Splicing Mutations Impairing CDKL5 Expression and Activity Can be Efficiently Rescued by U1snRNA-Based Therapy.

Mutations in the CDKL5 gene lead to an incurable rare neurological condition characterized by the onset of seizures in the first weeks of life and severe intellectual disability. Replacement gene or protein therapies could represent intriguing options, however, their application may be inhibited by the recent demonstration that CDKL5 is dosage sensitive. Conversely, correction approaches acting on pre-mRNA splicing would preserve CDKL5 physiological regulation. Since ~15% of CDKL5 pathogenic mutations are candidates to affect splicing, we evaluated the capability of variants of the spliceosomal U1 small nuclear RNA (U1snRNA) to correct mutations affecting +1 and +5 nucleotides at the 5' donor splice site and predicted to cause exon skipping. Our results show that CDKL5 minigene variants expressed in mammalian cells are a valid approach to assess CDKL5 splicing pattern. The expression of engineered U1snRNA effectively rescued mutations at +5 but not at the +1 nucleotides. Importantly, we proved that U1snRNA-mediated splicing correction fully restores CDKL5 protein synthesis, subcellular distribution and kinase activity. Eventually, by correcting aberrant splicing of an exogenously expressed splicing-competent CDKL5 transgene, we provided insights on the morphological rescue of CDKL5 null neurons, reporting the first proof-of-concept of the therapeutic value of U1snRNA-mediated CDKL5 splicing correction.

Oncology drugs in the crosshairs of pharmaceutical crime.

Oncology drugs clearly have become a target for pharmaceutical crime. In 2016, falsified oncology drugs ranked fifth in the most commonly falsified drug category among the reports received by the Pharmaceutical Security Institute. Although the prevalence of illicit oncology drugs in the legal supply chains appears to be small, these drugs are difficult to detect, particularly in clinical practice. Forthcoming countermeasures to detect illicit drugs in high-income countries include compulsory antitampering devices and product verification technology for a risk-based selection of medicines. Health-care professionals must implement these new procedures into their workflow and remain vigilant about those medicines that are not selected. Although countermeasures should firmly tighten supply chain security, there are concerns about how quickly pharmaceutical crime will adapt to these protections. Because patients and health-care professionals have shown a lenient attitude towards purchasing medicines from unreliable sources, measures against the highly accessible illegal medicine supply chain remain necessary. To improve detectability in clinical practice, reporting of ineffectiveness and unusual drug effects as adverse events or adverse drug reactions is essential.

Analysis of illicit dietary supplements sold in the Italian market: identification of a sildenafil thioderivative as adulterant using UPLC-TOF/MS and GC/MS.

Identification of pharmaceutical active ingredients sildenafil and tadalafil and the characterization of a dimethylated thio-derivative of sildenafil, called thioaildenafil or thiodimethylsildenafil, in illicit dietary supplements were described. A multi-residual ultra-performance liquid chromatography-time of flight mass spectrometry (UPLC-TOF/MS) method was developed to screen for the presence of the phosphodiesterase-5 (PDE-5) inhibitors sildenafil, tadalafil, and vardenafil and their analogues thioaildenafil and thiohomosildenafil in powders and pharmaceutical dosage forms. The study was developed in connection with an operation supervised by the Italian Medicines Agency (A.I.F.A.), aimed to monitor dietary supplements in the Italian market. In two of the eleven specimens under investigation, high-resolution mass spectrometry (HR-MS) allowed the identification of the PDE-5 inhibitors sildenafil and tadalafil, while another specimen proved to contain a unapproved dimethylated thioderivative of sildenafil, thioaildenafil or thiodimethylsildenafil, identified for the first time in Italy as adulterant in food supplements.

New regulatory strategies to manage medicines shortages in Europe.

Medicine shortages have been spreading in European countries. In many cases, the unavailability of medicinal products has a substantial impact on the capability of National Healthcare Systems in ensuring the continuity of care. Shortages originate from multifactorial causes. In particular, they can be due to supply-related factors (e.g., manufacturing issues, regulatory issues, logistics, distribution) and demand-related ones (e.g., fluctuating drug demand, parallel market, tendering, price and reimbursement policies). However, some extraordinary geopolitical events (e.g., Brexit) may also affect medicines' availability. The capability of European Regulatory Authorities and other stakeholders, which are involved in the pharmaceutical distribution chain and the healthcare assistance services, to define suitable problem-solving strategies has been limited for years by the fragmentation of the European regulatory framework, starting from the lack of a univocal definition of a medicine shortage. Only in 2019, the EMA and HMA joint task force released the first harmonized "shortage" definition in the European Economic Area (EEA) and guidance on public communication. This manuscript aims to review the current European regulatory framework on medicine shortages. To support the activities of regulators, manufacturers and other healthcare professionals, an algorithm was also proposed to be used as a harmonized procedure to determine the shortage/unavailability impact on public health and to rationalize the problem-solving strategies adopted in all different settings.