Addressing health equity for breastfeeding women: primaquine for Plasmodium vivax radical cure.

Plasmodium vivax malaria remains a global health challenge, with approximately 6.9 million estimated cases in 2022. The parasite has a dormant liver stage, the hypnozoite, which reactivates to cause repeated relapses over weeks, months, or years. These relapses erode patient health, contribute to the burden of malaria, and promote transmission. Radical cure to prevent relapses requires administration of an 8-aminoquinoline, either primaquine or tafenoquine. However, malaria treatment guidelines updated by the World Health Organization (WHO) in October 2023 restrict primaquine use for women breastfeeding children < 6 months of age, or women breastfeeding older children if their child is G6PD deficient or if the child's G6PD status is unknown. Primaquine restrictions assume that 8-aminoquinoline exposures in breast milk would be sufficient to cause haemolysis in the nursing infant should they be G6PD deficient. WHO recommendations for tafenoquine are awaited. Notably, the WHO recommends that infants are breastfed for the first 2 years of life, and exclusively until 6 months old. Repeated pregnancies, followed by extended breastfeeding leaves women in P. vivax endemic regions potentially vulnerable to relapses for many years. This puts women's health at risk, increases the malaria burden, and perpetuates transmission, hindering malaria control and elimination. The benefits of lifting restrictions on primaquine administration to breastfeeding women are significant, avoiding the adverse consequences of repeated episodes of acute malaria, such as severe anaemia. Recent data challenge the restriction of primaquine in breastfeeding women. Clinical pharmacokinetic data in breastfeeding infants ≥ 28 days old show that the exposure to primaquine is very low and less than 1% of the maternal exposure, indicating negligible risk to infants, irrespective of their G6PD status. Physiologically-based pharmacokinetic modelling complements the clinical data, predicting minimal primaquine exposure to infants and neonates via breast milk from early post-partum. This article summarizes the clinical and modelling evidence for a favourable benefit:risk evaluation of P. vivax radical cure with primaquine for breastfeeding women without the need for infant G6PD testing, supporting a change in policy. This adjustment to current treatment guidelines would support health equity in regard to effective interventions to protect women and their children, enhance malaria control strategies, and advance P. vivax elimination.

Regulatory Assessment of Casgevy for the Treatment of Transfusion-Dependent ß-Thalassemia and Sickle Cell Disease with Recurrent Vaso-Occlusive Crises.

Sickle cell disease (SCD) and transfusion-dependent ß-thalassemia (TDT) are hereditary haemoglobinopathies characterized by a reduction in functional ß-globin chains. Both conditions cause tiredness and increase susceptibility to infection, which can lead organ failure, significantly reducing life expectancy and typically requiring those affected to undergo regular erythrocyte transfusion. Recently, a novel therapeutic treatment for SCD and TDT was approved by the UK regulatory body (Medicines and Healthcare products Regulatory Agency; MHRA). Exagamglogene autotemcel (Casgevy) is the first licensed therapy globally to utilize CRIPSR/Cas9 technology and induces an increase in expression of γ-globin chains to compensate for the reduction in functional ß-globin. Casgevy represents a first-in-class therapeutic, and numerous considerations were made by the MHRA throughout its assessment of the medicine. These include, but are not limited to, the risk of tumorigenicity and off-target editing, a limited cohort size, the validity of proposed dosing and the conduction of only single-arm studies. The MHRA's analyses of the data to support the proposed indications are presented and discussed throughout this manuscript. Overall, the sponsors claims were considered well supported by their data, and Casgevy was licensed for the treatment of TDT or SCD in patients 12 years of age and older for whom hematopoietic stem cell (HSC) transplantation is appropriate, but a human leukocyte antigen-matched related HSC donor is not available.

In situ monolayer patch clamp of acutely stimulated human iPSC-derived cardiomyocytes promotes consistent electrophysiological responses to SK channel inhibition.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) represent an in vitro model of cardiac function. Isolated iPSC-CMs, however, exhibit electrophysiological heterogeneity which hinders their utility in the study of certain cardiac currents. In the healthy adult heart, the current mediated by small conductance, calcium-activated potassium (SK) channels (ISK) is atrial-selective. Functional expression of ISK within atrial-like iPSC-CMs has not been explored thoroughly. The present study therefore aimed to investigate atrial-like iPSC-CMs as a model system for the study of ISK. iPSCs were differentiated using retinoic acid (RA) to produce iPSC-CMs which exhibited an atrial-like phenotype (RA-iPSC-CMs). Only 18% of isolated RA-iPSC-CMs responded to SK channel inhibition by UCL1684 and isolated iPSC-CMs exhibited substantial cell-to-cell electrophysiological heterogeneity. This variability was significantly reduced by patch clamp of RA-iPSC-CMs in situ as a monolayer (iPSC-ML). A novel method of electrical stimulation was developed to facilitate recording from iPSC-MLs via In situ Monolayer Patch clamp of Acutely Stimulated iPSC-CMs (IMPASC). Using IMPASC, > 95% of iPSC-MLs could be paced at a 1 Hz. In contrast to isolated RA-iPSC-CMs, 100% of RA-iPSC-MLs responded to UCL1684, with APD50 being prolonged by 16.0 ± 2.0 ms (p < 0.0001; n = 12). These data demonstrate that in conjunction with IMPASC, RA-iPSC-MLs represent an improved model for the study of ISK. IMPASC may be of wider value in the study of other ion channels that are inconsistently expressed in isolated iPSC-CMs and in pharmacological studies.

A single coiled-coil domain mutation in hIKCa channel subunits disrupts preferential formation of heteromeric hSK1:hIKCa channels.

The expression of IKCa (SK4) channel subunits overlaps with that of SK channel subunits, and it has been proposed that the two related subunits prefer to co-assemble to form heteromeric hSK1:hIKCa channels. This implicates hSK1:hIKCa heteromers in physiological roles that might have been attributed to activation of SK channels. We have used a mutation approach to confirm formation of heterometric hSK1:hIKCa channels. Introduction of residues within hSK1 that were predicted to impart sensitivity to the hIKCa current blocker TRAM-34 changed the pharmacology of functional heteromers. Heteromeric channels formed between wildtype hIKCa and mutant hSK1 subunits displayed a significantly higher sensitivity and maximum block to addition of TRAM-34 than heteromers formed between wildtype subunits. Heteromer formation was disrupted by a single point mutation within one COOH-terminal coiled-coil domain of the hIKCa channel subunit. This mutation only disrupted the formation of hSK1:hIKCa heteromeric channels, without affecting the formation of homomeric hIKCa channels. Finally, the Ca2+ gating sensitivity of heteromeric hSK1:hIKCa channels was found to be significantly lower than the Ca2+ gating sensitivity of homomeric hIKCa channels. These data confirmed the preferred formation of heteromeric channels that results from COOH-terminal interactions between subunits. The distinct sensitivity of the heteromer to activation by Ca2+ suggests that heteromeric channels fulfil a distinct function within those neurons that express both subunits.

Palmitoylation regulates the magnitude of HCN4-mediated currents in mammalian cells.

The sinoatrial node (SAN) and subsidiary pacemakers in the cardiac conduction system generate spontaneous electrical activity which is indispensable for electrical and therefore contractile function of the heart. The hyperpolarisation-activated cyclic nucleotide-gated channel HCN4 is responsible for genesis of the pacemaker "funny" current during diastolic depolarisation. S-palmitoylation, the reversible conjugation of the fatty acid palmitate to protein cysteine sulfhydryls, regulates the activity of key cardiac Na+ and Ca2+ handling proteins, influencing their membrane microdomain localisation and function. We investigated HCN4 palmitoylation and its functional consequences in engineered human embryonic kidney 293T cells as well as endogenous HCN4 in neonatal rat ventricular myocytes. HCN4 was palmitoylated in all experimental systems investigated. We mapped the HCN4 palmitoylation sites to a pair of cysteines in the HCN4 intracellular amino terminus. A double cysteine-to-alanine mutation CC93A/179AA of full length HCN4 caused a ∼67% reduction in palmitoylation in comparison to wild type HCN4. We used whole-cell patch clamp to evaluate HCN4 current (IHCN4) in stably transfected 293T cells. Removal of the two N-terminal palmitoylation sites did not significantly alter half maximal activation voltage of IHCN4 or the activation slope factor. IHCN4 was significantly larger in cells expressing wild type compared to non-palmitoylated HCN4 across a range of voltages. Phylogenetic analysis revealed that although cysteine 93 is widely conserved across all classes of HCN4 vertebrate orthologs, conservation of cysteine 179 is restricted to placental mammals. Collectively, we provide evidence for functional regulation of HCN4 via palmitoylation of its amino terminus in vertebrates. We suggest that by recruiting the amino terminus to the bilayer, palmitoylation enhances the magnitude of HCN4-mediated currents, but does not significantly affect the kinetics.

Preferential formation of human heteromeric SK2:SK3 channels limits homomeric SK channel assembly and function.

Three isoforms of small conductance, calcium-activated potassium (SK) channel subunits have been identified (SK1-3) that exhibit a broad and overlapping tissue distribution. SK channels have been implicated in several disease states including hypertension and atrial fibrillation, but therapeutic targeting of SK channels is hampered by a lack of subtype-selective inhibitors. This is further complicated by studies showing that SK1 and SK2 preferentially form heteromeric channels during co-expression, likely limiting the function of homomeric channels in vivo. Here, we utilized a simplified expression system to investigate functional current produced when human (h) SK2 and hSK3 subunits are co-expressed. When expressed alone, hSK3 subunits were more clearly expressed on the cell surface than hSK2 subunits. hSK3 surface expression was reduced by co-transfection with hSK2. Whole-cell recording showed homomeric hSK3 currents were larger than homomeric hSK2 currents or heteromeric hSK2:hSK3 currents. The smaller amplitude of hSK2:hSK3-mediated current when compared with homomeric hSK3-mediated current suggests hSK2 subunits regulate surface expression of heteromers. Co-expression of hSK2 and hSK3 subunits produced a current that arose from a single population of heteromeric channels as exhibited by an intermediate sensitivity to the inhibitors apamin and UCL1684. Co-expression of the apamin-sensitive hSK2 subunit and a mutant, apamin-insensitive hSK3 subunit [hSK3(H485N)], produced an apamin-sensitive current. Concentration-inhibition relationships were best fit by a monophasic Hill equation, confirming preferential formation of heteromers. These data show that co-expressed hSK2 and hSK3 preferentially form heteromeric channels and suggest that the hSK2 subunit acts as a chaperone, limiting membrane expression of hSK2:hSK3 heteromeric channels.

The macrolide drug erythromycin does not protect the hERG channel from inhibition by thioridazine and terfenadine.

The macrolide antibiotic erythromycin has been associated with QT interval prolongation and inhibition of the hERG-encoded channels responsible for the rapid delayed rectifier K+ current I(Kr ). It has been suggested that low concentrations of erythromycin may have a protective effect against hERG block and associated drug-induced arrhythmia by reducing the affinity of the pore-binding site for high potency hERG inhibitors. This study aimed to explore further the notion of a potentially protective effect of erythromycin. Whole-cell patch-clamp experiments were performed in which hERG-expressing mammalian (Human Embryonic Kidney; HEK) cells were preincubated with low to moderate concentrations of erythromycin (3 or 30 µM) prior to whole-cell patch clamp recordings of hERG current (IhERG ) at 37°C. In contrast to a previous report, exposure to low concentrations of erythromycin did not reduce pharmacological sensitivity of hERG to the antipsychotic thioridazine and antihistamine terfenadine. The IC50 value for IhERG tail inhibition by terfenadine was decreased by ~32-fold in the presence of 3 µM erythromycin (p < .05 vs. no preincubation). Sensitivity to thioridazine remained unchanged (p > .05 vs. no preincubation). The effects of low concentrations of erythromycin were investigated for a series of pore blocking drugs, and the results obtained were consistent with additive and/or synergistic effects. Experiments with the externally acting blocker BeKm-1 on WT hERG and a pore mutant (F656V) were used to explore the location of the binding site for erythromycin. Our data are inconsistent with the use of erythromycin for the management of drug-induced QT prolongation.

Inadequate provision of postintubation anxiolysis and analgesia in the ED.

INTRODUCTION: Patients intubated in the emergency department (ED) often have extended ED stays. We hypothesize that ED intubated patients receive inadequate postintubation anxiolysis and analgesia after rapid sequence induction (RSI). METHODS: This was a retrospective cohort study of every adult intubated in a tertiary-care ED (July 2003-June 2004). Patients were included if they underwent RSI, remained in the ED for more than 30 minutes post intubation, and survived to admission. Presuming a mean patient weight of 70 kg, we defined adequacy of anxiolysis and analgesia on the provision postintubation of weight-based doses of lorazepam (0.77 mg/h) or midazolam (4.2 mg/h) and fentanyl (35 microg/h), referenced from pharmaceutical texts. Demographic data, time in ED, and dosage of each medication given were abstracted. The proportion, with 95% confidence intervals (CIs), of patients receiving inadequate anxiolysis and analgesia were computed. RESULTS: One hundred seventeen patients met the inclusion criteria. Mean time in the ED was 4.2 hours (SD +/- 3.1 hours). Thirty-nine patients received no anxiolytic (33%, CI 25%-43%), and 62 received no analgesic (53%, CI 44%-62%). Twenty-three patients received neither anxiolytic nor analgesic (20%, CI 13%-28%). Of 70 patients given postintubation vecuronium, 67 received either no or inadequate anxiolysis or analgesia (96%, CI 87%-99%). Overall, 87 of 117 patients received no or inadequate anxiolysis (74%, CI 65%-82%); and 88 of 117 received no or inadequate analgesia (75%, CI 66%-83%). CONCLUSION: Patients undergoing RSI in the ED frequently receive inadequate postintubation anxiolysis and analgesia.