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.

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.

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.

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.

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.