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It is well documented that caffeic acid (3,4-dihydroxycinnamic acid) (CA) interacts with and inhibits the oxidative reactions of myoglobin (Mb) and hemoglobin (Hb), and this interaction underlies its antioxidative action in meat. Sickle cell hemoglobin (HbS) is known for its tendency to oxidize more readily than normal HbA in the presence of hydrogen peroxide (H 2 O 2 ), which leads to a more persistent and highly oxidizing ferryl Hb (HbFe 4+ ). We have investigated the effects of CA on HbS oxidation intermediates, specifically on the ferric/ferryl forms. At a low concentration of H 2 O 2 (0.5-fold over heme), we observed a fivefold reduction in the amount of HbFe 4+ accumulated in a mixture of ferric and H 2 O 2 solution. Higher levels of H 2 O 2 (onefold and twofold over heme) led to a lesser threefold and twofold reduction in the content of HbFe 4+ , respectively, possibly due to the saturation of the binding sites on the Hb molecule. The most intriguing finding was that when 5-molar excess CA over heme was used, and a considerable increase in the delay time of HbS polymerization to approximately 200 s was observed. This delay in polymerization of HbS is theoretically sufficient to avoid microcapillary blockage and prevent vasoconstrictions in vivo. Mass spectrometry analysis indicated that CA was more extensively covalently bonded to βCys 93 than to βCys 112 and αCys 104 . The dual antioxidant and antisickling properties of CA may be explored further to maximize its therapeutic potential in SCD., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Therapeutic agents that increase the Hb affinity for oxygen (O 2 ) could, in theory, lead to decreased O 2 release from Hb and impose a hypoxic risk to tissues. In this study, GBT1118, an allosteric modifier of Hb affinity for O 2 , was used to assess the impact of increasing Hb affinity for O 2 on brain tissue oxygenation, blood pressure, heart rate, O 2 delivery, and tolerance to hypoxia in Townes transgenic sickle cell disease (SCD) mice. Brain oxygenation and O 2 delivery were studied during normoxia and severe hypoxic challenges. Chronic treatment with GBT1118 increased Hb affinity for O 2 , reducing the Po 2 for 50% HbO 2 saturation (P50) in SCD mice from 31 mmHg to 18 mmHg. This treatment significantly reduced anemia, increasing hematocrit by 33%, improved cardiac output (CO), and O 2 delivery and extraction. Chronically increasing Hb affinity for O 2 with GBT1118 preserved cortical O 2 tension during normoxia, improved cortical O 2 tension during hypoxia, and increased tolerance to severe hypoxia in SCD mice. Independent of hematological changes induced by chronic treatment, a single dose of GBT1118 significantly improved tolerance to hypoxia, highlighting the benefits of increasing Hb affinity for O 2 and consequently reducing sickling of RBCs in blood during hypoxia in SCD. NEW & NOTEWORTHY Chronic pharmacologically increased hemoglobin affinity for oxygen in sickle cell disease mice alleviated hematological consequences of sickle cell disease, increasing RBC half-life, hematocrit, and hemoglobin concentration, while also decreasing reticulocyte count. Additionally, chronically increased hemoglobin affinity for oxygen significantly improved survival as well as cortical tissue oxygenation in sickle cell disease mice during hypoxia, suggesting that oxygen delivery and utilization is improved by increased hemoglobin affinity for oxygen.

Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (β6 Glu → Val) on the β-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 ( 23 ). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23 , a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.

Aromatic aldehydes that bind to sickle hemoglobin (HbS) to increase the protein oxygen affinity and/or directly inhibit HbS polymer formation to prevent the pathological hypoxia-induced HbS polymerization and the subsequent erythrocyte sickling have for several years been studied for the treatment of sickle cell disease (SCD). With the exception of Voxelotor, which was recently approved by the U.S. Food and Drug Administration (FDA) to treat the disease, several other promising antisickling aromatic aldehydes have not fared well in the clinic because of metabolic instability of the aldehyde moiety, which is critical for the pharmacologic activity of these compounds. Over the years, our group has rationally developed analogs of aromatic aldehydes that incorporate a stable Michael addition reactive center that we hypothesized would form covalent interactions with Hb to increase the protein affinity for oxygen and prevent erythrocyte sickling. Although, these compounds have proven to be metabolically stable, unfortunately they showed weak to no antisickling activity. In this study, through additional targeted modifications of our lead Michael addition compounds, we have discovered other novel antisickling agents. These compounds, designated MMA, bind to the α-globin and/or β-globin to increase Hb affinity for oxygen and concomitantly inhibit erythrocyte sickling with significantly enhanced and sustained pharmacologic activities in vitro.

Voxelotor, a sickle hemoglobin polymerization inhibitor, was approved by the U.S. Food and Drug Administration to treat sickle cell disease (SCD) in November 2019. This article reviews published data about voxelotor treatment of SCD based on a search of MEDLINE, Embase, and International Pharmaceutical Abstracts. In a phase I/II trial, voxelotor demonstrated a dose-dependent pharmacokinetic and pharmacodynamic response and was well tolerated in healthy volunteers and patients with SCD. In a multi-center, randomized, double-blind, phase III trial (HOPE trial), a significantly higher percentage of patients randomized to voxelotor had increased hemoglobin (> 1 g/dl from baseline) compared to placebo. A greater reduction of hemolytic markers was also observed in the voxelotor-treated group, whereas the incidence of adverse effects was comparable. Three case series or reports also demonstrated the efficacy and safety of voxelotor use in a limited number of SCD patients in the real-world situation, although one patient with SCD, severe anemia, and a history of autoantibody-mediated hemolysis failed to respond to voxelotor. An ongoing trial (HOPE-KIDS) is designed to establish the use of voxelotor in younger pediatric patients with SCD. There is a theoretical concern that voxelotor may impair oxygen delivery, due to modification of the oxygen affinity of hemoglobin, which needs to be further evaluated. As a first-in-class hemoglobin modulator, voxelotor offers a new treatment option targeting the root cause of SCD., (© 2020 Pharmacotherapy Publications, Inc.)

Sickle cell disease afflicts millions of people worldwide and approximately 100,000 Americans. Complications are myriad and arise as a result of complex pathological pathways 'downstream' to a point mutation in DNA, and include red blood cell membrane damage, inflammation, chronic hemolytic anemia with episodic vaso-occlusion, ischemia and pain, and ultimately risk of cumulative organ damage with reduced lifespan of affected individuals. The National Heart, Lung, and Blood Institute's 2014 evidence-based guideline for sickle cell disease management states that additional research is needed before investigational curative therapies will be widely available to most patients with sickle cell disease. To date, sickle cell disease has been cured by hematopoietic stem cell transplantation in approximately 1,000 people, most of whom were children, and significantly ameliorated by gene therapy in a handful of subjects who have only limited follow-up thus far. During a timespan in which over 20 agents were approved for the treatment of cystic fibrosis by the Food and Drug Administration, similar approval was granted for only two drugs for sickle cell disease (hydroxyurea and L-glutamine) despite the higher prevalence of sickle cell disease. This trajectory appears to be changing, as the lack of multimodal agent therapy in sickle cell disease has spurred engagement among many in academia and industry who, in the last decade, have developed new drugs poised to prevent complications and alleviate suffering. Identified therapeutic strategies include fetal hemoglobin induction, inhibition of intracellular HbS polymerization, inhibition of oxidant stress and inflammation, and perturbation of the activation of the endothelium and other blood components (e.g. platelets, white blood cells, coagulation proteins) involved in the pathophysiology of sickle cell disease. In this article, we present a crash-course review of disease-modifying approaches (minus hematopoietic stem cell transplant and gene therapy) for patients with sickle cell disease currently, or recently, tested in clinical trials in the era following approval of hydroxyurea., (Copyright© 2019 Ferrata Storti Foundation.)

The complex, frequently devastating, multi-organ pathophysiology of sickle cell disease has a single root cause: polymerization of deoxygenated sickle hemoglobin. A logical approach to disease modification is, therefore, to interdict this root cause. Ideally, such interdiction would utilize small molecules that are practical and accessible for worldwide application. Two types of such small molecule strategies are actively being evaluated in the clinic. The first strategy intends to shift red blood cell precursor hemoglobin manufacturing away from sickle hemoglobin and towards fetal hemoglobin, which inhibits sickle hemoglobin polymerization by a number of mechanisms. The second strategy intends to chemically modify sickle hemoglobin directly in order to inhibit its polymerization. Important lessons have been learnt from the pre-clinical and clinical evaluations to date. Open questions remain, but this review summarizes the valuable experience and knowledge already gained, which can guide ongoing and future efforts for molecular mechanism-based, practical and accessible disease modification of sickle cell disease., (Copyright© 2019 Ferrata Storti Foundation.)

Background: Deoxygenated sickle hemoglobin (HbS) polymerization drives the pathophysiology of sickle cell disease. Therefore, direct inhibition of HbS polymerization has potential to favorably modify disease outcomes. Voxelotor is an HbS polymerization inhibitor., Methods: In a multicenter, phase 3, double-blind, randomized, placebo-controlled trial, we compared the efficacy and safety of two dose levels of voxelotor (1500 mg and 900 mg, administered orally once daily) with placebo in persons with sickle cell disease. The primary end point was the percentage of participants who had a hemoglobin response, which was defined as an increase of more than 1.0 g per deciliter from baseline at week 24 in the intention-to-treat analysis., Results: A total of 274 participants were randomly assigned in a 1:1:1 ratio to receive a once-daily oral dose of 1500 mg of voxelotor, 900 mg of voxelotor, or placebo. Most participants had sickle cell anemia (homozygous hemoglobin S or hemoglobin Sβ 0 -thalassemia), and approximately two thirds were receiving hydroxyurea at baseline. In the intention-to-treat analysis, a significantly higher percentage of participants had a hemoglobin response in the 1500-mg voxelotor group (51%; 95% confidence interval [CI], 41 to 61) than in the placebo group (7%; 95% CI, 1 to 12). Anemia worsened between baseline and week 24 in fewer participants in each voxelotor dose group than in those receiving placebo. At week 24, the 1500-mg voxelotor group had significantly greater reductions from baseline in the indirect bilirubin level and percentage of reticulocytes than the placebo group. The percentage of participants with an adverse event that occurred or worsened during the treatment period was similar across the trial groups. Adverse events of at least grade 3 occurred in 26% of the participants in the 1500-mg voxelotor group, 23% in the 900-mg voxelotor group, and 26% in the placebo group. Most adverse events were not related to the trial drug or placebo, as determined by the investigators., Conclusions: In this phase 3 randomized, placebo-controlled trial involving participants with sickle cell disease, voxelotor significantly increased hemoglobin levels and reduced markers of hemolysis. These findings are consistent with inhibition of HbS polymerization and indicate a disease-modifying potential. (Funded by Global Blood Therapeutics; HOPE ClinicalTrials.gov number, NCT03036813.)., (Copyright © 2019 Massachusetts Medical Society.)

The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) formation is poorly defined in sickle cell disease (SCD). Here we report that sickle Hb (HbS) oxidation, coupled with changes in cytosolic antioxidative proteins, is associated with membrane alterations and MP formation in homozygous Townes-sickle cell (Townes-SS) mice. Photometric and proteomic analyses confirmed the presence of high levels of Hb oxidation intermediates (ferric/ferryl) and consequent β-globin posttranslational modifications, including the irreversible oxidation of βCys93 and the ubiquitination of βLys96 and βLys145. This is the first report to our knowledge to link the UPS (via ubiquitinated Hb and other proteins) to oxidative stress. Ferryl Hb also induced complex formation with band 3 and RBC membrane proteins. Incubation of Townes-SS MPs with human endothelial cells caused greater loss of monolayer integrity, apoptotic activation, heme oxygenase-1 induction, and concomitant bioenergetic imbalance compared with control Townes-AA MPs. MPs obtained from Townes-SS mice treated with hydroxyurea produced fewer posttranslational Hb modifications. In vitro, hydroxyurea reduced the levels of ferryl Hb and shielded its target residue, βCys93, by a process of S-nitrosylation. These mechanistic analyses suggest potential antioxidative therapeutic modalities that may interrupt MP heme-mediated pathophysiology in SCD patients.

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 activities in 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.

CONTEXT AND OBJECTIVES Sickle cell disease (SCD) is the most common genetic disorder among people of African descent, affecting approximately 3,500 newborns each year in Brazil. Hydroxyurea (HU) is the only effective drug to treating patients with SCD, thereby reducing morbidity and mortality. The objective was to analyze the effects of HU on SCD patients at our institution. DESIGN AND SETTING Retrospective study conducted at a sickle cell centre in Ribeirão Preto, São Paulo, Brazil. METHODS We analyzed clinical and laboratory data on 37 patients. The hematological parameters and clinical events that occurred during the year before and the first year of treatment with HU were analyzed. The mean dose of HU was 24.5 ± 5.5 mg/kg/day. RESULTS There were rises in three parameters: hemoglobin (8.3 g/dl to 9.0 g/dl, P = 0.0003), fetal hemoglobin (HbF) (2.6% to 19.8%, P < 0.0001) and mean cell volume MCV (89 to 105 fl, P = 0.001); and reductions in the numbers of leukocytes (10,050/µl to 5,700/µl, P < 0.0001), neutrophils (6,200/µl to 3,400/µl, P = 0.001), platelets (459,000/µl to 373,000/µl, P = 0.0002), painful crises (1.86 to 0.81, P = 0.0014), acute chest syndromes (0.35 to 0.08, P = 0.0045), infections (1.03 to 0.5, P = 0.047), hospitalizations (1.63 to 0.53, P = 0.0013) and transfusions (1.23 to 0.1, P = 0.0051). CONCLUSION The patients presented clinical and hematological improvements, with an increase in HbF and a reduction in the infection rate, which had not been addressed in most previous studies.

We have developed a microfluidic approach to study the sickling of red blood cells associated with sickle cell anemia by rapidly varying the oxygen partial pressure within flowing microdroplets. By using the perfluorinated carrier oil as a sink or source of oxygen, the oxygen level within the water droplets quickly equilibrates through exchange with the surrounding oil. This provides control over the oxygen partial pressure within an aqueous drop ranging from 1 kPa to ambient partial pressure, i.e. 21 kPa. The dynamics of the oxygen exchange is characterized through fluorescence lifetime measurements of a ruthenium compound dissolved in the aqueous phase. The gas exchange is shown to occur primarily during and directly after droplet formation, in 0.1 to 0.5 s depending on the droplet diameter and speed. The controlled deoxygenation is used to trigger the polymerization of hemoglobin within sickle red blood cells, encapsulated in drops. This process is observed using polarization microscopy, which yields a robust criterion to detect polymerization based on transmitted light intensity through crossed polarizers.

Generation of transgenic mice have identified new pathophysiological mechanisms in sickle disease, including a permanent proinflammatory state and dysregulation of vascular tone. Treatment is no longer solely symptomatic. New agents target red cell hydration and the kinetics of deoxyhemoglobin S polymerization. Hydroxyurea, which reactivates fetal hemoglobin synthesis, is now widely used. Anti-adhesion molecules and agents modulating vascular tone are being tried in sickle mice. Bone marrow transplantation is widely used to cure patients with HLA-identical siblings, and gene therapy looks promising for those without a donor.

The understanding of the oxidative stress mechanisms helps to explain many of the processes of cellular lesion and death, especially those related to the hemolytic diseases. Sickle cell anemia, thalassemias and G6-PD deficiency are among the more frequent genetic anomalies accompanied by oxidative stress. In the sickle cells, one of the factors that predisposes to the hemolytic process is the oxidative degradation of the hemoglobin S due to its deoxigenation leading to hemichrome formation and precipitation as Heinz bodies. The oxidative stress contributes to the sickle process and shortening of the erythrocyte survival. Here we analyzed the oxidative process in erythrocytes of patients with two different genotypes for HbS (AS and SS). Units of blood from donors of the Center of Hematology and Hemotherapy of Paraná (HEMEPAR), from normal individuals (AA) and from heterozygote individuals (AS), and venous blood collected from patients with sickle cell anemia (SS) were analyzed. In order to evaluate the protective action of the vitamins C and E in oxidative stress, erythrocytes were treated with antioxidant substances, vitamin C and vitamin E, and then treated with the oxidant tert-butilhydroperoxide (TBHP). The oxidative action induced by TBHP was observed in erythrocytes AA

The vascular pathophysiology of sickle cell disease (SCD) is influenced by many factors, including adhesiveness of red and white blood cells to endothelium, increased coagulation, and homeostatic perturbation. The vascular endothelium is central to disease pathogenesis because it displays adhesion molecules for blood cells, balances procoagulant and anticoagulant properties of the vessel wall, and regulates vascular homeostasis by synthesizing vasoconstricting and vasodilating substances. The occurrence of intermittent vascular occlusion in SCD leads to reperfusion injury associated with granulocyte accumulation and enhanced production of reactive oxygen species. The participation of nitric oxide (NO) in oxidative reactions causes a reduction in NO bioavailability and contributes to vascular dysfunction in SCD. Therapeutic strategies designed to counteract endothelial, inflammatory, and oxidative abnormalities may reduce the frequency of hospitalization and blood transfusion, the incidence of pain, and the occurrence of acute chest syndrome and pulmonary hypertension in patients with SCD.

In an attempt to find new types of anti-sickling agents that specifically bind to intracellular sickle haemoglobin (HbS) without inhibition by plasma and tissue proteins or other undesirable consequences, we identified 5-hydroxymethyl-2-furfural (5HMF), a naturally occurring aromatic aldehyde, as an agent that fulfils this criterion. Preliminary studies in vitro showed that 5HMF forms a high-affinity Schiff-base adduct with HbS and inhibits red cell sickling by allosterically shifting oxygen equilibrium curves towards the left. Further studies with transgenic (Tg) sickle mice showed that orally administered 5HMF was rapidly absorbed into the bloodstream from the gastrointestinal tract without being destroyed, traversed the red blood cell membrane and specifically bound with, and modified, HbS molecules at levels as high as 90%. Pretreatment of Tg sickle mice with 5HMF inhibited the formation of sickle cells and significantly prolonged survival time under severe hypoxia, compared with untreated mice, which died within 15 min because of sickling-dependent pulmonary sequestration. These results indicate the feasibility of 5HMF as an attractive potential candidate for therapy of sickle cell disease.

As a result of an in vitro screening effort the antiviral agent acyclovir was found to inhibit aggregation of hemoglobin S and the sickling of erythrocytes from individuals with sickle cell disease. Sickling of the erythrocytes was significantly inhibited at 200 microg/ml under essentially anaerobic conditions, considerably more hypoxic than the conditions in which sickling occurs in sickle cell patients. The structurally related guanine-based antiviral agents ganciclovir, valacyclovir, and penciclovir were also tested. Valacyclovir and ganciclovir showed comparable anti-sickling activity at concentrations similar to that of acyclovir. An examination of the shared structural characteristics of the four guanine derivatives linked anti-sickling activity to the presence of an oxygen atom alpha to the N9 of the guanine moiety. These findings suggest a new approach in the search for new agents for the treatment of patients with sickle cell disease.

Previous studies have determined the laboratory alterations, clinical efficacy and toxicity profile associated with hydroxyurea (HU) therapy in patients with severe sickle cell anemia. We report the efficacy of HU treatment in the prevention of vaso-occlusive crises in an 11-years-old boy with severe sickle cell disease. The number of vaso-occlusive crises, hospital days and blood transfusions in the year before HU treatment were compared with the same parameters at 6, 12, 24, 36 and 72 months of treatment. A decrease in the frequency of vaso-occlusive crises, blood transfusions and days spent in hospital were demonstrated during the HU treatment period compared to the same period before hand. The clinical and laboratory response to HU was dramatic in this severely affected patient, allowing him a normal schooling and social life. The adverse effects observed were not serious and reversed after transient discontinuation of HU. We conclude that long-term chronic treatment with HU for seriously ill sickle cell patients appears feasible, and devoid of any major toxicity.

The dehydration of sickle red blood cells (RBCs) through the Ca-activated K channel depends on the parallel movement of Cl ions. To study whether Cl-conductance block might prevent dehydration of sickle RBCs, a novel Cl-conductance inhibitor (NS3623) was characterized in vitro using RBCs from healthy donors and sickle cell patients and in vivo using normal mice and a transgenic mouse model of sickle cell disease (SAD mice). In vitro, NS3623 reversibly blocked human RBC Cl-conductance (g(Cl)) with an IC(50) value of 210 nmol/L and a maximal block of 95%. In vivo, NS3623 inhibited RBC g(Cl) after oral administration to normal mice (ED(50) = 25 mg/kg). Although g(Cl), at a single dose of 100 mg/kg, was still 70% inhibited 5 hours after dosing, the inhibition disappeared after 24 hours. Repeated administration of 100 mg/kg twice a day for 10 days caused no adverse effects; therefore, this regimen was chosen as the highest dosing for the SAD mice. SAD mice were treated for 3 weeks with 2 daily administrations of 10, 35, and 100 mg/kg NS3623, respectively. The hematocrit increased, and the mean corpuscular hemoglobin concentration decreased in all groups with a concomitant increase in the intracellular cation content. A loss of the densest red cell population was observed in conjunction with a shift from a high proportion of sickled to well-hydrated discoid erythrocytes, with some echinocytes present at the highest dosage. These data indicate feasibility for the potential use of Cl-conductance blockers to treat human sickle cell disease.

Sickle transgenic mice expressing exclusively human globins are desirable for studying pathophysiology and testing gene therapy strategies, but they must have significant pathology and show evidence of amelioration by antisickling hemoglobins. Mice were generated that expressed exclusively human sickle hemoglobin with 3 levels of HbF using their previously described sickle constructs (cointegrated human miniLCRalpha2 and miniLCRbeta(S) [PNAS 89:12150, 1992]), mouse alpha- and beta-globin-knockouts, and 3 different human gamma-transgenes. It was found that, at all 3 levels of HbF expression, these mice have balanced chain synthesis, nearly normal mean corpuscular hemoglobin, and, in some cases, F cells. Mice with the least adult HbF expression were the most severe. Progressive increase in HbF from less than 3% to 20% to 40% correlated with progressive increase in hematocrit (22% to 34% to 40%) and progressive decrease in reticulocyte count (from 60% to 30% to 13%). Urine concentrating ability was normalized at high HbF, and tissue damage detected by histopathology and organ weight were ameliorated by increased HbF. The gamma-transgene that produces intermediate levels of HbF was introduced into knockout sickle mice described by Pàszty and coworkers that express the miniLCRalpha1(G)gamma(A)gammadeltabeta(S) transgene and have fetal but not adult expression of HbF. It was found that the level of HbF required to ameliorate low hematocrit and normalize urine concentrating defect was different for the miniLCRalpha2beta(S) and miniLCRalpha1(G)gamma(A)gammadeltabeta(S) mice. We conclude that knockout mice with the miniLCRalpha2beta(S) transgene and postnatal expression of HbF have sufficiently faithful sickle pathology to serve as a platform for testing antisickling interventions.

Effects of 3,5-dimethoxy-4-hydroxybenzoic acid and 2,3,4-trihydroxyacetophenone were studied on haemoglobin S (Hb S) polymerisation, analgesia and inflammation using Hb S solution, rats and mice. UV spectrophotometric procedure was used to monitor the polymerization of the Hb S. Acetic acid induced writhing in mice and egg albumin induced rat paw edema procedures were used to evaluate analgesic and anti-inflammatory activities of the compounds respectively. The results indicate that both drugs inhibit the process of polymerization significantly, possibly by direct action on the Hb S molecules. The drugs inhibited acetic acid induced pain and decreased egg albumin induced oedema. It is concluded that 3,5-dimethoxy-4-hydroxybenzoic acid and 2,3,4-trihydroxyacetophenone may have some value in the management of sickle cell disease.

Objectives: To report a case of acute chest syndrome (ACS) of sickle cell disease treated successfully with nitric oxide and to review the physiologic effects of nitric oxide and its potential ability to improve outcome in ACS., Design: Descriptive case report., Setting: Eighteen-bed pediatric intensive care unit in a university children's hospital., Patient: A 15-yr-old black male with sickle cell disease, bilateral pulmonary infiltrates, refractory hypoxemia, and unstable hemodynamics., Intervention: In addition to exchange transfusion, invasive hemodynamic monitoring, and aggressive ventilatory support, inhaled nitric oxide was administered in the gas mixture in a concentration of 20 ppm for 72 hrs., Measurements and Main Results: Cardiac output, pulmonary arterial pressure, pulmonary artery occlusion pressure, systemic vascular resistance, pulmonary vascular resistance, shunt fraction, and alveolar-arterial oxygen gradient were compared with and without inhaled nitric oxide. Marked reductions in pulmonary arterial pressure and pulmonary vascular resistance were noted. Cardiac output improved, and shunt fraction and alveolar-arterial oxygen gradient were markedly reduced. The patient required decreased ventilator and hemodynamic support and rapidly made a complete recovery., Conclusions: Nitric oxide may be beneficial for patients with ACS because of its ability to ameliorate pulmonary hypertension and ventilation/perfusion mismatch. Nitric oxide may confer some protection against polymerization of sickle hemoglobin and exert a reversible antiplatelet effect that may be beneficial in ACS. Further study is necessary to determine the safety and efficacy of inhaled nitric oxide as a treatment for ACS.

Hydroxyurea has been mixed with hemoglobin S and the reaction was studied using electronic absorption spectroscopy as a function of time and wavelength. The rate of conversion of oxyhemoglobin S to other species was determined and the nature of the reaction products was studied. We also report the formation of methemoglobin (and other reaction products) when deoxyhemoglobin S is combined with hydroxyurea. The probable increase in the formation of methemoglobin, and other potential reaction products such as nitric oxide-hemoglobin, in patients with sickle cell anemia who are taking hydroxyurea as a therapeutic drug is discussed in terms of the pathophysiology of the disease. It is proposed that methemoglobin and possibly nitric oxide-hemoglobin formation may partially explain beneficial effects observed in these patients before their levels of fetal hemoglobin have increased.

Use of hydroxyurea (HU) to treat sickle cell disease is usually associated with increments in fetal hemoglobin (Hb F) production; however, in vitro studies show that HU may also induce hemoglobin denaturation. Whole blood samples from Hb AA, Hb AS, and Hb SS patients were treated in vitro with 100, 150, 200, 250, and 300 micrograms/mL HU, incubated at 30 degrees C for up to 12 days, and analyzed by high-performance liquid chromatography (HPLC). Hb AA levels show decrements of 91 to 14% with 100 micrograms/mL and 89 to 4% with 150 micrograms/mL after 12 days; 86 to 2% with 200 micrograms/mL after 10 days; 86 to 8% with 250 and 300 micrograms/mL after 8 days. Similar treatment and incubation times for Hb AS whole blood demonstrate that HU equally degrades the A and S components of Hb AS. A comparable approach for Hb SS whole blood samples, using a 300 micrograms/mL HU treatment, showed a hemoglobin denaturing pattern that went from 93% to 1% after 12 days. Globin chain analysis of these samples by reverse-phase HPLC showed that the denaturing effects occur mostly on the beta-globin chain.

Dimer-tetramer assembly reactions of the 10 CN-met ligation microstates of hemoglobin (Hb) were analyzed as a function of NaCl concentration while maintaining constant water activity by the addition of compensating sucrose. The assembly free energy for fully ligated cyanomet Hb and for fully oxygenated Hb becomes less favorable by 1.8 kcal when [NaCl] is increased from 0.08 to 0.7 M, whereas that of unligated Hb is practically insensitive to changes in [NaCl]. Values of 1.6 and 0.3 mol chloride release were found for the assembly of fully ligated and deoxy Hb, respectively; i.e., a net release of 1.3 mol chloride is coupled to the ligation of tetramers for both oxygen and cyanomet ligation. The ligation-linked salt component at constant water activity was evaluated to be 1.0 mol for the full oxygenation of the Hb tetramer in agreement with the overall value previously reported. When the detailed salt linkages accompanying all 16 stepwise cyanomet ligation reactions were experimentally resolved, only two "chloride" effects were found. The first chloride effect correlates with the ligation steps, which create tertiary constraint, and the second effect is coupled to the six switchpoints of quaternary T-->R transition. The distribution of these chloride effects agrees closely with predictions of the "symmetry rule mechanism." The total chloride release for CN-met ligation is in good agreement with that for oxygenation. Free energy contributions to assembly and cooperativity arising from the osmotic effects of chloride were found to be small for all ligation species.

A study has been carried out into the effects of clinically important antisickling and anaesthetic substances and ionophoric antibiotics on the activities of (Na+, K+)- and (Ca+2, Mg2+)-ATPases of the human erythrocyte membrane. In general, these drugs, with the exception of nystatin, inhibit both types of enzymic activities but with varying degrees of efficacy. (Ca2+, Mg2+)-ATPases was more sensitive to the lipophilic anaesthetics and (Na+,K+)-ATPase to the ionophoric antibiotic, amphotericin B. These results are explained in the light of the partition coefficients of these drugs in erythrocyte membranes, their effects on the fluidity of the erythrocytes membranes, the changes they induce in the permeability properties of erythrocytes and the subsequent effect of procaine on sickling of erythrocytes, and their potential interaction with specific membrane components.

We have synthesized and tested a new series of compounds that inhibit the polymerization of deoxyhemoglobin S by noncovalent interaction. They consist of three structural elements: A p-aminobenzoyl residue to anchor the compound in the central cavity between the beta chains, a number of glutamates in gamma linkage to provide tight binding, and one or two hydrophobic amino acid residues which block the intermolecular hydrophobic interaction of valine beta 6. The most active compound was p-aminobenzoyl-(gamma-Glu)5-Phe-Phe. It increases the solubility of deoxy-HbS by a factor of 1.3 at a concentration of only 5-6 mM and is effective even in the presence of physiological concentrations of 2,3-diphosphoglycerate.

Methyl acetyl phosphate binds to the 2,3-diphosphoglycerate (2,3-DPG) binding site of hemoglobin and selectively acetylates three amino groups at or near that site. The subsequent binding of 2,3-DPG is thus impeded. When intact sickle cells are exposed to methyl acetyl phosphate, their abnormally high density under anaerobic conditions is reduced to the density range of oxygenated, nonsickling erythrocytes. This change is probably due to a combination of direct and indirect effects induced by the specific acetylation. The direct effect is on the solubility of deoxyhemoglobin S, which is increased from 17 g/dL for unmodified hemoglobin S to 22 g/dL for acetylated hemoglobin S at pH 6.8. Acetylated hemoglobin S does not gel at pH 7.4, up to a concentration of 32 g/dL. The indirect effect could be due to the decreased binding of 2,3-DPG to deoxyhemoglobin S within the sickle erythrocyte, thus hindering the conversion of oxyhemoglobin S to the gelling form, deoxyhemoglobin S.