Weight Bands Research Articles

Abstract 44: A novel, codon-based method to modulate off-tumor toxicity of chimeric antigen receptor (CAR) T cells

Abstract Chimeric antigen receptor (CAR) T cell therapies for hematologic malignancies, while promising and efficacious, can also result in a variety of adverse effects. Of note, on-target, off-tumor toxicity is one adverse effect that continues to limit the safety and efficacy of CAR-T cells and has even led to life-threatening or fatal outcomes in patients (Morgan et al. 2010.). To address this, we have developed a unique, codon-based approach by replacing strategically selected codons within CAR non-antigen binding domains with their rare synonymous isoforms. Hoekema et al. (1987) reported the ability of synonymous rare codons to alter gene and protein expression in other model systems. In CAR-T cells, we aim to use this novel, codon-based approach to fine tune and modulate CAR-T cell expression and function to elicit full on-tumor activity while lessening off-tumor toxicity. In our approach, we replaced specific endogenous codons within the non-antigen binding domains of Hu8F4-CAR, a TCR-like 2nd generation CAR developed by our group which targets the leukemia-associated antigen PR1/HLA-A2 (Ma et al. 2016.). Briefly, we created variants of Hu8F4-CAR containing rare codon substitutions in two subtypes: intracellular-based (type 1) and combined extracellular- and intracellular-based (type 2). Each variant subtype (type 1 or 2) had either between 6 and 30 or 27 and 57 rare codons introduced, respectively. Expression profiling and functional assessment of the Hu8F4-CAR variants was assessed in the Jurkat mutant cell line (J.RT3-T3.5) or human primary healthy peripheral blood mononuclear cells (PBMCs) that were enriched for T cells, respectively. Expression profiling results show that surface expression is reduced in J.RT3-T3.5 cells when assessed by flow cytometry by 1-1.5-fold compared to unmodified Hu8F4-CAR. When evaluated by immunoblotting, novel lower molecular weight bands emerge only in rare codon-modified variants, suggesting that rare codons may have altered translational or post-translational processing leading to truncated CAR protein isoforms (30, 45, or 60kDa vs 73kDa for unmodified Hu8F4-CAR), which may contribute to reduced cell surface CAR expression. Furthermore, we have conducted in vitro functional testing of the rare codon-modified variants and show reduced activity against non-malignant off-tumor targets that express PR1 and/or HLA-A2 including healthy donor PBMCs, mobilized hematopoietic cells, and bone marrow. Importantly, in addition to reduced off-tumor activity against healthy myeloid cells, rare codon-modified variants preserve their activity against on-tumor target cells, including the U937-A2+ cell line and primary acute myeloid leukemia. Altogether, these results suggest that our approach can modulate CAR expression and functionality creating a novel methodology to reduce the likelihood of off-tumor adverse effects typically associated with CAR therapies. Citation Format: Rolando A. Vedia, Sijie Lu, Anne V. Philips, Anna Sergeeva, Lisa S. St John, Karen Clise-Dwyer, Gheath Alatrash, Jeffrey J. Molldrem. A novel, codon-based method to modulate off-tumor toxicity of chimeric antigen receptor (CAR) T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 44.

Population pharmacokinetics, enzyme occupancy, and pharmacodynamic modeling of soticlestat in patients with developmental and epileptic encephalopathies.

Soticlestat (TAK-935) is a first-in-class, selective inhibitor of cholesterol 24-hydroxylase (CH24H) under phase III development for the treatment of the developmental and epileptic encephalopathies (DEEs), Dravet syndrome (DS), and Lennox-Gastaut syndrome (LGS). A previous model characterized the pharmacokinetics (PKs), CH24H enzyme occupancy (EO), and pharmacodynamics (PDs) of soticlestat in healthy volunteers. The present study extended this original model for patients with DEEs and investigated sources of variability. Model-based simulations were carried out to optimize dosing strategies for use in clinical trials. Data from eight phase I and II trials of healthy volunteers or patients with DEEs receiving oral soticlestat 15-1350 mg were included, encompassing 218 individuals for population PK (PopPK) analyses and 306 individuals for PK/PD analyses. Dosing strategies were identified through model-based simulations. The final mixed-effect PopPK/EO/PD model consisted of a two-compartment PK model and an effect-site compartment in the PK/EO model; soticlestat concentrations at the effect site were linked to 24S-hydroxycholesterol plasma concentrations using a semimechanistic inhibitory indirect response model. Covariates were included to account for sources of variability. Pediatric dosing strategies were developed for four body weight bands (10 to <15, 15 to <30, 30 to <45, and 45-100 kg) to account for covariate effects by body weight. The final PopPK and PK/EO/PD models accurately described PK, EO, and PD profiles of soticlestat in healthy volunteers and patients with DEEs. Covariate analyses and model-based simulations facilitated optimization of phase III trial dosing strategies for patients with DS or LGS.

Population Pharmacokinetics of Pediatric Lopinavir/Ritonavir Oral Pellets in Children Living with HIV in Africa.

Antiretroviral therapy for children living with HIV (CLHIV) under 3 years of age commonly includes lopinavir/ritonavir (LPV/r). However, the original liquid LPV/r formulation has taste and cold storage difficulties. To address these challenges, LPV/r oral pellets have been developed. These pellets can be mixed with milk or food for administration and do not require refrigeration. We developed the population pharmacokinetic (PK) model and assessed drug exposure of LPV/r oral pellets administered twice daily to CLHIV per World Health Organization (WHO) weight bands. The PK analysis included Kenyan and Ugandan children participating in the LIVING studies (NCT02346487) receiving LPV/r pellets (40/10 mg) and ABC/3TC (60/30 mg) dispersible tablets. Population PK models were developed for lopinavir (LPV) and ritonavir (RTV) to evaluate the impact of RTV on the oral clearance (CL/F) of LPV. The data obtained from the study were analyzed using nonlinear mixed-effects modeling approach. Data from 514 children, comprising a total of 2,998 plasma concentrations of LPV/r were included in the analysis. The LPV and RTV concentrations were accurately represented by a one-compartment model with first-order absorption (incorporating a lag-time) and elimination. Body weight influenced LPV and RTV PK parameters. The impact of RTV concentrations on the CL/F of LPV was characterized using a maximum effect model. Simulation-predicted target LPV exposures were achieved in children with this pellet formulation across the WHO weight bands. The LPV/r pellets dosed in accordance with WHO weight bands provide adequate LPV exposures in Kenyan and Ugandan children weighing 3.0 to 24.9 kg.

Coprecipitated Keratin/hydroxyapatite nanocomposites assisted by microwave/ultrasound irradiation, and its cytotoxic evaluation on NIH/3T3 fibroblast cells

This work is aimed to synthesize Hydroxyapatite (Hap) and Keratin/Hydroxyapatite (KerHap) nanocomposites by coprecipitation using the simultaneous microwave/ultrasound (Mw/Us) method to confer specific textural and morphological properties to the materials using keratin (Ker) extracted from human hair. Four materials were synthesized applying different powers of Mw/Us irradiation: Hap (1100 W Mw, 40 kHz Us), KerHap A (700 W Mw, 40 kHz Us), KerHap B (900 W Mw, 40 kHz Us) and KerHap C (1100 W Mw, 40 kHz Us) at 120 °C for 10 min. The materials were characterized according to their structure (FTIR, XRD, Raman spectroscopy), morphology and size (TEM), and thermal properties (TG). Their cytotoxic effects were studied at different concentrations (25, 50, 100, 200, and 400 μg/mL) using the fibroblast cell line (NIH/3T3) by MTT assay at 24, 48, and 72 h. The Ker extract exhibited a major molecular weight band between 11–10 kDa in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Morphology, particle, and crystallite sizes were dependent on the Mw irradiation power applied and keratin added during the synthesis. The MTT assay at 48 h demonstrated a significant increase in cell viability of 39% for Hap at 50 µg/mL, for KerHap A did not show a significant difference, while KerHap B-C showed a significant increase at 200 µg/mL of 30% in cell viability. It means that fibroblast presented better cell adhesion on Hap than on KerHap A-C composites; it may be due to keratin blocking Hap pores because of electrostatic interactions between keratin and Hap.

Delving into the significance of the His289Tyr single-nucleotide polymorphism in the glutamate ionotropic receptor kainate-1 (Grik1) gene of a genetically audiogenic seizure model.

Genetic abnormalities affecting glutamate receptors are central to excitatory overload-driven neuronal mechanisms that culminate in seizures, making them pivotal targets in epilepsy research. Increasingly used to advance this field, the genetically audiogenic seizure hamster from Salamanca (GASH/Sal) exhibits generalized seizures triggered by high-intensity acoustic stimulation and harbors significant genetic variants recently identified through whole-exome sequencing. Here, we addressed the influence of the missense single-nucleotide polymorphism (C9586732T, p.His289Tyr) in the glutamate receptor ionotropic kainate-1 (Grik1) gene and its implications for the GASH/Sal seizure susceptibility. Using a protein 3D structure prediction, we showed a potential effect of this sequence variation, located in the amino-terminal domain, on the stability and/or conformation of the kainate receptor subunit-1 protein (GluK1). We further employed a multi-technique approach, encompassing gene expression analysis (RT-qPCR), Western blotting, and immunohistochemistry in bright-field and confocal fluorescence microscopy, to investigate critical seizure-associated brain regions in GASH/Sal animals under seizure-free conditions compared to matched wild-type controls. We detected disruptions in the transcriptional profile of the Grik1 gene within the audiogenic seizure-associated neuronal network. Alterations in GluK1 protein levels were also observed in various brain structures, accompanied by an unexpected lower molecular weight band in the inferior and superior colliculi. This correlated with substantial disparities in GluK1-immunolabeling distribution across multiple brain regions, including the cerebellum, hippocampus, subdivisions of the inferior and superior colliculi, and the prefrontal cortex. Notably, the diffuse immunolabeling accumulated within perikarya, axonal fibers and terminals, exhibiting a prominent concentration in proximity to the cell nucleus. This suggests potential disturbances in the GluK1-trafficking mechanism, which could subsequently affect glutamate synaptic transmission. Overall, our study sheds light on the genetic underpinnings of seizures and underscores the importance of investigating the molecular mechanisms behind synaptic dysfunction in epileptic neural networks, laying a crucial foundation for future research and therapeutic strategies targeting GluK1-containing kainate receptors.

Pharmaceutical cost dynamics for the treatment of rifampicin-resistant tuberculosis in children and adolescents in South Africa, India, and the Philippines.

Rifampicin-resistant (RR) tuberculosis (TB) in children is a major global health concern but is often neglected in economics research. Accurate cost estimations across the spectrum of paediatric RR-TB treatment regimens are critical inputs for prioritisation and budgeting decisions, and an existing knowledge gap at local and international levels. This normative cost analysis was nested in a Phase I/II pharmacokinetics, safety, tolerability, and acceptability trial of TB medications in children in South Africa, the Philippines and India. It assessed the pharmaceutical costs of 36 childhood RR-TB regimens using combinations from 16 different medicines in 34 oral formulations (adult and child-friendly) in 11 weight bands in children <15 years of age. The analysis used local and Global Drug Facility pricing, and local and international guideline recommendations, including adaptions of BPaL and BPaLM regimens in adults. Costs varied significantly between regimen length, age/weight banding, severity of disease, presence of fluroquinolone resistance, and different country guideline recommendations. WHO recommended regimen costs ranged 12-fold: from US$232 per course (short regimen in non-severe disease) to US$2,761 (long regimen in severe, fluroquinolone-resistant disease). Regimen treating fluoroquinolone-resistant infection cost US$1,090 more than comparable WHO-recommended regimen. Providing child-friendly medicine formulations in <5-year-olds across all WHO-recommended regimens is expected to cost an additional $380 (range $212-$563) per child but is expected to have wider benefits including palatability, acceptability, adherence, tolerability, and dose accuracy. There were substantial differences in regimen affordability between countries when adjusted for purchasing power and domestic spending on health. Appropriate, effective, and affordable treatment options are an important component of the fight against childhood RR-TB. A comprehensive understanding of the cost and affordability dynamics of treatment options will enable national TB programs and global collaborations to make the best use of limited healthcare resources for the care of children with RR-TB.

Microtubule‐associated protein tau (MAPT) in the lens of the eye in non‐transgenic and transgenic tau mutant (P301S) mice

AbstractBackgroundWe previously discovered that Aβ accumulates in the cortical/supranuclear region of the lens in people with Alzheimer’s Disease (AD) (Goldstein et al., 2003) and Down Syndrome (DS; (Moncaster et al., 2010). We also demonstrated Aβ in the Tg2576 APP Swedish mutation AD mouse model (Moncaster et al., 2022). Another protein that is involved in AD is Microtubule‐Associated Protein Tau (MAPT). Tau is expressed in the brain and becomes hyperphosphorylated in AD eventually forming neurofibrillary tangles. Tau has previously been reported to be expressed in the mouse lens (Bai et al., 2007; Zhao et al., 2013) but the results vary depending on the mouse model. Here we investigated whether tau protein was expressed in non‐transgenic mouse lenses and in a transgenic tau mutant (P301S) mouse model. We also studied whether cataracts may develop in this P301S tau mutant mouse model.MethodP301S tau mutant transgenic and non‐transgenic mice were bred and maintained at Boston University. Breeder mice were purchased from The Jackson Laboratory, Bar Harbor, ME. Male and Female transgenic and non‐transgenic mice were sacrificed throughout their lifespan at ages 3‐12 months. Mice were perfused with phosphate buffer saline, lenses were isolated and then imaged under two different sources of light using a D70 digital Nikon camera and a custom‐adapted Zeiss stereophotomicroscope. Lenses were then snap frozen and analyzed by Western Blotting using tau antibodies.ResultP301S transgenic and non‐transgenic mouse lenses all expressed tau during their lifespan when analyzed by Western blot. P301S transgenic lenses demonstrated an additional higher molecular weight band compared to the non‐transgenic mice. No cataract phenotype was observed in transgenic or non‐transgenic mouse lenses.ConclusionThere was no overt difference in lens phenotype between the P301S transgenic and non‐transgenic mice. However, the banding pattern observed by Western blot for P301S transgenic mice compared to non‐transgenic was different. Our data suggest the P301S tau mutation affects tau processing but does not result in a cataract phenotype. Based on our current and previous results, the data suggests that Aβ may play a more significant role than tau in lens pathology in AD.

Genome-Wide CRISPR Screen Identifies MAD2L1BP, ANAPC15 and SLC39A7 As Intrinsic Regulators for Brentuximab Vedotin Sensitivity in Peripheral T-Cell Lymphoma Cells

Introduction: Brentuximab Vedotin (BV), an antibody-drug conjugate that specifically delivers the potent cytotoxic drug monomethyl auristatin E (MMAE) to CD30-positive cells, has been widely used as one of the key drugs for the treatment of peripheral T-cell lymphomas (PTCLs). BV shows relatively high response rate, however, the prognosis of PTCLs is not still satisfactory. The molecular mechanisms concerning sensitivity to BV remain to be clarified. Methods: To assess the cell-intrinsic genes affecting susceptibility to BV in PTCL cells, we performed a whole-genome loss-of-function CRISPR screen in DEL, an anaplastic large cell lymphoma (ALCL), ALK-positive cell line. DEL cells were transduced with the Brunello CRISPR knockout pooled library targeting 19,114 genes (4 sgRNAs/gene) followed by puromycin selection. Then, the transduced cells were treated with BV or DMSO control for 6 days. To rank the sgRNAs responsible for BV sensitivity, MAGeCK algorithm was used. Among the top 20 enriched genes, we further selected ones having at least 2 sgRNAs with a log2 fold change (BV/DMSO) &amp;lt; -0.5 or &amp;gt;0.5 as genes whose knock out confer BV sensitivity or resistance, respectively. Results: Through the whole-genome loss-of-function CRISPR screen, we identified 17 genes or 15 genes whose knock out confer BV sensitivity or resistance, respectively. TNF receptor superfamily member 8 (TNFRSF8, also known as CD30), a known BV target, was ranked as a top gene for BV resistance, confirming the reliability of our screening. Among the 17 enriched genes whose knock out confer BV sensitivity, endogenous mitotic checkpoint complex (MCC) inhibitors, namely MAD2L1BP and ANAPC15 were identified. As a confirmation, we knocked out MAD2L1BP or ANAPC15 in PTCL cell lines by CRISPR and found the enhanced susceptibility to BV of the cells. MAD2L1BP and ANAPC15, the endogenous MCC inhibitors, are known to facilitate anaphase-promoting complex/cyclosome (APC/C) activation. Therefore, we evaluated whether APC/C inhibition sensitize the PTCL cells to BV or not. Importantly, a combination use of proTAME, an APC/C inhibitor, and BV significantly suppressed cell proliferation of multiple PTCL lines, including ALCL, ALK-positive and adult T-cell leukemia/lymphoma (ATLL) (Fig. 1), compared to each single agent. In cell cycle analyses, PTCL lines treated with the combination demonstrated increased percentages of cells in G2/M phase, compared to each single agent. On the other hand, we found SLC39A7, a zinc channel in endoplasmic reticulum (ER), among 15 enriched genes whose knock out confer BV resistance. Since SLC39A7 has been reported to regulate TNFR1 cell surface expression (Fauster et al. Cell Death &amp; Differentiation 2019), we hypothesized SLC39A7 could also affect the CD30 cell surface expression levels. In fact, flow cytometry analyses revealed downmodulation of CD30 cell surface expression in SLC39A7 knocked out PTCL lines (Fig. 2). We further evaluated the amount of CD30 protein expression by immunoblotting and found that SLC39A7 knocked out cells reduced the amount of mature form of CD30, which was consistent with the findings in flow cytometry. Intriguingly, we observed increasement of lower molecular weight band, which was considered as immature precursor CD30, in SLC39A7 knocked out cells, suggesting that impared glycobiology in ER might be a cause for surface CD30 downregulation in SLC39A7 knocked out PTCL cells. Finally, we treated PTCL lines with NVS-ZP7-4, an SLC39A7 inhibitor and the similar results were obtained regarding to CD30 expression by flow cytometry and immunoblotting. Conclusions: We performed an unbiased genome-wide CRISPR/Cas9 library screen and identified cell-intrinsic regulators for BV sensitivity in PTCLs. Our screening results suggested that the disruption of MAD2L1BP/ANAPC15 or APC/C inhibition would be a potential strategy to enhance therapeutic activity of BV in PTCLs. We also identified SLC39A7 downregulation could confer BV resistance by downregulating CD30 cell surface expression.

Third time's a charm: band to sleeve to bypass.

Adjustable gastric bands (AGB) are frequently converted to sleeve gastrectomy (SG) or Roux-en-Y gastric bypass (RYGB) due to weight recurrence or band complications. Such conversions carry a higher-risk than primary procedures. Some patients undergo two conversions-from AGB to SG, and subsequently from SG to RYGB. This presents a unique situation with limited literature on indications and complication rates associated with these double conversions. We examined the 2020-2021 Metabolic and Bariatric Surgery Accreditation and Quality Improvement Program (MBSAQIP) Participant Use File to evaluate conversions from AGB to SG and then to RYGB. Patient and operative characteristics, along with outcomes, were evaluated. Descriptive statistics were applied. We identified 276 patients who underwent a conversion from AGB to SG and then to RYGB. The primary reason for the second conversion (SG to RYGB) was gastroesophageal reflux disease (GERD) at 55.1%, followed by inadequate weight loss or weight regain (IWL/WR) at 36.9%. The remaining reasons included dysphagia, nausea, vomiting, or others. Patients converted for IWL/WR demonstrated a higher baseline body mass index and prevalence of sleep apnea compared to other cohorts (both p < 0.001). Meanwhile, patients in the "other reasons" group had the highest rate of open surgical approaches (9.1%) and concurrent lysis of adhesions (p = 0.001 and p = 0.022), with correspondingly higher rates of anastomotic leak, reoperations, serious complications, and mortality. Patients undergoing double conversions (AGB to SG to RYGB) do so primarily for GERD or IWL/WR. Further research is required to better define the optimal primary operation for each patient, aiming to reduce the necessity for multiple conversions.

Optimizing dosing of the cycloserine pro-drug terizidone in children with rifampicin-resistant tuberculosis.

There are no pharmacokinetic data in children on terizidone, a pro-drug of cycloserine and a World Health Organization (WHO)-recommended group B drug for rifampicin-resistant tuberculosis (RR-TB) treatment. We collected pharmacokinetic data in children <15 years routinely receiving 15-20 mg/kg of daily terizidone for RR-TB treatment. We developed a population pharmacokinetic model of cycloserine assuming a 2-to-1 molecular ratio between terizidone and cycloserine. We included 107 children with median (interquartile range) age and weight of 3.33 (1.55, 5.07) years and 13.0 (10.1, 17.0) kg, respectively. The pharmacokinetics of cycloserine was described with a one-compartment model with first-order elimination and parallel transit compartment absorption. Allometric scaling using fat-free mass best accounted for the effect of body size, and clearance displayed maturation with age. The clearance in a typical 13 kg child was estimated at 0.474 L/h. The mean absorption transit time when capsules were opened and administered as powder was significantly faster compared to when capsules were swallowed whole (10.1 vs 72.6 min) but with no effect on bioavailability. Lower bioavailability (-16%) was observed in children with weight-for-age z-score below -2. Compared to adults given 500 mg daily terizidone, 2022 WHO-recommended pediatric doses result in lower exposures in weight bands 3-10 kg and 36-46 kg. We developed a population pharmacokinetic model in children for cycloserine dosed as terizidone and characterized the effects of body size, age, formulation manipulation, and underweight-for-age. With current terizidone dosing, pediatric cycloserine exposures are lower than adult values for several weight groups. New optimized dosing is suggested for prospective evaluation.

Population pharmacokinetic model of rifampicin for personalized tuberculosis pharmacotherapy: Effects of SLCO1B1 polymorphisms on drug exposure

Rifampicin (RIF) exhibits high pharmacokinetic (PK) variability among individuals; a low plasma concentration might result in unfavorable treatment outcomes and drug resistance. This study evaluated the contributions of non- and genetic factors to the interindividual variability of RIF exposure, then suggested initial doses for patients with different weight bands. This multicenter prospective cohort study in Korea analyzed demographic and clinical data, the solute carrier organic anion transporter family member 1B1 (SLCO1B1) genotypes, and RIF concentrations. Population PK modeling and simulations were conducted using nonlinear mixed-effect modeling. In total, 879 tuberculosis (TB) patients were divided into a training dataset (510 patients) and a test dataset (359 patients). A one-compartment model with allometric scaling for effect of body size best described the RIF PKs. The apparent clearance (CL/F) was 16.6% higher among patients in the SLCO1B1 rs4149056 wild-type group than among patients in variant group, significantly decreasing RIF exposure in the wild-type group. The developed model showed better predictive performance compared with previously reported models. We also suggested that patients with body weights of <40 kg, 40-55 kg, 55-70 kg, and >70 kg patients receive RIF doses of 450, 600, 750, and 1050 mg/day, respectively. Total body weight and SLCO1B1 rs4149056 genotypes were the most significant covariates that affected RIF CL/F variability in Korean TB patients. We suggest initial doses of RIF based on World Health Organization weight-band classifications. The model may be implemented in treatment monitoring for TB patients.

Abacavir Drug Exposures in African Children Under 14 kg Using Pediatric Solid Fixed Dose Combinations According to World Health Organization Weight Bands.

The pharmacokinetics of abacavir (ABC) in African children living with HIV (CLHIV) weighing <14 kg and receiving pediatric fixed dose combinations (FDC) according to WHO weight bands dosing are limited. An ABC population pharmacokinetic model was developed to evaluate ABC exposure across different World Health Organization (WHO) weight bands. Children enrolled in the LIVING study in Kenya and Uganda receiving ABC/lamivudine (3TC) dispersible tablets (60/30 mg) according to WHO weight bands. A population approach was used to determine the pharmacokinetic parameters. Monte Carlo simulations were conducted using an in silico population with demographic characteristics associated with African CLHIV. ABC exposures (AUC0-24) of 6.4-50.4 mg h/L were used as targets. Plasma samples were obtained from 387 children. A 1-compartment model with allometric scaling of clearance (CL/F) and volume of distribution (V/F) according to body weight best characterized the pharmacokinetic data of ABC. The maturation of ABC CL/F was characterized using a sigmoidal Emax model dependent on postnatal age (50% of adult CL/F reached by 0.48 years of age). Exposures to ABC were within the target range for children weighing 6.0-24.9 kg, but children weighing 3-5.9 kg were predicted to be overexposed. Lowering the ABC dosage to 30 mg twice daily or 60 mg once daily for children weighing 3-5.9 kg increased the proportion of children within the target and provided comparable exposures. Further clinical study is required to investigate clinical implications and safety of the proposed alternative ABC doses.

Dose rationale for gabapentin and tramadol in pediatric patients with chronic pain.

Despite off-label use, the efficacy and safety of gabapentin and tramadol in pediatric patients (3 months to <18 years old) diagnosed with chronic pain has not been characterized. However, generating evidence based on randomized clinical trials in this population has been extremely challenging. The current investigation illustrates the use of clinical trial simulations (CTSs) as a tool for optimizing doses and protocol design for a prospective investigation in pediatric patients with chronic pain. Pharmacokinetic (PK) modeling and CTSs were used to describe the PKs of gabapentin and tramadol in the target population. In the absence of biomarkers of analgesia, systemic exposure (AUC, Css) was used to guide dose selection under the assumption of a comparable exposure-response (PKPD) relationship for either compound between adults and children. Two weight bands were identified for gabapentin, with doses titrated from 5 to 63 mg/kg. This yields gabapentin exposures (AUC0-8 ) of approximately 35 mg/L*h (1200 mg/day adult dose equivalent). For tramadol, median steady state concentrations between 200 and 300 ng/mL were achieved after doses of 2-5 mg/kg, but concentrations showed high interindividual variability. Simulation scenarios showed that titration steps are required to explore therapeutically relevant dose ranges taking into account the safety profile of both drugs. Gabapentin can be used t.i.d. at doses between 7-63 and 5-45 mg/kg for patients receiving gabapentin weighing <15 and ≥15 kg, respectively, whereas a t.i.d. regimen with doses between 1 and 5 mg/kg can be used for tramadol in patients who are not fast metabolisers.

Anti-nuclear valosin-containing protein-like autoantibody is associated with calcinosis and higher risk of cancer in systemic sclerosis.

Systemic sclerosis (SSc)-specific autoantibodies allow the diagnosis and predict the prognosis of SSc patients with different clinical characteristics. The aim of this study was to describe new SSc-related autoantibodies by a novel protein immunoprecipitation (IP) assay. Serum samples and clinical data were collected from 307 SSc patients. Antinuclear autoantibodies were tested in all patients by indirect immunofluorescence (IIF) on HEp-2 cells. SSc-specific autoantibodies were evaluated with a commercial immunoblot and chemiluminescence immunoassay, and traditional RNA-IP. Patients negative for all these autoantibodies (n = 51) were further tested with a non-radioactive protein IP assay. Protein bands detected on SDS-PAGE were then analysed by mass spectrometry (MS) and confirmed by western blot (WB). Additional 56 patients with nucleolar pattern by IIF were tested by protein IP-WB. Five patients who underwent protein IP testing showed a 110-115kDa molecular weight band on SDS-PAGE and a homogeneous nucleolar pattern by IIF. MS identified the bands as nuclear valosin-containing protein-like (NVL). An additional positive patient was detected by IP-WB. As compared with the remaining 101 negative patients, anti-NVL positive patients showed a greater prevalence of calcinosis (100% vs 18.9%, P < 0.001), and cancer (66.7% vs 8.9%, P = 0.002), with a particular association with synchronous cancer (OR = 16.3; P = 0.024). We identified NVL as a new autoantibody target by a novel protein IP assay in SSc patients with a homogeneous nucleolar IIF pattern, testing negative for all known SSc-specific autoantibodies by commercial assays and RNA IP. Anti-NVL identifies a new clinical phenotype, characterized by calcinosis and cancer.

Diagnostic Reference Levels in Interventional Pediatric Cardiology: Two-Year Experience in a Tertiary Referral Hospital in Latin America.

The goal of the present study was to propose the first local diagnostic reference levels (DRLs) for interventional pediatric cardiology procedures in a large hospital in Colombia. The data collection period was from April 2020 to July 2022. The local DRLs were calculated as the 3rd quartile of patient-dose distributions for the kerma-area product (Pka) values. The sample of collected clinical procedures (255) was divided into diagnostic and therapeutic procedures and grouped into five weight and five age bands. The Pka differences found between diagnostic and therapeutic procedures were statistically significant in all weight and age bands, except for the 1-5-year age group. The local DRLs for weight bands were 3.82 Gy·cm2 (<5 kg), 7.39 Gy·cm2 (5-<15 kg), 19.72 Gy·cm2 (15-<30 kg), 28.99 Gy·cm2 (30-<50 kg), and 81.71 Gy·cm2 (50-<80 kg), respectively. For age bands, the DRLs were 3.97 Gy·cm2 (<1 y), 9.94 Gy·cm2 (1-<5 y), 20.82 Gy·cm2 (5-<10 y), 58.00 Gy·cm2 (10-<16 y), and 31.56 Gy·cm2 (<16 y), respectively. In conclusion, when comparing our results with other existing DRL values, we found that they are similar to other centers and thus there is scope to continue optimizing the radiation dose values. This will contribute to establishing national DRLs for Colombia in the near future.

Population pharmacokinetics of ivermectin after mass drug administration in lymphatic filariasis endemic communities of Tanzania.

Ivermectin (IVM) is a drug of choice used with albendazole for mass drug administration (MDA) to halt transmission of lymphatic filariasis. We investigated IVM pharmacokinetic (PK) variability for its dose optimization during MDA. PK samples were collected at 0, 2, 4, and 6 h from individuals weighing greater than 15 kg (n = 468) receiving IVM (3-, 6-, 9-, or 12 mg) and ALB (400 mg) during an MDA campaign in Tanzania. Individual characteristics, including demographics, laboratory/clinical parameters, and pharmacogenetic variations were assessed. IVM plasma concentrations were quantified by liquid-chromatography tandem mass spectrometry and analyzed using population-(PopPK) modeling. A two-compartment model with transit absorption kinetics, and allometrically scaled oral clearance (CL/F) and central volume (Vc /F) was adapted. Fitting of the model to the data identified 48% higher bioavailability for the 3 mg dose compared to higher doses and identified a subpopulation with 97% higher mean transit time (MTT). The final estimates for CL/F, Vc /F, intercompartment clearance, peripheral volume, MTT, and absorption rate constant for a 70 kg person (on dose other than 3 mg) were 7.7 L/h, 147 L, 20.4 L/h, 207 L, 1.5 h, and 0.71/h, respectively. Monte-Carlo simulations indicated that weight-based dosing provides comparable exposure across weight bands, but height-based dosing with capping IVM dose at 12 mg for individuals with height greater than 160 cm underdoses those weighing greater than 70 kg. Variability in IVM PKs is partly explained by body weight and dose. The established PopPK model can be used for IVM dose optimization. Height-based pole dosing results in varying IVM exposure in different weight bands, hence using weighing scales for IVM dosing during MDA is recommended.

Population Pharmacokinetic Modeling of Dolutegravir to Optimize Pediatric Dosing in HIV-1-Infected Infants, Children, and Adolescents.

HIV treatment options remain limited in children. Dolutegravir is a potent and well-tolerated, once-daily HIV-1 integrase inhibitor recommended for HIV-1 infection in both adults and children down to 4 weeks of age. To support pediatric dosing of dolutegravir in children, we used a population pharmacokinetic model with dolutegravir data from the P1093 and ODYSSEY clinical trials. The relationship between dolutegravir exposure and selected safety endpoints was also evaluated. A population pharmacokinetic model was developed with data from P1093 and ODYSSEY to characterize the pharmacokinetics and associated variability and to evaluate the impact of pharmacokinetic covariates. The final population pharmacokinetic model simulated exposures across weight bands, doses, and formulations that were compared with established adult reference data. Exploratory exposure-safety analyses evaluated the relationship between dolutegravir pharmacokinetic parameters and selected clinical laboratory parameters and adverse events. A total of N = 239 participants were included, baseline age ranged from 0.1 to 17.5 years, weight ranged from 3.9 to 91 kg, 50% were male, and 80% were black. The final population pharmacokinetic model was a one-compartment model with first-order absorption and elimination, enabling predictions of dolutegravir concentrations in the pediatric population across weight bands and doses/formulations. The predicted geometric mean trough concentration was comparable to the adult value following a 50-mg daily dose of dolutegravir for all weight bands at recommended doses. Body weight, age, and formulation were significant predictors of dolutegravir pharmacokinetics in pediatrics. Additionally, during an exploratory exposure-safety analysis, no correlation was found between dolutegravir exposure and selected safety endpoints or adverse events. The dolutegravir dosing in children ≥ 4 weeks of age on an age/weight-band basis provides comparable exposures to those historically observed in adults. Observed pharmacokinetic variability was higher in this pediatric population and no additional safety concerns were observed. These results support the weight-banded dosing of dolutegravir in pediatric participants currently recommended by the World Health Organization.

Pharmacokinetics, safety, and tolerability of dispersible and immediate-release abacavir, dolutegravir, and lamivudine tablets in children with HIV (IMPAACT 2019): week 24 results of an open-label, multicentre, phase 1–2 dose-confirmation study

Child-friendly fixed-dose combination (FDC) antiretroviral therapy (ART) options are limited. We evaluated the pharmacokinetics, safety, and tolerability of dispersible and immediate-release FDC abacavir, dolutegravir, and lamivudine taken once per day in children younger than 12 years with HIV. IMPAACT 2019 was an international, phase 1-2, multisite, open-label, non-comparative dose-confirmation study of abacavir, dolutegravir, and lamivudine in children younger than 12 years. Participants were enrolled across five weight bands: those weighing 6 kg to less than 25 kg received abacavir (60 mg), dolutegravir (5 mg), and lamivudine (30 mg) dispersible tablets (three to six tablets depending on body weight), and those weighing 25 kg to less than 40 kg received abacavir (600 mg), dolutegravir (50 mg), and lamivudine (300 mg) in an immediate-release tablet. At entry, participants were ART naive or ART experienced and virologically suppressed on stable ART for 6 months or more. Dose confirmation was based on pharmacokinetic and safety criteria in the first five to seven participants in each weight band to week 4; all participants were followed up to week 48. We present the results for the primary objectives to assess pharmacokinetics, confirm dosing, and evaluate safety through 24 weeks across all weight bands. The trial is registered with ClinicalTrials.gov (NCT03760458). 57 children were enrolled and initiated study drug (26 [46%] female and 31 [54%] male; 37 [65%] Black, 18 [32%] Asian, and 1 [2%] had race reported as unknown). Within each weight band, 6 kg to less than 10 kg, 10 kg to less than 14 kg, 14 kg to less than 20 kg, 20 kg to less than 25 kg, and 25 kg or higher: the geometric mean dolutegravir area under the concentration time curve over the 24 h dosing interval (AUC0-24 h) was 75·9 h·μg/mL (33·7%), 91·0 h·μg/mL (36·5%), 71·4 h·μg/mL (23·5%), 84·4 h·μg/mL (26·3%), and 71·8 h·μg/mL (13·9%); dolutegravir concentrations 24 h after dosage (C24 h) were 0·91 μg/mL (67·6%), 1·22 μg/mL (77·5%), 0·79 μg/mL (44·2%), 1·35 μg/mL (95·5%), and 0·98 μg/mL (27·9%); abacavir AUC0-24 h was 17·7 h·μg/mL (38·8%), 19·8 h·μg/mL (50·6%), 15·1 h·μg/mL (40·3%), 17·4 h·μg/mL (19·4%), and 25·7 h·μg/mL (14·6%); lamivudine AUC0-24 h was 10·7 h·μg/mL (46·0%), 14·2 h·μg/mL (23·9%), 13·0 h·μg/mL (15·6%), 14·5 h·μg/mL (16·6%), and 21·7 h·μg/mL (26·2%), respectively. Pharmacokinetic targets and safety criteria were met within each weight band, and thus dosing of abacavir, dolutegravir, and lamivudine was confirmed at the originally selected doses. 54 (95%) of participants were treatment experienced and all who continued taking the study drug remained virologically suppressed (<200 copies per mL) through week 24. Virological suppression was achieved in two of three participants who were ART naive by week 24. There were no grade 3 or higher adverse events related to abacavir, dolutegravir, and lamivudine and no discontinuations because of toxicity to week 24. Both formulations were well tolerated. Dosing of abacavir, dolutegravir, and lamivudine was confirmed in children weighing 6 kg to less than 40 kg, and both FDC formulations were safe, well tolerated, and efficacious through 24 weeks of treatment. These findings support global efforts to expand the availability of FDC abacavir, dolutegravir, and lamivudine to children with HIV. National Institute of Allergy and Infectious Diseases, the Eunice Kennedy Shriver National Institute of Child Health and Human Development, the National Institute of Mental Health, ViiV Healthcare, and GlaxoSmithKline.

How fractionation procedure of mung bean protein affects transglutaminase crosslinking

Transglutaminase is often used to improve functional properties of plant proteins. Here, we report on the effects of the fractionation procedure on transglutaminase susceptibility of mung bean proteins at three length scales: molecular, colloidal and bulk. Dispersions of 4 wt% mung bean protein fractions were treated with transglutaminase. The four fractions were obtained through (1) dry fractionation (fine fraction); (2) dry fractionation with additional heating; (3) lab-scale wet fractionation; and (4) a commercial protein isolate. Analysis of rheological properties (at 9.5 wt% dry matter) revealed that transglutaminase crosslinking formed tougher materials when using the fine fraction, the heated fine fraction, and the wet fractionated isolate, while the rheological properties of the protein isolate were hardly changed. Proteins in all fractions were crosslinked by transglutaminase at molecular scale, as confirmed by an increase in large molecular weight bands in gel electrophoresis, but the extent of crosslinking depended on the fraction used. The molecular changes also resulted in altered physical chemical properties for some fractions, as revealed by protein dispersibility, confocal laser scanning microscopy, and particle size distribution. At the colloidal scale, transglutaminase crosslinking led to an increased particle size in the fine fraction and heated fine fraction, while particle sizes of the wet fractionated isolate and commercial protein isolate were mostly unaffected. The differences between the fractions were explained by three mechanistic crosslinking routes. To conclude, protein fractionation procedure greatly affected susceptibility to transglutaminase.

Production and evaluation of monovalent anti-snake immunoglobulins from chicken egg yolk using Ghanaian puff adder (Bitis arietans) Venom: Isolation, purification, and neutralization efficacy

Snakebites are rampant in Ghana, especially among the farmers, herdsmen, military recruits, hunters, and rural dwellers, and the antisnake venoms (ASV) use to treat these bites are not locally produced but rather imported, which come with a high cost, lack of constant supply and low specificity. The study was therefore aimed at isolating, purifying, and evaluating the efficacy of monovalent ASV from chicken egg yolk using puff adder (Bitis arietans) venom from Ghana. The major pathophysiological properties of the venom and the efficacy of the locally produced ASV were evaluated. The results showed that the snake venom (LD50 of 0.85 mg/kg body weight) had anticoagulant, haemorrhagic, and edematic activities in mice which were effectively neutralized using the purified egg yolk immunoglobulin Y (IgY), with two distinct molecular weight bands (∼70 and 25 kDa). The cross-neutralization studies also showed that the venom/IgY mixture (2.55 mg/kg body weight: 90 mg/kg body weight) offered 100% protection to the animals with ED50 of IgY being 22.66 mg/kg body weight. However, the applied dose (11.36 mg/kg body weight) of the available polyvalent ASV offered 25% protection compared with the 62% protection of the IgY at the same dose. The findings showed successful isolation and purification of a Ghanaian monovalent ASV with a better neutralization efficacy compared with the clinically available polyvalent drug.