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

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 218-218
Author(s):  
Osheiza Abdulmalik ◽  
Tanvi Deshpande ◽  
Mohini Ghatge ◽  
Yan Zhang ◽  
Jurgen Venitz ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Phase I ◽  
Sickle Cell ◽  
Clinical Studies ◽  
Ex Vivo ◽  
Dependent Manner ◽  
Cell Disease ◽  
Hb S ◽  
Dose Dependent

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

Hemoglobin Blocks Von Willebrand Factor Proteolysis by ADAMTS-13: A Mechanism Associated with Acquired ADAMTS-13 Deficiency in Patients with Sickle Cell Disease

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3919-3919
Author(s):  
Zhou Zhou ◽  
Han Hyojeong ◽  
Miguel A. Cruz ◽  
Jose A. Lopez ◽  
Jing-fei Dong ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Von Willebrand Factor ◽  
Elevated Level ◽  
Dependent Manner ◽  
Cell Disease ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract One of the hallmark events of sickle cell disease (SCD) is vasoocclusion and episodic pain crisis. Although the mechanism of vascular occlusion is very complicated, processes like thrombosis and thromboembolism have been recognized to play an important role in the development of such clinical manifestation in SCD. Studies have shown that the von Willebrand factor (VWF), especially the ultra-large (UL) multimers play a major role in vasoocclusion, which clearly indicates a possible impairment of the VWF-cleaving metalloproteae ADAMTS-13 in these patients with SCD. In a recent work, indeed we have mentioned that the plasma ADAMTS-13 in patients with SCD having normal antigen level showed 35% less protease activity than the normal. There may be several plasma factors responsible for the acquired deficiency of ADAMTS-13 in SCD. Since, the increasing evidences suggest that the elevated level of extracellular hemoglobin (Hb) in plasma parallely associated with the pathogenesis of SCD, we investigated the effects of extracellular Hb on VWF proteolysis by ADAMTS-13. We observed that purified Hb dose-dependently inhibited the ADAMTS-13 cleavage of recombinant(r) VWF and endothelial ULVWF multimers under static and flow conditions. Hb bound to VWF multimers in a saturation-dependent manner and more potently to the rVWFA2 domain (affinity Kd~24nM), which contains the cleavage site for ADAMTS-13. Hb bound also to the ADAMTS-13 (Kd~65nM), with 2.7 times less affinity than to VWFA2. The bindings were neither calcium-dependent nor affected by haptoglobin. However, it is the Hb-binding to VWF that prevented the substrate from being cleaved by ADAMTS-13. These in vitro findings are consistent with the in vivo observations in patients with SCD. An elevated level of extracellular Hb in plasma was inversely correlated (linear regression, r2 =0.6354) with the low activity of ADAMTS-13 in a cohort of ten adult patients with SCD (mean±SE, Hb 346±138 mg/l; activity 33.3±30%) compared to age and gender-matched normal individuals (n=10; Hb 24±8 mg/l; activity 76.2±16%). The data together suggest that patients with SCD suffer from acquired ADAMTS-13 deficiency, primarily because Hb competitively binds and inhibits the proteolysis of VWF multimers, leading to ULVWF accumulation on vascular endothelium and in circulation. The Hb-VWF interaction may therefore be considered as a therapeutic target for reducing thrombotic and vasoocclusive complications in patients with severe hemolysis such as those with SCD.

Quantification of Anti-Sickling Effect of Aes-103 in Sickle Cell Disease Using an in Vitro Microfluidic Assay

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2699-2699 ◽  
Author(s):  
E. Du ◽  
Laurel Mendelsohn ◽  
James S. Nichols ◽  
Ming Dao ◽  
Gregory J. Kato
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Dependent Manner ◽  
Cell Disease ◽  
Pdms Film ◽  
Patient Sample ◽  
Hydroxyurea Treatment ◽  
O2 Concentration

Abstract Background: Under hypoxic conditions, sickle hemoglobin (HbS) polymerizes, causing morphologic distortion (sickling) of red blood cells (RBCs) in sickle cell disease (SCD). Aes-103 (5-hydroxymethylfurfural, 5-HMF) can stabilize the R-state and increase the oxygen affinity of hemoglobin, inhibiting the intracellular polymerization of HbS. Using a microfluidics-based hypoxia assay, we were able to track sickling of individual cells and quantify the anti-sickling effect of Aes-103 at millimolar (mM) levels in blood from SCD patients on hydroxyurea treatment (on-HU) and not on hydroxyurea treatment (off-HU). Method: We have developed a microfluidic assay that utilizes a gas permeable polydimethylsiloxane (PDMS) film 150 µm in thickness, to create a severe hypoxia microenvironment in a 5 µm deep chamber to measure cell sickling in vitro at 37°C. The hypoxia condition was 5 minutes in total, consisting of an initial oxygen-rich stage (20% O2), a transient deoxygenating stage (O2 concentration decreased to 5% within 15 second), and a steady-stage stage (O2 concentration decreased further and maintained at 2% for the rest of time). Blood samples from 3 on-HU and 3 off-HU patients were incubated with Aes-103 at concentrations of 0.5, 1, 2, and 5 mM for one hour at 37 degrees C, washed with Phosphate Buffered Saline and suspended in RPMI-1640 containing 1% w/v Bovine Serum Albumin for in vitro testing. Sickle RBCs undergoing sickling typically form spiky edges and a dark coarse texture due to intracellular HbS polymerization visually enhanced by a bandpass filter (Fig. 1A). The anti-sickling effect of Aes-103 was then quantified by the maximum sickled fraction (fraction of all RBCs that were morphologically distorted) under the hypoxia condition. Results: In the absence of Aes-103, the sickled fractions varied from 34% to 73% (Mean ± SD: 54% ± 18%). With the presence of Aes-103, the mean sickled fraction decreased with drug concentration (Fig. 1B), which can be well fitted with linear regression (R2= 0.95). With 2 mM Aes-103 incubation, each patient sample showed a significant decrease in cell sickling from its baseline. Addition of Aes-103 at 5 mM concentration prevented majority of RBCs from sickling (sickled fraction ≤ 5%). The sickled fraction of one patient sample was nearly zero. The distribution of sickled fractions does not completely correlate with the patient's HU status in this limited sample size (Fig. 1C). We also observed that hypoxia-induced sickling at baseline showed an apparent bimodal distribution, although the slope of response to Aes-103 concentration was similar. Conclusions: Our microfluidic assay enabled a rapid, quantitative characterization of cell sickling in vitro within a few minutes and using a single drop of whole blood patient sample. We confirmed the anti-sickling efficacy of Aes-103 for both on-HU and off-HU patient samples in a dosage-dependent manner. This assay has potential as a biomarker for drug development and monitoring for in vivo effect of potential anti-sickling therapeutics. Figure 1. (A) Identification of cell sickling from a microscopic image (arrows indicate the sickled RBCs). (B) Sickled fraction as a function of Aes-103 concentration. (C) Variation in response among different on-HU and off-HU patient samples. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Regulation of Na+/Mg2+ Exchange in Sickle Erythrocytes By Endothelin-1

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4064-4064
Author(s):  
Pablo A. Rivera ◽  
Yaritza Inostroza ◽  
Jose R. Romero ◽  
Alicia Rivera
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Ex Vivo ◽  
Red Cells ◽  
Cell Disease ◽  
Magnesium Homeostasis ◽  
Increased Activity ◽  
Exchange Activity

Abstract Excess levels of endothelin-1 (ET-1), erythrocyte sickling and chronic inflammation have been proposed as important contributors to the pathophysiology of sickle cell disease (SCD). We have shown that ET-1 receptor antagonists improve hematological parameters by reducing Gardos channel activity in two transgenic mouse models of SCD while reducing oxidant stress by decreasing circulating levels of protein disulfide isomerase. Magnesium (Mg2+) deficiency, mediated in part via increased erythrocyte Na+/Mg2+ exchanger activity, has been demonstrated to contribute to erythrocyte dehydration, K+ loss and sickling in SCD. However, the relationship between ET-1 and the Na+/Mg2+ exchanger in SCD remains unclear. We measured Na+/Mg2+ exchange activity in ex vivo red cells and observed increased activity following in vitro incubation of human (2.2 ± 0.2 to 3.2 ± 0.1 mmol/1013 cell x h, P<0.03, n=5) and mouse red blood cells with ET-1 (P<0.001, n=5); events that were significantly blocked by pre-incubation of cells with 1 μM BQ788, a selective inhibitor of ET-1 type B receptors. In addition, in vitro deoxygenation of sickle red cells led to increased exchanger activity that was inhibited by impramine, a Na+/Mg2+ exchange inhibitor, and associated with reduced deoxygenation-stimulated sickle cell dehydration. These results suggest an important role for ET-1 and cellular magnesium homeostasis in sickle cell disease. To this end, we studied Na+/Mg2+ exchange activity in ex vivo erythrocytes from three transgenic sickle mouse models and observed increased activity in these cells when compared to red cells from either Hb A transgenic or C57BL/J6 wild-type mice (P<0.03, n=4). We then tested the in vivo effects of ET-1 receptor antagonists on erythrocyte Na+/Mg2+ exchange activity in the BERK mouse, a transgenic model of SCD. We blocked ET-1 receptors type A and B by in vivo treatment with BQ-788 and BQ-123 (360mg/Kg/Day) for 14 days and observed lower erythrocyte exchanger activity when compared to cells from vehicle treated BERK mice (P<0.02, n=6). Thus our results suggest that ET-1 receptor blockade represents an important therapeutic approach to control erythrocyte volume and magnesium homeostasis that may lead to improved inflammatory and vascular complications observed in SCD. Supported by NIH R01HL090632 to AR. Disclosures No relevant conflicts of interest to declare.

scholarly journals Evaluation of persistence and fate of ex vivo edited-HSC modified with donor template and Its role in correcting Sickle Cell Disease

2021 ◽  
Author(s):  
Sowmya Pattabhi ◽  
Samantha N Lotti ◽  
Mason P Berger ◽  
David J Rawlings
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Ex Vivo ◽  
Cell Disease ◽  
Gene Correction ◽  
Healthy Human ◽  
Peripheral Blood Stem Cells ◽  
Donor Template

Sickle cell disease (SCD) is caused by a single nucleotide transversion in exon 1 of the HBB gene that changes the hydrophobicity of adult globin (βA), leading to substantial morbidity and reduced lifespan. Ex vivo autologous gene editing utilizing co-delivery of a designer nuclease along with a DNA donor template allows for precise homology-directed repair (HDR). These gene corrected cells when engrafted into the bone marrow (BM) can prove to be therapeutic and serves as an alternative to HLA-matched BM transplantation. In the current study, we extensively explored the role of single stranded oligonucleotide (ssODN) and recombinant adeno-associated 6 (rAAV6) donor template delivery to introduce a codon-optimized change (E6optE) or a sickle mutation (E6V) change following Crispr/Cas9-mediated cleavage of HBB in healthy human mobilized peripheral blood stem cells (mPBSCs). We achieved efficient HDR in vitro in edited cells and observed robust human CD45+ engraftment in the BM of NBSGW mice at 16-17 weeks. Notably, recipients of ssODN-modified HSC exhibited a significantly higher proportion of HDR-modified cells within individual BM, CD34+ and CD235+ compartments of both E6optE and E6V cohorts. We further assessed key functional outcomes including RNA transcripts analysis and globin sub-type expression. Our combined findings demonstrate the capacity to achieve clinically relevant HDR in vitro and in vivo using both donor template delivery method. The use of ssODN donor template-delivery is consistently associated with higher levels of gene correction in vivo as demonstrated by sustained engraftment of HDR-modified HSC and erythroid progeny. Finally, the HDR-based globin protein expression was significantly higher in the E6V ssODN-modified animals compared to the rAAV6-modified animals confirming that the ssODN donor template delivery outperforms rAAV6-donor template delivery.

scholarly journals GTx011, a Potent Allosteric Modifier of Hemoglobin Oxygen Affinity, Prevents RBC Sickling in Whole Blood and Prolongs RBC Half-Life in Vivo in a Murine Model of Sickle Cell Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 217-217 ◽  
Author(s):  
Kobina Dufu ◽  
Donna Oksenberg ◽  
Chengjing Zhou ◽  
Athiwat Hutchaleelaha ◽  
David R. Archer
Keyword(s):  
Flow Cytometry ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Half Life ◽  
Ex Vivo ◽  
Cell Disease ◽  
Employment Equity ◽  
Hypoxic Conditions ◽  
Equity Ownership

Abstract Sickle cell disease (SCD) is caused by a point mutation in the β-globin gene leading to production of hemoglobin S (HbS) that polymerizes under hypoxic conditions with subsequent formation of sickled red blood cells (RBCs). We have developed a novel small molecule, GTx011, which attains effective concentrations in blood upon oral dosing in multiple species. GTx011 increases the affinity of oxygen (O2) for HbS, delays in vitro HbS polymerization and prevents sickling of isolated RBCs under hypoxic conditions. We report here that GTx011 prevents in vitro sickling of RBCs in blood from sickle cell patients. Moreover, in a murine model of sickle cell disease (Townes SS mice), GTx011 prevents ex vivo sickling of RBCs and prolongs RBC half-life. We previously reported that GTx011 prevents sickling of isolated sickle cell RBCs (SSRBCs) subjected to a fixed hypoxic condition (pO2 of ~30 mm Hg) for 30 min. For a more physiologically relevant evaluation, we determined the anti-sickling activity of GTx011 in blood under variable hypoxic conditions over a shorter duration of time. Sickling of SSRBCs in blood was evaluated using a combination of hemoximetry and morphometric measurements. Whole blood from sickle cell patients was modified in vitro with GTx011 prior to hemoximetry. Conversely, blood from SS mice with GTx011 orally dosed acutely or chronically for 10-12 days was used for hemoximetry. SSRBCs were harvested during hemoximetry at various O2 tensions and immediately fixed in a deoxygenated solution of 2% glutaraldehyde/PBS prior to morphological quantitative analysis with CellVigene software or imaging flow cytometry (AMNIS ImageStreamX MkII). To evaluate the effect of GTx011 on RBC half-life in SS mice, N-hydroxysuccinimide biotin was injected into SS mice on day 5 of chronic dosing, producing a pulse-label. Flow cytometry was performed using fluorescently labeled streptavidin to determine the decay of biotinylation and RBC half-life. Reticulocyte counts were measured at different intervals during the dosing regimen by determining the percentage of blood cells that were Ter-119+, Thiazole-Orange+ and CD45- by flow cytometry. In a dose-dependent manner, GTx011 decreased the p50 value of human blood indicating an increase in Hb-O2 affinity. In parallel, GTx011 dose-dependently reduced the number of sickled SSRBCs under all hypoxic conditions (pO2 of <40 mm Hg) evaluated. Moreover, at an O2 tension mimicking typical hypoxic conditions in tissue capillaries (40 mm Hg), 300 µM of GTx011 was sufficient to prevent sickling of human SSRBCs in whole blood (20% Hct). Similarly, ex vivo sickling analysis indicated that, relative to blood from vehicle-treated SS mice, blood from GTx011-treated SS mice showed a pronounced reduction in the number of sickled RBCs under hypoxic conditions with a concurrent reduction in p50. For example, at a pO2 of 10 mm Hg, 19% of SSRBCs in blood from GTx011-treated mice sickled ex vivo compared with 56% in blood from vehicle-treated SS mice. In SS mice chronically dosed with GTx011, a prolongation of the RBC half-life from 2.4 days to 3.8 days was achieved together with a marked decrease in reticulocyte count. This increase in RBC half-life and accompanying reduction in reticulocyte count was observed in mice with GTx011 concentrations in blood that corresponded to >30% calculated Hb target occupancy. Taken together, these data suggest that GTx011 has the potential to be a beneficial therapeutic agent for the chronic treatment of SCD. Table SS mice RBC half life Reticulocytes Sickled RBCs Hemoximetry Chronic treatment, PO, BID, 10-12 days (Days) (%) (% at 10 mm Hg) p20 (mm Hg) p50 (mm Hg) Vehicle-treated 2.4 53 56 18 32 GBT440-treated (100mg/kg) 3.8 32 19 4.5 21 Disclosures Dufu: Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Zhou:Global Blood Therapeutics: Research Funding. Hutchaleelaha:Global Blood Therapeutics: Employment, Equity Ownership. Archer:Global Blood Therapeutics: Consultancy, 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.

Finding of 8 New Therapeutic Agents by the Use of New High-Throughput Drug Screening Methods Established by the Nhlbi Sicke Cell Disease Reference Laboratory.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3751-3751
Author(s):  
Toshio Asakura ◽  
Osheiza Abdulmalik ◽  
Efe Iyamu ◽  
Qiukan Chen ◽  
Jisheng Yang ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
High Throughput ◽  
Drug Evaluation ◽  
In Vitro Tests ◽  
Screening Methods ◽  
Reference Laboratory ◽  
Cell Disease ◽  
Hb S

Abstract The NHLBI Sickle Cell Disease (SCD) Reference Laboratory (Ref Lab) was established in 1997 to facilitate rapid, efficient, accurate and economical evaluation of agents or therapies that may be beneficial for the treatment of patients with sickle cell disease. On the basis of the assumption that the unsuccessful finding of antisickling agents in the past was attributed to insufficient pre-clinical drug evaluation, the SCD Ref Lab proposed to screen drugs using more than 10 in vitro tests to study if a drug inhibits or promotes sickling, hydrates or dehydrates SS cells, prolongs or shortens the delay time prior to deoxy-Hb S polymerization, induces Hb F, increases or decreases the solubility of deoxy-Hb S; Hb oxygen affinity; membrane ion transport, adhesion of SS cells to endothelial cells; met-Hb S formation; hemolysis and the amount of intracellular denatured Hb S. In addition, drugs that showed a beneficial effect without significant adverse effects in these tests were further studied using transgenic mice that produce human sickle hemoglobin. To perform many in vitro tests efficiently, the SCD Ref Lab established a new high-throughput drug evaluation protocol by which many tests are conducted simultaneously using a small volume of SS blood samples or Hb S. To date, we have found 8 new antisickling agents. They are NS3623 (Blood97:1451–7, 2001); MX-1520 (Brit. J. Haematol.125:788–795,2004); NIPRISAN [Brit. J. Haematol.118: 337–343, 2002 and 122: 1001–8 (2003)]; FLOCOR (manuscript in preparation); and 5HMF which forms a Schiff base adduct with Hb S. In vivo studies using transgenic sickle mice showed that pretreatment with each of these drugs prevented the formation of sickled cells in the blood and prolonged the survival time under severe hypoxic conditions. We also found that Amidox, Didox and Trimidox increased the synthesis of Hb F in K562 cells (Am J. Hematol.63: 176–183, 2000).

scholarly journals Effects of Targeting the Interleukin-1 Receptor in a Murine Model of Sickle Cell Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2259-2259
Author(s):  
Jessica Venugopal ◽  
Jintao Wang ◽  
Chiao Guo ◽  
Daniel Eitzman
Keyword(s):  
Sickle Cell Disease ◽  
Stroke Volume ◽  
Sickle Cell ◽  
Whole Blood ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
Cell Disease ◽  
Post Stroke ◽  
Formalin Fixed

Background: Sickle cell disease (SCD) is associated with chronic hemolytic anemia and a heightened inflammatory state. Multiple inflammatory pathways have been shown to be activated in SCD, however the causal role of these pathways in mediating organ damage and contributing to stroke is unclear. Methods: Bone marrow transplantation (bmt) from SCD (Townes) to wild-type (WT) recipient mice was used to generate SCD mice (WTSCD). To generate mice with non-hematopoietic deficiency of the interleukin-1 receptor (IL1R), SCD marrow was transplanted into IL1R deficient recipients (IL1R-/- SCD). Other WTSCD mice were given the IL1R inhibitor, anakinra, or PBS for 2 or 4 weeks before sacrifice. Iron and collagen staining were performed in multiple formalin-fixed tissues with Prussian blue and Masson's Trichrome, respectively. Complete blood counts and reticulocyte percentages were analyzed 15 weeks following BMT. Plasma levels of soluble E-selectin (sE-sel) and soluble P-selectin (sP-sel) were measured 14 weeks following bmt with ELISA. Ischemic stroke was induced by middle cerebral artery (MCA) photothrombosis at 16 weeks of age. WTSCD mice were given anakinra following stroke induction. Seventy-two hours after MCA occlusion, stroke volume was assessed by staining brain sections with 2, 3, 5-triphenyltetrazolium chloride. Formalin-fixed brain sections were also stained for macrophages. Results: All SCD mice were anemic and the severity of anemia was not different between WTSCD and IL1R-/- SCD mice. Increased circulating erythrocytes (9.01 ± 0.78 vs 6.77 ± 0.18 M/uL; p<0.05) and decreased reticulocytes (25.31 ± 1.79 vs 32.45 ± 2.74%; p<0.05) were observed in WTSCD mice treated with anakinra for 4 weeks. Two week anakinra treatment was sufficient to decrease organ iron deposition and lung collagen deposition. In vivo treatment with anakinra also decreased erythrocyte sickling in a whole blood ex vivo sickling assay (15.14 ± 0.94 vs 24.18 ± 1.95 % sickled; p<0.01). Further, in vitro treatment of WTSCD whole blood with IL-1β increased sickling (26.70 ± 0.39 vs 22.34 ± 0.95; p<0.05), and this was ameliorated with in vitro pretreatment with anakinra. Alterations to ex vivo sickling were not observed when conditions were repeated on erythrocytes alone. Stroke volume was significantly reduced in IL1R-/- SCD mice compared to WTSCD mice 3 days following MCA occlusion (8.08 ± 0.91 vs 11.10 ± 1.16 % hemisphere; p<0.05). Post-stroke treatment of WTSCD mice with anakinra also decreased stroke size compared to vehicle-treated mice (14.45 ± 1.44 vs 9.7 ± 3.23 % hemisphere; p=0.05). Non-hematopoietic deficiency of IL1R decreased sE-sel (vs 50.10 ± 2.31 vs 32.12 ± 2.08 pg/mL; p<0.05) and sP-sel (155.98 ± 24.44 vs 327.23 ± 18.31 ng/mL; p<0.05) levels in post-stroke animals and decreased the number of macrophages observed in the peri-stroke area (0.26 ± 0.05 vs 0.722 ± 0.15 % hemisphere; p<0.05). Conclusions: Deficiency of non-hematopoietic IL1R or treatment with an IL1R antagonist was appears beneficial towards several phenotypes in a mouse model of SCD. Given the safety of IL1R inhibitors in various patient populations, this study reveals a potential therapeutic intervention for treatment of sickle cell patients. Disclosures No relevant conflicts of interest to declare.

New Approach for Finding Novel Antisickling Agents at the NIH NHLBI Sickle Cell Disease Reference Laboratory.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1219-1219
Author(s):  
Toshio Asakura ◽  
Jisheng Yang ◽  
Qiukan Chen ◽  
Greg Evans ◽  
Osheiza Abdulmalik
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Aromatic Aldehydes ◽  
The Body ◽  
Therapeutic Modality ◽  
Reference Laboratory ◽  
Side Chains ◽  
Cell Disease ◽  
Hb S

Abstract Although numerous antisickling agents that chemically bind with sickle hemoglobin (Hb S) and inhibit sickling of sickle erythrocytes (SS cells) in vitro were reported and some of them were brought to clinical trials, none were satisfactory for clinical use. The major problem with this type of drugs is the difficulty of delivering a sufficient amount of antisickling drug to intracellular Hb S because:chemicals that bind with the side chains of hemoglobin also bind with the side chains of other proteins in the gastrointestinal tract, blood and red blood cells (RBCs) prior to binding with intracellular Hb S; andthe concentration of Hb S in SS cells is very high (5 mM of tetrameric Hb S, 20 mM of Hb subunits, or a total amount of approximately 500 grams of Hb S in an adult patient), and the amount of chemical that reaches SS cells upon oral administration cannot modify a significant portion of Hb S to inhibit sickling of SS cells. For these reasons, drugs that chemically bind with Hb S including those that form Schiff base adducts with Hb S seem to have no clinical value. We found that certain aromatic aldehydes that bind with Hb S allosterically do not have these problems. Since these compounds combine with Hb S physically like a key-and-lock relationship, a low concentration (such as less than 2 mM) of such compound in the blood can bind with a large portion of intracellular Hb S, shift the oxygen equilibrium curve toward the left and inhibit sickling of SS cells (Brit. J. Hematol.128:552–561, 2005). This type of allosteric binding between aromatic aldehydes and Hb is analogous to the binding between 2,3-diphosphoglycerate and Hb. Quite interestingly, our investigations indicate that NIPRISAN (NICOSAN), an herbal medicine that shows a strong antisickling effect both in vitro and in vivo (in transgenic sickle mice) (Brit. J. Haematol.118:337–343, 2002; Brit. J. Haematol.122:1001–1008 2003), contains various aromatic aldehydes (unpublished data). Allosteric drugs designed to interact with specific proteins in the body may be useful as a new type of therapeutic modality for the treatment of sickle cell disease and other diseases. [Researchers with candidate drugs that may have beneficial effects for SCD are welcome to contact the SCD Reference Laboratory for free in vitro evaluation of their agents (for more information, please see our website at www.tatcom.com/sickle-cell).

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