Panorama of the distal myopathies

Distal myopathies are genetic primary muscle disorders with a prominent weakness at onset in hands and/or feet. The age of onset (from early childhood to adulthood), the distribution of muscle weakness (upper versus lower limbs) and the histological findings (ranging from nonspecific myopathic changes to myofibrillar disarrays and rimmed vacuoles) are extremely variable. However, despite being characterized by a wide clinical and genetic heterogeneity, the distal myopathies are a category of muscular dystrophies: genetic diseases with progressive loss of muscle fibers. Myopathic congenital arthrogryposis is also a form of distal myopathy usually caused by focal amyoplasia. Massive parallel sequencing has further expanded the long list of genes associated with a distal myopathy, and contributed identifying as distal myopathy-causative rare variants in genes more often related with other skeletal or cardiac muscle diseases. Currently, almost 20 genes (ACTN2, CAV3, CRYAB, DNAJB6, DNM2, FLNC, HNRNPA1, HSPB8, KHLH9, LDB3, MATR3, MB, MYOT, PLIN4, TIA1, VCP, NOTCH2NLC, LRP12, GIPS1) have been associated with an autosomal dominant form of distal myopathy. Pathogenic changes in four genes (ADSSL, ANO5, DYSF, GNE) cause an autosomal recessive form; and disease-causing variants in five genes (DES, MYH7, NEB, RYR1 and TTN) result either in a dominant or in a recessive distal myopathy. Finally, a digenic mechanism, underlying a Welander-like form of distal myopathy, has been recently elucidated. Rare pathogenic mutations in SQSTM1, previously identified with a bone disease (Paget disease), unexpectedly cause a distal myopathy when combined with a common polymorphism in TIA1. The present review aims at describing the genetic basis of distal myopathy and at summarizing the clinical features of the different forms described so far. ©2020 Gaetano Conte Academy - Mediterranean Society of Myology, Naples, Italy.Peer reviewe

Metabolism of a Bioorthogonal PET Tracer Candidate [F-19/F-18]SiFA-Tetrazine in Mouse Liver Microsomes: Biotransformation Pathways and Defluorination Investigated by UHPLC-HRMS

Organofluorosilicon based F-18-radiolabeling is an efficient method for incorporating fluorine-18 into BF-radiopharmaceuticals for positron emission tomography (PET) by F-19/F-18 isotopic exchange (1E). The first PET radiopharmaceutical, F-18 SiFAlin-TATE, radiolabeled with a silicon-based [F-18]fluoride acceptor (SiFA), namely, a para-substituted di-tert-butyl[F-18]-fluorosilylbenzene, has entered clinical trials, and is paving the way for other potential [F-18]SiFA-labeled radiopharmaceuticals for diagnostic use. In this study, we report the in vitro metabolism of an oxime-linked SiFA tetrazine (SiFA-Tz), a new PET-radiotracer candidate, recently evaluated for pretargeted PET imaging and macromolecule labeling. Metabolism of SiFA-Tz was studied in mouse liver microsomes (MLM) for elucidating its major biotransformation pathways. Nontargeted screening by ultrahigh performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) was utilized for detection of unknown metabolites. The oxime bond between the SiFA and Tz groups forms two geometric (E/Z) isomers, which underwent the same biotransformations, but unexpectedly with different kinetics. In total, nine proposed metabolites of SiFA-Tz from phase I and II reactions were detected, five of which were defluorinated in MLMs, elucidating the metabolic pathway leading to previously reported defluorination of [F-18]SiFA-Tz in vivo. Based on the HRMS studies a biotransformation pathway is proposed: hydroxylation (+O) to tert-butyl group adjacent to the silicon, followed by oxidative defluorination (+OH/-F) cleaving the fluorine off the silicon. Interestingly, eight proposed metabolites of a reduced dihydrotetrazine analogue, SiFA-H,Tz, from phase I and II reactions were additionally detected. To the best of our knowledge, this is the first reported comprehensive investigation of enzyme mediated metabolic pathway of tetrazines and para-substituted di-tertbutylfluorosilylbenzene fluoride acceptors, providing novel structural information on the biotransformation and fragmentation patterns of radiotracers bearing these structural motifs. By investigating the metabolism preceding defluorination, structurally optimized new SiFA compounds can be designed for expanding the portfolio of efficient F-19/F-18 isotopic exchange labeling probes for PET imaging

A Theranostic Cellulose Nanocrystal-based Drug Delivery System with Enhanced Retention in Pulmonary Metastasis of Melanoma

Metastatic melanoma can be difficult to detect until at the advanced state that decreases the survival rate of patients. Several FDA-approved BRAF inhibitors have been used for treatment of metastatic melanoma, but overall therapeutic efficacy has been limited. Lutetium-177 (Lu-177) enables simultaneous tracking of tracer accumulation with single-photon emission computed tomography and radiotherapy. Therefore, the codelivery of Lu-177 alongside chemotherapeutic agents using nanoparticles (NPs) might improve the therapeutic outcome in metastatic melanoma. Cellulose nanocrystals (CNC NPs) can particularly deliver payloads to lung capillaries in vivo. Herein, Lu-177-labeled CNC NPs loaded with vemurafenib ([Lu-177]Lu-CNC-V NPs) is developed and the therapeutic effect in BRAF V600E mutation-harboring YUMM1.G1 murine model of lung metastatic melanoma is investigated. The [Lu-177]Lu-CNC-V NPs demonstrate favorable radiolabel stability, drug release profile, cellular uptake, and cell growth inhibition in vitro. In vivo biodistribution reveals significant retention of the [Lu-177]Lu-CNC-V NPs in the lung, liver, and spleen. Ultimately, the median survival time of animals is doubly increased after treatment with [Lu-177]Lu-CNC-V NPs compared to control groups. The enhanced therapeutic efficacy of [Lu-177]Lu-CNC-V NPs in the lung metastatic melanoma animal model provides convincing evidence for the potential of clinical translation for theranostic CNC NP-based drug delivery systems after intravenous administration.Peer reviewe

Biocompatibility of Thermally Hydrocarbonized Porous Silicon Nanoparticles and their Biodistribution in Rats

Peer reviewe

Synthesis, in vitro and in vivo evaluation of 1,3,5-triazines as cannabinoid CB2 receptor agonists

The cannabinoid receptors type 2 (CBR2) are attractive therapeutic targets of the endocannabinoid signaling system (ECS) as they are not displaying the undesired psychotropic and cardiovascular side-effects seen with cannabinoid receptor type 1 (CB1R) agonists. In continuation of our previous work on 2,4,6-trisubstituted 1,3,5-triazines as potent CB2 agonists, we synthesized an additional series of more polar analogues (1-10), which were found to possess high CB2R agonist activity with enhanced water solubility. The most potent compound in the series was N-(adamantan-1-yl)-4-ethoxy-6-(4-(2-fluoroethyl)piperazin-1-yl)-1,3,5-triazin-2-amine (9) with EC50 value of 0.60nM. To further evaluate the biological effects of the compounds, the selected compounds were tested in vitro against four different cell lines. A human retinal pigment epithelial cell line (ARPE-19) was used to evaluate the cytotoxicity of the compounds whereas an androgen-sensitive human prostate adenocarcinoma cell line (LNCaP), a Jurkat leukemia cell line and a C8161 melanoma cell line were used to assess the antiproliferative activity of the compounds. The most interesting results were obtained for N-(adamantan-1-yl)-4-ethoxy-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (6), which induced cell viability decrease in prostate and leukemia cell lines, and diminished proliferation of C8161 melanoma cells. The results could be reversed in leukemia cells with the selective CB2R antagonist AM630, whereas in prostate cells the AM630 induced a significant cell viability decrease with a mechanism probably unlinked to CB2 cannabinoid receptor. The antiproliferative effect of 6 on the melanoma cells seemed not to be mediated via the CB1R or CB2R. No cytotoxicity was detected against ARPE-19 cell line at concentrations of 1 and 10μM for compound 6. However, at 30μM concentration the compound 6 decreased the cell viability. Finally, in order to estimate in vivo behavior of these compounds, (18)F labeled PET ligand, N-cyclopentyl-4-ethoxy-6-(4-(2-fluoro-18-ethyl)piperazin-1-yl)-1,3,5-triazin-2-amine ([(18)F]5), was synthesized and its biodistribution was determined in healthy male Sprague-Dawley rats. As a result, the tracer showed a rapid (<15min) elimination in urine accompanied by a slower excretion via the hepatobiliary route. In conclusion, we further demonstrated that 1,3,5-triazine scaffold serves as a suitable template for the design of highly potent CB2R agonists with reasonable water solubility properties. The compounds may be useful when studying the role of the endocannabinoid system in different diseases. The triazine scaffold is also a promising candidate for the development of new CB2R PET ligands

Pretargeting of internalizing trastuzumab and cetuximab with a 18F-tetrazine tracer in xenograft models

Pretargeting-based approaches are being investigated for radioimmunoimaging and therapy applications to reduce the effective radiation burden to the patient. To date, only a few studies have used short-lived radioisotopes for pretargeting of antibodies, and such examples with internalizing antibodies are even rarer. Herein, we have investigated pretargeting methodology using inverse electron-demand Diels-Alder (IEDDA) for tracing two clinically relevant, internalizing monoclonal antibodies, cetuximab and trastuzumab. Bioorthogonal reaction between tetrazine and trans-cyclooctene (TCO) was used for tracing cetuximab and trastuzumab in vivo with a fluorine-18 (t (A 1/2) = 109.8 min) labelled tracer. TCO-cetuximab or TCO-trastuzumab was administered 24, 48, or 72 h prior to the injection of tracer to A431 or BT-474 tumour-bearing mice, respectively. With cetuximab, the highest tumour-to-blood ratios were achieved when the lag time between antibody and tracer injections was 72 h. With trastuzumab, no difference was observed between different lag times. For both antibodies, the tumour could be clearly visualized in the PET images with the highest tumour uptake of 3.7 +/- 0.1%ID/g for cetuximab and 1.5 +/- 0.1%ID/g for trastuzumab as quantified by ex vivo biodistribution. In vivo IEDDA reaction was observed in the blood for both antibodies, but with trastuzumab, this was to a much lower degree than with cetuximab. We could successfully visualize the tumours by using cetuximab and trastuzumab in pretargeted PET imaging despite the challenging circumstances where the antibody is internalized and there is still some unbound antibody circulating in the blood flow. This clearly demonstrates the potential of a pretargeted approach for targeting internalizing antigens and warrants development of pharmacokinetic optimization of the biorthogonal reactants to this end.Peer reviewe

Pretargeting of internalizing trastuzumab and cetuximab with a 18F-tetrazine tracer in xenograft models

Background: Pretargeting-based approaches are being investigated for radioimmunoimaging and therapy applications to reduce the effective radiation burden to the patient. To date, only a few studies have used short-lived radioisotopes for pretargeting of antibodies, and such examples with internalizing antibodies are even rarer. Herein, we have investigated pretargeting methodology using inverse electron-demand Diels-Alder (IEDDA) for tracing two clinically relevant, internalizing monoclonal antibodies, cetuximab and trastuzumab. Results: Bioorthogonal reaction between tetrazine and trans-cyclooctene (TCO) was used for tracing cetuximab and trastuzumab in vivo with a fluorine-18 (t½ = 109.8 min) labelled tracer. TCO-cetuximab or TCO-trastuzumab was administered 24, 48, or 72 h prior to the injection of tracer to A431 or BT-474 tumour-bearing mice, respectively. With cetuximab, the highest tumour-to-blood ratios were achieved when the lag time between antibody and tracer injections was 72 h. With trastuzumab, no difference was observed between different lag times. For both antibodies, the tumour could be clearly visualized in the PET images with the highest tumour uptake of 3.7 ± 0.1%ID/g for cetuximab and 1.5 ± 0.1%ID/g for trastuzumab as quantified by ex vivo biodistribution. In vivo IEDDA reaction was observed in the blood for both antibodies, but with trastuzumab, this was to a much lower degree than with cetuximab. Conclusions: We could successfully visualize the tumours by using cetuximab and trastuzumab in pretargeted PET imaging despite the challenging circumstances where the antibody is internalized and there is still some unbound antibody circulating in the blood flow. This clearly demonstrates the potential of a pretargeted approach for targeting internalizing antigens and warrants development of pharmacokinetic optimization of the biorthogonal reactants to this end

Exploring the Biochemical Foundations of a Successful GLUT1-Targeting Strategy to BNCT: Chemical Synthesis and In Vitro Evaluation of the Entire Positional Isomer Library of ortho-Carboranylmethyl-Bearing Glucoconjugates

Boron neutron capture therapy (BNCT) is a noninvasive binary therapeutic modality applicable to the treatment of cancers. While BNCT offers a tumor-targeting selectivity that is difficult to match by other means, the last obstacles preventing the full harness of this potential come in the form of the suboptimal boron delivery strategies presently used in the clinics. To address these challenges, we have developed delivery agents that target the glucose transporter GLUT1. Here, we present the chemical synthesis of a number of ortho-carboranylmethyl-substituted glucoconjugates and the biological assessment of all positional isomers. Altogether, the study provides protocols for the synthesis and structural characterization of such glucoconjugates and insights into their essential properties, for example, cytotoxicity, GLUT1-affinity, metabolism, and boron delivery capacity. In addition to solidifying the biochemical foundations of a successful GLUT1-targeting approach to BNCT, we identify the most promising modification sites in d-glucose, which are critical in order to further develop this strategy toward clinical use.Peer reviewe

Dominant Distal Myopathy 3 (MPD3) Caused by a Deletion in the HNRNPA1 Gene

Background and Objectives To determine the genetic cause of the disease in the previously reported family with adult-onset autosomal dominant distal myopathy (myopathy, distal, 3; MPD3). Methods Continued clinical evaluation including muscle MRI and muscle pathology. A linkage analysis with single nucleotide polymorphism arrays and genome sequencing were used to identify the genetic defect, which was verified by Sanger sequencing. RNA sequencing was used to investigate the transcriptional effects of the identified genetic defect. Results Small hand muscles (intrinsic, thenar, and hypothenar) were first involved with spread to the lower legs and later proximal muscles. Dystrophic changes with rimmed vacuoles and cytoplasmic inclusions were observed in muscle biopsies at advanced stage. A single nucleotide polymorphism array confirmed the previous microsatellite-based linkage to 8p22-q11 and 12q13-q22. Genome sequencing of three affected family members combined with structural variant calling revealed a small heterozygous deletion of 160 base pairs spanning the second last exon 10 of the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) gene, which is in the linked region on chromosome 12. Segregation of the mutation with the disease was confirmed by Sanger sequencing. RNA sequencing showed that the mutant allele produces a shorter mutant mRNA transcript compared with the wild-type allele Immunofluorescence studies on muscle biopsies revealed small p62 and larger TDP-43 inclusions. Discussion A small exon 10 deletion in the gene HNRNPA1 was identified as the cause of MPD3 in this family. The new HNRNPA1-related phenotype, upper limb presenting distal myopathy, was thus confirmed, and the family displays the complexities of gene identification.Peer reviewe

PET Imaging of Extracellular pH in Tumors with \u3csup\u3e64\u3c/sup\u3eCu- and \u3csup\u3e18\u3c/sup\u3eF-Labeled pHLIP Peptides: A Structure–Activity Optimization Study

pH (low) insertion peptides (pHLIP peptides) target acidic extracellular environments in vivo due to pH-dependent cellular membrane insertion. Two variants (Var3 and Var7) and wild-type (WT) pHLIP peptides have shown promise for in vivo imaging of breast cancer. Two positron emitting radionuclides (64Cu and 18F) were used to label the NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivo biodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. All of the constructs were radiolabeled with 64Cu or [18F]-AlF in good yield. The in vivo biodistribution of the 12 constructs in 4T1 orthotopic allografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeled with either 18F (4T1 uptake; 8.9 ± 1.7%ID/g at 4 h p.i.) or 64Cu (4T1 uptake; 8.2 ± 0.9%ID/g at 4 h p.i. and 19.2 ± 1.8% ID/g at 24 h p.i.), shows the most promise for clinical translation. Additional studies to investigate other tumor models (melanoma, prostate, and brain tumor models) indicated the universality of tumor targeting of these tracers. From this study, future clinical translation will focus on 18F- or 64Cu-labeled NO2A-cysVar3