Lessons learned from COVID-19 for clinical research operations in Italy: what have we learned and what can we apply in the future?

The coronavirus disease 2019 (COVID-19) pandemic has stressed the importance of health research as never before. In the specific domain of clinical research, the effort to rapidly find responses to health challenges and therapeutic hypotheses has highlighted the need for efficient, timely, ethically correct research. The guidelines published by the Agenzia Italiana del Farmaco have shown that some useful changes are feasible: simple and rapid methods have been implemented to conduct clinical research in the emergency conditions of the pandemic, maintaining high levels of quality. In this perspective, four Italian scientific associations operating in clinical research have worked together to evaluate which measures, among the ones implemented during the pandemic, have been particularly significant and potentially effective under normal conditions or in case of emergencies, and that therefore will be useful in the future as well.

Amifampridine phosphate in the treatment of muscle-specific kinase myasthenia gravis: a phase IIb, randomized, double-blind, placebo-controlled, double crossover study.

OBJECTIVE: The aim of this study is to determine the safety and the efficacy of amifampridine phosphate in muscle-specific kinase antibody-positive myasthenia gravis, in a 1:1 randomized, double-blind, placebo-controlled, switchback, double crossover study. METHODS: Eligible patients had muscle-specific kinase myasthenia gravis, >18 years of age, and Myasthenia Gravis Foundation of America class II-IV with a score of ⩾9 on Myasthenia Gravis Composite scale. After the run-in phase, during which amifampridine phosphate was titrated to a tolerable and effective dosage, patients were randomized to receive placebo-amifampridine-placebo sequence or amifampridine-placebo-amifampridine sequence daily for 7 days. Then, patients switched treatment arms twice, for a total of 21 days of double-blind treatment. Safety was determined by serial assessments of adverse events/serious adverse events, physical examinations, and clinical and laboratory tests. The co-primary outcome measures included changes from baseline of Quantitative Myasthenia Gravis score and Myasthenia Gravis-specific Activities of Daily Living Profile score. The secondary outcome measures comprised changes from baseline of Myasthenia Gravis Composite score, Myasthenia Gravis Quality of Life scale-15 questions, Fatigue Severity Scale, and Carlo Besta Neurological Institute-Myasthenia Gravis scale. Statistical analyses were assessed using a switchback model for three-period, two-treatment crossover design. RESULTS: A total of 10 patients were screened, enrolled, and treated. Transient paresthesias (60%) were the only amifampridine phosphate-related adverse events reported. Four patients were randomized to receive placebo-amifampridine-placebo sequence and three patients to receive amifampridine-placebo-amifampridine sequence. The co-primary objectives were statistically met (Quantitative Myasthenia Gravis score: p = 0.0003 and Myasthenia Gravis-specific Activities of Daily Living Profile score: p = 0.0006), as well as all the secondary endpoints (Myasthenia Gravis Composite score: p < 0.0001, Myasthenia Gravis Quality of Life scale-15 questions: p = 0.0025, Fatigue Severity Scale: p = 0.0061, and Carlo Besta Neurological Institute-Myasthenia Gravis scale: p = 0.0014). CONCLUSION: Despite the low number of patients, MuSK-001 study provided evidence that amifampridine phosphate, in the range of 30-60 mg daily dose, was safe and effective in treating muscle-specific kinase myasthenia gravis, suggesting the need for a large multi-center trial to confirm these results.

Blockade of A2A adenosine receptors prevents basic fibroblast growth factor-induced reactive astrogliosis in rat striatal primary astrocytes.

Previous literature data show that blockade of A(2A) adenosine receptors via selective antagonists induces protection in various models of neurodegenerative diseases. The mechanisms underlying this effect are still largely unknown. Since it is known that excessive reactive astrogliosis is a factor contributing to cell death in diseases characterized by neurodegenerative events, the present study has been aimed at determining whether selective A(2A) receptor antagonists can counteract the formation of reactive astrocytes induced in vitro by basic fibroblast growth factor (bFGF), a typical trigger of this reaction. Exposure of primary rat striatal astrocytes to the selective A(2A) antagonist SCH58261 resulted in concentration-dependent abolition of bFGF induction of astrogliosis in vitro. This effect could also be reproduced with the chemically unrelated A(2A) antagonist KW-6002. The direct activation of A(2A) adenosine receptors by selective receptor agonists was not sufficient per se to induce astrogliosis, suggesting that the A(2A) receptor needs to act in concert with other bFGF-induced genes to trigger the formation of reactive astrocytes. These results provide a mechanism at the basis of the neuroprotection induced by A(2A) receptor antagonists in models of brain damage and highlight this adenosine receptor subtype as a novel target for the pharmacological modulation of the gliotic reaction.

Induction of COX-2 and reactive gliosis by P2Y receptors in rat cortical astrocytes is dependent on ERK1/2 but independent of calcium signalling.

The present study has been aimed at characterizing the ATP/P2 receptor (and transductional pathways) responsible for the morphological changes induced in vitro by alphabetamethyleneATP on rat astrocytes obtained from cerebral cortex, a brain area highly involved in neurodegenerative diseases. Exposure of cells to this purine analogue resulted in elongation of cellular processes, an event reproducing in vitro a major hallmark of in vivo reactive gliosis. alphabetamethyleneATP-induced gliosis was prevented by the P2X/P2Y blocker pyridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid, but not by the selective P2X antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP, ruling out a role for ligand-gated P2X receptors. Conversely, the Gi/Go protein inactivator pertussis toxin completely prevented alphabetamethyleneATP-induced effects. No effects were induced by alphabetamethyleneATP on intracellular calcium concentrations. RT-PCR and western blot analysis showed that alphabetamethyleneATP-induced gliosis involves up-regulation of cyclooxygenase-2 (but not lipooxygenase). Also this effect was fully prevented by pyridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid. Experiments with inhibitors of mitogen-activated protein kinases (MAPK) suggest that extracellular signal regulated protein kinases (ERK)1/2 mediate both cyclooxygenase-2 induction and the associated in vitro gliosis. These findings suggest that purine-induced gliosis involves the activation of a calcium-independent G-protein-coupled P2Y receptor linked to ERK1/2 and cyclooxygenase-2. Based on the involvement of cyclooxygenase-2 and inflammation in neurodegenerative diseases, these findings open up new avenues in the identification of novel biological targets for the pharmacological manipulation of neurodegeneration.