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Biomarkers of menopause
No full textNatural menopause is defined as the permanent cessation of menstruation resulting from
the loss of ovarian follicular activity. This significant phase in women’s reproductive aging has
notable effects on fat distribution, dyslipidemia, and neurodegeneration. These changes contribute
to an increased risk of dementia and cardiovascular disease as women age. Estimating the age at
which natural menopause will occur is crucial for cancer survivors, women with a family history
of premature ovarian insufficiency or early menopause, and those delaying their first pregnancy.
Additionally, the timing of natural menopause is an important clinical indicator of longevity and
a risk factor for morbidity and mortality. Beyond age and menstrual cycle characteristics,
biomarkers related to menopause are essential for confirming its onset and predicting its
premature occurrence.Prirodna menopauza se definiše kao trajni prestanak menstruacije usled gubitka folikularne
aktivnosti jajnika. Ova značajna faza u reproduktivnom starenju žena ima značajne efekte na
distribuciju masti, dislipidemiju i neurodegeneraciju. Ove promene doprinose povećanom riziku
od demencije i kardiovaskularnih bolesti kako žene stare. Procena starosti u kojoj će nastupiti
prirodna menopauza je ključna za preživele od kancera, žene sa porodičnom istorijom preuranjene
insuficijencije jajnika ili rane menopauze i one koje odlažu prvu trudnoću. Pored toga, vreme
prirodne menopauze je važan klinički pokazatelj dugovečnosti i faktor rizika za morbiditet i
mortalitet. Osim starosti i karakteristika menstrualnog ciklusa, biomarkeri koji se odnose na
menopauzu su od suštinskog značaja za potvrđivanje njenog početka i predviđanje njenog
preranog nastupanja
Application of Separation Methods in Biomimetic Studies and the Determination of Physicochemical Properties of Imidazoline and α Adrenergic Receptor Ligands
No full textObjective
The assessment of key pharmacokinetic and physicochemical parameters in the early stages of drug
discovery using traditional “gold standard” methods can be expensive and time consuming. With
advances in stationary phase technology, biomimetic chromatography has emerged as a valuable
alternative. The aim of this study is to investigate the potential of different separation methods as models
for the assessment of blood-brain barrier permeability, plasma protein binding, lipophilicity and
ionisation constants of ligands targeting imidazoline and alpha-adrenergic receptors.
Methods
Drug–protein interactions were studied using a chromatographic system comprising a stationary phase
with immobilised α1-acid glycoprotein and a mobile phase with Sørensen phosphate buffer (pH 7.0) and
2-propanol as organic modifier. Blood–brain barrier permeability was analysed by biopartitioning
micellar chromatography using different ratios of Brij 35 (pH 7.4) and methanol as mobile phase and a
Zorbax Extend-C18 column as stationary phase. The lipophilicity of the compound was determined
using mobile phases with different methanol/water/ammonia ratios and stationary phases of different
polarity (CN, RP2, RP8 and RP18), while the ionisation constants were determined by capillary
electrophoresis.
Results
Quantitative structure–retention relationships (QSRR) models were developed using the retention data
of the investigated biomimetic systems and the calculated molecular descriptors. The predictive power
of the models was confirmed by both internal and external validation. The influence of the selected
molecular descriptors on the dependent variables was analysed and discussed. Among the systems tested,
the stationary phase RP8 in thin-layer chromatography proved to be the most suitable for assessing the
lipophilicity of compounds. Capillary electrophoresis proved to be a fast, simple and accurate method
for determining the pKa values of the analysed compounds.
Conclusions
The separation techniques investigated and the QSRR models developed provide valuable tools for
optimising the pharmacokinetic and physicochemical properties of newly synthesized, pharmaceutically
relevant compounds targeting the central nervous system
Development of separation methods for the investigation of retention characteristics, assay and impurities of ziprasidone
No full textObjective
Ziprasidone (ZIP) is second generation, high-efficiency antipsychotic drug with unique multipotent
G-protein-coupled (GPCR) receptor binding profile, which may be the reason for its large number
of additional therapeutic application or some adverse effects [1]. The impurity profile of drug
substance and drug product is critical for safety assessment as impurities (Imps) can affect the
quality, efficacy and safety of the drug. Since ZIP is one of the most lipophilic antipsychotic with
high incidence for tautomerisation and photodegradation, development of efficient method for the
simultaneous determination of ZIP and its main Imps, significantly differing in polarity, was a very
challenging task. This prompt us to develop high performance liquid chromatography method with
the ultraviolet detection (HPLC-UV)[2]. The unknown degradant was detected and after that we
developed a highly sensitive and fast ultra high performance liquid chromatography (UHPLC)
method with tandem mass detection (UHPLC-MS/MS)[3] for the structure characterisation. Also,
the structure was investigated with nuclear magnetic resonance (NMR) spectroscopy.
Methods
Two chromatographic analyses, HPLC-UV and UHPLC-MS/MS, for the concurrent analysis of
assay and purity profile of ZIP were performed. Retention characteristics of 10 structurally related
ZIP compounds (metabolites/Imps/degradants) in the HPLC system were investigated by
Quantitative Structure Retention Relationship (QSRR) model.
Results
Based on the obtained HPLC chromatographic retention data, the QSRR model for the prediction
of the retention times for the other ZIP derivatives, including one earlier observed and an unknown
detected degradant was developed[4]. The successful separation and determination of ZIP and its
five main Imps, and identification of a novel ZIP degradant have been performed by UHPLC
MS/MS method and the NMR studies [3]. Both, HPLC-UV and UHPLC-MS/MS, methods were
validated and their performances were compared for separation and determination of the
investigated set of compounds[5]. It was proved that two separation methods are convenient for
the assay and purity control of ZIP in raw materials and pharmaceuticals, but still with enhanced
green profile of UHPLC-MS/MS method.
Conclusions
The whole study is important for the further estimation of structures of other unknown Imps of ZIP
which could greatly affect GPCR affinity and consequently therapeutic effect or might be of the
possible toxicological concern
Successful phtalate monitoring – challanges in ESI-MS parameters tuning
No full textObjective:
Recently, pthalates or phthalic acid esters (PAEs) have become the focus of regulatory and
scientific attention due to their ubiquitous occurrence and adverse health effects [1]. PAEs are
commonly analyzed by liquid chromatography - mass spectrometry (LC-MS) mainly with an
electrospray ionization (ESI) source in positive mode. As they are relatively non-polar, weak bases
analytes, the PAEs ES ionisation is a great challenge. The aim of this work was therefore to
understand how different instrumental parameters of the ESI ion source affect ionization of selected
PAEs and how to maximize their response.
Methods:
LC-MS analysis was performed using Accela HPLC system connected to TSQ Quantum Access MAX
mass spectrometer. The separation of phthalates was achieved by the previously optimized HPLC
method. The ions were analysed in ESI (+) Multiple Reaction Monitoring (MRM) mode. Spray voltage
(3500-5000 V), capillary temperature (250-350 °C) and vaporizer temperature (250-450 °C) were varied
according to the Central Composite Design (CCD) to maximize the phthalate responses.
Results:
PAEs with longer side chains (e.g. diisononyl phthalate, DINP) have shown a relatively low response
compared to those with short side chains (e.g. diethyl phthalate, DEP). Of the instrument parameters
investigated, capillary temperature and vaporizer temperature had a statistically significant influence (p
< 0.05) on the responses of all PAEs tested. Although high spray voltages such as 4500 or 5500 V are
commonly used in the literature [2], it has been shown that all values between 3500 and 5500 V are
suitable. The empirical models created were of high quality in terms of the predicted and adjusted
coefficients of determination (values above 0.85), with the exception of DEP, for which the LC-MS
response is less affected by the variation of the ion source parameters. For all PAEs tested, it was crucial
to set a lower capillary temperature to maximise the response.
Conclusions:
A lower capillary temperature appears to allow efficient desolvation without considerable in-source
fragmentation of parent ions. Tuning ESI source parameters is particularly important for high molecular
weight phthalates, with relatively weak response. Further studies should focus on other challenges
associated with phthalate analysis, such as appropriate sample handling to minimize contamination
Analytical quality by design compliant development of RP-HPLC method for the determination of riociguat and its four impurities in tablets
No full textObjective:
The main drawbacks of the methods described in the literature for the analysis of riociguat and its
impurities can be summarized as follows: gradient elution mode of RP-HPLC method for the
separation of related substances in the bulk drug and the use of an aqueous phosphoric acid
solution with an extremely low pH in the mobile phase for the analysis of impurities A and C in
the dosage forms. Thus, the aim of this investigation was development and robust optimization of
a reliable and robust isocratic RP-HPLC method for the qualitative and quantitative analysis of
riociguat and its four impurities in tablets following the Analytical Quality by Design (AQbD)
concept.
Methods:
The risk analysis strategy consisted of an Ishikawa diagram, followed by a Control, Noise and
eXperimental test (CNX) and a Failure Mode and Effects Analysis (FMEA). After this threestage
risk assessment, the critical method parameters (CMPs) were selected and their effects on
the critical method attributes (CMAs) were analyzed using the Design of Experiments (DoE)
methodology using the Box-Behnken optimization design. In order to achieve the pre-defined
quality, Monte Carlo simulations were applied, in which the error originating from the calculated
coefficients of the mathematical models was propagated and a Design Space (DS) was
constructed. A Plackett-Burman desing was used for quantitative robustness tests.
Results:
As a CMPs, acetonitrile content in the mobile phase, concentration of ammonium acetate in
aqueous part of mobile phase and column temperature were selected and their effect on CMAs
(retention factor of the last eluting peak (kimp D) and separation of critical peak pairs (Rimp B /
riociguat, Rimp C /imp D)) were investigated. It was followed by computation of the DS as the area
within which the robustness of the qualitative performance of the developed method is confirmed
(with the probability π = 95 % of reaching the pre-defined targets (Rimp B/riociguat ≥ 2.1, Rimp C/imp D
≥ 1.9 and kimp D ≤ 10)). Finally, working point was selected from the middle part of the DS, and
the optimal chromatographic conditions corresponding to the selected working point were: 34 %
of acetonitrile and 66 % of 15 mM ammonium acetate in the aqueous phase whose pH was
adjusted at 5.5 with glacial acetic acid, column temperature 36C. Then, verification experiments
were carried out and the analysis was performed on an XTerra® RP18 column (150 mm × 4.6
mm, particle size 3.5 μm). The flow rate was 1 mL/min, the detection wavelength was 210 nm,
while the injection volume was 20 μL. CMAs achieved satisfactory values (Rimp B / riociguat = 2.49,
Rimp C /imp D = 2.10 and kimp D = 8.5) under optimal chromatographic conditions. For this chosen
optimal chromatographic conditions method validation was implemented successfully and
included the investigation of stability of solutions, selectivity, linearity, accuracy and precision,
and additionally the limit of quantification and the limit of detection were determined for the
impurities A, B, C and D.
Conclusions:
The isocratic RP-HPLC method was successfully developed using the AQbD approach and subsequently
validated to confirm its reliability for routine quality control of riociguat and its four impurities in tablets
Progress toward Implementing Multiomic Approaches in Atherosclerotic Cardiovascular Disease: Update from the 4 th AtheroNET Meeting in Sarajevo (Bosnia and Herzegovina)
No full textThe COST Action CA21153 “Network for Implementing Multiomic Approaches in Atherosclerotic Cardiovascular Disease Prevention and Research” (AtheroNET; https://atheronet.eu) was launched in October 2022 with the mission to accelerate the application of multiomics in atherosclerosis research and to foster collaboration among experts from diverse disciplines. Born from the pressing need to advance atherosclerosis management and overcome existing challenges, AtheroNET creates a collaborative arena where innovation thrives through dialogue. The initiative was spearheaded by Prof. Miron Sopić (Faculty of Pharmacy, University of Belgrade), the primary proposer of the Action. The Action is led by Chair Prof. Paolo Magni (Università degli Studi di Milano) and Vice-Chair Prof. Yvan Devaux (Luxembourg Institute of Health), with strategic support from a dedicated leadership team:
• Grant Holder Scientific Representative: Dr. Ines Potočnjak
• Science Communication Coordinator: Prof. Georgios Kararigas
• Grant Awarding Coordinator: Dr. Susana Novella
• WG1 - Pathophysiological mechanisms: applying multiomics to uncover novel pathogenic players and processes; Leader: Prof. Dimitris Kardassis
• WG2 - Personalized clinical models: translating omics insights into improved management of ASCVD; Leader: Prof. Alberico Catapano
• WG3 - Standardization and harmonization: developing SOPs and guidelines to enhance reproducibility across omics research; Leader: Dr. Marie Mardal
• WG4 - Data integration and ML/AI: optimizing algorithms for integrating complex multiomic datasets; Leader: Dr. Aleksandra Gruca
• WG5 - Dissemination and communication: sharing advances with scientists, clinicians, patients, and the public; Leader: Prof. Georgios Kararigas
Since its inception, AtheroNET has grown to 475 members from across Europe, embracing COST’s core values of inclusiveness and excellence across geography, gender, and career stage. A significant proportion of members come from Inclusive Target Countries (n=282), with strong representation of women (n=277) and early-career researchers (n=223)
Chitosan–Hydrazone-Modified Calcium Phosphate Scaffolds: Fabrication, Characterization, and Drug Delivery Potential
No full textBackground/Objectives: Recent advancements in biomaterials aimed at closely mimicking natural biological tissues hold great promise for hard tissue regeneration and controlled drug release due to their superior physical, chemical, and biological properties. This study aimed to develop multi-ion doped calcium hydroxyapatite (HAp) scaffolds with chitosan-based coatings for localized drug delivery, incorporating a novel hydrazone compound with potential anticancer activity. Methods: HAp powders doped with magnesium (Mg2+), strontium (Sr2+), and varying fluoride (F−) contents (0–2 mol.%) were synthesized via a hydrothermal method. Scaffolds were fabricated using the sponge replica technique and subsequently coated with chitosan or a chitosan–hydrazone blend. Dopant incorporation was confirmed by electron dispersive X-ray spectroscopy (EDS). Phase composition and morphology were analyzed via X-ray diffraction (XRD) and scanning electron microscopy (SEM). Mechanical properties, bioactivity, cytotoxicity, and hydrazone release profiles were systematically evaluated. Results: EDS confirmed successful incorporation of Mg2+ and Sr2+ in all powders, while F− was detected only in powders with 1 and 2 mol.% fluoride. XRD and SEM revealed the phase composition and scaffold microstructure. Chitosan coatings significantly improved scaffold compressive strength and reduced degradation rate, indicating enhanced stability in biological environments. The coated scaffolds supported MRC-5 fibroblast viability. The hydrazone compound exhibited dose-dependent antitumor cytotoxicity comparable to cisplatin and showed sustained release from scaffolds for up to 15 days. Conclusions: The combination of multi-ion doped HAp scaffolds and chitosan–hydrazone coatings provides a promising platform for bone tissue engineering and localized cancer therapy, demonstrating both mechanical stability and controlled, sustained drug release
Deciphering Morris water maze behavior in aging Sprague-Dawley rats: Taking care of age- and sex-specific effects in preclinical research
No full textAs aging is recognized as one of the risk factors for cognitive impairment contributing to
thedecline in daily functioning in the aging population, its impact should be better assessed and
characterizedin preclinical models. Therefore, we investigated the differences in learning and
memory behavior in the Morris water maze (MWM) between aging and young adults.
15- and 7-month-old Sprague-Dawley rats of both sexes were subjected to extended
behavioral battery including the MWM. The animals were trained to find the platform for 4 days.
Further, probe test was performed in which platform was removed and the animals were allowed
to explore MWM.
Aging animals covered greater distance than young adults during the training phase. Only
young adult males and females covered shorter distance on training day 4 vs day 1. In addition, an
interaction between sex and age for distance was observed during the training phase. On training
day 2, aging males showed longer latency to find the platform compared to young adult males.
Aging and young adult females showed higher percentage of peripheral time than young adult
males on the training day 4. In the probe test, the latency to find the platform did not differ between
groups, but the young adult females traveled greater distance than the young adult males. In
addition, these animals had higher percentage of peripheral time compared to all other groups,
except for the young adult males, where the difference was at trend level.
Learning deficits in aging animals were partially associated with motor impairments in
both sexes and were pronounced in aging males at the beginning of the training phase. Sex-specific
differences were reflected in the increased anxiety observed in females, which may also contribute
to the reduced learning performance. In the probe test, young adult females showed the
hyperactivity characteristic of this age group, but also increased anxiety. The latter was reversed
in the aging females, indicating their possible emotional maturation. The complex cognitive
behavior in older age, which is influenced by sex, should be taken into account in drug discovery
research of dementia and other age-related diseases
Quercetin, Catechin, and Diosmin as Modulators of Haloperidol–HSA Interactions: A Biophysical and Computational Study
No full textPotential interactions of haloperidol with food ingredients such as flavonoids may be of great importance both for understanding the pharmacokinetic interactions of xenobiotics with human serum albumin and for clinical practice itself. In this study, the effect of the flavonoids quercetin, catechin, and diosmin on the interaction of haloperidol and human serum albumin was examined. These flavonoids are very common in foods of plant origin. Haloperidol is a typical antipsychotic that has a pronounced binding affinity for human serum albumin. Fluorescence spectroscopy, molecular docking analysis, and molecular dynamics simulations were used for these tests. Previous studies have shown that all test substances bind to the same binding site on human serum albumin (Sudlow site I, Subdomain IIA). Fluorescence spectroscopy revealed that the tested flavonoids reduce the value of the haloperidol binding constant to human serum albumin (from 4.45 × 103 in the binary system to 3.75 × 102, 5.40 × 102 and 6.24 × 102 in the ternary systems, respectively), due to competition for the same binding site. Experimental results were confirmed by molecular docking analysis and molecular dynamics simulations
Polifenoli i antioksidativni kapacitet odabranih vrsta iz familije Compositae
No full textSpecies belonging to the Compositae family are known for their rich content
of phenolic compounds and flavonoids with notable antioxidant properties.
Given the increasing demand for natural antioxidants in various industries,
this study aims to evaluate the total phenolic and flavonoid content alongside
antioxidant activities of ethanolic extracts of six Compositae species; to
identify interspecific differences and highlight their potential for use in
pharmaceutical, cosmetic, and food sectors. The investigated species include
Centaurea salonitana, C. atropurpurea, Achillea atrata, A. clavennae, Filago
arvensis, and Xeranthemum annuum. Phenolic content was quantified utilizing
the Folin–Ciocalteu method (GAE), while flavonoid content was measured as
rutin equivalent (RuE). The antioxidant activity was determined via the DPPH
assay (IC 50 values). Significant interspecific variation was observed; notably,
Achillea atrata exhibited the highest phenolic content, whereas Centaurea
salonitana was distinguished by its elevated flavonoid content.Vrste iz familije Compositae poznate su po visokom sadržaju fenolnih
jedinjenja i flavonoida, koji poseduju izražena antioksidativna svojstva.
Imajući u vidu sve veću potražnju za prirodnim antioksidansima u raznim
industrijama, cilj ovog istraživanja je bio da se proceni ukupan sadržaj fenola
i flavonoida, kao i antioksidativna aktivnost etanolnih ekstrakata šest vrsta
familije Compositae; da se identifikuju interspecijske razlike i istakne njihov
potencijal za upotrebu u famaceutskom, kozmetičkom i prehrambenom
sektoru. U okviru istraživanja ispitivani su etanolni ekstrakti sledećih vrsta:
Centaurea salonitana, C. atropurpurea, Achillea atrata, A. clavennae,
Filago arvensis i Xeranthemum annuum. Ukupan sadržaj fenola određen je
primenom Folin–Ciocalteu metode i izražen kao ekvivalent galne kiseline
(GAE), dok je sadržaj flavonoida izražen kao ekvivalent rutinu (RuE).
Antioksidativna aktivnost određena je DPPH metodom putem IC50 vrednosti.
Rezultati su pokazali značajne interspecijske varijacije, pri čemu je Achillea
atrata imala najviši sadržaj fenolnih jedinjenja, dok se Centaurea salonitana
istakla visokim sadržajem flavonoid