Inorganic Chemistry of the Tripodal Picolinate Ligand Tpaa with Gallium(III) and Radiolabeling with Gallium-68

We report here the improved synthesis of the tripodal picolinate chelator Tpaa, with an overall yield of 41% over five steps, in comparison to the previously reported 6% yield. Tpaa was investigated for its coordination chemistry with Ga(III) and radiolabeling properties with gallium-68 (68Ga). The obtained crystal structure for [Ga(Tpaa)] shows that the three picolinate arms coordinate to the Ga(III) ion, fully occupying the octahedral coordination geometry. This is supported by 1H NMR which shows that the three arms are symmetrical when coordinated to Ga(III). Assessment of the thermodynamic stability through potentiometry gives log KGa-Tpaa = 21.32, with a single species being produced across the range of pH 3.5-7.5. Tpaa achieved >99% radiochemical conversion with 68Ga under mild conditions ([Tpaa] = 6.6 μM, pH 7.4, 37 °C) with a molar activity of 3.1 GBq μmol-1. The resulting complex, [68Ga][Ga(Tpaa)], showed improved stability over the previously reported [68Ga][Ga(Dpaa)(H2O)] in a serum challenge, with 32% of [68Ga][Ga(Tpaa)] remaining intact after 30 min of incubation with fetal bovine serum

Amino acid based gallium-68 chelators capable of radiolabeling at neutral pH

Gallium-68 ( 68 Ga) has been the subject of increasing interest for its potential in the production of radiotracers for diagnosis of diseases. In this work we report the complexation of 68 Ga by the amino acid based tripodal chelate H 3 Dpaa, and two bifunctional derivatives, H 3 Dpaa.dab and H 4 Dpaa.ga, under a range of conditions with particular emphasis on the rapid complexation of 68 Ga at pH 7.4. 100 μM H 3 Dpaa achieved a radiochemical yield of 95% at pH 7.4 in 5 minutes at 37 °C. The bifunctional derivatives H 4 Dpaa.ga and H 3 Dpaa.dab achieved 94% and 84% radiochemical yields, respectively, under the same conditions. The resulting Ga(iii) complexes show thermodynamic stabilities of logK GaDpaa = 18.53, logK GaDpaa.dab = 22.08, logK GaDpaa.ga = 18.36. Unfortunately, the resulting radiolabelled species do not present sufficient serum stability for in vivo application. Herein we show a flexible synthesis for bifunctional chelators based on amino acids that rapidly complex 68 Ga under physiological conditions

SMN Protein Can Be Reliably Measured in Whole Blood with an Electrochemiluminescence (ECL) Immunoassay: Implications for Clinical Trials

Spinal muscular atrophy (SMA) is caused by defects in the survival motor neuron 1 (SMN1) gene that encodes survival motor neuron (SMN) protein. The majority of therapeutic approaches currently in clinical development for SMA aim to increase SMN protein expression and there is a need for sensitive methods able to quantify increases in SMN protein levels in accessible tissues. We have developed a sensitive electrochemiluminescence (ECL)-based immunoassay for measuring SMN protein in whole blood with a minimum volume requirement of 5μL. The SMN-ECL immunoassay enables accurate measurement of SMN in whole blood and other tissues. Using the assay, we measured SMN protein in whole blood from SMA patients and healthy controls and found that SMN protein levels were associated with SMN2 copy number and were greater in SMA patients with 4 copies, relative to those with 2 and 3 copies. SMN protein levels did not vary significantly in healthy individuals over a four-week period and were not affected by circadian rhythms. Almost half of the SMN protein was found in platelets. We show that SMN protein levels in C/C-allele mice, which model a mild form of SMA, were high in neonatal stage, decreased in the first few weeks after birth, and then remained stable throughout the adult stage. Importantly, SMN protein levels in the CNS correlated with SMN levels measured in whole blood of the C/C-allele mice. These findings have implications for the measurement of SMN protein induction in whole blood in response to SMN-upregulating therapy

Phase II Open Label Study of Valproic Acid in Spinal Muscular Atrophy

UNLABELLED:Preliminary in vitro and in vivo studies with valproic acid (VPA) in cell lines and patients with spinal muscular atrophy (SMA) demonstrate increased expression of SMN, supporting the possibility of therapeutic benefit. We performed an open label trial of VPA in 42 subjects with SMA to assess safety and explore potential outcome measures to help guide design of future controlled clinical trials. Subjects included 2 SMA type I ages 2-3 years, 29 SMA type II ages 2-14 years and 11 type III ages 2-31 years, recruited from a natural history study. VPA was well-tolerated and without evident hepatotoxicity. Carnitine depletion was frequent and temporally associated with increased weakness in two subjects. Exploratory outcome measures included assessment of gross motor function via the modified Hammersmith Functional Motor Scale (MHFMS), electrophysiologic measures of innervation including maximum ulnar compound muscle action potential (CMAP) amplitudes and motor unit number estimation (MUNE), body composition and bone density via dual-energy X-ray absorptiometry (DEXA), and quantitative blood SMN mRNA levels. Clear decline in motor function occurred in several subjects in association with weight gain; mean fat mass increased without a corresponding increase in lean mass. We observed an increased mean score on the MHFMS scale in 27 subjects with SMA type II (p<or=0.001); however, significant improvement was almost entirely restricted to participants <5 years of age. Full length SMN levels were unchanged and Delta7SMN levels were significantly reduced for 2 of 3 treatment visits. In contrast, bone mineral density (p<or=0.0036) and maximum ulnar CMAP scores (p<or=0.0001) increased significantly. CONCLUSIONS:While VPA appears safe and well-tolerated in this initial pilot trial, these data suggest that weight gain and carnitine depletion are likely to be significant confounding factors in clinical trials. This study highlights potential strengths and limitations of various candidate outcome measures and underscores the need for additional controlled clinical trials with VPA targeting more restricted cohorts of subjects. TRIAL REGISTRATION:ClinicalTrials.gov

SMA CARNI-VAL Trial Part I: Double-Blind, Randomized, Placebo-Controlled Trial of L-Carnitine and Valproic Acid in Spinal Muscular Atrophy

Valproic acid (VPA) has demonstrated potential as a therapeutic candidate for spinal muscular atrophy (SMA) in vitro and in vivo.Two cohorts of subjects were enrolled in the SMA CARNIVAL TRIAL, a non-ambulatory group of "sitters" (cohort 1) and an ambulatory group of "walkers" (cohort 2). Here, we present results for cohort 1: a multicenter phase II randomized double-blind intention-to-treat protocol in non-ambulatory SMA subjects 2-8 years of age. Sixty-one subjects were randomized 1:1 to placebo or treatment for the first six months; all received active treatment the subsequent six months. The primary outcome was change in the modified Hammersmith Functional Motor Scale (MHFMS) score following six months of treatment. Secondary outcomes included safety and adverse event data, and change in MHFMS score for twelve versus six months of active treatment, body composition, quantitative SMN mRNA levels, maximum ulnar CMAP amplitudes, myometry and PFT measures.At 6 months, there was no difference in change from the baseline MHFMS score between treatment and placebo groups (difference = 0.643, 95% CI = -1.22-2.51). Adverse events occurred in >80% of subjects and were more common in the treatment group. Excessive weight gain was the most frequent drug-related adverse event, and increased fat mass was negatively related to change in MHFMS values (p = 0.0409). Post-hoc analysis found that children ages two to three years that received 12 months treatment, when adjusted for baseline weight, had significantly improved MHFMS scores (p = 0.03) compared to those who received placebo the first six months. A linear regression analysis limited to the influence of age demonstrates young age as a significant factor in improved MHFMS scores (p = 0.007).This study demonstrated no benefit from six months treatment with VPA and L-carnitine in a young non-ambulatory cohort of subjects with SMA. Weight gain, age and treatment duration were significant confounding variables that should be considered in the design of future trials.Clinicaltrials.gov NCT00227266

High Interlaboratory Reproducibility and Accuracy of Next-Generation-Sequencing- Based Bacterial Genotyping in a Ring Trial

Today, next-generation whole-genome sequencing (WGS) is increasingly used to determine the genetic relationships of bacteria on a nearly whole-genome level for infection control purposes and molecular surveillance. Here, we conducted a multicenter ring trial comprising five laboratories to determine the reproducibility and accuracy of WGS-based typing. The participating laboratories sequenced 20 blind-coded Staphylococcus aureus DNA samples using 250-bp paired-end chemistry for library preparation in a single sequencing run on an Illumina MiSeq sequencer. The run acceptance criteria were sequencing outputs >5.6 Gb and Q30 read quality scores of > 75%. Subsequently, spa typing, multilocus sequence typing (MLST), ribosomal MLST, and core genome MLST (cgMLST) were performed by the participants. Moreover, discrepancies in cgMLST target sequences in comparisons with the included and also published sequence of the quality control strain ATCC 25923 were resolved using Sanger sequencing. All five laboratories fulfilled the run acceptance criteria in a single sequencing run without any repetition. Of the 400 total possible typing results, 394 of the reported spa types, sequence types (STs), ribosomal STs (rSTs), and cgMLST cluster types were correct and identical among all laboratories; only six typing results were missing. An analysis of cgMLST allelic profiles corroborated this high reproducibility; only 3 of 183,927 (0.0016%) cgMLST allele calls were wrong. Sanger sequencing confirmed all 12 discrepancies of the ring trial results in comparison with the published sequence of ATCC 25923. In summary, this ring trial demonstrated the high reproducibility and accuracy of current next-generation sequencing-based bacterial typing for molecular surveillance when done with nearly completely locked-down methods

A single-pot template reaction towards a manganese-based T1 contrast agent

Manganese-based contrast agents (MnCAs) have emerged as suitable alternatives to gadolinium-based contrast agents (GdCAs). However, due to their kinetic lability and laborious synthetic procedures, only a few MnCAs have found clinical MRI application. In this work, we have employed a highly innovative single-pot template synthetic strategy to develop a MnCA, MnL Me, and studied the most important physicochemical properties in vitro. MnL Me displays optimized r 1 relaxivities at both medium (20 and 64 MHz) and high magnetic fields (300 and 400 MHz) and an enhanced r 1 b=21.1 mM −1 s −1 (20 MHz, 298 K, pH 7.4) upon binding to BSA (K a=4.2×10 3 M −1). In vivo studies show that MnL Me is cleared intact into the bladder through renal excretion and has a prolonged blood half-life compared to the commercial GdCA Magnevist. MnL Me shows great promise as a novel MRI contrast agent

Analysis of mortality metrics associated with a comprehensive range of disorders in Denmark, 2000 to 2018: A population-based cohort study

Background: The provision of different types of mortality metrics (e.g., mortality rate ratios [MRRs] and life expectancy) allows the research community to access a more informative set of health metrics. The aim of this study was to provide a panel of mortality metrics associated with a comprehensive range of disorders and to design a web page to visualize all results. Methods and findings: In a population-based cohort of all 7,378,598 persons living in Denmark at some point between 2000 and 2018, we identified individuals diagnosed at hospitals with 1,803 specific categories of disorders through the International Classification of Diseases-10th Revision (ICD-10) in the National Patient Register. Information on date and cause of death was obtained from the Registry of Causes of Death. For each of the disorders, a panel of epidemiological and mortality metrics was estimated, including incidence rates, age-of-onset distributions, MRRs, and differences in life expectancy (estimated as life years lost [LYLs]). Additionally, we examined models that adjusted for measures of air pollution to explore potential associations with MRRs. We focus on 39 general medical conditions to simplify the presentation of results, which cover 10 broad categories: circulatory, endocrine, pulmonary, gastrointestinal, urogenital, musculoskeletal, hematologic, mental, and neurologic conditions and cancer. A total of 3,676,694 males and 3,701,904 females were followed up for 101.7 million person-years. During the 19-year follow-up period, 1,034,273 persons (14.0%) died. For 37 of the 39 selected medical conditions, mortality rates were larger and life expectancy shorter compared to the Danish general population. For these 37 disorders, MRRs ranged from 1.09 (95% confidence interval [CI]: 1.09 to 1.10) for vision problems to 7.85 (7.77 to 7.93) for chronic liver disease, while LYLs ranged from 0.31 (0.14 to 0.47) years (approximately 16 weeks) for allergy to 17.05 (16.95 to 17.15) years for chronic liver disease. Adjustment for air pollution had very little impact on the estimates; however, a limitation of the study is the possibility that the association between the different disorders and mortality could be explained by other underlying factors associated with both the disorder and mortality. Conclusions: In this study, we show estimates of incidence, age of onset, age of death, and mortality metrics (both MRRs and LYLs) for a comprehensive range of disorders. The interactive data visualization site (https://nbepi.com/atlas) allows more fine-grained analysis of the link between a range of disorders and key mortality estimates.publishedVersio