Search Results (252)

A robust and unified reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous quantitative analysis of pitavastatin calcium and fenofibrate in dual-layer tablet formulations. Although individual analytical methods for each active pharmaceutical ingredient have been reported, a single analytical procedure applicable to both assay and dissolution testing of this fixed-dose combination has not been sufficiently established. In this study, a single RP-HPLC method was optimized to support both quality control purposes, thereby improving analytical efficiency and reducing method-related variability. Chromatographic separation was achieved using a C18 column (4.6 × 150 mm, 5 µm) under isocratic conditions, with a flow rate of 1.1 mL/min, an injection volume of 40 µL, and UV detection at 245 nm. The total run time was 10 min. The method was validated in accordance with ICH guideline Q2(R1) for system suitability, specificity, linearity and range, accuracy, precision (repeatability and intermediate precision), limits of detection and quantitation, and solution stability. Validation was conducted for both assay and dissolution samples using the same chromatographic conditions. The method demonstrated excellent linearity over the validated concentration ranges for both assay and dissolution analyses (r² ≥ 0.99). Accuracy and precision results satisfied the predefined acceptance criteria for assay (98.0–102.0%) and dissolution-related quantification (95.0–105%), with relative standard deviation values not exceeding 2.0%. The method showed adequate sensitivity, specificity, and solution stability, confirming its suitability for routine analysis. Application of the validated method to finished dual-layer tablets demonstrated reliable assay results and clearly distinguished the rapid dissolution of pitavastatin calcium from the delayed dissolution behavior of fenofibrate. Overall, the developed RP-HPLC method provides a validated analytical platform capable of supporting both assay and dissolution testing of pitavastatin–fenofibrate dual-layer tablets. Beyond routine validation, the proposed analytical framework demonstrates how a single chromatographic condition can support multiple quality attributes, offering an analytically integrated approach for supporting multiple quality attributes in complex combination drug products. Full article

Background/Objectives: Among the antiretroviral therapies (ARTs) recently introduced for people living with HIV (PLWH), the fixed-dose combination of bictegravir, emtricitabine and tenofovir alafenamide (B/F/TAF) became reimbursable in Italy in June 2019. Methods: This study evaluated drug utilization, healthcare resource consumption and direct costs among ART-naïve adults initiating B/F/TAF or other non-bictegravir-based regimens, identified from June 2019 to September 2022 within administrative databases of healthcare entities covering approximately nine million citizens. Baseline clinical characteristics at first ART prescription were compared across B/F/TAF-treated patients, those receiving other ART regimens, and non-HIV controls, while treatment outcomes during follow-up were evaluated among PLWH receiving B/F/TAF or other ARTs; healthcare consumption and costs were assessed after propensity score matching within the PLWH cohorts only. Results: Overall, 374 individuals initiated B/F/TAF and 5576 other ARTs. Patients treated with B/F/TAF showed greater adherence and persistence, with multivariate analyses confirming a lower risk of discontinuation or switching (HR = 0.66, 95% CI 0.57–0.76, p < 0.001) and a higher likelihood of adherence (HR = 2.40, 95% CI 1.58–3.64, p < 0.001). After matching, the B/F/TAF group exhibited lower 12-month consumption of non-HIV medications, fewer non-HIV hospitalizations, and reduced total healthcare costs, particularly for non-HIV drug prescriptions compared to other ART users. Conclusions: Overall, B/F/TAF was associated with better treatment continuity and meaningful cost savings. Full article

Background: Fixed-dose combination drugs (FDCs) are combinations of two or more active ingredients in a single dosage form. These formulations have proven effective in combating the development of resistance in diseases such as tuberculosis and malaria. Despite the benefits observed in the aforementioned cases, fixed-dose antibiotics combinations (FDACs) are increasingly raising questions about their rationality. This is the case for several FDACs listed in the AWaRe classification as not recommended, which unfortunately remain available on the pharmaceutical market, particularly in low- and middle-income countries like the Democratic Republic of Congo (DRC). Objectives: To identify the essential medicines available in pharmacies open to the public in the city of Kinshasa and to assess their inclusion in the DRC’s National List of Essential Medicines (NLEM) and in the World Health Organization’s (WHO) List of Essential Medicines (LEM). The rationality of the FDACs circulating in the city of Kinshasa were also evaluated based on the 2023 AWaRe classification. Methods: A cross-sectional and descriptive study was conducted between February and October 2025 in Kinshasa. For this purpose, fifty registered pharmacies open to the public were selected by systematic random sampling as the research sample. Data collection consisted of completing a data collection form after we had provided the pharmacies’ owners with the necessary explanations regarding the importance of the study and guaranteed their anonymity. Results: The controlled FDACs encountered comprised 27 specialties across 15 different formulations. Out of 15 formulations, 12 (80%) were included on the WHO list of non-recommended antibiotics and were not included in the DRC’s NLEM nor in the WHO’s LEM. Some had been withdrawn from the market in their countries of manufacture. Of the 15 FDACs evaluated for their rationality and compliance, the injectable FDACs presented problems related to the relevance and completeness of information contained on their packaging. On their primary packaging, there was a significant difference in the expiration dates of the powder and sterile water for injection contained in the combination pack, ranging from 6 to 36 months. Furthermore, the secondary packaging lacked data related to the sterile water for injection contained in the combination pack. In addition, several medications contained the same therapeutic combination. For injectable FDAC, for example, the combination Ceftriaxone-Sulbactam was represented by eight medications. For oral FDACs, the combination Sulfamethoxazole-Trimethoprim was represented by seven medications. Globally, 100% of these drug combinations originated from India. Conclusions: Fifteen varieties of FDACs were available in Kinshasa, most of which (80%) were unsuitable. It is important that public health authorities address this situation and develop stricter guidelines for granting marketing authorizations, particularly for FDACs. Full article

Urinary gluten immunogenic peptides (u-GIPs) have been proposed as a complementary marker to classical intestinal permeability tests based on lactulose, mannitol, and the lactulose:mannitol ratio (LMR). However, the effects of gluten dose, urine collection interval, and sampling strategy on their performance remain insufficiently defined. This study evaluated these variables to support protocol standardization. Data from four standardized protocols including 46 healthy adults exposed to 0, 2, 4, or 10 g of gluten were analyzed. All participants ingested fixed doses of lactulose and mannitol. Urine was collected cumulatively (0–6 h and 0–15 h) or by individual voids. u-GIP levels were measured by lateral-flow immunoassay, and lactulose and mannitol by ion chromatography. u-GIP excretion showed a clear dose dependence. Lactulose excretion increased transiently only at the 10 g dose during the 0–6 h interval, while mannitol excretion and LMR were unaffected. The u-GIP excretion index showed linear proportionality at the 2 g and 4 g doses but exhibited saturation kinetics at the 10 g dose. The 4 g dose showed the lowest interindividual variability. Sampling strategies yielded equivalent results. A 4 g gluten challenge combined with a 6 h urine collection demonstrated effectiveness in healthy volunteers and may be suitable for clinical application. Further research involving larger cohorts of both healthy individuals and patients with intestinal hyperpermeability is required to validate this method. Full article

Neonicotinoid insecticides and fluoroquinolone antibiotics frequently co-occur in aquatic and terrestrial environments, posing a threat to human health, yet their combined neurotoxic potential remains poorly characterized. This study aimed to assess the cytotoxicity of typical neonicotinoids and fluoroquinolones as well as their mixtures in human neuroblastoma SK-N-SH cells and identify affected pathways. SK-N-SH cells were exposed to clothianidin (CLO), imidacloprid (IMI), enrofloxacin (ENR), and ofloxacin (OFX) individually and in fixed-ratio mixtures (50% of each compound’s IC₅₀) for 24 h and 48 h, and cell viability was quantified using the alamarBlue^® method. Single-compound dose–response testing showed time-dependent cytotoxicity, with higher potency for fluoroquinolones (24 h IC₅₀: ENR 1.446 mM, OFX 2.742 mM; 48 h IC₅₀: ENR 0.826 mM, OFX 2.005 mM) than neonicotinoids (24 h IC₅₀: IMI 4.754 mM, CLO 5.356 mM; 48 h IC₅₀: IMI 3.631 mM, CLO 4.029 mM). Concentration-addition analysis indicated that most mixtures produced synergistic interaction in reduction in cell viability, with ENR+OFX showing the strongest effect at 48 h (Observed viability 7.138% vs. Predicated viability 82.368%). RNA-seq (24 h) revealed that binary mixtures generally induced more differentially expressed genes than single exposures, and ENR-containing mixtures showed the largest transcriptomic shifts, enriching pathways related to cellular stress and injury as well as neuronal signaling and connectivity. RT-qPCR validated the changes in expressions of five key neurobiology-relevant genes (LMO3, NOS1, ADCY8, FGF7 and TNFRSF12A). These findings highlight the importance of assessing insecticide–antibiotic mixtures when evaluating their hazards in environment. Full article

Background: Food rich in tocopherols (T) and tocotrienols (T3) are considered functional due to their ability to reduce oxidative stress and modulate anti-viability and pro-apoptotic pathways with anticancer potential; however, their efficacy differs between T and T3 and among isoforms (α and γ) likely due to differences in intracellular uptake and, consequently, in the activation of anticancer signaling pathways. To address these isoform-dependent differences, HeLa and MCF7 cancer cell lines were used to assess the antiproliferative activity of α-tocopherol (αT), γ-tocopherol (γT) and tocotrienols (Tocomin) as well as their pharmacological interactions according to Loewe and Chou–Talalay models. Methods: The tocol profile of the commercial mixture of T3 (Tocomin) was quantified by normal-phase HPLC. HeLa, MCF7, and ARPE-19 cells were cultured in DMEM supplemented with 10% FBS and exposed to αT, γT, or Tocomin (50–800 µg/mL; DMSO vehicle) for 48 h; viability was measured by the MTT assay and EC50 values were obtained from log(dose)–response fits (n = 3). Fixed-ratio (1:1) combinations were evaluated in HeLa and MCF7, and interactions were quantified using Loewe additivity and Chou–Talalay combination indices, supported by isobologram analysis. Results: Tocomin showed greater potency with αT and γT, and synergy with αT/γT; however, the combination of αT + γT showed antagonism in both cell lines. Conclusions: The higher potency of Tocomin and its synergistic interactions with αT or γT suggest that tocotrienol-rich mixtures may enhance the antiproliferative response, whereas combining αT and γT together may reduce efficacy under the tested conditions. Full article

Resistance to apoptosis represents a major limitation of platinum-based chemotherapy in colorectal carcinoma, frequently arising from impaired p53 signaling and inefficient execution of programmed cell death. In this study, we investigated the anticancer activity of Erica spiculifolia extract (ESE) and its ability to synergistically enhance cisplatin cytotoxicity in HT-29 colorectal carcinoma cells. Cell viability was assessed using the MTT assay, followed by formal combination analysis based on the Chou–Talalay methodology. Combination experiments employed a non-constant ratio regimen in which a fixed ESE concentration (45 µg/mL) was combined with serial cisplatin dilutions (45.0–2.8 µg/mL) to define interaction behavior across multiple effect levels. Quantitative analysis revealed a strong superadditive effect, with Combination Index values well below 1 and markedly elevated Dose Reduction Indices for cisplatin, indicating substantial dose-sparing across effect levels. To elucidate the molecular basis of this synergism, apoptosis-related protein expression was profiled using a membrane-based immunoassay. Combined ESE and cisplatin treatment induced full-scale p53 reactivation, including restoration of phosphorylated p53 isoforms associated with DNA damage-dependent apoptotic signaling. Acridine orange/propidium iodide staining confirmed a pronounced increase in early and late apoptotic/necrotic cells following combination treatment. UHPLC-HRMS analysis identified kaempferol 3-O-glucoside (8830.19 ± 11.01 ng/mg dw) and myricitrin (3074 ± 3.12 ng/mg) as predominant flavonols, followed by naringenin 7-O-glucoside (5958.96 ± 9.98 ng/mg), while chlorogenic, cinnamic, quinic, and gallic acids were the main phenolic acids detected. These constituents may contribute to HT-29 cell sensitization to cisplatin. Full article

Food systems expose bacteria to repeated nonlethal stresses during primary production, processing, storage, and sanitation. Depending on the type, intensity, and sequence of exposure, these stresses may weaken cells, act synergistically to promote inactivation, or fail to eliminate contamination. Instead, they can alter bacterial physiology in ways that affect survival, recovery, detection, and responses to control measures. This review examines how stress history contributes to persistent food safety challenges. Listeria monocytogenes is used as a central biological model, with relevant comparisons to other foodborne pathogens. Evidence from food-processing and environmental studies shows that repeated sublethal stress can shift bacterial populations toward stress-hardened states. Here, “stress-hardened” refers to reversible physiological changes and the survival of more tolerant cells, not permanent genetic adaptation. These states include sublethal injury, delayed growth, viable but nonculturable cells, biofilm formation, and increased tolerance to later stresses. These outcomes contribute to, but do not fully explain, the persistence of L. monocytogenes in food environments; intrinsic traits such as psychrotrophic growth and interactions with endogenous microflora also play important roles. These factors help explain repeated recovery of L. monocytogenes after sanitation and the underestimation of viable cells by routine culture-based methods, which do not reliably indicate whether pre-stressed cells retain the potential to cause foodborne illness. Many monitoring and validation approaches rely on unstressed laboratory cultures and fixed enrichment protocols. These conditions do not reflect the physiological states encountered in real food systems. As a result, negative test results may reflect limited recovery rather than true absence, and control performance may be overestimated when stress-conditioned populations are not considered. Across the farm-to-fork continuum, stress responses, persistence mechanisms, and detection limitations are closely linked, indicating that stress history should be considered a core element of hazard characterization, monitoring, and control validation. Incorporating stress biology into food safety assessment can improve the realism of verification strategies when combined with risk characterization that considers infectious dose and host susceptibility, and support control strategies under real-world processing and environmental conditions. Full article

Background/Objectives: Topical treatment of cutaneous tuberculosis (CTB) requires reliable models to evaluate dermal drug release and diffusion, particularly for fixed-dose combinations (FDCs) with contrasting physicochemical properties. Human skin remains the reference standard but poses ethical, logistical, and reproducibility challenges. This study investigated the suitability of Strat-M^® synthetic membranes as an alternative to human skin for assessing the simultaneous release and diffusion of clofazimine (CFZ) and pyrazinamide (PZA) from a topical FDC, and aimed to develop an optimized dermal emulsion using a Quality-by-Design (QbD)-informed formulation development tool. Methods: Self-emulsifying dermal emulsions containing CFZ and PZA were developed following QbD principles. Preformulation studies included drug solubility screening, oil phase selection, and pseudoternary phase diagram construction to identify stable emulsion regions. Formulations were characterized for droplet size, polydispersity index, zeta potential, viscosity, self-emulsification efficiency, and thermodynamic stability. Eight stable emulsions were identified, of which four were selected for in vitro drug release studies. The peppermint oil-based emulsion (PPO415) was further evaluated in comparative diffusion studies using Strat-M^® membranes and ex vivo human skin (Caucasian and African). Results: PPO415 demonstrated favorable physicochemical properties, including high CFZ solubility, uniform droplet distribution, and suitability for dermal application. Comparative diffusion studies showed that Strat-M^® underestimated the partitioning of lipophilic CFZ while overestimating the diffusion of hydrophilic PZA relative to human skin. These differences were attributed to compositional and structural disparities between synthetic membranes and biological skin. Conclusions: Strat-M^® membranes show potential as a reproducible and ethical in vitro screening tool during early-stage formulation development for topical FDCs. However, ex vivo human skin remains essential for accurately predicting dermal drug distribution and therapeutic performance. Full article

Background and Objectives: Sodium-glucose co-transporter 2 (SGLT2) inhibitors have emerged as key agents in the management of type 2 diabetes mellitus (T2DM), with expanding indications in heart failure and chronic kidney disease. This study assessed national trends in SGLT2 inhibitor utilization in Croatia between 2014 and 2024 using data from IQVIA. Materials and Methods: Drug use was quantified in defined daily doses per 1000 inhabitants per day (DDD/1000/day), alongside financial expenditure and prescribing patterns. Results: Since their market introduction in 2014, SGLT2 inhibitor utilization increased from 0.49 to 11.63 DDD/1000/day by 2024. Fixed-dose combinations with metformin accounted for a growing share of prescribing, reflecting a shift toward adherence-friendly regimens. Dapagliflozin was the most prescribed agent, likely due to broad therapeutic versatility and favorable pricing. Despite these trends, SGLT2 inhibitors (monotherapy) seem to be underutilized, accounting for just 12% of non-insulin antidiabetic prescriptions in 2024. Conclusions: These findings highlight the gradual integration of SGLT2 inhibitors into national clinical practice and emphasize the need for targeted educational and policy efforts to overcome therapeutic inertia and align prescribing with evidence-based cardio-renal-metabolic care. Full article

Background/Objectives: Relapsed and refractory (rr) primary large B-cell lymphoma of the central nervous system (PCNSL) has a dismal prognosis, and the standard of care is not established. The most common genetic imbalance includes the B-cell lymphoma 2 (BCL-2) locus. Methods: We planned a bi-centric phase Ib dose-escalation study with the chemotherapy-free combination of the BCL-2 inhibitor venetoclax and CD20 antibody obinutuzumab for rrPCNSL patients in Germany. The intended treatment consisted of 6 cycles of fixed-dose obinutuzumab at 1000 mg intravenously every 3 weeks, and an oral daily dose of 600, 800, or 1000 mg venetoclax, depending on the planned dosing group, followed by a 12-month venetoclax maintenance period. The primary endpoint was the pharmacokinetics of venetoclax and obinutuzumab in cerebrospinal fluid (CSF). Results: This study was prematurely terminated after registration of 5/15 (33%) patients in dosing group 1 (600 mg oral daily dose of venetoclax) between May 2020 and November 2021. The mean ratio of the concentration of venetoclax in CSF over peripheral blood was 0.55% (±0.28 standard deviation (SD)) and 0.25% (±0.23 SD) for obinutuzumab. Two of five patients achieved complete remission, and each one patient achieved partial remission and stable disease as best response. The median duration of response was 6.5 months (range 0.7–47). Conclusions: Venetoclax and obinutuzumab can penetrate into the central nervous system, but the CSF concentration did not correlate with the outcome. The combination is feasible, tolerable, and may lead to durable responses in selected rrPCNSL patients. Full article

Background: Cefepime/enmetazobactam (FEP/META) is a novel fixed-dose β-lactam/β-lactamase inhibitor combination. The objective was to study the physicochemical stability of the approved daily dose in polypropylene syringes and elastomeric devices over a 24 or 72 h period to understand the feasibility of using FEP/META in prolonged infusions and its use for outpatient parenteral antibiotic therapy (OPAT). Methods: Solutions of FEP/META were prepared in 0.9% NaCl or 5% dextrose (D5W) and stored in syringes (6 g/1.5 g/48 mL) or silicone and polyisoprene elastomeric devices (EDs) at 6 g/1.5 g/120 mL and 6 g/1.5 g/240 mL: syringes were tested at 22–25 °C over a 24 h period, polyisoprene EDs at 2–8 °C over 72 h period, and silicone and polyisoprene EDs at 32 °C over a 24 h period. The solution was considered stable if it retained more than 90% of its initial concentration (Ci), no pH variation (±1 unit), no significant visual change, and with compliant subvisual examination. Liquid Chromatography–Electrospray Ionization–Quadrupole Time-of-Flight–Mass Spectrometry was utilized to identify intermediate degradation products. Results: At the daily dose, FEP/META retained >90% of its Ci up to 12 h in 0.9% NaCl and 24 h in D5W when stored in syringes. In silicone ED, stability was enhanced up to 24 h in D5W at all concentrations. The solution was chemically stable for 24 h when stored in polyisoprene ED in 0.9% NaCl at 2–8 °C. Conclusions: FEP/META combination showed prolonged stability with physicochemical integrity up to 12–24 h in all containers and conditions. It appears to be stable for prolonged infusions and for OPAT. Full article

Background: In internal medicine, the management of type 2 diabetes mellitus (T2DM) is challenged by multimorbidity and polypharmacy. The fixed-dose combination of sitagliptin and extended-release metformin (SITA/MET ER) is a valuable option for frail and comorbid patients. Methods: This multicenter, retrospective, observational study involved five Italian Internal Medicine units. Consecutive patients with T2DM who initiated SITA/MET ER were included. Demographic, clinical, and laboratory data were collected at baseline (T0) and at follow-up (T1, 3–4 months). The primary endpoint was change in HbA1c; secondary endpoints included fasting plasma glucose (FPG), treatment adherence, adverse events, and modifications in concomitant antidiabetic therapies. Results: A total of 292 patients (mean age 70.8 ± 10.6 years; 43% female) were analyzed. At baseline, mean HbA1c was 7.4 ± 1.0% and FPG 150.2 ± 42.5 mg/dL, with significant reductions observed at follow-up (HbA1c 7.0 ± 0.8%, FPG 136.8 ± 29.6 mg/dL; both p < 0.05). SITA/MET ER was predominantly prescribed to patients with a complex clinical profile, as reflected by the high prevalence of microvascular (37%) and macrovascular (42%) complications. The use of sulfonylureas decreased from 11% to 3% (p < 0.001), while SGLT2 inhibitor and insulin use remained stable. Treatment adherence to SITA/MET ER was excellent, with full compliance reported and no adverse events recorded. Conclusions: In this real-world internal medicine study, SITA/MET ER improved glycemic control and was well tolerated among patients with complex clinical profiles. These findings support the role of SITA/MET ER as a flexible and practical therapeutic choice in this setting. Full article

Reversed-phase high-performance liquid chromatography (RP-HPLC) remains one of the most widely applied analytical techniques in the development and quality control testing of finished pharmaceutical products. The combination of gradient chromatographic methods with diode-array detection (DAD) enhances selectivity, ensuring accuracy and reliability when testing drugs with diverse chemical properties in a single dosage form (i.e., fixed-dose combination (FDC) products). In this study, an RP-DAD-HPLC method was developed for the quantitative analysis of clofazimine (CFZ) and pyrazinamide (PZA) for inclusion in an FDC topical drug delivery system. Chromatographic separation was achieved using a C18 column (4.6 mm × 150 mm, 5 µm particle size) with gradient elution at 1 mL/min, employing 0.1% aqueous formic acid and acetonitrile (mobile phases). PZA and CFZ were detected at 254 nm and 284 nm, respectively. The method was validated in accordance with ICH Q2 guidelines, assessing specificity (considering interference from solvents, product matrix, and degradation products), linearity (7.8–500.0 µg/mL, r² = 0.9999), system repeatability (%RSD ≤ 2.7%), and intermediate precision (25–500 µg/mL, %RSD ≤ 0.85%). Method robustness was evaluated using a three-level Box–Behnken design (BBD) with response surface methodology (RSM) to assess the effects of variations in detection wavelength, mobile phase flow rate, and column temperature. Full article

Background/Objectives: Cutaneous leishmaniasis (CL) remains a major neglected tropical disease, and current chemotherapeutic options are limited by toxicity and emerging resistance. Repurposing azole antifungals is a promising approach, as they target ergosterol biosynthesis, a pathway also essential in Leishmania spp. This study investigated the antileishmanial potential of econazole through in vitro and in vivo assays. Methods: Econazole activity was evaluated against Leishmania amazonensis promastigotes and intracellular amastigotes using MTT and luminescence-based methods. Cytotoxicity in NCTC cells was determined to calculate the selectivity index (SI). Drug interactions with miltefosine were assessed by fixed-ratio isobologram analysis. In vivo efficacy was examined in BALB/c mice infected with L. amazonensis and orally treated with econazole (2.5, 5, or 10 mg/kg/day) for 28 days. Lesion development and parasite burden were monitored. Molecular docking simulations were performed using SwissDock. Results: Econazole showed potent in vitro activity, with EC₅₀ values of 8.9 µM for promastigotes and 11 µM for intracellular amastigotes, and a CC₅₀ of 31 µM. Isobologram analysis revealed additive interactions with miltefosine (ΣFIC 0.5–1.2; mean 0.95). In vivo, econazole reduced lesion size and parasite load, achieving up to 75% reduction at 10 mg/kg/day. Docking results suggested that econazole may inhibit sterol biosynthesis, potentially through interaction with 14α-demethylase. Conclusions: These findings provide the first evidence of econazole activity against L. amazonensis in vitro and in vivo. Its exploratory efficacy and compatibility with miltefosine support further investigation of econazole as a repurposed candidate for CL, including optimization of dosing strategies and combination regimens. Full article