD) is a genetic disorder characterized by abnormal hemoglobin S (HbS) that polymerizes under hypoxic conditions leading to sickled-shape red blood cells (RBCs). Vaso-occlusive crisis (VOC) is one of the major clinical manifestations of the disease, very painful for patients and causing irreversible organ damages. RBC exchange is commonly used as preventive and curative treatment of the disease. However, the therapeutic action of RBC exchange only relies on the removal of HbS-containing RBCs (SS-RBCs) and their transient replacement by normal RBCs (AA-RBCs). Recent works have shown that sickled reticulocytes, activated platelets and leukocytes play a critical role in the onset of VOC. They aggregate with endothelial cells creating local hypoxia, enhancing sickling and thus capillary blockade. Oxygen deprivation that occurs in venous capillaries may widely contribute to the severity of the occlusion. Therefore, increasing the oxygenation level in capillaries could help to prevent SS-RBCs from sickling and avoid crisis. This may be possible by transfusing patients with AA-RBCs loaded with Inositol HexaPhosphate (IHP), an allosteric effector that binds tightly to hemoglobin. The resulting suspension (IHP-RBCs) has the ability to increase oxygen release by 2 to 3 fold compared to normal AA-RBCs. The objective of the present study was to evaluate in vivo the benefit of using IHP-RBCs treatment in SCD. We used BERK transgenic mouse model that fully mimics human SCD in childhood with specific features of splenomegaly, reticulocytosis and leukocytosis. IHP-RBCs were prepared by loading IHP into murine C57BL6J RBCs using reversible hypotonic lysis method. RBCs subjected to reversible hypotonic lysis but without IHP were used as control suspension. Study was designed with repeated RBC exchanges scheduled every 2 weeks. First RBC exchange using IHP-RBCs or control suspension was performed on 6–7 week-old mice followed by 2 further injections. Mice were sacrificed one week after last RBC exchange and critical hematological parameters (reticulocyte, leukocyte, platelet counts and sickled cells) as well as serum inflammation markers were used as readouts to evaluate the risk of VOC. The first study was performed in normoxic conditions. After the therapy, approximately 42% of mouse RBCs had been replaced by IHP-RBCs or control suspension. Strong reduction of spleen weight (50%) and circulating sickled RBCs was observed in both cases due to the dilution of SS-RBCs. Interestingly, IHP-RBCs treatment enabled to significantly lower reticulocytes (18% vs 31%), leukocytes (5.3 vs 8.4 103/μl) and platelet counts (1057 vs 1518 103/μl) compared to not treated mice. Additionally, Serum Amyeloid Protein (SAP), an inflammation marker analogous of human C-Reactive Protein was also significantly reduced with IHP-RBCs (450 vs 750 μg/ml) indicating lowered severity of inflammation. The analysis of VCAM and HIF-1 factors in both spleen and lungs were very low in both treated and not treated mice. Further experiments demonstrated that hypoxic stress is needed to induce significative inflammation at the organ level. The study will thus be repeated in hypoxic conditions to evaluate the effect of IHP-RBCs treatment on organ damaging. We had in a previous study demonstrated in vitro the ability of IHP-RBCs to reduce sickling of human SS-RBCs (Bourgeaux et al, Transfusion, in press). The present in vivo study brings new evidence of the therapeutic potential of IHP-RBCs with the observation of a significant reduction of VOC risk factors and SAP level in treated mice. These results strongly support the fact that loading IHP into AA-RBCs may improve the effectiveness of conventional transfusion therapy. Disclosures: No relevant conflicts of interest to declare.

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