Involvement of CB1 and CB2 receptors in neuroprotective effects of cannabinoids in experimental TDP-43 related frontotemporal dementia using male mice.

BACKGROUND: The elevation of endocannabinoid levels through inhibiting their degradation afforded neuroprotection in CaMKIIα-TDP-43 mice, a conditional transgenic model of frontotemporal dementia. However, which cannabinoid receptors are mediating these benefits is still pending to be elucidated. METHODS: We have investigated the involvement of the CB1 and the CB2 receptor using chronic treatments with selective ligands in CaMKIIα-TDP-43 mice, analysis of their cognitive deterioration with the Novel Object Recognition test, and immunostaining for neuronal and glial markers in two areas of interest in frontotemporal dementia. RESULTS: Our results confirmed the therapeutic value of activating either the CB1 or the CB2 receptor, with improvements in the animal performance in the Novel Object Recognition test, preservation of pyramidal neurons, in particular in the medial prefrontal cortex, and attenuation of glial reactivity, in particular in the hippocampus. In addition, the activation of both CB1 and CB2 receptors reduced the elevated levels of TDP-43 in the medial prefrontal cortex of CaMKIIα-TDP-43 mice, an effect exerted by mechanisms that are currently under investigation. CONCLUSIONS: These data reinforce the notion that the activation of CB1 and CB2 receptors may represent a promising therapy against TDP-43-induced neuropathology in frontotemporal dementia. Future studies will have to confirm these benefits, in particular with one of the selective CB2 agonists used here, which has been thoroughly characterized for clinical development.

A novel antibiotic class targeting the lipopolysaccharide transporter.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.

Safety, Tissue Distribution, and Metabolism of LNA-Containing Antisense Oligonucleotides in Rats.

Antisense oligonucleotides (ASOs) are chemically modified nucleic acids with therapeutic potential, some of which have been approved for marketing. We performed a study in rats to investigate mechanisms of toxicity after administration of 3 tool locked nucleic acid (LNA)-containing ASOs with differing established safety profiles. Four male rats per group were dosed once, 3, or 6 times subcutaneously, with 7 days between dosing, and sacrificed 3 days after the last dose. These ASOs were either unconjugated (naked) or conjugated with N-acetylgalactosamine for hepatocyte-targeted delivery. The main readouts were in-life monitoring, clinical and anatomic pathology, exposure assessment and metabolite identification in liver and kidney by liquid chromatography coupled to tandem mass spectrometry, ASO detection in liver and kidney by immunohistochemistry, in situ hybridization, immune electron microscopy, and matrix-assisted laser desorption/ionization mass spectrometry imaging. The highly toxic compounds showed the greatest amount of metabolites and a low degree of tissue accumulation. This study reveals different patterns of cell death associated with toxicity in liver (apoptosis and necrosis) and kidney (necrosis only) and provides new ultrastructural insights on the tissue accumulation of ASOs. We observed that the immunostimulatory properties of ASOs can be either primary from sequence-dependent properties or secondary to cell necrosis.

Clinical Micro-Dose Studies to Explore the Human Pharmacokinetics of Four Selective Inhibitors of Human Nav1.7 Voltage-Dependent Sodium Channels.

BACKGROUND: The emergence of genetic data linking Nav1.7 sodium channel over- and under- expression to human pain signalling has led to an interest in the treatment of chronic pain through inhibition of Nav1.7 channels. OBJECTIVE: We describe the pharmacokinetic (PK) results of a clinical microdose study performed with four potent and selective Nav1.7 inhibitors and the subsequent modelling resulting in the selection of a single compound to explore Nav1.7 pharmacology at higher doses. METHODS: A clinical microdose study to investigate the intravenous and oral PK of four compounds (PF-05089771, PF-05150122, PF-05186462 and PF-05241328) was performed in healthy volunteers. PK parameters were derived via noncompartmental analysis. A physiologically-based PK (PBPK) model was used to predict exposure and multiples of Nav1.7 50 % inhibitory concentration (IC50) for each compound at higher doses. RESULTS: Plasma clearance, volume of distribution and bioavailability ranged from 45 to 392 mL/min/kg, 13 to 36 L/kg and 38 to 110 %, respectively. The PBPK model for PF-05089771 predicted a 1 g oral dose would be required to achieve exposures of approximately 12× Nav1.7 IC50 at maximum concentration (C max), and approximately 3× IC50 after 12 h (minimum concentration [C min] for a twice-daily regimen). Lower multiples of Nav1.7 IC50 were predicted with the same oral doses of PF-05150122, PF-05186462, and PF-05241328. In a subsequent single ascending oral dose clinical study, the predictions for PF-05089771 compared well with observed data. CONCLUSION: Based on the human PK data obtained from the microdose study and subsequent modelling, PF-05089771 provided the best opportunity to explore Nav1.7 blockade for the treatment of acute or chronic pain conditions.

Feedback from a European Bioanalysis Forum survey on bioanalysis of drugs in tissues.

Tissue analysis has always been a difficult discipline of bioanalysis. Laboratories that perform bioanalysis in tissue are facing a lot of challenges and questions before starting experiments, from a scientific/technical point of view regarding more regulated aspects. Actually, literature is poor regarding the more technical and scientific aspects but also beyond that no clear guidance is available on this topic and laboratories performing tissue analysis face real ambiguity regarding regulatory requirements, always with the risk of under- or over-validation of the assay. For all of these reasons bioanalysis in tissue became a frequently discussed topic within the European Bioanalytical Forum (EBF) organization. The EBF then decided to treat this as a specific topic, and carried out a survey that was done in two steps between 2012 and 2013. This paper represents an exhaustive summary of the result of this survey that includes themost important aspects of tissue bioanalysis. This survey provided the team a good starting point for their discussions and resulted in an EBF recommendation paper published separately.

Recommendations from the European Bioanalysis Forum on method establishment for tissue homogenates.

Tissue analysis has always been a difficult discipline of bioanalysis. Differences in scientific approaches or level of adherence to regulated guidelines have led to a growing ambiguity on how to perform tissue analysis, an ambiguity that starts with the question of if we analyze tissue or tissue homogenates. The European Bioanalysis Forum (EBF) is proposing a recommendation on how to perform method establishment and analysis of tissue homogenates. The recommendation is based on broad discussions and survey data from the EBF community and, as for many EBF recommendations, focuses on finding the right balance between science, technology and regulations.

Efficient resolution of racemic N-benzyl beta3-amino acids by iterative liquid-liquid extraction with a chiral (salen)cobalt(III) complex as enantioselective selector.

The efficient (up to 93% ee) resolution of racemic N-benzyl beta(3)-amino acids has been achieved by an iterative (two cycle) liquid-liquid extraction process using a lipophilic chiral (salen)cobalt(III) complex [Co(III)(1)(OAc)]. As a result of the resolution by extraction, one enantiomer of the N-benzyl beta(3)-amino acid predominated in the aqueous phase, while the other enantiomer was driven into the organic phase by complexation to cobalt. The complexed amino acid was then quantitatively released into an aqueous phase, by a reductive (Co(III)--> Co(II)) counter-extraction using l-ascorbic acid. The reductive cleavage allowed for the recovery of the cobalt(II) selector in up to 90% yield (easily re-oxidable to Co(III) with air/AcOH).

Evaluation of water-compatible molecularly imprinted polymers as solid-phase extraction sorbents for the selective extraction of sildenafil and its desmethyl metabolite from plasma samples.

The evaluation of molecularly imprinted polymers (MIPs) as selective sorbents for the solid-phase extraction of sildenafil and its principal metabolite, desmethylsildenafil, was investigated. Two MIPs were synthesised using structural analogues of sildenafil as templates, and a comparison of the performance of the two MIP sorbents in organic and aqueous media was performed. Additionally, the feasibility of applying molecularly imprinted solid-phase extraction (MISPE) to the clean-up of plasma samples containing sildenafil and desmethylsildenafil was investigated. A preliminary, quantitative MISPE for the determination of both compounds in plasma was also performed. The results showed that the MIPs used for the selective extraction of sildenafil gave better compound recovery when aqueous samples were used in comparison to organic-based samples. A preliminary, quantitative MISPE of sildenafil and desmethylsildenafil indicated that the imprinted materials could be used successfully as SPE sorbents for sample pre-treatment for the determination of sildenafil, and related compounds, in plasma.

Enantiodifferentiation of N-benzyloxycarbonylaminophosphonic and phosphinic acids and their esters using cyclodextrins by means of capillary electrophoresis.

Capillary electrophoresis was successfully applied for separation of the enantiomers of N-benzyloxycarbonyl-alpha-aminophosphonic and alpha-aminophosphinic acids as well as their ethyl and phenyl monoesters with the use of a range of commercially available cyclodextrins (alpha, beta and hydroxypropyl-gamma-cyclodextrins) as chiral selectors. The dependence of effectiveness of separation on type and concentration of these chiral selectors as well as on pH of background electrolyte was examined in some detail.

Combination of supported liquid membrane and solid-phase extraction for sample pretreatment of triazine herbicides in juice prior to capillary electrophoresis determination.

A possibility of a combination of supported liquid membrane (SLM) and solid-phase extraction (SPE) for the determination of atrazine at microgram level in different type of fruit juices is presented. In comparison to SPE extraction from juice samples, the application of SLM-SPE enrichment provides much cleaner extracts and the possibility of lowering the limit of detection as low as 30 microg/l. However, it was also shown that by appropriate manipulation of SLM extraction conditions mainly flow-rate of donor phase and volume ratio between donor and acceptor phase, the level of detection can be further decreased to 10 microg/l. The results suggest that the application of SLM extraction prior to SPE is an alternative method for atrazine enrichment from complicated liquid matrices and could be used as routine method for the clean-up of such samples.

Separation of aromatic aminophosphonic acid enantiomers by capillary electrophoresis with the application of cyclodextrins.

The detailed studies concerning capillary electrophoresis separation of aminophosphonic acid enantiomers with various commercially available cyclodextrins are presented. The obtained results show that the separation of these stereoisomers is dependent on pH of background electrolyte, concentration of cyclodextrin as well as on the type of applied chiral selector. The separation mechanism is based on the co-operative effect of hydrogen bond type interactions enhanced by hydrophobic forces and sterical constrains between aminophosphonate and cyclodextrin. With application of elaborated method, enantiomeric baseline or partial separation of 18 alpha-aminophosphonic acids was achieved. This separation can be successfully used for routine aminophosphonic acids enantiopurity determination.