Ivacaftor pharmacokinetics and lymphatic transport after enteral administration in rats.

Background: Ivacaftor is a modern drug used in the treatment of cystic fibrosis. It is highly lipophilic and exhibits a strong positive food effect. These characteristics can be potentially connected to a pronounced lymphatic transport after oral administration. Methods: A series of studies was conducted to describe the basic pharmacokinetic parameters of ivacaftor in jugular vein cannulated rats when dosed in two distinct formulations: an aqueous suspension and an oil solution. Additionally, an anesthetized mesenteric lymph duct cannulated rat model was studied to precisely assess the extent of lymphatic transport. Results: Mean ± SD ivacaftor oral bioavailability was 18.4 ± 3.2% and 16.2 ± 7.8%, respectively, when administered as an aqueous suspension and an oil solution. The relative contribution of the lymphatic transport to the overall bioavailability was 5.91 ± 1.61% and 4.35 ± 1.84%, respectively. Conclusion: Lymphatic transport plays only a minor role in the process of ivacaftor intestinal absorption, and other factors are, therefore, responsible for its pronounced positive food effect.

Analysis of the Ibotenic Acid, Muscimol, and Ergosterol Content of an Amanita Muscaria Hydroalcoholic Extract with an Evaluation of Its Cytotoxic Effect against a Panel of Lung Cell Lines In Vitro.

The fungus Amanita muscaria is universally recognizable for its iconic appearance; it is also widely regarded as poisonous, inedible, and even deadly. In spite of that, there have been documented cases of use of A. muscaria-containing preparations against various diseases, including cancer, to no apparent ill effect. The search for compounds that can be used to treat cancer among various plants and fungi has been intensifying in recent years. In light of this, we describe an HPLC HILIC analytical method for the evaluation of the content of the anticancer compound ergosterol (ERG) and the neuroactive alkaloids ibotenic acid (IBO) and muscimol (MUS) that contribute significantly to the unpleasant physiological syndrome associated with A. muscaria consumption. A 'homemade' A. muscaria tincture made using 80-proof rye vodka as the solvent, an A. muscaria extract made with a standardized water-ethanol solution as the solvent, and fractions obtained from the second extract via liquid-liquid extraction with nonpolar solvents were analyzed. The study also presents the results of capillary zone electrophoresis with contactless conductivity detection and UHPLC-MS/MS analyses of the IBO and MUS content of the two native A. muscaria extracts and an evaluation of the standardized extract's cytotoxic effect against a small panel of lung cell cultures in vitro. Our results show that the standardized extract has a significant cytotoxic effect and does not contain the compounds of interest in any significant quantity.

Casein as protein and hydrolysate: Biostimulant or nitrogen source for Nicotiana tabacum plants grown in vitro?

In contrast to inorganic nitrogen (N) assimilation, the role of organic N forms, such as proteins and peptides, as sources of N and their impact on plant metabolism remains unclear. Simultaneously, organic biostimulants are used as priming agents to improve plant defense response. Here, we analysed the metabolic response of tobacco plants grown in vitro with casein hydrolysate or protein. As the sole source of N, casein hydrolysate enabled tobacco growth, while protein casein was used only to a limited extent. Free amino acids were detected in the roots of tobacco plants grown with protein casein but not in the plants grown with no source of N. Combining hydrolysate with inorganic N had beneficial effects on growth, root N uptake and protein content. The metabolism of casein-supplemented plants shifted to aromatic (Trp), branched-chain (Ile, Leu, Val) and basic (Arg, His, Lys) amino acids, suggesting their preferential uptake and/or alterations in their metabolic pathways. Complementarily, proteomic analysis of tobacco roots identified peptidase C1A and peptidase S10 families as potential key players in casein degradation and response to N starvation. Moreover, amidases were significantly upregulated, most likely for their role in ammonia release and impact on auxin synthesis. In phytohormonal analysis, both forms of casein influenced phenylacetic acid and cytokinin contents, suggesting a root system response to scarce N availability. In turn, metabolomics highlighted the stimulation of some plant defense mechanisms under such growth conditions, that is, the high concentrations of secondary metabolites (e.g., ferulic acid) and heat shock proteins.

Serum and lymph pharmacokinetics of nilotinib delivered by yeast glucan particles per os.

Nilotinib is a selective tyrosine-kinase inhibitor approved for the treatment of chronic myeloid leukemia. It is poorly soluble in aqueous media and has a low oral bioavailability. Nilotinib encapsulation into yeast glucan particles (GPs) was investigated in this work as a means of increasing bioavailability. The amorphization of nilotinib in GPs resulted in an increased dissolution rate, which was confirmed by in vitro experiments using biorelevant dissolution media. Simultaneously, GPs containing nilotinib were effectively taken up by macrophages, which was quantified in vitro on cell cultures. The overall oral bioavailability in a rat model was approximately 39 % for nilotinib delivered in a reference formulation (Tasigna) and was almost doubled when delivered in GPs. The contribution of glucan particles to the lymphatic transport of nilotinib was quantified. When delivered by GPs, cumulative nilotinib absorption via the lymphatic system increased by a factor of 10.8 compared to the reference, but still represented arelative bioavailability of only 1.12 %. The cumulative uptake of GPs in the lymph was found to be 0.54 mg after a single dose of 50 mg. Yeast glucan particles can therefore serve as a drug delivery vehicle with a dual function: dissolution rate enhancement by amorphization, and, to asmaller extent, lymphatic delivery due to macrophage uptake.

Application of Oil-in-Water Cannabidiol Emulsion for the Treatment of Rheumatoid Arthritis.

Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune disease with unknown cause. It mainly affects joints and, without proper treatment, negatively impacts their movement, causes painful deformities, and reduces the patients' quality of life. Current treatment options consist of various types of disease-modifying antirheumatic drugs (DMARDs), however 20-30% of patients are partially resistant to them. Therefore, development of new drugs is necessary. Possible option are compounds exhibiting their action via endocannabinoid system, which plays an important role in pain and inflammation modulation. One such compound - cannabidiol (CBD) has already been shown to attenuate synovitis in animal model of RA in in vivo studies. However, it has low bioavailability due to its low water solubility and lipophilicity. This issue can be addressed by preparation of a lipid containing formulation targeting lymphatic system, another route of absorption in the body. Materials and Methods: CBD-containing emulsion was prepared by high-shear homogenization and its droplet size distribution was analysed by optical microscopy. The relative oral bioavailability compared to oil solution as well as total availability of CBD were assessed in a cross-over study in rats and absorption of CBD via lymphatic system was observed. The effect of CBD on the animal model of RA was determined. Results: Compared to oil solution, the emulsion exhibited higher absolute oral bioavailability. Significant lymphatic transport of CBD was observed in all formulations and the concentrations in lymph were calculated. The therapeutic effect of CBD on RA was confirmed as an improvement in clinical symptoms as well as morphological signs of disease activity were observed during the study. Conclusion: In this work, we prepared a simple stable emulsion formulation, determined the pharmacokinetic parameters of CBD and calculated its absolute bioavailability in rats. Moreover, we successfully tested the pharmaceutical application of such a formulation and demonstrated the positive effect of CBD in an animal model of RA.

Pharmaceutical Product Characterization and Manufacturability of Surfactant-Enriched Oil Marbles with Abiraterone Acetate.

The present study investigates the physicochemical properties and stability of a novel lipid-based formulation-surfactant-enriched oil marbles containing abiraterone acetate. While the biopharmaceutical performance of this formulation has been reported recently, this study aims to fill the gap between a promising in vivo performance and industrial applicability. A series of techniques were employed to assess the solid-state characteristics of oil marble cores along with their physicochemical properties upon stability testing. The chemical stability of abiraterone acetate in the formulation was also investigated. The core of the formulation was found to be stable both physically and chemically over 12 months of storage. The in vitro performance of stressed samples was evaluated using a dissolution experiment. The formulation has successfully self-emulsified upon incubation in bio-relevant media, resulting in a fast and complete API release. An important issue connected with the excipient used as a covering material of oil marbles has been identified. The seemingly insignificant water sorption caused agglomeration of the oil marbles and consequently compromised the dissolution rate in some of the stressed samples. Replacing HPMC with lactose as a covering material resulted in more favorable properties upon storage. Overall, it has been shown that oil marbles are an industrially applicable concept of the solidified lipid-based formulation.

Human ultrarare genetic disorders of sulfur metabolism demonstrate redundancies in H2S homeostasis.

Regulation of H2S homeostasis in humans is poorly understood. Therefore, we assessed the importance of individual enzymes in synthesis and catabolism of H2S by studying patients with respective genetic defects. We analyzed sulfur compounds (including bioavailable sulfide) in 37 untreated or insufficiently treated patients with seven ultrarare enzyme deficiencies and compared them to 63 controls. Surprisingly, we observed that patients with severe deficiency in cystathionine ß-synthase (CBS) or cystathionine γ-lyase (CSE) - the enzymes primarily responsible for H2S synthesis - exhibited increased and normal levels of bioavailable sulfide, respectively. However, an approximately 21-fold increase of urinary homolanthionine in CBS deficiency strongly suggests that lacking CBS activity is compensated for by an increase in CSE-dependent H2S synthesis from accumulating homocysteine, which suggests a control of H2S homeostasis in vivo. In deficiency of sulfide:quinone oxidoreductase - the first enzyme in mitochondrial H2S oxidation - we found normal H2S concentrations in a symptomatic patient and his asymptomatic sibling, and elevated levels in an asymptomatic sibling, challenging the requirement for this enzyme in catabolizing H2S under physiological conditions. Patients with ethylmalonic encephalopathy and sulfite oxidase/molybdenum cofactor deficiencies exhibited massive accumulation of thiosulfate and sulfite with formation of large amounts of S-sulfocysteine and S-sulfohomocysteine, increased renal losses of sulfur compounds and concomitant strong reduction in plasma total cysteine. Our results demonstrate the value of a comprehensive assessment of sulfur compounds in severe disorders of homocysteine/cysteine metabolism and provide evidence for redundancy and compensatory mechanisms in the maintenance of H2S homeostasis.

Alternative method for canagliflozin oxidation analysis using an electrochemical flow cell - Comparative study.

This paper highlights the potential of electrochemical flow cells for oxidative-stress testing of active pharmaceutical ingredients using canagliflozin as a model substance. Based on design of experiments, we developed our method through a reduced combinatorial design, optimizing the following independent variables: cell size, electrolyte flow rate, electrolyte concentration, and electrolyte pH. Using ammonium phosphate buffer with methanol in a 50/50 vol ratio as a working electrolyte, we electrochemically oxidized samples and analyzed them by high-performance liquid chromatography, considering the following dependent variables: peak area of each impurity, peak area of canagliflozin, and the percentage of the corresponding peak areas. Our results showed that the most significant independent variables were electrolyte pH and flow rate. By data optimization, we determined the most suitable conditions for electrochemical oxidation of canagliflozin, namely 50 µm cell size, 300 mM electrolyte concentration, 0.1 mL/h electrolyte flow rate, and electrolyte pH = 4. The repeatability of the method, expressed as the relative standard deviation of the canagliflozin peak area, measured in ten separately oxidized samples, was 1.64%. For comparison purposes, we performed a degradation experiment using hydrogen peroxide, identifying five identical impurities in both cases, as confirmed by mass spectrometry. The degradation products formed when using the chemical method after 1, 3, and 7 days totaled 0.09%, 0.75%, and 3.75%, respectively, and the degradation products formed when using the electrochemical method after 3 h totaled 3.11%. Oxidation with hydrogen peroxide required 7 days, whereas electrochemical oxidation was completed in 3 h. Overall, the electrochemical method significantly saves time and reduces the consumption of active ingredients and solvents thanks to the miniaturized size of the electrochemical cell, thereby minimizing the costs of forced degradation studies.

Bioavailability Enhancement and Food Effect Elimination of Abiraterone Acetate by Encapsulation in Surfactant-Enriched Oil Marbles.

Abiraterone acetate has limited bioavailability in the fasted state and exhibits a strong positive food effect. We present a novel formulation concept based on the so-called oil marbles (OMs) and show by in vitro and in vivo experiments that the food effect can be suppressed. OMs are spherical particles with a core-shell structure, formed by coating oil-based droplets that contain the dissolved drug by a layer of powder that prevents the cores from sticking and coalescence. OMs prepared in this work contained abiraterone acetate in the amorphous form and showed enhanced dissolution properties during in vitro experiments when compared with originally marketed formulation of abiraterone acetate (Zytiga®). Based on in vitro comparison of OMs containing different oil/surfactant combinations, the most promising formulation was chosen for in vivo studies. To ensure relevance, it was verified that the food effect previously reported for Zytiga® in humans was translated into the rat animal model. The bioavailability of abiraterone acetate formulated in OMs in the fasted state was then found to be enhanced by a factor of 2.7 in terms of AUC and by a factor of 4.0 in terms of Cmax. Crucially, the food effect reported in the literature for other abiraterone acetate formulations was successfully eliminated and OMs showed comparable extent of bioavailability in a fed-fasted study. Oil marbles therefore seem to be a promising formulation concept not only for abiraterone acetate but potentially also for other poorly soluble drugs that reveal a positive food effect.

Preclinical evaluation of new formulation concepts for abiraterone acetate bioavailability enhancement based on the inhibition of pH-induced precipitation.

Abiraterone acetate is a potent drug used for the treatment of metastatic castration resistant prostate cancer. However, currently marketed product containing crystalline abiraterone acetate exhibits strong positive food effect which results in strict dosing regimen. In the present work, a rational approach towards design of novel abiraterone acetate formulations that would allow increased bioavailability on a fasting stomach and thus decreased food effect is presented. Precipitation experiments in biorelevant media were designed to assess pH induced precipitation of the drug and a pool of polymeric excipients was then screened for their potential to inhibit precipitation. The best performing polymeric excipients were subsequently used as carriers for the preparation of amorphous solid dispersions. Two main approaches were followed in order to formulate the drug. The first approach relies on the suppression of precipitation from a supersaturated solution whereas the second one is based on the hypothesis that when the release of the drug is tuned, optimal uptake of the drug can be reached. Optimized formulation prototypes were tested in a rat animal model in an incomplete block, randomized bioequivalence study to assess their relative bioavailability under fasting conditions. We show that both formulation approaches lead to increased bioavailability of abiraterone acetate on a fasting stomach with bioavailability in rats being enhanced up to 250% compared to the original drug product containing crystalline drug.

Lipophosphonoxins II: Design, Synthesis, and Properties of Novel Broad Spectrum Antibacterial Agents.

The increase in the number of bacterial strains resistant to known antibiotics is alarming. In this study we report the synthesis of novel compounds termed Lipophosphonoxins II (LPPO II). We show that LPPO II display excellent activities against Gram-positive and -negative bacteria, including pathogens and multiresistant strains. We describe their mechanism of action-plasmatic membrane pore-forming activity selective for bacteria. Importantly, LPPO II neither damage nor cross the eukaryotic plasmatic membrane at their bactericidal concentrations. Further, we demonstrate LPPO II have low propensity for resistance development, likely due to their rapid membrane-targeting mode of action. Finally, we reveal that LPPO II are not toxic to either eukaryotic cells or model animals when administered orally or topically. Collectively, these results suggest that LPPO II are highly promising compounds for development into pharmaceuticals.

Design of experiments for amino acid extraction from tobacco leaves and their subsequent determination by capillary zone electrophoresis.

In this study, we optimized a method for the determination of free amino acids in Nicotiana tabacum leaves. Capillary electrophoresis with contactless conductivity detector was used for the separation of 20 proteinogenic amino acids in acidic background electrolyte. Subsequently, the conditions of extraction with HCl were optimized for the highest extraction yield of the amino acids because sample treatment of plant materials brings some specific challenges. Central composite face-centered design with fractional factorial design was used in order to evaluate the significance of selected factors (HCl volume, HCl concentration, sonication, shaking) on the extraction process. In addition, the composite design helped us to find the optimal values for each factor using the response surface method. The limits of detection and limits of quantification for the 20 proteinogenic amino acids were found to be in the order of 10-5 and 10-4 mol l-1, respectively. Addition of acetonitrile to the sample was tested as a method commonly used to decrease limits of detection. Ambiguous results of this experiment pointed out some features of plant extract samples, which often required specific approaches. Suitability of the method for metabolomic studies was tested by analysis of a real sample, in which all amino acids, except for L-methionine and L-cysteine, were successfully detected. The optimized extraction process together with the capillary electrophoresis method can be used for the determination of proteinogenic amino acids in plant materials. The resulting inexpensive, simple, and robust method is well suited for various metabolomic studies in plants. As such, the method represents a valuable tool for research and practical application in the fields of biology, biochemistry, and agriculture.

Purification and enzymatic characterization of tobacco leaf ß-N-acetylhexosaminidase.

The kinetic properties of ß-N-acetylhexosaminidase purified from tobacco (Nicotiana tabacum L.) leaves have been investigated. In addition to chromogenic pNP derivates, N,N'-diacetylchitobiose and N,N',N″-triacetylchitotriose were also used as substrates of ß-N-acetylhexosaminidase. The highest reaction rate and the affinity for the substrate were observed for pNP-GlcNAc; however, an excess of this substrate inhibits the reaction. The reaction rate with pNP-GalNAc as the substrate was found to be about 85% of that obtained with pNP-GlcNAc. The hydrolysis of acetylated chitooligomers by ß-N-acetylhexosaminidase followed by separation and quantification using capillary electrophoresis was slower compared to pNP-GlcNAc. The pH optimum of ß-N-acetylhexosaminidase for individual substrates was found at 4.3-5.0 and the temperature optimum was 50-55 °C. Gel permeation chromatography and red native electrophoresis determined the relative molecular weight as 280 000 and the isoelectric point as 5.3. The inhibition of ß-N-acetylhexosaminidase by monosaccharides GlcN, GalN, GlcNAc, GalNAc in combination with substrates pNP-GlcNAc and pNP-GalNAc was studied and the type of inhibition and the inhibition constants were determined.

Overcharging in biological systems: reversal of electrophoretic mobility of aqueous polyaspartate by multivalent cations.

Charge reversal as an extreme case of charge compensation is directly observed by capillary electrophoresis for a negatively charged peptide in aqueous solutions of trivalent cations. Atomistic and coarse-grained simulations provide molecular interpretation of this effect showing that it is largely of electrostatic origin with a minor contribution of chemical specificity of the salt ions.

Effect of association with sulfate on the electrophoretic mobility of polyarginine and polylysine.

Domains rich in cationic amino acids are ubiquitous in peptides with the ability to cross cell membranes, which is likely related to the binding of such polypeptides to anionic groups on the membrane surface. To shed more light on these interactions, we investigated specific interactions between basic amino acids and oligopeptides thereof and anions by means of electrophoretic experiments and molecular dynamics simulations. To this end, we measured the electrophoretic mobilities of arginine, lysine, tetraarginine, and tetralysine in sodium chloride and sodium sulfate electrolytes as a function of ionic strength. The mobility was found to be consistently lower in sodium sulfate than in sodium chloride at the same ionic strength. The decrease in mobility in sodium sulfate was greater for tetraarginine than for tetralysine and was larger for tetrapeptides compared to the corresponding free amino acids. On the basis of molecular dynamics simulations and Bjerrum theory, we rationalize these results in terms of enhanced association between the amino acid side chains and sulfate. Simulations also predict a greater affinity of sulfate to the guanidinium side chain groups of arginine than to the ammonium groups of lysine, as the planar guanidinium geometry allows simultaneous strong hydrogen bonding to two sulfate oxygens. We show that the sulfate binding to arginine, but not to lysine, is cooperative. These results are consistent with the greater decrease in the mobility of arginine compared to that of lysine upon addition of sulfate salt. The nonspecific mobility retardation by sulfate is ascribed to its electrostatic interaction with the cationic amino acid side chain groups.

Pluronic F-127 as the buffer additive in capillary entangled polymer electrophoresis: some fundamental aspects.

Polymeric macromolecules of well-designed structures and specific properties open promising directions in the capillary entangled polymer electrophoresis. Pluronic F-127, as a thermoassociating polymer, possesses some unique properties that can be utilized in capillary entangled polymer electrophoresis of amino acids, peptides and proteins. In this study, we studied properties of Pluronic F-127 polymer as an additive to BGE for the separation of peptides and proteins. The influence of the thermoassociation on separation selectivity was studied. The addition of Pluronic caused severe instabilities of the electrical current and the signal of the UV detector. This study reveals remarkable positive effect of a low pressure applied to the inlet buffer vial during the analysis, which apparently stabilizes the electrical current and the detector signal. The effect of hydrodynamic flow induced by the pressure applied on the separation efficiency was studied and the significance of this effect was discussed. Pluronic F-127, as a representative of synthetic macromolecules, was compared with dextran, as a representative of natural polymers, in terms of separation power, selectivity and repeatability of migration times.