Treatment of sickle cell disease by increasing oxygen affinity of hemoglobin.

The issue of treating sickle cell disease with drugs that increase hemoglobin oxygen affinity has come to the fore with the US Food and Drug Administration approval in 2019 of voxelotor, the only antisickling drug approved since hydroxyurea in 1998. Voxelotor reduces sickling by increasing the concentration of the nonpolymerizing, high oxygen affinity R (oxy) conformation of hemoglobin S (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. In this study, we used 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 results of experiments using a new robust assay, which shows the 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. However, reduction of sickling mitigates 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 Hb or decrease mean corpuscular hemoglobin concentration (MCHC) should be more therapeutically effective than drugs that increase oxygen affinity.

MWC allosteric model explains unusual hemoglobin-oxygen binding curves from sickle cell drug binding.

An oxygen-affinity-modifying drug, voxelotor, has very recently been approved by the FDA for treatment of sickle cell disease. The proposed mechanism of action is by preferential binding of the drug to the R quaternary conformation, which cannot copolymerize with the T conformation to form sickle fibers. Here, we report widely different oxygen dissociation and oxygen association curves for normal blood in the presence of voxelotor and interpret the results in terms of the allosteric model of Monod, Wyman, and Changeux with the addition of drug binding. The model does remarkably well in quantitatively explaining a complex data set with just the addition of drug binding and dissociation rates for the R and T conformations. Whereas slow dissociation of the drug from R results in time-independent dissociation curves, the changing association curves result from slow dissociation of the drug from T, as well as extremely slow binding of the drug to T. By calculating true equilibrium curves from the model parameters, we show that there would be a smaller decrease in oxygen delivery from the left shift in the dissociation curve caused by drug binding if drug binding and dissociation for both R and T were rapid. Our application of the Monod, Wyman, and Changeux model demonstrates once more its enormous power in explaining many different kinds of experimental results for hemoglobin. It should also be helpful in analyzing oxygen binding and in vivo delivery in future investigations of oxygen-affinity-modifying drugs for sickle cell disease.

Interactions of an Anti-Sickling Drug with Hemoglobin in Red Blood Cells from a Patient with Sickle Cell Anemia.

The pathophysiology associated with sickle cell disease (SCD) includes hemolytic anemia, vaso-occlusive events, and ultimately end organ damage set off by the polymerization of deoxygenated hemoglobin S (HbS) into long fibers and sickling of red blood cells (RBCs). One approach toward mitigating HbS polymerization is to pharmacologically stabilize the oxygenated (R) conformation of HbS and thereby reduce sickling frequency and SCD pathology. GBT440 is an α-subunit-specific modifying agent that has recently been reported to increase HbS oxygen binding affinity and consequently delay in vitro polymerization. In addition, animal model studies have demonstrated the potential for GBT440 to be a suitable therapeutic for daily oral dosing in humans. Here, we report an optimized method for detecting GBT440 intermediates in human patient hemolysate using a combination of HPLC and mass spectrometry analysis. First, oxygen dissociation curves (ODCs) analyzed from patient blood showed that oxygen affinity increased in a dose dependent manner. Second, HPLC and integrated mass spectrometric analysis collectively confirmed that GBT440 labeling was specific to the α N-terminus thereby ruling out other potential ligand binding sites. Finally, the results from this optimized analytical approach allowed us to detect a stable α-specific GBT440 adduct in the patient's hemolysate in a dose dependent manner. The results and methods presented in this report could therefore potentially help therapeutic monitoring of GBT440 induced oxygen affinity and reveal critical insight into the biophysical properties of GBT440 Hb complexes.

Awakening from the listeriosis crisis: Food safety challenges, practices and governance in the food retail sector in South Africa.

Background: The recent listeriosis outbreak in South Africa brought food safety concerns to the fore in terms of both policy and practice. These concerns encompass both health and nutrition aspects, as well as the economy, because the food system in South Africa contributes significantly to economic growth and food security. However, the food sector is challenged with food safety risks, such as foodborne diseases, food fraud and a general lack of effective enforcement of regulation. The inability of government to effectively regulate the food sector is a contributing factor to increased food safety risks. Focusing on the formal sector, which is subject to regulation, this review provides an overview of the current state of food safety policies and regulations, food safety challenges, and food safety practices in the food system, after the listeriosis crisis of 2017 and 2018. Method: This study used a systematic process to review three sets of data in South Africa: food safety related public policies and regulations, company reports (2013-2018) and media articles (May 2017-May 2018). Food safety policies were selected from a food system policy database created by the research team. The company reports were retrieved from their websites. Factivia was used to search for the media articles. The data were thematically analyzed. The analysis framework was informed by the Food and Agriculture Organization's (FAO) food safety risk analysis. Activities related to food safety risk analysis: risk assessment, risk management, and risk communication were searched for in each material included in the study. Results: Seventy-four documents made up of 13 policies, 47 media articles and 15 company reports were reviewed. Food safety is regulated by three governments departments: Department of Health (DOH), Department of Agriculture, Forestry and Fisheries (DAFF), and Department of Trade and Industry (DTI) through bylaws and regulations. The departments are directly (DAFF) or indirectly (DOH through municipal or metro Environmental Health Professional) involved in food safety enforcement, surveillance, and education. The enforcement of different regulatory processes is often poorly coordinated. Responding to this regulatory environment, food safety activities of the food retail industry include a self-regulatory system reliant on internal and third-party food audits, worker training, external testing, and consumer education. Given this fragmented framework and the lack of interaction, it is clear that the governance of the South African food safety system is not "fit for purpose" in that there is a gap in the effectiveness of government regulation and the self-regulation of the formal sector, and a growing risk from an inability to regulate the large informal sector. Food safety challenges identified in our analysis included disease outbreaks, concerns over mislabeling, and lack of regulation for food handling and distribution. Conclusion: The findings suggest that there should be a combination of responsibility from all levels of stake-holders in the food retail sector in order to improve food safety and prevent food safety breaches. In addition, strong governance of the food safety system is required to enable effective legislation and enforcement.