Effect of zamicastat on blood pressure and heart rate response to cold pressor test: A double-blind, randomized, placebo-controlled study in healthy subjects.

AIMS: Dopamine beta-hydroxylase (DßH) inhibitors, like zamicastat, hold promise for treating pulmonary arterial hypertension. This study aimed to validate the mechanism of action of zamicastat by studying its effect on the overdrive of the sympathetic nervous system (SNS). METHODS: A single-centre, prospective, double-blind, randomized, placebo-controlled, crossover study evaluated the effect of 400 mg zamicastat in 22 healthy male subjects. Cold pressor test (CPT) was performed at screening and each treatment period on Days -1 and 10. Plasma and 24 h-urine levels of dopamine (DA), epinephrine (EPI) and norepinephrine (NE), and plasma DßH activity, were measured. RESULTS: Compared to placebo, zamicastat showed a - 4.62 mmHg decrease in systolic blood pressure during the cold stimulus vs. rest phases on Day 10 of CPT (P = .020). Zamicastat decreased mean arterial pressure response to cold stimulus during CPT (-2.62 mmHg; P = .025). At Day 10, zamicastat significantly increased plasma DA, before CPT (12.63 ng/L; P = .040) and after CPT (19.22 ng/L; P = .001) as well as the estimated plasma EPI change from baseline after CPT (P = .040). Inhibition of plasma DßH activity ranged from 19.8% to 25.0%. At Day 10, significant reductions in 24-h urinary excretion of EPI (P = .002) and NE (P = .001) were observed. Zamicastat Cτ geometric mean ± GSD ranged from 45.86 ± 1.46 ng/mL on Day 3 to 58.64 ± 1.52 ng/mL on Day 10, with moderate inter-individual variability (CV: 32.6%-36.6%). Steady state was already achieved on Day 6. CONCLUSIONS: Our results demonstrated the effect of zamicastat on the overdrive sympathetic response to cold stimulus, confirming its potential as SNS modulator.

Drug-Drug Interaction between Oral Zamicastat and Continuous Epoprostenol Infusion at Steady-State Conditions in Healthy Subjects.

This study intended to evaluate the interactions between zamicastat and epoprostenol in healthy human subjects. This was a single-center, open-label, two-period study. In period 1, epoprostenol 8 ng/kg/min was administered alone. In period 2, epoprostenol 8 ng/kg/min was administered following an 8-day treatment with zamicastat. Since the initial dose of epoprostenol showed to be insufficiently tolerated, it was decreased to 6 ng/kg/min. Blood samples were collected to determine the metabolites of epoprostenol and concentrations of zamicastat and its metabolites. A total of 54 subjects were enrolled and data from 28 subjects were available for pharmacokinetic analysis. The epoprostenol plus zamicastat-to-epoprostenol geometric means ratio (GMR) and corresponding 90% confidence interval (CI) for Cav,ss and area under the plasma concentration-time curve from time 0 up to 16 h at steady state (AUC0-16,ss) of the metabolites of epoprostenol were within the acceptance bioequivalence range (80.00%-125.00%). The intrasubject coefficient of variation (ISCV) was below 10% for both parameters, on both metabolites. For zamicastat AUC0-τ,ss, the zamicastat plus epoprostenol-to-zamicastat GMR and corresponding 90% CI were within the bioequivalence acceptance range, while for zamicastat Cmax,ss, the lower limit of the 90% CI was slightly below the acceptance range. For zamicastat metabolites, Cmax,ss and AUC0-τ,ss and the zamicastat plus epoprostenol-to-zamicastat GMR were below the acceptance bioequivalence range. ISCV was between 30% and 41% for Cmax,ss and between 21% and 41% for AUC0-τ,ss, for zamicastat and both metabolites. This study showed that the administration of zamicastat did not significantly modify the cardiovascular effects of epoprostenol and that the interactions between zamicastat and epoprostenol are not expected to be clinically relevant.

Antibiotic Residues and Zinc Concentrations in the Livers and Kidneys of Portuguese Piglets-Relationship to Antibiotic and Zinc Resistance in Intestinal Escherichia coli.

Metal ions such as zinc and copper have been used as alternatives to antibiotics, to improve animal health and growth rates in pig farming. This study aims to determine antibiotic residues and Zn concentration in piglets' livers (n = 56) and kidneys (n = 60); and to examine the correlation between the use of Zn and antibiotics, and resistance to Zn and antibiotics of Escherichia coli isolated from piglets' faeces (n = 60). Samples were collected from randomly selected healthy piglets (n = 60); antibiotic residues were quantified by ultra-high-performance-liquid-chromatography time-of-flight mass spectrometry (UHPLC-ToF-MS); Zn was quantified using flame atomic absorption spectrometry (FAAS); microbiological methods were used for E. coli isolation, antibiotic susceptibility, and Zn minimal inhibitory concentration; and Real-Time PCR was used for gene detection. The presence of antibiotic residues and Zn concentrations in the liver was found to be negatively correlated, whilst no significant difference was observed in the kidney. In E. coli isolated from piglet faeces considered to be susceptible or multi-drug-resistant, no significant difference was found between Zn concentrations in the liver and in the kidney, which appears to indicate that Zn accumulated in the liver and in the kidney does not promote resistance to antibiotics in E. coli. The isolates showed tolerance to Zn which would suggest that antibiotic resistance and phenotypic tolerance to Zn in these isolates are not related. The genes zitB and zntA associated to Zn tolerance, were predominantly found in the more resistant Zn isolates. The findings provide insights on how Zn use in pig production maintains antibiotic resistance and metal tolerance in bacteria, with implications for One Health.

Predictive Potential of Cmax Bioequivalence in Pilot Bioavailability/Bioequivalence Studies, through the Alternative ƒ2 Similarity Factor Method.

Pilot bioavailability/bioequivalence (BA/BE) studies are downsized trials that can be conducted prior to the definitive pivotal trial. In these trials, 12 to 18 subjects are usually enrolled, although, in principle, a sample size is not formally calculated. In a previous work, authors recommended the use of an alternative approach to the average bioequivalence methodology to evaluate pilot studies' data, using the geometric mean (Gmean) ƒ2 factor with a cut off of 35, which has shown to be an appropriate method to assess the potential bioequivalence for the maximum observed concentration (Cmax) metric under the assumptions of a true Test-to-Reference Geometric Mean Ratio (GMR) of 100% and an inter-occasion variability (IOV) in the range of 10% to 45%. In this work, the authors evaluated the proposed ƒ2 factor in comparison with the standard average bioequivalence in more extreme scenarios, using a true GMR of 90% or 111% for truly bioequivalent formulations, and 80% or 125% for truly bioinequivalent formulations, in order to better derive conclusions on the potential of this analysis method. Several scenarios of pilot BA/BE crossover studies were simulated through population pharmacokinetic modelling, accounting for different IOV levels. A redefined decision tree is proposed, suggesting a fixed sample size of 20 subjects for pilot studies in the case of intra-subject coefficient of variation (ISCV%) > 20% or unknown variability, and suggesting the assessment of study results through the average bioequivalence analysis, and additionally through Gmean ƒ2 factor method in the case of the 90% confidence interval (CI) for GMR is outside the regulatory acceptance bioequivalence interval of [80.00-125.00]%. Using this alternative approach, the certainty levels to proceed with pivotal studies, depending on Gmean ƒ2 values and variability scenarios tested (20-60% IOV), were assessed, which is expected to be helpful in terms of the decision to proceed with pivotal bioequivalence studies.

Alternative Analysis Approaches for the Assessment of Pilot Bioavailability/Bioequivalence Studies.

Pilot bioavailability/bioequivalence (BA/BE) studies are usually conducted and analysed similarly to pivotal studies. Their analysis and interpretation of results usually rely on the application of the average bioequivalence approach. However, due to the small study size, pilot studies are inarguably more sensitive to variability. The aim of this work is to propose alternative approaches to the average bioequivalence methodology, in a way to overcome and reduce the uncertainty on the conclusions of these studies and on the potential of test formulations. Several scenarios of pilot BA/BE crossover studies were simulated through population pharmacokinetic modelling. Each simulated BA/BE trial was analysed using the average bioequivalence approach. As alternative analyses, the centrality of the test-to-reference geometric least square means ratio (GMR), bootstrap bioequivalence analysis, and arithmetic (Amean) and geometric (Gmean) mean ƒ2 factor approaches were investigated. Methods performance was measured with a confusion matrix. The Gmean ƒ2 factor using a cut-off of 35 was the most appropriate method in the simulation conditions frame, enabling to more accurately conclude the potential of test formulations, with a reduced sample size. For simplification, a decision tree is also proposed for appropriate planning of the sample size and subsequent analysis approach to be followed in pilot BA/BE trials.

Defining milestones for the study of remyelination using the cuprizone mouse model: How early is early?

BACKGROUND: Cuprizone (CPZ) is a copper chelator used to produce a reversible oligodendrocytopathy in animals, which has some similarities to the pathology found in human multiple sclerosis (MS). This model is attractive to study remyelination. AIMS: To demonstrate that a two-week period after cessation of CPZ exposure is sufficient to establish changes compatible with remyelination, without accompanying behavior or brain magnetic resonance imaging (MRI) disturbances. METHODS: Two groups of male C57BL/6 mice were fed an oral solution of CPZ (0.2%) for 5 weeks (W5); half of the animals were kept under the vehicle for another 2 weeks (W7). After 5 and 7 weeks, animals were subjected to a battery of behavioural tests and 18 animals to brain MRI. Animals' cerebellar samples were studied for gene expression and/or protein levels of GFAP, myelin proteolipid protein (PLP), TNF-α and IL-1ß. RESULTS: No differences were observed between CPZ-exposed and control animals, regarding behavior and MRI, both at W5 and W7. However, myelin PLP levels decreased in CPZ (W5) treated animals, and these changes reverted at W7. GFAP levels varied in the opposite direction. CONCLUSIONS: Observed changes validate the use of W5 and W7 temporal moments for the study of demyelination and early remyelination in this model.

Current Evidence in Migraine Surgery: A Systematic Review.

INTRODUCTION: Migraine headache is a widespread neurovascular disorder with an enormous social and economic impact. A subgroup of patients cannot be managed with pharmacological therapy. Although surgical decompression of extracranial sensory nerves has been proposed as a valid alternative treatment option, the medical community remains reluctant to accept it. MATERIALS AND METHODS: This systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. An electronic search was performed in September 2020 on PubMed, ScienceDirect, CENTRAL, and Google Scholar databases for original articles reporting outcomes on migraine surgery. RESULTS: The search strategy revealed a total of 922 studies, of which 52 were included in the review. Significant improvement was reported in 58.3% to 100% and complete elimination in 8.3% to 86.8% of patients across studies. No major complications were reported. DISCUSSION: This systematic review demonstrates that migraine surgery is an effective and safe procedure, with a positive impact in patients' quality of life and a reduction in long-term costs. CONCLUSION: There is considerable scientific evidence suggesting extracranial migraine surgery is an effective and safe procedure. This surgery should be considered in properly selected migraineurs refractory to medical treatment.

Untangling Absorption Mechanisms and Variability in Bioequivalence Studies Using Population Analysis.

PURPOSE: Both inter-individual (IIV) and inter-occasion (IOV) variabilities are observed in bioequivalence studies. High IOV may be a cause of problems on the demonstration of bioequivalence, despite strict measures are taken to control it. The objective of this study is to investigate further means of controlling IIV by optimizing study design of crossover studies. METHODS: Data from 18 bioequivalence studies were used to develop population pharmacokinetics (popPK) models to characterize the absorption and disposition processes of 14 drugs, to estimate IOV for each drug substance and to evaluate possible correlations with biopharmaceutical properties of drug substances, classified in accordance to the Biopharmaceutics Drug Disposition Classification System (BDDCS). RESULTS: Plasma-pharmacokinetics profiles for the 14 drugs analyzed were successfully described using popPK. The pharmacokinetic parameters that showed greater variability were first-order rate constant of absorption, duration of the zero-order absorption process, relative bioavailability and time of latency. ISCV% estimated for Cmax seems to correlate with the log-Dose-Number for Class 1, 2 and 3, despite no direct correlation was observed between popPK model residual variability (RUV) and ISCV%. Nevertheless, higher RUV estimates were observed for Class 2 drugs in comparison to Class 1 and 3. CONCLUSION: Pharmacokinetic parameters related to drug absorption showed greater variability. Ingestion of the IMP along with 240 mL of water showed to standardize gastric emptying. Given the dependency between Cmax variability and dose-solubility ratio, for classes 2 and 4, ad libitum water intake may increase Cmax and AUC ISCV%. A water ingestion standardization until the expected Tmax of the drug is suggested.

Designer Cathinones N-Ethylhexedrone and Buphedrone Show Different In Vitro Neurotoxicity and Mice Behaviour Impairment.

N-Ethylhexedrone (NEH) and buphedrone (Buph) are emerging synthetic cathinones (SC) with limited information about their detrimental effects within central nervous system. Objectives: To distinguish mice behavioural changes by NEH and Buph and validate their differential harmful impact on human neurons and microglia. In vivo safety data showed the typical induced behaviour of excitation and stereotypies with 4-64 mg/kg, described for other SC. Buph additionally produced jumping and aggressiveness signs, while NEH caused retropulsion and circling. Transient reduction in body-weight gain was obtained with NEH at 16 mg/kg and induced anxiolytic-like behaviour mainly with Buph. Both drugs generated place preference shift in mice at 4 and 16 mg/kg, suggestive of abuse potential. In addition, mice withdrawn NEH displayed behaviour suggestive of depression, not seen with Buph. When tested at 50-400 µM in human nerve cell lines, NEH and Buph caused neuronal viability loss at 100 µM, but only NEH produced similar results in microglia, indicating different cell susceptibilities. NEH mainly induced microglial late apoptosis/necrosis, while Buph caused early apoptosis. NEH was unique in triggering microglia shorter/thicker branches indicative of cell activation, and more effective in increasing microglial lysosomal biogenesis (100 µM vs. 400 µM Buph), though both produced the same effect on neurons at 400 µM. These findings indicate that NEH and Buph exert neuro-microglia toxicities by distinct mechanisms and highlight NEH as a specific inducer of microglia activation. Buph and NEH showed in vivo/in vitro neurotoxicities but enhanced specific NEH-induced behavioural and neuro-microglia dysfunctionalities pose safety concerns over that of Buph.

Metabolism of N-ethylhexedrone and buphedrone: An in vivo study in mice using HPLC-MS/MS.

N-ethylhexedrone (NEH) and buphedrone (BUPH) are synthetic drugs structurally related to natural cathinone. These synthetic cathinones (SC) are members of the heterogenous family of new psychoactive substances (NPS), which have caused major concern in scientific and forensic communities over the past years, due to their widespread consume. Thus, there is a constant need for monitoring the use of these new substances and gather knowledge on their metabolism and excretion profiles, in order to try to identify markers of NPS consumption. This study aimed at the identification and quantification of NEH, BUPH and selected phase I metabolites using HPLC-MS/MS. NEH, BUPH and some related metabolites were synthesized in-house and quantified in 24 h mice urine, following single dose administration of each drug (64 mg kg-1, i.p.). NEH and BUPH were quantified in mice urine at 58.3 ± 14.4 and 146.2 ± 14.9 µg mL-1, respectively. Similar metabolic pathways were observed for both drugs. Among the metabolites studied, the most excreted ones derived from N-dealkylation of either NEH or BUPH (at around 80 µg mL-1 of urine). Other metabolites resulting from ketone reduction and ketone reduction combined with N-dealkylation or 4-aryl hydroxylation (detected for the first time in non-ring substituted SC) were also identified and quantified. Urine samples were screened using liquid chromatography-high resolution mass spectrometry and various phase II metabolites, including N-acetylated, glucuronides and dicarboxylic acid conjugates were tentatively identified, some of them for the first time. This work is a contribution to the identification of metabolites from SC that can become potential markers to estimate drug consumption.

Expression of receptor activator of NFkB (RANK) drives stemness and resistance to therapy in ER+HER2- breast cancer.

The role of RANKL-RANK pathway in progesterone-driven mammary carcinogenesis and triple negative breast cancer tumorigenesis has been well characterized. However, and despite evidences of the existence of RANK-positive hormone receptor (HR)-positive breast tumors, the implication of RANK expression in HR-positive breast cancers has not been addressed before. Here, we report that RANK pathway affects the expression of cell cycle regulators and decreases sensitivity to fulvestrant of estrogen receptor (ER)-positive (ER+)/HER2- breast cancer cells, MCF-7 and T47D. Moreover, RANK overexpressing cells had a staminal and mesenchymal phenotype, with decreased proliferation rate and decreased susceptibility to chemotherapy, but were more invasive in vivo. In silico analysis of the transcriptome of human breast tumors, confirmed the association between RANK expression and stem cell and mesenchymal markers in ER+HER2- tumors. Importantly, exposure of ER+HER2- cells to continuous RANK pathway activation by exogenous RANKL, in vitro and in vivo, induced a negative feedback effect, independent of RANK levels, leading to the downregulation of HR and increased resistance to hormone therapy. These results suggest that ER+HER2- RANK-positive cells may constitute an important reservoir of slow cycling, therapy-resistance cancer cells; and that RANK pathway activation is deleterious in all ER+HER2- breast cancer cells, independently of RANK levels.

Functional and cellular localization diversity associated with Fukutin-related protein patient genetic variants.

Genetic variants in Fukutin-related protein (FKRP), an essential enzyme of the glycosylation pathway of α-dystroglycan, can lead to pathologies with different severities affecting the eye, brain, and muscle tissues. Here, we generate an in vitro cellular system to characterize the cellular localization as well as the functional potential of the most common FKRP patient missense mutations. We observe a differential retention in the endoplasmic reticulum (ER), the indication of misfolded proteins. We find data supporting that mutant protein able to overcome this ER-retention through overexpression present functional levels comparable to the wild-type. We also identify a specific region in FKRP protein localized between residues 300 and 321 in which genetic variants found in patients lead to correctly localized proteins but which are nevertheless functionally impaired or catalytically dead in our model, indicating that this particular region might be important for the enzymatic activity of FKRP within the Golgi. Our system thus allows the functional testing of patient-specific mutant proteins and the identification of candidate mutants to be further explored with the aim of finding pharmacological treatments targeting the protein quality control system.

Identification of thiostrepton as a pharmacological approach to rescue misfolded alpha-sarcoglycan mutant proteins from degradation.

Limb-girdle muscular dystrophy type 2D (LGMD2D) is characterized by a progressive proximal muscle weakness. LGMD2D is caused by mutations in the gene encoding α-sarcoglycan (α-SG), a dystrophin-associated glycoprotein that plays a key role in the maintenance of sarcolemma integrity in striated muscles. We report here on the development of a new in vitro high-throughput screening assay that allows the monitoring of the proper localization of the most prevalent mutant form of α-SG (R77C substitution). Using this assay, we screened a library of 2560 FDA-approved drugs and bioactive compounds and identified thiostrepton, a cyclic antibiotic, as a potential drug to repurpose for LGMD2D treatment. Characterization of the thiostrepton effect revealed a positive impact on R77C-α-SG and other missense mutant protein localization (R34H, I124T, V247M) in fibroblasts overexpressing these proteins. Finally, further investigations of the molecular mechanisms of action of the compound revealed an inhibition of the chymotrypsin-like activity of the proteasome 24 h after thiostrepton treatment and a synergistic effect with bortezomib, an FDA-approved proteasome inhibitor. This study reports on the first in vitro model for LGMD2D that is compatible with high-throughput screening and proposes a new therapeutic option for LGMD2D caused by missense mutations of α-SG.

Different outcome of sarcoglycan missense mutation between human and mouse.

Sarcoglycanopathies are rare autosomic limb girdle muscular dystrophies caused by mutations in one of the genes coding for sarcoglycan (α, ß, δ, and γ-sarcoglycans). Sarcoglycans form a complex, which is an important part of the dystrophin-associated glycoprotein complex that protects sarcolemma against muscle contraction-induced damages. Absence of one of the sarcoglycan at the plasma membrane induces the disappearance of the whole complex and perturbs muscle fiber membrane integrity. We previously demonstrated that point mutations in the human sarcoglycan genes affects the folding of the corresponding protein, which is then retained in the endoplasmic reticulum by the protein quality control and prematurely degraded by the proteasome. Interestingly, modulation of the quality control using pharmacological compounds allowed the rescue of the membrane localization of the mutated sarcoglycan. Two previously generated mouse models, knock-in for the most common sarcoglycan mutant, R77C α-sarcoglycan, failed in reproducing the dystrophic phenotype observed in human patients. Based on these results and the need to test therapies for these fatal diseases, we decided to generate a new knock-in mouse model carrying the missense mutation T151R in the ß-sarcoglycan gene since this is the second sarcoglycan protein with the most frequently reported missense mutations. Muscle analysis, performed at the age of 4 and 9-months, showed the presence of the mutated ß-sarcoglycan protein and of the other components of the dystrophin-associated glycoprotein complex at the muscle membrane. In addition, these mice did not develop a dystrophic phenotype, even at a late stage or in condition of stress-inducing exercise. We can speculate that the absence of phenotype in mouse may be due to a higher tolerance of the endoplasmic reticulum quality control for amino-acid changes in mice compared to human.

Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D.

Limb-girdle muscular dystrophy type 2D (LGMD2D) is a rare autosomal-recessive disease, affecting striated muscle, due to mutation of SGCA, the gene coding for α-sarcoglycan. Nowadays, more than 50 different SGCA missense mutations have been reported. They are supposed to impact folding and trafficking of α-sarcoglycan because the defective polypeptide, although potentially functional, is recognized and disposed of by the quality control of the cell. The secondary reduction of α-sarcoglycan partners, ß-, γ- and δ-sarcoglycan, disrupts a key membrane complex that, associated to dystrophin, contributes to assure sarcolemma stability during muscle contraction. The complex deficiency is responsible for muscle wasting and the development of a severe form of dystrophy. Here, we show that the application of small molecules developed to rescue ΔF508-CFTR trafficking, and known as CFTR correctors, also improved the maturation of several α-sarcoglycan mutants that were consequently rescued at the plasma membrane. Remarkably, in myotubes from a patient with LGMD2D, treatment with CFTR correctors induced the proper re-localization of the whole sarcoglycan complex, with a consequent reduction of sarcolemma fragility. Although the mechanism of action of CFTR correctors on defective α-sarcoglycan needs further investigation, this is the first report showing a quantitative and functional recovery of the sarcoglycan-complex in human pathologic samples, upon small molecule treatment. It represents the proof of principle of a pharmacological strategy that acts on the sarcoglycan maturation process and we believe it has a great potential to develop as a cure for most of the patients with LGMD2D.

Exploiting the CRISPR/Cas9 system to study alternative splicing in vivo: application to titin.

The giant protein titin is the third most abundant protein in striated muscle. Mutations in its gene are responsible for diseases affecting the cardiac and/or the skeletal muscle. Titin has been reported to be expressed in multiple isoforms with considerable variability in the I-band, ensuring the modulation of the passive mechanical properties of the sarcomere. In the M-line, only the penultimate Mex5 exon coding for the specific is7 domain has been reported to be subjected to alternative splicing. Using the CRISPR-Cas9 editing technology, we generated a mouse model where we stably prevent the expression of alternative spliced variant(s) carrying the corresponding domain. Interestingly, the suppression of the domain induces a phenotype mostly in tissues usually expressing the isoform that has been suppressed, indicating that it fulfills (a) specific function(s) in these tissues allowing a perfect adaptation of the M-line to physiological demands of different muscles.

Adenoviral vector DNA for accurate genome editing with engineered nucleases.

Engineered sequence-specific nucleases and donor DNA templates can be customized to edit mammalian genomes via the homologous recombination (HR) pathway. Here we report that the nature of the donor DNA greatly affects the specificity and accuracy of the editing process following site-specific genomic cleavage by transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nucleases. By applying these designer nucleases together with donor DNA delivered as protein-capped adenoviral vector (AdV), free-ended integrase-defective lentiviral vector or nonviral vector templates, we found that the vast majority of AdV-modified human cells underwent scarless homology-directed genome editing. In contrast, a significant proportion of cells exposed to free-ended or to covalently closed HR substrates were subjected to random and illegitimate recombination events. These findings are particularly relevant for genome engineering approaches aiming at high-fidelity genetic modification of human cells.