In vitro dissolution testing is commonly performed to ensure that oral solid dosage medicines are of high quality and will achieve their targeted in vivo performance. However, this testing is time and material consuming. Therefore, pharmaceutical companies have been developing predictive dissolution models (PDMs) for drug product release based on fast at- and/or on-line measurements, including real-time release testing of dissolution (RTRT-D). Recently, PDMs have seen acceptance by major regulatory bodies as release tests for the dissolution critical quality attribute. In this paper, several methodologies are described to develop and validate a fit-for-purpose model, then to implement it as a surrogate release test for dissolution. These approaches are further exemplified by real-life case studies, which demonstrate that PDMs for release are not only viable but more sustainable than in vitro dissolution testing and can significantly accelerate drug product release. The rise of continuous manufacturing within the pharmaceutical industry further favors the implementation of real-time release testing. Therefore, a steep uptake of PDMs for release is expected once this methodology is globally accepted. To that end, it is advantageous for global regulators and pharmaceutical innovators to coalesce around a harmonized set of expectations for development, validation, implementation, and lifecycle of PDMs as part of drug product release testing.

• Klíčová slova
• dissolution, predictive dissolution modelling, real-time release testing,
• MeSH
• aplikace orální MeSH
• farmaceutická chemie metody   MeSH
• léčivé přípravky chemie   aplikace a dávkování   MeSH
• lidé MeSH
• příprava léků MeSH
• rozpustnost MeSH
• schvalování léčiv MeSH
• uvolňování léčiv * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• léčivé přípravky MeSH

This manuscript reports on a fully automatic sequential injection system incorporating a 3D printed module for real-time monitoring of the release of Metridia luciferase from a modified liver epithelial cell line. To this end, a simple and effective approach for the automation of flash-type chemiluminescence assays was developed. The 3D printed module comprised an apical and a basal compartment that enabled monitoring membrane processes on both sides of the cell monolayer aimed at elucidating the direction of luciferase release. A natural release was observed after transfection with the luciferase plasmid by online measurement of the elicited light from the reaction of the synthesized luciferase with the coelenterazine substrate. Model substances for acute toxicity from the group of cholic acids - chenodeoxycholic and deoxycholic acids - were applied at the 1.0 and 0.5 mmol L-1 levels. The tested cholic acids caused changes in cell membrane permeability that was accompanied by an increased luciferase release. The obtained kinetic profiles were evaluated based on the delay between the addition of the toxic substance and the increase of the chemiluminescence signal. All experiments were carried out in a fully automatic system in ca. 5 min per sample in 30 min intervals and no manual interventions were needed for a sampling period of at least 6 h.

• Klíčová slova
• 3D-printing, Automation, Cell toxicity, Metridia luciferase, Real-time monitoring, Sequential injection analysis,
• MeSH
• Copepoda * metabolismus   MeSH
• kinetika MeSH
• kyseliny cholové MeSH
• luciferasy genetika   metabolismus   MeSH
• luminiscenční měření MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kyseliny cholové MeSH
• luciferasy MeSH

The crayfish plague pathogen, Aphanomyces astaci, is one of the most serious threats to indigenous European crayfish species. The North American invasive spiny-cheek crayfish, Orconectes limosus, is an important source of this pathogen in central and western Europe. We evaluated potential changes in A. astaci spore release rate from infected individuals of this species by experiments investigating the pathogen transmission to susceptible noble crayfish, Astacus astacus. We filtered defined volumes of water regularly to quantify spore concentration, and sampled crayfish tissues at the end of the experiment. The filters and tissues were then tested for the presence of A. astaci DNA by species-specific quantitative PCR. Additionally, we tested the efficiency of horizontal transmission to apparently uninfected O. limosus. The experiments confirmed that A. astaci can be transmitted to susceptible crayfish during intermoult periods, and that the pathogen was more frequently detected in noble crayfish recipients than in American ones. The pathogen spore concentrations substantially varied in time, and significantly increased during moulting of infected hosts. Our study strengthens the evidence that although the likelihood of crayfish plague transmission by water transfer from localities with infected American crayfish might increase when these are moulting or dying, no time-periods can be proclaimed safe.

• MeSH
• Aphanomyces genetika   izolace a purifikace   fyziologie   MeSH
• druhová specificita MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• severní raci parazitologie   MeSH
• spory MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Maintaining a stable glycaemia in diabetes mellitus type 1 requires flexible insulin administration and carbohydrate intake to affected individuals. In real life, there might be some situations limiting the insulin-sugar balance control, e.g. night sleep or prolonged sporting activities. Glucose pellets with a pre-determined time lag between the pellet administration and glucose release were developed to mimic a 'snack eaten in advance'. In this article, a 13 C-glucose breath test was introduced to translate laboratory dissolution testing to clinical confirmation of the glucose release pattern using 5% δ abundance to differentiate the appearance of in 13 C exhaled breath. An independent two-sample t-test (p = 0.20) confirmed an average clinical lag time of 300 min and an in vitro time of 338 min to be identical at a level of significance of α = 0.05. Moreover, using the same statistical method, the clinical tmax (564 min) and the in vitro t50 (594 min) were also considered identical (p = 0.34). It was concluded that dissolution testing is a relevant method to determine the time lags of dosage forms with controlled release of glucose and that the 13 C-glucose breath test is a suitable clinical tool for lag time verification in clinical studies.

• Klíčová slova
• 13C-breath test, controlled release dosage form, diabetes mellitus type 1, hypoglycemia, in vivo/in vitro evaluation,
• MeSH
• dechové testy * MeSH
• dospělí MeSH
• glukosa aplikace a dávkování   chemie   farmakokinetika   MeSH
• izotopy uhlíku farmakokinetika   MeSH
• léky s prodlouženým účinkem aplikace a dávkování   chemie   farmakokinetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• tobolky MeSH
• uvolňování léčiv MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky MeSH
• Názvy látek
• glukosa MeSH
• izotopy uhlíku MeSH
• léky s prodlouženým účinkem MeSH
• tobolky MeSH

Terminally-differentiated cells cease to proliferate and acquire specific sets of expressed genes and functions distinguishing them from less differentiated and cancer cells. Mature granulocytes show lobular structure of cell nuclei with highly condensed chromatin in which HP1 proteins are replaced by MNEI. These structural features of chromatin correspond to low level of gene expression and the loss of some important functions as DNA damage repair, shown in this work and, on the other hand, acquisition of a new specific function consisting in the release of chromatin extracellular traps in response to infection by pathogenic microbes. Granulocytic differentiation is incomplete in myeloid leukemia and is manifested by persistence of lower levels of HP1γ and HP1β isoforms. This immaturity is accompanied by acquisition of DDR capacity allowing to these incompletely differentiated multi-lobed neutrophils of AML patients to respond to induction of DSB by γ-irradiation. Immature granulocytes persist frequently in blood of treated AML patients in remission. These granulocytes contrary to mature ones do not release chromatin for NETs after activation with phorbol myristate-12 acetate-13 and do not exert the neutrophil function in immune defence. We suggest therefore the detection of HP1 expression in granulocytes of AML patients as a very sensitive indicator of their maturation and functionality after the treatment. Our results show that the changes in chromatin structure underlie a major transition in functioning of the genome in immature granulocytes. They show further that leukemia stem cells can differentiate ex vivo to mature granulocytes despite carrying the translocation BCR/ABL.

• MeSH
• akutní myeloidní leukemie genetika   metabolismus   patologie   MeSH
• buněčná diferenciace * MeSH
• chromatin genetika   MeSH
• chromozomální proteiny, nehistonové genetika   metabolismus   MeSH
• fluorescenční protilátková technika MeSH
• granulocyty metabolismus   patologie   MeSH
• hematopoetické kmenové buňky metabolismus   patologie   MeSH
• homolog proteinu s chromoboxem 5 MeSH
• kultivované buňky MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• neutrofily patologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• poškození DNA * MeSH
• proliferace buněk MeSH
• tetradekanoylforbolacetát MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• CBX1 protein, human MeSH Prohlížeč
• chromatin MeSH
• chromozomální proteiny, nehistonové MeSH
• homolog proteinu s chromoboxem 5 MeSH
• messenger RNA MeSH
• tetradekanoylforbolacetát MeSH

In the current study, the results of an intradermal tuberculin test and a gamma interferon (IFN-γ) release assay were compared. IFN-γ release assay is based on the detection of IFN-γ production after in vitro stimulation with Mycobacterium avium subsp. avium-specific antigen for the discrimination of pigs naturally infected with M. avium subsp. hominissuis. Fifty-five clinically healthy pigs were used in the study. Three of these were proven by culture and real-time quantitative polymerase chain reaction methods to be infected with M. avium subsp. hominissuis (2 animals) and Mycobacterium xenopi (1 animal). No animals were positive by the tuberculin test. Both M. avium subsp. hominissuis-positive pigs were evaluated as positive by the IFN-γ release assay. Bacteriologically negative and M. xenopi-positive pigs were unresponsive in the IFN-γ release assay, indicating the specificity of the method. The results suggest that the IFN-γ release assay has a higher sensitivity than the tuberculin test and that the assay can be used for diagnosis of M. avium infections in live, naturally infected pigs.

• MeSH
• Mycobacterium avium izolace a purifikace   MeSH
• nemoci prasat diagnóza   mikrobiologie   MeSH
• prasata MeSH
• test pomocí interferonu gama veterinární   MeSH
• tuberkulinový test veterinární   MeSH
• tuberkulóza diagnóza   mikrobiologie   veterinární   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

PURPOSE: The evaluation of four post-pandemic influenza seasons 2009-2013 in the Faculty hospital Hradec Králové and comparison of used rapid antigen tests (RATs) with real time RT-PCR. MATERIAL AND METHODS: Between November 2009 and June 2013 were examined 3845 samples from patients with respiratory tract infections by RATs (Influenza A/B 2 Panel Test (GECKO® Pharma, Germany), Rapid VIDITEST Influenza A+B Card (VIDIA®, Czech Republic) and BinaxNOW Influenza A&B Test (ALERE®, USA) or real time RT-PCR (RTR InfA/H1N1 Detection Set (Roche®), RealStar® Influenza S and T RT-PCR Kit 3.0 (Altona ®, Germany). RESULTS: A totally 1059 samples were examined simultaneously by RAT and real time RT-PCR. The overall sensitivity and specificity of RATs compared with real time RT-PCR were 32,2 % and 98,1 % for influenza A and 17,6 % and 99,4 % for influenza B. Higher sensitivity of RATs were in children (66,6 %) compared with adults (14,3 - 40,0 %). In the first three post-pandemic seasons were continuously decrease of positive samples from 23,5 % in season 2009-2010 to 3,3% in season 2011-2012, but in season 2012-2013 were rapidly in-crease of positive results, to 31,5%, with high share of influenza -A/H1N1/2009 (79,6%). CONCLUSION: Our results shown insufficient sensitivity of all used RATs and necessity of having other confirmatory test, like RT-PCR. It was also shown unexplained increase of case and influenza severity in season 2012-2013.

• MeSH
• antigeny virové analýza   MeSH
• časové faktory MeSH
• chřipka lidská diagnóza   epidemiologie   MeSH
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• retrospektivní studie MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• translačně kontrolovaný nádorový protein 1 MeSH
• virus chřipky A, podtyp H1N1 * MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• antigeny virové MeSH
• Tpt1 protein, rat MeSH Prohlížeč
• translačně kontrolovaný nádorový protein 1 MeSH

Transferring an existing marketed pharmaceutical product from batch to continuous manufacturing (CM) without changes in regulatory registration is a challenging task in the pharmaceutical industry. Continuous manufacturing can provide an increased production rate and better equipment utilisation while retaining key quality attributes of the final product. Continuous manufacturing necessitates the monitoring of critical quality attributes in real time by appropriate process analytical tools such as near infra-red (NIR) probes. The present work reports a successful transfer of an existing drug product from batch to continuous manufacturing process without changing the formulation. A key step was continuous powder blending, whose design and operating parameters including weir type, agitation rate, dynamic hold-up and residence time were systematically investigated with respect to process repeatability. A NIR-based multivariate data model for in-line composition monitoring has been developed and validated against an existing quality control method for measuring tablet content uniformity. A continuous manufacturing long-run with a throughput of 30 kg/h (approx. 128,000 tablets per hour), uninterrupted for 320 min, has been performed to test and validate the multivariate data model as well as the batch to continuous process transfer. The final disintegration and dissolution properties of tablets manufactured by the continuous process were found to be equivalent to those manufactured by the original batch process.

• Klíčová slova
• Continuous manufacturing, Continuous powder blending, Direct compression, Near-infrared probe, Residence time,
• MeSH
• blízká infračervená spektroskopie metody   MeSH
• farmaceutická chemie metody   MeSH
• farmaceutická technologie * metody   MeSH
• pomocné látky chemie   MeSH
• prášky, zásypy, pudry chemie   MeSH
• příprava léků metody   MeSH
• řízení kvality MeSH
• rozpustnost MeSH
• tablety * MeSH
• uvolňování léčiv MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• pomocné látky MeSH
• prášky, zásypy, pudry MeSH
• tablety * MeSH

One third of the world's population is latently infected with Mycobacterium tuberculosis (Mtb) and up to 10% of infected individuals develop active tuberculosis (TB) in their lifetime. Among the major challenges in the control of TB is the implementation of sensitive methods for detection of latent tuberculosis infection (LTBI). Currently, in vitro interferon gamma release assays, yielding single value readout, are used as an alternative to the traditional tuberculin skin test for the diagnosis of LTBI. More complex characterization of immune status of LTBI individuals, however, is desirable for indication of LTBI subjects for preventative chemotherapy. Here we describe a quantitative polymerase chain reaction (qPCR) for determination of expression levels of 14 genes, additional to interferon gamma, which was applied for comparison of the specific Mtb-antigen immune response of blood cells from healthy, latently infected, and TB individuals. With the use of principal component analysis and discriminant analysis, a pattern of mRNA levels of 6 genes was identified, allowing discrimination of healthy individuals from active TB and LTBI subjects. These results open the way to development of multimarker qPCR for the detection of LTBI.

• MeSH
• antigeny bakteriální imunologie   MeSH
• diagnostické techniky molekulární metody   MeSH
• dospělí MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• latentní tuberkulóza diagnóza   MeSH
• leukocyty mononukleární imunologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• Mycobacterium tuberculosis imunologie   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• stanovení celkové genové exprese metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny bakteriální MeSH

Drug amorphisation by loading to inorganic mesoporous carriers represents an emerging area of improving the dissolution rate and bioavailability of poorly water-soluble active pharmaceutical ingredients (APIs). In this work, for the first time, a molecular-level insight into the process of API loading to mesoporous SiO2 (silica) carriers by the hot-melt impregnation method and its subsequent release during dissolution was obtained using ATR-FTIR spectroscopic imaging. A physical mixture of ibuprofen crystals and mesoporous silica particles was heated and the dynamics of melt loading into the silica pore structure was directly observed in situ by ATR-FTIR spectroscopic imaging. The loss of crystallinity, the redistribution of the API in the silica pore network and the subsequent stabilisation of the amorphous form upon cooling were proven. The API was involved in two different kinds of molecular-level interactions: API dimers in the amorphous bulk, and individual API molecules adsorbed on the silica surface. The melt-loaded silica carriers were comprehensively characterised by DSC, SEM and dissolution tests, which proved dissolution rate enhancement due to amorphisation of the API. Drug release form the hot-melt loaded mesoporous silica carriers was observed in real time and the conditions leading to local re-crystallisation of super-saturated solution of the API were identified.

• MeSH
• adsorpce MeSH
• biologická dostupnost MeSH
• diferenciální skenovací kalorimetrie MeSH
• farmaceutická chemie metody   MeSH
• ibuprofen aplikace a dávkování   chemie   MeSH
• krystalizace MeSH
• mikroskopie elektronová rastrovací MeSH
• nosiče léků chemie   MeSH
• oxid křemičitý chemie   MeSH
• poréznost MeSH
• rozpustnost MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• uvolňování léčiv MeSH
• voda chemie   MeSH
• vysoká teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ibuprofen MeSH
• nosiče léků MeSH
• oxid křemičitý MeSH
• voda MeSH