Pharmacokinetic principles of dose adjustment of mTOR inhibitors in solid organ transplanted patients.

WHAT IS KNOWN AND OBJECTIVES: mTOR inhibitors possess narrow therapeutic range and substantial pharmacokinetic variability and the consequences from suboptimal dosing are serious. The aim of this review is to summarize the current knowledge about the factors influencing mTOR inhibitors pharmacokinetics and the possibility of using these relationships in order to improve its therapy individualization in solid organ transplanted patients. METHODS: Literature search from Pubmed and Web of Science databases were performed using Boolean search operators in order to identify relevant studies. RESULTS AND DISCUSSION: A total of 701 reports were identified from the initial literature search. Out of which 40 studies dealt with relationships between various factors and pharmacokinetics of mTOR inhibitors and with relevance of these associations for dosage optimization. WHAT IS NEW AND CONCLUSION: The overview of the current covariates for pharmacokinetic variability of mTOR inhibitors has been provided on the level of absorption, distribution and elimination, and consequences of these relationships for dosing optimization has been summarized.

Lymphatic Transport of Drugs after Intestinal Absorption: Impact of Drug Formulation and Physicochemical Properties.

PURPOSE: To provide a comprehensive and up-to-date overview focusing on the extent of lymphatic transport of drugs following intestinal absorption and to summarize available data on the impact of molecular weight, lipophilicity, formulation and prandial state. METHODS: Literature was searched for in vivo studies quantifying extent of lymphatic transport of drugs after enteral dosing. Pharmacokinetic data were extracted and summarized. Influence of molecular weight, log P, formulation and prandial state was analyzed using relative bioavailability via lymph (FRL) as the parameter for comparison. The methods and animal models used in the studies were also summarized. RESULTS: Pharmacokinetic data on lymphatic transport were available for 103 drugs. Significantly higher FRL [median (IQR)] was observed in advanced lipid based formulations [54.4% (52.0)] and oil solutions [38.9% (60.8)] compared to simple formulations [2.0% (27.1)], p < 0.0001 and p = 0.004, respectively. Advanced lipid based formulations also provided substantial FRL in drugs with log P < 5, which was not observed in simple formulations and oil solutions. No relation was found between FRL and molecular weight. There were 10 distinct methods used for in vivo testing of lymphatic transport after intestinal absorption so far. CONCLUSION: Advanced lipid based formulations provide superior ability to increase lymphatic absorption in drugs of various molecular weights and in drugs with moderate to low lipophilicity.

Gastric pH in Rats: Key Determinant for Preclinical Evaluation of pH-dependent Oral Drug Absorption.

Data on gastric pH in rats to be used in preclinical models for pH-dependent drug absorption are still limited or contradictory. The aim of this study was to describe gastric pH in rats at fasted state and to evaluate its changes induced by pentagastrin or omeprazole in order to mimic gastric pH at fasted and fed human subjects. Twenty Wistar rats, fasting for 12 h, were randomly assigned into four treatment groups (n=5): control, pre-treated with omeprazole 2 h before pH measurement, pre-treated with omeprazole 12 h before pH measurement, and pre-treated with pentagastrin 20 min before pH measurement. An incision on the stomach wall was made in anesthetized animals, and pH of gastric juice was measured. The observed pH values were significantly different among groups (p=0.0341), with the median (IQR) values of gastric pH of 3.5 (2.7-4.2), 6.7 (4.7-7.0), 5.6 (3.5-6.4) and 2.2 (1.6-3.1) in control, omeprazole 2 h, omeprazole 12 h and pentagastin group, respectively. We recommend using short interval pentagastrin and 2 h omeprazole pre-treatment in fasting animals to model similar gastric pH as is expected in human fasted and fed state pharmacokinetic studies, respectively.

The immunophilin repertoire of Plasmodiophora brassicae and functional analysis of PbCYP3 cyclophilin.

Plasmodiophora brassicae is a soil-borne pathogen that belongs to Rhizaria, an almost unexplored eukaryotic organism group. This pathogen requires a living host for growth and multiplication, which makes molecular analysis further complicated. To broaden our understanding of a plasmodiophorid such as P. brassicae, we here chose to study immunophilins, a group of proteins known to have various cellular functions, including involvement in plant defense and pathogen virulence. Searches in the P. brassicae genome resulted in 20 putative immunophilins comprising of 11 cyclophilins (CYPs), 7 FK506-binding proteins (FKBPs) and 2 parvulin-like proteins. RNAseq data showed that immunophilins were differentially regulated in enriched life stages such as germinating spores, maturing spores, and plasmodia, and infected Brassica hosts (B. rapa, B. napus and B. oleracea). PbCYP3 was highly induced in all studied life stages and during infection of all three Brassica hosts, and hence was selected for further analysis. PbCYP3 was heterologously expressed in Magnaporthe oryzae gene-inactivated ΔCyp1 strain. The new strain ΔCyp1+ overexpressing PbCYP3 showed increased virulence on rice compared to the ΔCyp1 strain. These results suggest that the predicted immunophilins and particularly PbCYP3 are activated during plant infection. M. oryzae is a well-studied fungal pathogen and could be a valuable tool for future functional studies of P. brassicae genes, particularly elucidating their role during various infection phases.

Cyclophilins: Less Studied Proteins with Critical Roles in Pathogenesis.

Cyclophilins (EC 5.2.1.8) belong to a subgroup of proteins known as immunophilins, which also include FK506-binding proteins and parvulins. Members of the immunophilins have two main characteristic properties: (i) peptidyl-prolyl cis-trans isomerase activity and (ii) the ability to bind immunosuppressant molecules of fungal origin. Cyclophilins are some of the most conserved proteins present in eukaryotes and prokaryotes, and they have been implicated in diverse cellular processes and responses to multiple biotic and abiotic stresses. Cyclophilins have been exploited in humans and plants extensively, but they have only recently received attention in regard to phytopathogens. In Phellinus sulphurascens and species of the genus Leptosphaeria and Phytophthora, high expression of cyclophilins was found to be related to infection. Moreover, recent studies of cyclophilins in certain phytopathogens, such as Magnaporthe oryzae, Botrytis cinerea, Cryphonectria parasitica, and Puccinia triticina, demonstrated their roles as a pathogenicity factors. In addition to pathogenicity, cyclophilins have high affinity for the immunosuppressive drug cyclosporin A, which is a potent antifungal agent. Although cyclophilins are highly conserved in phytopathogens, because they have been less studied, their role remains largely unknown. In this review, we provide detailed information on the cyclophilins in several phytopathogens, including fungi and oomycetes, as well as their role in suppressing plant immunity.

Suppression of Meloidogyne incognita by the Entomopathogenic Fungus Lecanicillium muscarium.

The entomopathogenic fungus Lecanicillium muscarium (Petch) Zare and Gams is currently being developed as a biocontrol agent against insect pests, as well as some plant-pathogenic fungi and bacteria. Data about its activity against plant-parasitic nematodes exist, but are relatively limited. To expand this understanding, we investigated the biocontrol efficiency of three isolates of L. muscarium (Lm) against the root knot nematode, Meloidogyne incognita, in both in vitro and in vivo conditions. In our experiments, the maximum number of nematode eggs, juveniles (J2s), females, and egg masses that were parasitized were quantified after a 72-h exposure to the fungus. The isolate Lm1 was designated as the best biocontrol agent against nematode eggs as well as J2s. It showed the highest colonization of eggs and significantly decreased egg hatching events. The results from two additional isolates, Lm2 and Lm3, were also significant (P = 0.05) but less pronounced than those observed with Lm1. L. muscarium treatments had significant (P = 0.05) positive effects on plant shoot and root growth compared with the growth of control plants. These results suggest the effectiveness of the fungus may be due to either the infection of eggs and J2s, or the production of secondary metabolites that induced plant defense mechanisms and lead to systemic resistance. Our study demonstrates that L. muscarium could be used as a potential biocontrol agent against root knot nematodes.

Exploitation of FTA cartridges for the sampling, long-term storage, and DNA-based analyses of plant-parasitic nematodes.

The use of DNA-based analyses in molecular plant nematology research has dramatically increased over recent decades. Therefore, the development and adaptation of simple, robust, and cost-effective DNA purification procedures are required to address these contemporary challenges. The solid-phase-based approach developed by Flinders Technology Associates (FTA) has been shown to be a powerful technology for the preparation of DNA from different biological materials, including blood, saliva, plant tissues, and various human and plant microbial pathogens. In this work, we demonstrate, for the first time, that this FTA-based technology is a valuable, low-cost, and time-saving approach for the sampling, long-term archiving, and molecular analysis of plant-parasitic nematodes. Despite the complex structure and anatomical organization of the multicellular bodies of nematodes, we report the successful and reliable DNA-based analysis of nematode high-copy and low-copy genes using the FTA technology. This was achieved by applying nematodes to the FTA cards either in the form of a suspension of individuals, as intact or pestle-crushed nematodes, or by the direct mechanical printing of nematode-infested plant tissues. We further demonstrate that the FTA method is also suitable for the so-called "one-nematode-assay", in which the target DNA is typically analyzed from a single individual nematode. More surprisingly, a time-course experiment showed that nematode DNA can be detected specifically in the FTA-captured samples many years after initial sampling occurs. Collectively, our data clearly demonstrate the applicability and the robustness of this FTA-based approach for molecular research and diagnostics concerning phytonematodes; this research includes economically important species such as the stem nematode (Ditylenchus dipsaci), the sugar beet nematode (Heterodera schachtii), and the Northern root-knot nematode (Meloidogyne hapla).

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.

Genome-wide identification of the opsin protein in Leptosphaeria maculans and comparison with other fungi (pathogens of Brassica napus).

The largest family of transmembrane receptors are G-protein-coupled receptors (GPCRs). These receptors respond to perceived environmental signals and infect their host plants. Family A of the GPCR includes opsin. However, there is little known about the roles of GPCRs in phytopathogenic fungi. We studied opsin in Leptosphaeria maculans, an important pathogen of oilseed rape (Brassica napus) that causes blackleg disease, and compared it with six other fungal pathogens of oilseed rape. A phylogenetic tree analysis of 31 isoforms of the opsin protein showed six major groups and six subgroups. All three opsin isoforms of L. maculans are grouped in the same clade in the phylogenetic tree. Physicochemical analysis revealed that all studied opsin proteins are stable and hydrophobic. Subcellular localization revealed that most isoforms were localized in the endoplasmic reticulum membrane except for several isoforms in Verticillium species, which were localized in the mitochondrial membrane. Most isoforms comprise two conserved domains. One conserved motif was observed across all isoforms, consisting of the BACTERIAL_OPSIN_1 domain, which has been hypothesized to have an identical sensory function. Most studied isoforms showed seven transmembrane helices, except for one isoform of V. longisporum and four isoforms of Fusarium oxysporum. Tertiary structure prediction displayed a conformational change in four isoforms of F. oxysporum that presumed differences in binding to other proteins and sensing signals, thereby resulting in various pathogenicity strategies. Protein-protein interactions and binding site analyses demonstrated a variety of numbers of ligands and pockets across all isoforms, ranging between 0 and 13 ligands and 4 and 10 pockets. According to the phylogenetic analysis in this study and considerable physiochemically and structurally differences of opsin proteins among all studied fungi hypothesized that this protein acts in the pathogenicity, growth, sporulation, and mating of these fungi differently.

Dasatinib anhydrate containing oral formulation improves variability and bioavailability in humans.

Dasatinib monohydrate indicated for the treatment of chronic myeloid leukemia displays pH-dependent solubility. The aim of reported development program of novel dasatinib anhydrate containing formulation was to demonstrate improved absorption and lower pharmacokinetic variability compared to dasatinib monohydrate. In a bioavailability study comparing formulations containing 110.6 mg and 140 mg of dasatinib as anhydrate and monohydrate, respectively, both Cmax and AUC of dasatinib were within standard 80.00-125.00% range, while the intra- and inter-subject variability for AUC0-inf after the test product was approximately 3-fold and 1.5-fold less than after the reference, respectively.In a drug-drug interaction study, omeprazole 40 mg reduced the mean AUC0-inf of dasatinib by 19%, when the test was ingested 2 h before the 5th omeprazole dose. This decrease of exposure is clinically irrelevant and substantially less than after the reference. Co-prescription analysis supports the importance of pH-dependent solubility of dasatinib, as >21% of patients were treated concomitantly with a PPI and dasatinib despite warnings against this co-medication in the SmPC.The novel dasatinib anhydrate containing formulation demonstrated improved absorption and less pharmacokinetic variability compared to dasatinib monohydrate product, which may translate into improved clinical outcomes, although this needs to be proven by an appropriate trial.

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.

Explaining dissolution properties of rivaroxaban cocrystals.

The aim of this study was to improve rivaroxaban water-solubility by cocrystal preparation and to understand this process. The screening with water-soluble coformers was performed via both mechanochemical and solution-mediated techniques. Two cocrystals of rivaroxaban with malonic acid and oxalic acid were prepared, and the structure of the cocrystal with oxalic acid was solved. Both cocrystals exhibit improved dissolution properties. The mechanism of the supersaturation maintenance was studied by in-situ Raman spectroscopy. The transformation into rivaroxaban dihydrate was identified as the critical step in the improved dissolution properties of both cocrystals. Moreover, the transformation kinetics and solubilization effects of the coformers were identified as responsible for the differences in the dissolution behavior of the cocrystals. In-vivo experiments proved that the use of cocrystal instead of form I of free API helped to increase the bioavailability ofrivaroxaban.

Pharmacokinetic Variability in Pre-Clinical Studies: Sample Study with Abiraterone in Rats and Implications for Short-Term Comparative Pharmacokinetic Study Designs.

One of the major concerns for all in vivo experiments is intra- and inter-subject variability, which can be a great source of inaccuracy. The aim of this study is, therefore, to estimate the ability of parallel vs. cross-over design studies in order to describe the relative pharmacokinetic performance of the studied drug formulations. We analyzed the data from a drug development program that examined the performance of innovative abiraterone acetate formulations against the identical reference product in three stages. In stages 1-3, groups A-F were dosed with the reference product once in a parallel manner. Stage 4 was performed to evaluate the intra-individual variability (IIV) by repeated administration of the reference product to the same animals. Although the geometric mean (90% CI) values of abiraterone AUClast in groups A-F were similar to the IIV group (24.36 (23.79-41.00) vs. 26.29 (20.56-47.00) mg/mL·min·g), the results generated in the isolated parallel groups provided imprecise estimates of the true AUClast values ranging from 9.62 to 44.62 mg/mL·min·g due to chance. Notably, in 4 out of 15 possible pair comparisons between the parallel groups, the confidence intervals did not include 100%, which is the true ratio for all comparisons tested after identical formulation administration to all groups. A cross-over design can significantly improve the methodology in short-term comparative pre-clinical pharmacokinetic studies, and can provide more precise and accurate results in comparison to more traditional pre-clinical study designs.

Factors Affecting the Metabolic Conversion of Ciprofloxacin and Exposure to Its Main Active Metabolites in Critically Ill Patients: Population Pharmacokinetic Analysis of Desethylene Ciprofloxacin.

The objective of this prospective study was to examine the exposure to the main active metabolites of ciprofloxacin in critically ill patients and to examine the factors (demographic, laboratory and genetic) that could potentially affect the drug metabolic conversion of ciprofloxacin. The secondary aim was to develop a population pharmacokinetic model for the metabolite showing the most associations with the abovementioned factors. A total of 29 patients were treated with intravenous infusion of ciprofloxacin and enrolled on this trial. Blood samples for pharmacokinetic analysis were taken at 1, 4, and 11.5 h following the completion of the infusion. Sex, age, body weight, height, serum creatinine and bilirubin levels, and creatinine clearance (CLCR) were recorded, and polymorphisms rs2032582 and rs1045642 in the ABCB1 gene, rs4148977 in the SLCO1A2 gene and rs762551 in the CYP1A2 gene were analyzed. A three-stage parent drug-metabolite population pharmacokinetic model was developed. Median (IQR) metabolite/parent ratios of the desethylene ciprofloxacin, formyl ciprofloxacin and oxociprofloxacin were 5.86 (4.09-9.87)%, 4.08 (3.38-6.92)% and 5.91 (3.42-13.65)%, respectively. The desethylene ciprofloxacin metabolic ratio was positively associated with height (r2 = 0.2277, p = 0.0089) and CLCR (r2 = 0.2023, p = 0.0144) and negatively associated with age (r2 = 0.2227, p = 0.0112). Males had a significantly higher oxociprofloxacin metabolic ratio than females (9.14 vs 3.42%, p = 0.0043). In the desethylene ciprofloxacin population PK model, the volume of distribution decreased with age, the parent drug-metabolite transfer rate constant increased with CLCR, and the metabolite elimination rate constant decreased with age and is increased in CYP1A2 rs762551 variant allele carriers. We therefore hypothesized that the CYP1A2 inhibition by ciprofloxacin is mediated by its metabolite desethylene ciprofloxacin.

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.

No Time Dependence of Ciprofloxacin Pharmacokinetics in Critically Ill Adults: Comparison of Individual and Population Analyses.

The aim of this prospective PK study was to evaluate the pharmacokinetics of ciprofloxacin dosed within the first 36 h (early phase) and after 3 days of treatment (delayed phase) using individual and population PK analysis. The secondary aim of the study was to evaluate possible dosing implications of the observed PK differences between early and delayed phases to achieve a PK/PD target for ciprofloxacin of AUC24/MIC ≥ 125. Blood concentrations of ciprofloxacin (1 and 4 h after dose and trough) were monitored in critically ill adults in the early and delayed phases of the treatment. Individual and population PK analyses were performed. Complete concentration-time profiles in the early phase, delayed phase, and both phases were obtained from 29, 15, and 14 patients, respectively. No systematic changes in ciprofloxacin PK parameters between the early and delayed phases were observed, although variability was higher at the early phase. Both individual and population analyses provided similar results. Simulations showed that after standard dosing, it is practically impossible to reach the recommended ciprofloxacin PK/PD target (AUC/MIC ≥ 125) for pathogens with MIC ≥ 0.5 mg/L. A dosing nomogram utilizing patients' creatinine clearance and MIC values was constructed. Both individual and population analyses provided similar results. Therapeutic drug monitoring should be implemented to safeguard the optimal ciprofloxacin exposure.

Characterization of the Molecular Mechanisms of Resistance against DMI Fungicides in Cercospora beticola Populations from the Czech Republic.

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most important foliar pathogen of sugar beet worldwide. Extensive reliance on fungicides to manage CLS has resulted in the evolution of fungicide resistance in C. beticola worldwide, including populations in the Czech Republic. One important class of fungicides used to manage CLS is the sterol demethylation inhibitors (DMI). The aim of our study was to assess DMI resistance in C. beticola from the Czech Republic and elucidate the molecular basis of DMI resistance in this population. A total of 50 isolates were collected in 2018 and 2019 from the major sugar beet growing regions of the Czech Republic and assessed for in vitro sensitivity to the DMI fungicides propiconazole, prochloraz, and epoxiconazole. These analyses identified three strains that exhibited 50% effective concentration (EC50) values > 1.0 µg mL-1 against respective fungicides, which were therefore considered resistant. In contrast, strains that exhibited lowest EC50 values were considered sensitive. To explore the molecular basis of resistance in these three strains, the cytochrome P450-dependent sterol 14α-demethylase (Cyp51) gene was sequenced. Sequence analysis identified a Y464S mutation in all three resistant strains. To assess whether Cyp51 gene expression may play a role in DMI resistance, selected strains were grown in vitro with and without fungicide treatment. These analyses indicated that Cyp51 gene expression was significantly induced after fungicide treatment. Thus, we conclude that Y464S point mutation along with induced Cyp51 gene overexpression is likely responsible for resistance against DMI fungicides in C. beticola from the Czech Republic.

Validity of cycloheximide chylomicron flow blocking method for the evaluation of lymphatic transport of drugs.

BACKGROUND AND PURPOSE: Lymphatic transport of drugs after oral administration is an important mechanism for absorption of highly lipophilic compounds. Direct measurement in lymph duct cannulated animals is the gold standard method, but non-invasive cycloheximide chylomicron flow blocking method has gained popularity recently. However, concerns about its reliability have been raised. The aim of this work was to investigate the validity of cycloheximide chylomicron flow blocking method for the evaluation of lymphatic transport using model compounds with high to very high lipophilicity, that is, abiraterone and cinacalcet. EXPERIMENTAL APPROACH: Series of pharmacokinetic studies were conducted with abiraterone acetate and cinacalcet hydrochloride after enteral/intravenous administration to intact, lymph duct cannulated and/or cycloheximide pre-treated rats. KEY RESULTS: Mean total absolute oral bioavailability of abiraterone and cinacalcet was 7.0% and 28.7%, respectively. There was a large and significant overestimation of the lymphatic transport extent by the cycloheximide method. Mean relative lymphatic bioavailability of abiraterone and cinacalcet in cycloheximide method was 28-fold and 3-fold higher than in cannulation method, respectively. CONCLUSION AND IMPLICATIONS: Cycloheximide chylomicron flow blocking method did not provide reliable results on lymphatic absorption and substantially overestimated lymphatic transport for both molecules, that is, abiraterone and cinacalcet. This non-invasive method should not be used for the assessment of lymphatic transport and previously obtained data should be critically revised.

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.

Efficient Confirmation of Plant Viral Proteins and Identification of Specific Viral Strains by nanoLC-ESI-Q-TOF Using Single-Leaf-Tissue Samples.

Plant viruses are important pathogens that cause significant crop losses. A plant protein extraction protocol that combines crushing the tissue by a pestle in liquid nitrogen with subsequent crushing by a roller-ball crusher in urea solution, followed by RuBisCO depletion, reduction, alkylation, protein digestion, and ZipTip purification allowed us to substantially simplify the sample preparation by removing any other precipitation steps and to detect viral proteins from samples, even with less than 0.2 g of leaf tissue, by a medium resolution nanoLC-ESI-Q-TOF. The presence of capsid proteins or polyproteins of fourteen important viruses from seven different families (Geminiviridae, Luteoviridae, Bromoviridae, Caulimoviridae, Virgaviridae, Potyviridae, and Secoviridae) isolated from ten different economically important plant hosts was confirmed through many identified pathogen-specific peptides from a protein database of host proteins and potential pathogen proteins assembled separately for each host and based on existing online plant virus pathogen databases. The presented extraction protocol, combined with a medium resolution LC-MS/MS, represents a cost-efficient virus protein confirmation method that proved to be effective at identifying virus strains (as demonstrated for PPV, WDV) and distinct disease species of BYDV, as well as putative new viral protein sequences from single-plant-leaf tissue samples. Data are available via ProteomeXchange with identifier PXD022456.