Application of machine learning and molecular docking to the modelling and design of HDAC6/ROCK dual inhibitors with in vitro experimental validation

Background & Aims: Biliary tract cancer (BTC) is a rare but aggressive malignancy with limited treatment options and poor prognosis, necessitating novel therapeutic strategies. Histone deacetylases (HDACs) play a critical role in the epigenetic regulation of BTC progression, and HDAC inhibitors have shown promise in preclinical studies. However, their clinical efficacy as monotherapies remains limited, driving the exploration of multitarget approaches. Recent studies by Djokovic et al. highlight the potential of combining HDAC and Rho-associated protein kinase (ROCK) inhibitors as an antimetastatic strategy. This study aimed to develop machine learning (ML) models for predicting the activity of HDAC6 and ROCK2 inhibitors and to apply these models in the design and evaluation of novel dual HDAC/ROCK inhibitors. Materials & Methods: Datasets for HDAC6 and ROCK2 inhibitors were extracted from the ChEMBL database, preprocessed, and used to calculate 2D molecular descriptors. Predictive models were developed using random forest regression to establish quantitative structure–activity relationship (QSAR) models for both targets. The models were applied for virtual screening of newly designed dual inhibitors, followed by molecular docking studies to assess binding potential. Results: The ML-driven QSAR models demonstrated high predictive accuracy for HDAC6 and ROCK2 inhibitors. Virtual screening identified promising dual HDAC/ROCK candidates, which were further evaluated through molecular docking. The top candidates were synthesized and tested in enzyme assays confirming a lead compound with potent activity, accurately predicted by the ML models. Further in vitro studies in BTC cell lines are ongoing to assess anticancer efficacy. Conclusion: This study presents an ML-guided approach for the rational design of dual HDAC6/ROCK inhibitors, offering a promising therapeutic strategy for BTC. The integration of computational modeling with experimental validation demonstrates the potential of AI-driven drug discovery in oncology.Monothematic Conference, Liquid Biopsy in BTC: from bench to bedside, May 22-23, 2025, Mallorca, Spai

Assessment of Suppressive Effects of Negative Air Ions on Fungal Growth, Sporulation and Airborne Viral Load

Spores of filamentous fungi are common biological particles in indoor air that can negatively impact human health, particularly among immunocompromised individuals and patients with chronic respiratory conditions. Airborne viruses represent an equally pervasive threat, with some carrying the potential for pandemic spread, affecting both healthy individuals and the immunosuppressed alike. This study investigated the abundance and diversity of airborne fungal spores in both hospital and residential environments, using custom designed air samplers with or without the presence of negative air ions (NAIs) inside the sampler. The main purpose of investigation was the assessment of biological effects of NAIs on fungal spore viability, deposition, mycelial growth, and sporulation, as well as airborne viral load. The precise assessment of mentioned biological effects is otherwise difficult to carry out due to low concentrations of studied specimens; therefore, specially devised and designed, ion-bioaerosol interaction air samplers were used for prolonged collection of specimens of interest. The total fungal spore concentrations were quantified, and fungal isolates were identified using cultural and microscopic methods, complemented by MALDI-TOF mass spectrometry. Results indicated no significant difference in overall spore concentration between environments or treatments; however, presence of NAIs induced a delay in the sporulation process of Cladosporium herbarum, Aspergillus flavus, and Aspergillus niger within 72 h. These effects of NAIs are for the first time demonstrated in this work; most likely, they are mediated by oxidative stress mechanisms. A parallel experiment demonstrated a substantially reduced concentration of aerosolized equine herpesvirus 1 (EHV-1) DNA within 10–30 min of exposure to NAIs, with more than 98% genomic load reduction beyond natural decay. These new results on the NAIs interaction with a virus, as well as new findings regarding the fungal sporulation, resulted in part from a novel interaction setup designed for experiments with the bioaerosols. Our findings highlight the potential of NAIs as a possible approach for controlling fungal sporulation and reducing airborne viral particle quantities in indoor environments

Population pharmacokinetics of linezolid and correlation with efficacy and safety parameters in patients with acute respiratory distress syndrome on veno-venous extracorporal membrane oxygenation

Veno-venska ekstrakorporalna membranska oksigenacija (V-V ECMO) je sve više zastupljena u zbrinjavanju respiratorne insuficijencije kod kritično oboljelih. Ipak, uticaj ovog oblika terapijske podrške na farmakokinetiku (FK) i doziranje lijekova, posebno linezolida, još uvijek nije dovoljno istražena oblast. Cilj istraživanja je da se procijeni opravdanost primjene viših doza linezolida, u poređenju sa standardnim, kod kritično oboljelih sa akutnim respiratornim distres sindromom (ARDS) uzrokovanim COVID-19, koji su istovremeno bili na V-V ECMO podršci. U istraživanje su prospektivno uključeni bolesnici sa dijagnostikovanim COVID-19 i ARDS uz terapijski model vvECMO podrške sa istovremenom intravenskom primjenom linezolida 600 mg/8 h. Kriterijumi za isključivanje su bili: osobe mlađe od 18 godina, poznata alergija na linezolid, trudnoća, terapijska izmjena plazme u posljednjih 24 sata i nadomjesna bubrežna terapija. Za analizu FK linezolida od svakog bolesnika u stanju ravnoteže lijeka prikupljeno je po šest uzoraka krvi u definisanim vremenskim tačkama. Prikupljeni su svi demografski i klinički podaci neophodni za procjenu uticaja istih na FK linezolida. Nelinearnim modeliranjem kombinovanih efekata razvijen je i validiran populacioni FK model, korišten za Monte Karlo simulacije (5000 virtualnih bolesnika) za generisanje individualnih FK parametara i koncentracijskih profila nakon režima doziranja 600 mg/8 h i 600 mg/12 h. Za procjenu vjerovatnoće postizanja ciljnih vrijednosti farmakokinetičkog-farmakodinamičkog (FK-FD) indeksa (PTA) korišteni su sljedeći targeti: 85%T>MIC, fAUC24/MIC≥80 i fAUC24/MIC≥100. Izračunata je i kumulativna frakcija odgovora (CFR) za različite Gram-pozitivne bakterije. Praćeni su i bezbjednosni aspekti na osnovu promjene nivoa trombocita i razvoj trombocitopenije u odnosu na vrijeme započinjanja vvECMO i linezolida uz istovremenu primjenu. Ukupno su analizirane 53 koncentracije linezolida. Pokazana je visoka korelacija izmjeđu Cmin i AUC24. FK parametri linezolida nisu značajno odstupali u odnosu na ne-ECMO bolesnike i nije pronađen značajan uticaja kovarijata na FK parameter. Nakon doznog režima linezolida 600 mg/8 h predviđeno je jednako i veće postizanje FK-FD ciljne vrijednosti 85%T>MIC za MIC=2 mg/L kod 90%, dok je kod bolesnika koji su primali standardni dozni režim 85%T>MIC zabilježen kod dvije trećine bolesnika. fAUC24/MIC≥80 postignut je kod skoro tri puta većeg broja bolesnika primjenom linezolida 600 mg/8 h za istu vrijednost MIC-a. Prisustvo trombocitopenije sa značajnim smanjenjem broja tombocita zabilježeno je kod ukupno 81,8% bolesnika. Rezultati istraživanja ukazaju da dozni režim linezolida 600 mg/8 h u odnosu na standardni ima prednost kod istovremene primjene vvECMO terapijskog modela za postizanje FK-FD ciljnih vrijednosti linezolida i dobijanja adekvatnog terapijskog odgovora. Terapijsko praćenje koncentracije linezolida u serumu i broja trombocita je neophodno kako bi se zadovoljili bezbjednosni aspekti uz maksimalan terapijski efekat linezolida kod kritično oboljelih sa ARDS na V-V ECMO podršci.Veno-venous extracorporeal membrane oxygenation (V-V ECMO) is increasingly used in managing respiratory failure in critically ill patients. However, the impact of this form of therapeutic support on the pharmacokinetics (PK) and dosing of medications, especially linezolid, remains an insufficiently explored area. This study aims to evaluate the rationale for administering higher doses of linezolid, compared to standard doses, in critically ill patients with acute respiratory distress syndrome (ARDS) caused by COVID-19, who are concurrently supported by V-V ECMO. The study prospectively included patients with COVID-19, ARDS, on a therapeutic V-V ECMO support model with concurrent intravenous administration of linezolid 600 mg every 8 hours. Exclusion criteria were: individuals under 18 years, known allergy to linezolid, pregnancy, therapeutic plasma exchange in the last 24 hours, and renal replacement therapy. For the analysis of linezolid PK, six blood samples were collected from each patient at steady state at defined time points. All demographic and clinical data necessary for assessing their impact on linezolid PK were collected. A population PK model was developed and validated using nonlinear modeling of combined effects, which was then employed in Monte Carlo simulations (5,000 virtual patients) to generate individual PK parameters and concentration profiles after dosing regimens of 600 mg every 8 hours and 600 mg every 12 hours. The probability of achieving target pharmacokinetic-pharmacodynamic (PK-PD) index values (PTA) was assessed using the following targets: 85%T>MIC, fAUC24/MIC≥80 i fAUC24/MIC≥100. The cumulative fraction of response (CFR) was also calculated for various Gram-positive bacteria. Safety aspects were monitored based on changes in platelet levels and development of thrombocytopenia in relation to the initiation of V-V ECMO and linezolid with concurrent application. A total of 53 linezolid concentrations were analyzed. A high correlation was found between Cmin and AUC24. Linezolid PK parameters did not significantly deviate from those in non-ECMO patients, and no significant impact of covariates on PK parameters was found. After the linezolid dosing regimen of 600 mg every 8 hours, the probability of achieving PK-PD target value of 85%T>MIC for MIC =2 mg/L was predicted to be 90%, while two-thirds of the patients on the standard dosing regimen reached 85%T>MIC. fAUC24/MIC≥80 was achieved in nearly three times more patients with the 600 mg every 8 hours linezolid regimen for the same MIC value. Thrombocytopenia occurred in 81.8% of patients with a significant reduction in platelet count. This study indicates that the linezolid dosing regimen of 600 mg every 8 hours, compared to the standard, is advantageous when using the V-V ECMO therapeutic model to achieve linezolid PK-PD target values and adequate therapeutic response. Therapeutic monitoring of linezolid serum concentrations and platelet counts is necessary to satisfy safety aspects while maximizing therapeutic effects in critically ill patients with ARDS on V-V ECMO

Comparing overlapping mean response, desirability functions, and analytical quality by design approaches to the optimization of chaotropic chromatography method for the separation of citalopram and its impurities

Tradicionalni pristupi multikriterijumskoj optimizaciji, kao što su primena funkcija poželjnih odgovora i preklapanje prosečnih odgovora, nisu u skladu sa definicijom prostora dizajna datom u ICH Q14, jer ne pružaju meru osiguranja kvaliteta, tj. verovatnoću da ključni atributi kvaliteta ispunjavaju granične vrednosti prihvatljivosti [1]. Dodatni problem je što ovi pristupi ne uzimaju u obzir nesigurnost u procenjenim vrednostima koeficijenata modela, niti korelaciju reziduala regresionih modela [2]. Cilj ovog rada bio je da se ispita koliko se pomenute teorijske razlike odražavaju na rezultate optimizacije na primeru metode za ispitivanje stabilnosti citaloprama u tabletama. U ovom slučaju, retencioni factor nečistoće A (k) i rezolucija između citaloprama i nečistoće E ( Rs) izabrani su kao kritični atributi kvaliteta sa sledećim graničnim vrednostima: k > 1 i Rs > 2. Kritični parametri analitičke metode bili su sadržaj acetonitrila i koncentracija perhlorata u mobilnoj fazi i temperatura kolone. Upoređeni su region dobijen preklapanjem odgovora, region pri kojem je vrednost funkcije poželjnih odgovora veća od 0,6 i prostor dizajna određen Bajesovom analizom. Na osnovu dvodimenzionalnih grafičkih prikaza utvrđeno je da se optimalni region dobijen tradicionalnim pristupima u velikoj meri poklapa sa prostorom dizajna. Razlog za to može biti slaba korelacija reziduala (–0,26) i male standardne greške koeficijenata modela (0,0015–0,0240). S obzirom na odsustvo komercijalno dostupnih softvera za određivanje prostora dizajna prema ICH Q14, moguća alternativa je da se prikažu mere nesigurnosti parametara i korelacije reziduala zajedno sa rezultatima tradicionalne optimizacije, čime se obezbeđuje dodatna garancija kvaliteta analitičke metode.Traditional approaches to multicriteria optimization, including desirability functions and overlapping mean responses (OMRs), are not aligned with definition of design space in ICH Q14 because they do not provide a measure of quality assurance, i.e., the probability that critical quality attributes (CQAs) meet acceptance limits [1]. Furthermore, these approaches neglect uncertainty of the model coefficient estimates and correlation of the regression residuals [2]. The aim of this work was to evaluate how such theoretical differences affect the optimization results using the example of a stability-indicating method for citalopram tablets. In this case, the retention factor of impurity A (k) and the resolution between citalopram and impurity E (Rs) were selected as CQAs with the following acceptance limits: k > 1 and Rs > 2. Critical parameters of the analytical method were acetonitrile content, perchlorate concentration in the mobile phase, and column temperature. The region obtained by OMR, the region where the global desirability function exceeded 0.6, and the design space derived by Bayesian analysis were compared. Two-dimensional contour plots showed that the optimal regions obtained by traditional approaches largely overlapped with the design space. This can be explained by the low correlation of the residuals (−0.26) and the low standard errors of the model coefficients (0.0015–0.0240). Since there is no available software to determine the design space according to ICH Q14, a possible alternative is to report the measures of parameters uncertainty and residual correlation along with traditional optimization results, providing an additional measure of analytical method quality

Exploring the effect of bioenvironment on the ionization of drugs using the membrane mimetic properties of differently charged micelles

Objective The membrane-mimicking approach is grounded in the idea of organizing molecules into distinct compartments within systems, which can influence reaction rates, physicochemical properties and stereochemistry of pharmacologicaly active compounds, in ways that differ from those observed in "pure" water [1]. There isn't a perfect model that can replicate all the complexities of biological membranes, but a large variety of membrane mimetics are available [2]. Among them, micellar solutions of differently charged surfactants are systems the most commonly used to mimic the desired functions of cells membranes, since their properties are well understood at the chemical level [1]. Methods The pKa values of pharmacologically active compounds, with and without the presence of anionic (SDS), cationic (CTAB) and nonionic (TX-100, Brij 35) micelles, were determined potentiometrically, under the same experimental conditions (temperature 25°C and ionic strength 0.1 M NaCl). The experimental results were analyzed in the Hyperquad program. Compounds which are distereomers are additionally analysed in RP-HPLC system using Chromolith Performance using RP-18e column and phosphate buffer pH 7.0-acetonitrile mixture as a mobile phase. Results The presence of micelles caused the most significant shifts in the protolytic equilibria (∆pKa) of amino (-2.80 to +1.44) and carboxyl groups (-0.92 to +1.90). A more pronounced effect on the change in ionization mode was observed in the presence of anionic and cationic micelles compared to nonionic micelles. A change in the distribution of equilibrium forms of investigated drugs, in the presence of micelles, was observed at biopharmaceutically significant pH values (1.2; 4.5; 6.8; 7.4), where the content of ionized forms changed in the range from -74% to +66% compared to "pure" water. Conclusions Small changes in solution conditions can significantly affect the protolytic equilibria of drugs, suggesting that the ionization of drugs under physiological conditions may be completely different than expected exclusively on the basis of the pKa values determined in “pure” water

Enhancing Transcutaneous Drug Delivery: Advanced Perspectives on Skin Models

Skin acts as a dynamic interface with the environment. Pathological alterations in the skin barrier are associated with skin diseases. These conditions are characterized by specific impairments in epidermal barrier functions. Despite its protective nature, the skin can be a relevant route of drug administration, both for topical and transdermal therapy, allowing for improved drug delivery and reducing the incidence of adverse reactions. This manuscript reviews transcutaneous drug delivery as a strategy for treating localized and systemic conditions, highlighting the importance of skin models in the evaluation of drug efficacy and barrier function. It explores advances in in vitro, ex vivo, in vivo, and in silico models for studying cellular uptake, wound healing, oxidative stress, anti-inflammatory and immune modulation activities. Disease-specific skin models are also discussed

Inovacije u 3D štampanim farmaceutskim oblicima lekova za pedijatrijsku populaciju - primer lozengi za žvakanje

Three-dimensional (3D) printing offers a versatile platform for producing personalized, age-appropriate dosage forms that address the specific therapeutic and administration needs of the pediatric population. Among several oral dosage forms that can be prepared by 3D printing, chewing lozenges offer numerous advantages, especially for the pediatric population. This study illustrates a formulation development and selection of process parameters for 3D-printed chewing lozenges containing propranolol hydrochloride as a model drug, with potential application in the pediatric population. It also highlights the advantages of 3D printing using the semi-solid material extrusion method. Gelatin and sodium alginate were used as carriers for 3D printing. Immersion time in the calcium chloride solution (tim) and lozenges shape were varied, while the following tests were performed: the assessment of organoleptic properties, mass and thickness variations, melting and disintegration time, drug content and dissolution rate. Initially, the key 3D printing parameters for lozenge production were identified, and then the appropriate formulation was selected. Subsequent testing demonstrated that the shape of lozenges, along with variations in tim, influenced the pharmaceutical-technological characteristics. A formulation based on the combination of gelatin and sodium alginate in 1:3 ratio, immersed for 60 seconds in a calcium chloride solution, was found to be suitable for 3D printing of chewable lozenges.Trodimenzionalna (3D) štampa pruža široku platformu za proizvodnju personalizovanih farmaceutskih oblika prilagođenih uzrastu, koji odgovaraju specifičnim terapijskim potrebama pedijatrijske populacije. Među različitim oralnim farmaceutskim oblicima koji se mogu pripremiti pomoću 3D štampe, lozenge za žvakanje nude brojne prednosti, naročito za pedijatrijske pacijente. Ovaj rad prikazuje razvoj formulacije i izbor procesnih parametara za 3D-štampane lozenge za žvakanje koje sadrže propranolol-hidrohlorid kao model supstancu, sa potencijalnom primenom u pedijatrijskoj populaciji. Takođe, ističu se prednosti 3D štampe korišćenjem metode ekstruzije polučvrstog materijala. Želatina i natrijum-alginat korišćeni su kao nosači za 3D štampu. Varirani su vreme uranjanja u rastvor kalcijum-hlorida (tim) i oblik lozengi, pri čemu su sprovedena sledeća ispitivanja: procena organoleptičkih svojstava, variranje mase i debljine, vreme topljenja i raspadljivost lozengi, sadržaj i brzina rastvaranja propranolol-hidrohlorida. U početnoj fazi identifikovani su ključni parametri 3D štampe za izradu lozengi, a zatim je odabrana odgovarajuća formulacija. Dalja ispitivanja su pokazala da oblik lozengi, u kombinaciji sa varijacijama u vremenu uranjanja, utiče na farmaceutsko-tehnološke karakteristike izrađenih preparata. Formulacija zasnovana na kombinaciji želatine i natrijum-alginata u odnosu 1:3, uronjena 60 sekundi u rastvor kalcijum-hlorida, pokazala se pogodnom za 3D štampu lozengi za žvakanje

Inulin-Stabilised Vegetable Oil Emulsions as Fat Replacers in Chicken Frankfurters: Technological and Textural Evaluation

This study investigated the complete replacement of pork backfat in frankfurters with inulin-based emulsion gels made from linseed, walnut or algal oil and structured in two ratios (1:2:0.5 and 1:2:1, oil–water–inulin). Proximate composition, water holding capacity, emulsion stability and colour were assessed after production, while texture profile analysis (TPA) was monitored during 45 days of vacuum storage. The reformulated sausages showed a significant reduction in fat content (from 21.91% to 3.81%, p < 0.001) and increased water and carbohydrate levels (p < 0.001). These shifts in composition resulted in a slightly lower pH, higher cooking and purge losses and lower emulsion stability (p < 0.001), particularly when treated with algal oil. Colour measurements revealed lighter (higher L*, p = 0.008) and more yellowish sausages (p < 0.001), with walnut oil at a 1:2:0.5 emulsion ratio showing the least deviation from the control (ΔE = 7.45). The TPA showed that oil type was the dominant factor. Walnut formulations, especially in the 1:2:1 ratio, had hardness and chewiness values closest to those of the control, while algal sausages were softer and less cohesive (p < 0.05). PCA and heatmap analyses confirmed clustering by oil type and storage time, underlining the technological suitability of the walnut gels. Overall, inulin–oil gels enable nutritional reformulation but pose a technological challenge, with walnut oil proving to be the most promising substitute and algal oil requiring additional stabilisation

Single-cell RNA sequencing (scRNA-seq) and its insights into cellular heterogeneity in atherosclerosis

Single-cell RNA sequencing (scRNA-seq) has transformed our understanding of cellular diversity in human biology, providing novel insights into disease mechanisms. In cardiovascular disease (CVD), scRNA-seq enables precise mapping of complex cell populations, uncovering unique cell types and states that influence disease progression and suggest new therapeutic targets. In atherosclerosis (AS), scRNA-seq has redefined plaque pathology by identifying distinct cell types, including endothelial cells (ECs), smooth muscle cells (SMCs), fibroblasts, macrophages, T cells, and B cells, each with specific roles in plaque stability, inflammation, and disease progression. In our review, we summarized these major cellular populations and their cellular heterogeneity in non-diseased and atherosclerotic aorta, as identified by scRNA-seq in mice and human tissues. We discussed conserved and species-specific subpopulations, their defining markers, and their functional implications in plaque progression. In addition, we integrated findings from scRNA-seq with experimental studies to highlight key molecular targets with therapeutic potential. In the future, these insights offer a refined cellular and molecular framework of atherosclerosis and may help the development of targeted interventions to promote plaque stabilization and reduce cardiovascular risk