An ultra-high performance liquid chromatography method for simultaneous determination of tacrolimus impurities in pharmaceutical dosage forms has been developed. Appropriate chromatographic ...separation was achieved on a BEH C
column using gradient elution with a total run time of 14 min. The method was applied to analyses of commercial samples and was validated in terms of linearity, precision, accuracy, sensitivity and specificity. It was found to be linear, precise and accurate in the range of 0.05 to 0.6 % of the impurities level in pharmaceutical dosage forms. Stability indicating power of the method was demonstrated by the results of forced degradation studies. The forced degradation study in solution revealed tacrolimus instability under stress alkaline, thermal, light and photolytic conditions and in the presence of a radical initiator or metal ions. The drug was stable at pH 3–5. Solid-state degradation studies conducted on amorphous tacrolimus demonstrated its sensitivity to light, elevated temperature, humidity and oxidation.
•Solid state compatibility study.•Isolation and characterization of new tacrolimus degradation impurity.•Structural elucidation using NMR and MS.•Degradation pathway of tacrolimus in the presence of ...divalent cations proposed.
Tacrolimus is macrolide drug that is widely used as a potent immunosuppressant. In the present work compatibility testing was conducted on physical mixtures of tacrolimus with excipients and on compatibility mixtures prepared by the simulation of manufacturing process used for the final drug product preparation. Increase in one major degradation product was detected in the presence of magnesium stearate based upon UHPLC analysis. The degradation product was isolated by preparative HPLC and its structure was elucidated by NMR and MS studies. Mechanism of the formation of this degradation product is proposed based on complementary degradation studies in a solution and structural elucidation data. The structure was proven to be alpha-hydroxy acid which is formed from the parent tacrolimus molecule through a benzilic acid type rearrangement reaction in the presence of divalent metallic cations. Degradation is facilitated at higher pH values.
The objective of this study is to present a practical example of a scale-independent design space development using a step-wise approach. A detailed description of the development process with a ...systematic outline of the main steps is provided. Design space is developed for film coating of tablets with moisture protective polyvinyl alcohol (PVA) based coating. The impact of scale-independent coating process parameters on the properties of film-coated tablets (FCT), i.e. water activity and film coating protection ability, and consequently on product long-term stability is explored. The main finding is that with model simplifications, a step-wise approach and rational development of scale-independent design space for the coating process, it is possible to efficiently predict, control, and optimize the long-term stability of a moisture sensitive product. However, the PVA moisture protective coating itself is recognized as having conflicting effects on product stability.
Reduction or elimination of chemically synthesized additives from foods is a current demand in food industry. A new approach to prevent the proliferation of microorganisms or protect food from ...oxidation is the use of essential oils or plant extracts as natural additives in foods. We have studied antimicrobial activity of rosemary extracts (Rosmarinus officinalis L.) against different species of Listeria and against different strains of L. monocytogenes. We used two extracts of rosemary, VivOX 20 and VivOX 40 (Vitiva d.d., Slovenia) containing different levels of carnosic acid. We wanted to proof an antimicrobial activity of selected rosemary extracts with two most commonly used methods: disc diffusion method and broth dilution method. With the disc diffusion method we have obtained the inhibition zone and at the lowest concentrations, where no visible bacterial growth was recorded, were assumed as minimal inhibitory concentration values (MIC). We determined MIC values in the ranges from 625 μg extract/ml EtOH to 5000 μg extract/ml EtOH for VivOX 20 and from 312.5 μg extract/ml EtOH do 2500 μg extract/ml EtOH for VivOX 40 in the medium. We have established that the resistance of Listeria species against rosemary extracts depends on: selected extract, selected concentration, various species and strain of Listeria. With broth dilution method we have determined minimal bactericidal concentration (MBC), as the concentration giving 0.1% bacterial survival. With this method we have tested two strains of L. monocytogenes and in determinate MBC values in the range from 15.63 μg/ml TSB to 98.5 μg/ml TSB for both tested extracts. Results have confirmed our assumption that resistance of Listeria against rosemary extracts depended on the selected strain.
Zahteve potrošnikov po celem svetu so zmanjšati oz. izločiti kemično sintetizirane konzervanse iz živil. Novejše metode preprečevanja mikrobne kontaminacije in oksidacije uporabljajo eterična olja ali rastlinske ekstrakte kot naravne konzervanse. Proučevali smo protimikrobno delovanje ekstraktov rožmarina (Rosmarinus officinalis L.) na različne vrste bakterij rodu Listeria in seve bakterij L. monocytogenes. Uporabili smo dva različna komercialno pripravljena ekstrakta rožmarina, VivOX 20 in VivOX 40 (Vitiva d.d., Slovenija), ki sta vsebovala različno koncentracijo karnozolne kisline. Protimikrobni učinek izbranih ekstraktov smo želeli dokazati z dvema najpogosteje uporabljenima metodama: metoda difuzije v trdnem gojišču in metoda razredčevanja v tekočem gojišču. Pri metodi difuzije v trdnem gojišču smo po inkubaciji odčitali nastale inhibicijske cone, s katerimi smo določili minimalne inhibitorne koncentracije (MIC), kot tiste koncentracije, pri katerih ni bilo vidne rasti bakterij na gojišču. Vrednosti MIC smo določili v območju med 625 μg ekstrakta/ml EtOH do 5000 μg ekstrakta/ml EtOH za ekstrakt VivOX 20 in med 312,5 μg ekstrakta/ml EtOH do 2500 μg ekstrakta/ml EtOH za ekstrakt VivOX 40. Ugotovili smo, da je odpornost listerij proti ekstraktoma rožmarina odvisna od izbranega ekstrakta, izbrane koncentracije ter vrste in seva listerij. Z metodo razredčevanja v tekočem gojišču smo določali minimalne baktericidne koncentracije (MBC), kot tiste koncentracije, pri katerih preživi 0,1 % testnih bakterij. Uporabili smo dva različna seva bakterij vrste L. monocytogenes in vrednosti MBC v večini poskusov določili med 15,63 μg/ml gojišča TSB in 98,5 μg/ml gojišča za oba uporabljena ekstrakta. Rezultati so ponovno potrdili našo domnevo, da je odpornost listerij proti ekstraktoma rožmarina odvisna od seva.
Different types of factorial experimental designs can be used in compatibility studies of drug development, where many different factors and their interactions should be evaluated to predict their ...effects on the degradation of the drug substance under study. All possible main and interaction effects of different potential excipients that can constitute the drug product should be evaluated in order to select the best combination of excipients that give the lowest possible degradation, i.e., the most stable drug product. Statistical experimental designs enable the user to obtain the maximum amount of information, i.e., the degradation effects of excipients and their interactions on the stability of the drug substance, on the basis of the smallest possible number of experiments. The use of full and two different fractional factorial designs is described using a real example where the excipients that stabilize the drug substance or cause as little degradation as possible are selected for a solid dosage formulation. It was shown that the type and the sequence of design used during the studies are also important to get reliable and valuable results. A thorough explanation of the statistical evaluation of data and different presentations of final solutions are given.
The liver is to date the best example of a sexually dimorphic non-reproductive organ. Over 1,000 genes are differentially expressed between sexes indicating that female and male livers are two ...metabolically distinct organs. The spectrum of liver diseases is broad and is usually prevalent in one or the other sex, with different contributing genetic and environmental factors. It is thus difficult to predict individual's disease outcomes and treatment options. Systems approaches including mathematical modeling can aid importantly in understanding the multifactorial liver disease etiology leading toward tailored diagnostics, prognostics and therapy. The currently established computational models of hepatic metabolism that have proven to be essential for understanding of non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) are limited to the description of gender-independent response or reflect solely the response of the males. Herein we present
, the first sex-based multi-tissue and multi-level liver metabolic computational model. The model was constructed based on
liver model
and the object-oriented modeling. The crucial factor in adaptation of liver metabolism to the sex is the inclusion of estrogen and androgen receptor responses to respective hormones and the link to sex-differences in growth hormone release. The model was extensively validated on literature data and experimental data obtained from wild type C57BL/6 mice fed with regular chow and western diet. These experimental results show extensive sex-dependent changes and could not be reproduced
with the uniform model
.
represents the first large-scale liver metabolic model, which allows a detailed insight into the sex-dependent complex liver pathologies, and how the genetic and environmental factors interact with the sex in disease appearance and progression. We used the model to identify the most important sex-dependent metabolic pathways, which are involved in accumulation of triglycerides representing initial steps of NAFLD. We identified PGC1A, PPARα, FXR, and LXR as regulatory factors that could become important in sex-dependent personalized treatment of NAFLD.
Circular RNAs (circRNAs) are increasingly recognized as having a role in cancer development. Their expression is modified in numerous cancers, including hepatocellular carcinoma (HCC); however, ...little is known about the mechanisms of their regulation. The aim of this study was to identify regulators of circRNAome expression in HCC. Using publicly available datasets, we identified RNA binding proteins (RBPs) with enriched motifs around the splice sites of differentially expressed circRNAs in HCC. We confirmed the binding of some of the candidate RBPs using ChIP-seq and eCLIP datasets in the ENCODE database. Several of the identified RBPs were found to be differentially expressed in HCC and/or correlated with the overall survival of HCC patients. According to our bioinformatics analyses and published evidence, we propose that NONO, PCPB2, PCPB1, ESRP2, and HNRNPK are candidate regulators of circRNA expression in HCC. We confirmed that the knocking down the epithelial splicing regulatory protein 2 (ESRP2), known to be involved in the maintenance of the adult liver phenotype, significantly changed the expression of candidate circRNAs in a model HCC cell line. By understanding the systemic changes in transcriptome splicing, we can identify new proteins involved in the molecular pathways leading to HCC development and progression.
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