Metformin enhances response to chemotherapy combined with immunotherapy in a triple negative breast cancer in vivo model.

Metformin (MET) is one of the most prescribed antidiabetic drugs worldwide, and its recurrent detection in aquatic environments raises concerns regarding ecological and human health risks. In this work, a multifunctional composite based on natural zeolite functionalized with iron nanoparticles (NZ-FeNPs) was synthesized via a green route using Guazuma ulmifolia extract. The material was thoroughly characterized, confirming successful functionalization, structural stability, and preservation of the zeolitic framework. Comprehensive characterization techniques, including SEM, TEM, EDX, FTIR, XRD, XRF, leaching tests, and zeta potential measurements, confirmed the successful formation, homogeneous dispersion, and chemical stability of Fe nanoparticles within the zeolitic framework. Adsorption studies revealed a maximum capacity of 9.66 mg/g for MET at 45 °C, with the Sips and pseudo-second-order models accurately describing the equilibrium and kinetic data. Thermodynamic analysis indicated an endothermic and spontaneous process, governed by hydrogen bonding, electrostatic interactions, and metal–ligand complexation. The composite also demonstrated robust performance in multicomponent systems, achieving complete removal of chloroquine and neutral red while maintaining high MET adsorption efficiency. Desorption tests with NaOH confirmed the catalyst's reusability over multiple cycles. Furthermore, NZ-FeNPs exhibited antibacterial activity against Escherichia coli , with MIC and MBC values of 62.5 and 125 mg/mL, respectively. These results demonstrate the potential of NZ-FeNPs as a sustainable, versatile material that can couple adsorption and antibacterial functionalities for advanced water purification. • Green synthesis of Fe-based nanoparticles using Guazuma ulmifolia extract. • Natural zeolite acted as an efficient support for nanoparticle formation and stabilization. • NZ–FeNPs preserved zeolitic structure and mesoporosity after green functionalization. • NZ–FeNPs showed effective removal of metformin, chloroquine, and neutral red in multicomponent systems. • NZ-FeNPs exhibited antibacterial activity against Escherichia coli.

Combining lactate oxidase and metformin in cancer cell membrane-biomimetic liposomes for synergistic ferroptosis induction and hypoxia-alleviated cancer therapy.

Kaempferol ameliorates PCOS by alleviating metabolic and endocrine abnormalities as well as oxidative stress.

Analysis of thermophysical properties of l-alanine/l-histidine in aqueous metformin hydrochloride solutions at varying temperatures

Branebrutinib (BRNB) is a selective, oral Bruton's Tyrosine Kinase (BTK) inhibitor under clinical development. Metformin (MTF) exhibits antidiabetic properties and is recognized for enhancing chemotherapy efficacy by inhibiting cell proliferation and facilitating apoptosis via critical genes like TP53 and caspases. Consequently, research into diabetic patients co-morbid with B-cell malignancies has gained significance. This study compared invivo pharmacokinetics of MTF and BRNB to elucidate their metabolic and transporter level interactions. A sensitive, reliable ultra-high performance liquid chromatography mass spectrometric (UHPLC-MS/MS) method was validated for efficiently quantifying analytes on Poroshell 120 EC-C18 column using ammonium bicarbonate and methanol. Mass transitions revealed m/z 130.0 → 60.0 (MTF), m/z 371.1 → 150.0 (BRNB), and m/z 260.1 → 56.1 (propranolol), and protein precipitation provided effective extraction. The method was validated as per USFDA, and it was found specific, selective, and sensitive at 10 ng/mL concentration and noticed linear, accurate, precise, and free from carry over and matrix effects. Invivo oral MTF (25 mg/kg) and BRNB (5 mg/kg) co-administration significantly increased MTF's Cmax (3.4-fold), AUC0-t (3.32-fold), and half-life (1.65-fold). Similarly, systemic exposure of BRNB followed an increased trend for Cmax (1.59-fold) and AUC0-t (2.1-fold), while its elimination rate remained unaltered. Integrated PK/PD studies support further concurrent MTF-BRNB therapies.

Objective This study aims to explore the targets and mechanisms by which metformin (MET) exacerbates diabetic amyotrophy (DA) using a streptozotocin (STZ)-induced diabetic SD rat model. Method The targets of MET were acquired from 10 databases, including HIT and TCMSP . The DA targets were obtained from the GeneCards database. The associated targets were imported into the Venn analysis platform to draw Venn diagrams, and their intersections were visualized. A target protein–protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Database for Annotation, Visualization, and Integrated Discovery (DAVID) databases and Cytoscape software, and the core targets were identified. GO enrichment and KEGG pathway analyses were performed on the targets, and the construction of the “MET exacerbated DA target-pathway” network was carried out. Molecular docking was performed using Schrödinger Maestro 13.5 software to determine the binding free energy and binding mode of MET with the target protein. The expression of NOS1 and 3-nitrotyrosine proteins in gastrocnemius muscle tissue was detected by WB. The expression of 3-nitrotyrosine protein in gastrocnemius muscle tissue was detected using an enzyme-linked immunosorbent assay (ELISA). The localization of NOS1 in the gastrocnemius muscle tissue was detected using immunohistochemistry (IHC). 16S rDNA gene sequencing was performed to detect structural changes in the fecal microbiota. Results Network toxicology analysis revealed 19 intersecting proteins. GO functional enrichment and KEGG pathway analyses demonstrated that the obtained targets were involved in molecular functions, biological processes, and cellular components and were involved in pathways such as drug metabolism—other enzymes, peroxisomes, and nucleotide metabolism. Based on the crystal structures of potential target proteins, complex structural models of target–MET were constructed through molecular docking (extra precision [XP] mode of flexible docking), followed by MM-GBSA analysis. The IHC results indicated that MET significantly promoted the redistribution of NOS1 from the membrane region to the cytoplasm. WB results showed that MET significantly ( p < 0.001) promoted the expression of 3-nitrotyrosine in the diabetic SD rat model but had no significant effect on the expression of NOS1. ELISA results demonstrated that MET significantly ( p < 0.001) promoted the expression of 3-nitrotyrosine in the diabetic SD rat model. The 16S rDNA gene sequencing results showed that MET led to a significant ( p < 0.05) increase in the abundance of Desulfovibrio in the intestinal tract of rats. Conclusion MET exacerbates diabetic amyotrophy by promoting the increased relative abundance of Desulfovibrio , inducing muscle oxidative stress, and causing NOS1 dysregulation.

Although metformin (MET), the well-known antidiabetic drug, exhibits clear antineoplastic effects and is reported to target mitochondria, several issues are still open in this regard, thus limiting its utilization as an anticancer drug alone or in combination with other molecules. Here a functional investigation was carried out to reveal how MET impacted on mitochondrial functions and cell energy metabolism in human cultured clear cell renal carcinoma cells (ccRCCs), in which the anticancer effect of MET is already known. The in vitro effect of increasing MET concentrations on cell viability, necrosis and apoptosis of ccRCCs was checked and compared to normal immortalized HK2 cells. At the same time, the effect of MET on mitochondrial functions, ATP synthesis via oxidative phosphorylation, cellular ATP level, L-lactate (L-LAC) production and export, glucose consumption and key mitochondrial and cytosolic enzyme activities was also investigated in cancer cells. MET affected ccRCC viability and impaired mitochondrial respiration, membrane potential generation and ATP production by targeting complex I (CI), III and IV of the respiratory chain at a concentration near to the IC50 value (25 mM). Importantly, we first identified a significant inhibition of the adenine nucleotide translocator (ANT) activity in response to MET treatment. Notably, the sensitivity of ANT and CI activity to increasing MET concentrations differed markedly, the former being considerably inhibited already at a low, near-clinically relevant concentrations, while the latter only at concentrations ≥ 1 mM. The drug also induced a glycolytic shift in ccRCCs and increased the activity of the mitochondrial flavoenzymes succinate dehydrogenase (SDH) and D-lactate dehydrogenase (D-LDH), and of the key enzymes of the pay-off phase of glycolysis, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK) and pyruvate kinase (PK). Nevertheless, cellular ATP level dropped markedly, and the intracellular L-LAC amount was almost doubled in the presence of MET. Interestingly, MET-induced glycolytic shift showed a drug concentration dependence similar to that seen for CI inhibition, suggesting not ANT but rather CI inhibition may be the trigger for metabolic rewiring. These findings give new insights into MET mechanisms of action which may potentially improve its application and outcome in cancer as well as in other pathologies.

Metformin (MET), a first-line antidiabetic drug, has been increasingly implicated in cellular protection under oxidative stress, yet its mechanisms in lens epithelial cells (LECs) remain incompletely defined. Using a tert-butyl hydroperoxide (TBHP)-induced acute oxidative injury model in human HLE-B3 cells, we investigated whether SIRT1/FOXO1-related autophagy contributes to MET-associated cytoprotection. MET pretreatment reduced intracellular reactive oxygen species, preserved antioxidant defenses, improved cell viability, and decreased apoptosis after TBHP challenge. MET also enhanced autophagy markers and, under lysosomal blockade with chloroquine or bafilomycin A1, showed LC3-II/p62 changes consistent with increased autophagic flux. Pharmacologic inhibition of SIRT1 (EX-527) or early-stage autophagy (3-methyladenine) partially attenuated MET-associated improvements across oxidative stress and survival endpoints, supporting a role for SIRT1/FOXO1-related autophagy in this response. Although limited to an in vitro setting and pharmacological perturbation, these findings suggest that MET may mitigate oxidative injury in lens epithelium, highlighting SIRT1/FOXO1-autophagy as a potential pathway relevant to oxidative stress processes in cataractogenesis.

To improve clinical and radiographic results in patients with periodontitis, this systematic review assessed the efficacy of metformin (MF) as an adjuvant to surgical and nonsurgical periodontal therapy. Patients with periodontitis (P), MF as an adjuvant (I), periodontal therapy alone, or placebo (C), and outcomes such as bone regeneration (O), clinical attachment level (CAL), and probing depth (PD), were evaluated with the PICO framework. Between June 15 and July 1, 2025, we searched for in vitro, in vivo, and randomized controlled trials (RCTs) published between June 2015 and June 2025 in PubMed, Google Scholar, ScienceDirect, and Semantic Scholar. Data were narratively synthesized according to MF concentration (0.5-1.5%), outcome measures, and patient type. The Cochrane tools SYRCLE and QUIN were used to assess risk of bias for RCTs, animal studies, and in vitro studies. The analysis included 15 RCTs, 10 in vitro studies, and 8 in vivo studies among 4005 identified records. The most commonly used formulation was local MF gel. Follow-up times ranged from 1 to 9 months. MF, in contrast to scaling and root planing or placebo, produced vertical bone fill as high as 26.8%, a CAL gain of 1.5-2.7 mm, and a PD decrease of 1.5-3.4 mm. Experimental investigations had moderate risk of bias, whereas RCTs had low risk of bias. MF's osteogenic and anti-inflammatory qualities make it a promising adjuvant in periodontal therapy. However, the need for additional long-term research was highlighted by the study heterogeneity, limited follow-up periods, and a lack of meta-analysis.

An adapted new cyano-assisted salting out CASO-HPLC method for simultaneous determination of linagliptin and metformin hydrochloride binary mixture in pure and tablet dosage form

Allicin, a bioactive compound derived from garlic, exhibits therapeutic potential against metabolic disorders but is hindered by instability and a pungent odor, limiting its clinical application. This study develops an allicin oleogel (OG) formulation, leveraging molecular interactions with unsaturated fatty acids and gelators to stabilize allicin and mitigate its odor for transcutaneous delivery. The skin analysis confirmed superior transdermal delivery and accumulation in subcutaneous fat with OG, highlighting its advantages for topical application. In a high-fat diet (HFD)-induced mouse model of obesity, OG demonstrated superior efficacy in suppressing weight gain, reducing food intake, and improving metabolic parameters, including fasting blood glucose, oral glucose tolerance, and insulin sensitivity, compared to orally administered allicin oleogel (OGO) and metformin (MET). OG also ameliorated non-alcoholic fatty liver disease (NAFLD), as evidenced by reduced hepatic lipid accumulation and normalized adipose tissue metabolism. Notably, OG treatment shifted macrophage polarization toward an anti-inflammatory M2 phenotype and increased mitochondrial activity, facilitating adipose tissue remodeling and enhancing energy expenditure and thermogenesis. Safety evaluations revealed no systemic toxicity, supporting its potential for long-term use. This study underscores transcutaneously delivered OGas a promising therapeutic strategy for obesity and related metabolic disorders.

Objective: The present study aimed to comparatively evaluate floating and non-floating controlled-release formulations of Metformin HCl to understand the influence of formulation composition and processing variables on gastroretentive behavior, drug release characteristics, and pharmacokinetic performance. The study particularly focused on the role of rate-controlling polymer, binder grade, and granulation process in modulating formulation performance and bioavailability. Methodology: Floating gastroretentive tablets of Metformin HCl were developed using Eudragit NE 30D as the rate-controlling polymer and polyvinyl alcohol (PVA) as the granulating binder through a low-shear fluid bed granulation process. Both floating and non-floating controlled-release systems were formulated and optimized to assess the impact of binder grade and formulation parameters on tablet performance. The prepared formulations were evaluated for physicochemical properties, in vitro buoyancy behaviour, swelling index, matrix erosion, and dissolution profile. In addition, in vivo gastric retention and pharmacokinetic performance were assessed in healthy human volunteers. Results: The optimised floating formulation (MTH3) demonstrated rapid flotation within 20 seconds and sustained buoyancy for more than 16 hours. In contrast, the non-floating optimized formulation (MTH6) prepared with lower grades of PVA as a binder with the addition of controlled release polymer, maintained controlled drug release characteristics. Dissolution studies showed prolonged and near-complete drug release from both the formulations. Drug release kinetics for both the formulations followed the Higuchi model, indicating a combined diffusion and matrix erosion mechanism. Stability studies confirmed consistent dissolution similarity (f₂), demonstrating formulation robustness. In vivo pharmacokinetic evaluation revealed higher systemic exposure for the floating formulation compared with the non-floating system. The optimized floating tablets met bioequivalence criteria relative to the reference listed drug, at 90% confidence intervals for Cmax (87.60%–97.77%), AUC₀–t (84.95%–97.73%), and AUC₀–inf (85.17%–97.47%) were entirely contained within the predefined acceptance limits of 80–125%, whereas the non-floating formulation failed to achieve bioequivalence. Conclusions: The study demonstrates that formulation variables such as rate-controlling polymer selection, binder grade, and granulation conditions significantly influence the gastroretentive properties, drug release kinetics, and bioavailability of controlled-release Metformin HCl tablets. The optimised floating system provided improved gastric retention and enhanced systemic exposure, successfully achieving bioequivalence with the reference product, highlighting its potential as an effective gastroretentive controlled-release formulation.

Viruses rely on energy and biosynthetic materials of host cell from glucose metabolism to support their replication, and glucose plays a crucial role in viral infection. In this study, we found that porcine epidemic diarrhea virus (PEDV) infection significantly increased cellular glucose uptake and stimulated the production of the glycometabolite lactate. Exogenous supplementation of glucose or L-lactate confirmed that it significantly promoted PEDV proliferation, indicating that replication of PEDV was enhanced by regulating host glucose metabolism, particularly reprogramming glycolysis. Based on these findings, we explored the potential antiviral approach targeting the virus through regulating glycolytic processes. Metformin hydrochloride (MH) is a well-known hypoglycemic agent, which has shown notable anti-PEDV activity. After MH treatment, the transcriptome analysis showed the differential genes were mainly enriched in PI3K-AKT signaling pathway, and the expression levels of its downstream molecule GSK3B and MYC were significantly upregulated and downregulated, respectively. The gene expression related to glycolysis was also significantly inhibited. Further experiments showed that MH significantly inhibited the phosphorylation of AKT and its translocation to plasma membrane, while reducing the phosphorylation level of GSK3B. MH maintained GSK3B in a non-phosphorylated state by blocking the activation of the EGFR/PI3K/AKT/GSK3B pathway, mediated the degradation of c-MYC through phosphorylation, inhibited the glycolysis process, reduced the production of lactic acid, and finally exerted its antiviral effect. This study demonstrated that PEDV infection could induce glycolysis through metabolic reprogramming, thereby promoting viral replication; whereas, MH was able to effectively reverse this process, significantly inhibiting the virus-induced glycolysis pathway and exhibiting antiviral activity.IMPORTANCEThis study aims to elucidate the antiviral effects and molecular mechanisms of MH against PEDV. The results show that MH can inhibit the activation of the PI3K-AKT signaling pathway induced by PEDV infection, thereby suppressing the production of the glycolytic product L-lactic acid and ultimately resisting PEDV infection. This research provides new insights into the prevention and control of PEDV and offers scientific evidence for the application of MH in veterinary medicine.

Environmentally benign and straightforward spectrophotometric methods were developed and validated for the simultaneous determination of saxagliptin (SAX), metformin (MET), and the pharmacopeial impurity of MET, melamine (MEL), in bulk powder and pharmaceutical formulations. These proposed approaches provide reliable, low-cost, and accessible alternatives to conventional chromatographic techniques, which often require complex instrumentation, extended analysis times, and significant solvent consumption. Two complementary spectrophotometric methods were established. Method A, the ratio-difference approach, utilized MEL as a divisor for the quantification of SAX, while SAX served as a divisor for determining MET and MEL. Method B, based on the first derivative of ratio spectra, applied MEL as a divisor under optimized conditions (scaling factor = 10, Δλ = 4 nm). However, MEL could not be quantified by Method B because its derivative ratio signals in the selected spectral region were too weak and overlapped extensively with those of SAX and MET, preventing the establishment of a reliable linear calibration curve for this impurity. The methods exhibited linearity over the ranges of 5–90 µg/mL for SAX, 1–40 µg/mL for MET, and 0.5–10 µg/mL for MEL. Validation in accordance with ICH Q2(R1) guidelines confirmed their accuracy, precision, selectivity, and specificity. The results obtained by the proposed methods were statistically compared with those of a reported RP‑HPLC method, confirming the reliability of the proposed procedures. The environmental impact was assessed using the principles of green analytical chemistry (GAC), which incorporates sustainability by promoting methods that limit hazardous reagents, reduce energy consumption, and minimize waste while maintaining suitable analytical performance. Holistic evaluation tools such as the analytical greenness metric (AGREE), the green analytical procedure index (GAPI), the national environmental methods index (NEMI), the blue applicability grade index (BAGI), and the RGB 12 model enable simultaneous assessment of analytical quality, environmental impact, and practical applicability. These tools foster the development of “white” analytical chemistry and complement emerging indices like the environmental and practical performance index (EPPI).Overall, these spectrophotometric approaches represent sustainable, efficient, and accessible analytical alternatives for the routine quality control of Saxagliptin and Metformin in combined pharmaceutical formulations.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-026-38952-3.

Preconception care is globally recognized as essential for optimizing pregnancy outcomes; however, in Japan, comprehensive data on medication-related potential exposure to pregnancy-contraindicated medications among women of reproductive age remain limited. We conducted a retrospective cross-sectional descriptive study using data from Japan’s National Database of Health Insurance Claims (fiscal year 2022) to assess the potential exposure to pregnancy-contraindicated medications among women of reproductive age. Outpatient prescriptions for oral medications dispensed to women aged 15–49 years were analyzed. In total, 270 medications classified as contraindicated during pregnancy were identified, of which 75 were also contraindicated for women planning pregnancy. Of these, 58 active ingredients were restricted in both phases. Notably, 212 medications were uniquely contraindicated during pregnancy, highlighting the broader contraindication profiles during fetal development than during the preconception period. Despite these contraindications, high prescription volumes were observed for medications such as loxoprofen sodium hydrate, sodium valproate, and metformin hydrochloride among women of reproductive age. These findings illustrate a high baseline utilization of pregnancy-contraindicated medications among women of reproductive age. As most women in this demographic are neither pregnant nor actively planning conception, these volumes primarily reflect standard care rather than inappropriate prescribing. In conclusion, pharmacists serve as an important supplementary safety net by routinely confirming pregnancy status to prevent inadvertent exposure.

Diabetes mellitus remains a worldwide health burden, associated with a significant co-morbidity, including heart disease, nerve damage, kidney failure, vision loss, and diminished quality of life. Ensuring therapeutic quality of the drugs is vital to safeguard the treatment efficacy, patient safety, and public health outcomes. Therefore, the aim of the study was to establish the pharmaceutical equivalence of various brands of metformin hydrochloride available in tertiary care hospital in Kathmandu. In this study a systematic quality assessment of weight variation, friability, assay, and related substance impurities were evaluated as per the Indian pharmacopeia (IP 2022). The weight variation test revealed significant mass consistency, within the ±5% accepted range. Friability results were markedly lower than the 1.0% ceiling, falling between 0.041% and 0.286%. All tablets met pharmacopeial disintegration threshold (≤15 min), but times varied significantly (1.57–6.30 min) across the formulations. Assay analysis depicted drug contents ranging from 95.91% to 97.22% of the 500 mg label aligning the pharmacopeial limit. Impurity analysis further validated product`s integrity, with concentrations of dicyandiamide and other unspecified impurities within the threshold’s limits. In conclusion, all evaluated metformin brands met the pharmacopeial standards for uniformity, potency, disintegration time, and purity. These results confirm their pharmaceutical equivalence and standard quality, accrediting their substitutability for clinical use within an institutional formulary. This research supplies pivotal quality verification of various brands of the marketed metformin tablets, informing the procurement and formulary management decisions in the tertiary care hospitals.

Background: Combination therapy is widely prescribed in Type II diabetes mellitus to maintain effective glycemic control. The rising use of multidrug regimens demands selective and reliable bioanalytical methods capable of simultaneously quantifying multiple antidiabetic agents in human plasma for pharmacokinetic and bioequivalence studies. Methodology: A rapid, sensitive, and cost-effective LC–MS/MS method was developed and validated in accordance with ICH M10, USFDA, and EMA guidelines for the simultaneous estimation of metformin hydrochloride, teneligliptin hydrobromide hydrate, and pioglitazone hydrochloride. The assay enabled triple-drug quantification within a single 7-minute chromatographic run, showing an estimated 12–40% reduction in analysis time versus previously reported 8–15-minute single- or dual-analyte methods. Separation was achieved on a Cosmosil CN column (150 × 4.6 mm, 5 μm) using 10 mM ammonium acetate and acetonitrile (40:60 %v/v). Plasma samples were prepared by protein precipitation followed by liquid–liquid extraction, and detection was performed in positive electrospray ionization multiple-reaction-monitoring mode. Results and Discussion: Strong linearity was obtained for all analytes (r² > 0.995). LLOQs were 10.0 ng/mL for metformin, 1.25 ng/mL for teneligliptin, and 5.0 ng/mL for pioglitazone. Metformin-D6 served as the internal standard for metformin, while saxagliptin was used as the internal standard for teneligliptin and pioglitazone to ensure appropriate normalization across chemical classes. Precision remained below 10% CV, recovery was consistent, and stability stayed within ±15% under tested conditions. Reduced runtime and unified multi-analyte detection improved analytical throughput and minimized solvent consumption without compromising regulatory compliance. Conclusion: The validated LC–MS/MS method provides a reliable, resource-efficient platform for concurrent quantification of combined antidiabetic drugs in pharmacokinetic, bioequivalence, and clinical studies.

Background/Objectives: Aspasomes (ASPs) are composed of ascorbyl palmitate (AP), which has antioxidant activity. The objective of this study was the formulation of aspasomes (ASPs) loaded with metformin hydrochloride (MFC) for the topical treatment of melasma. Methods: MFC-ASPs were prepared using the thin-film method with different amounts of phospholipid and ascorbyl palmitate (AP) in the absence or presence of ethanol surfactant. The prepared formulations were optimized using a D-optimal mixture. The assessed responses included entrapment efficiency (%EE), particle size (PS), polydispersity index (PDI), and zeta potential (ZP). Results: The optimum OASPs, composed of 193.121 mg PC and 30 mg AP, exhibited spherical vesicles with an EE% of 87.50 ± 0.33%, PS of 264.47 ± 0.02 nm, PDI of 0.423 ± 0.001, and ZP of -21.67 ± 0.12 mV. The optimum formula represented a spherical morphology using transmission electron microscopy, along with sustained release behavior compared with MFC. Also, it showed good stability for up to 90 days. Furthermore, a clinical appraisal of patients with melasma confirmed the superiority of the cream compared to the other cream in clinical study. Conclusions: The optimum OASPs present a promising approach for the treatment of melasma topically.

The objective of this in vitro study was to examine the impact of metformin (MET) at different concentrations (0.1, 1, 10, 50, and 100 mM) on rat primary osteoblasts, as the results obtained so far are inconsistent. Osteoblast apoptosis, viability, alkaline phosphatase (ALPL) activity, production of osteoblast-specific biomarkers, including ALPL, osteocalcin (BGLAP), type I collagen alpha 1 (COL1A1), integrin-binding sialoprotein (IBSP), bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor (VEGF), tumor necrosis factor ligand superfamily member 11 (TNFSF11 or RANKL), as well as calcium/collagen deposition were assessed. Our results revealed that a dose of 100 mM was cytotoxic to osteoblasts and resulted in a complete loss of their viability. Therefore, this concentration was excluded from further analyses. In general, MET exhibited a dose-dependent impact on multiple osteoblast-specific functional biomarkers, with beneficial effects noted on ALPL activity (at 0.1 and 1 mM) as well as on the levels of ALPL (0.1 and 1 mM), BGLAP (at 0.1-50 mM), IBSP (at 0.1-50 mM), BMP2 (at 0.1, 10 and 50 mM), VEGF (at 0.1 and 1 mM), and RANKL (at 0.1 mM). Calcium/collagen deposition at concentrations of 0.1 and 1 mM reached the same level as control cells, higher doses (10 and 50 mM) dramatically reduced cell viability after 21 days and the aforementioned parameters could not be evaluated. It can be concluded that MET at concentrations up to 1 mM can promote osteoblast viability, osteogenic differentiation, angiogenic signaling, and reduce osteoclastogenesis. Key words Metformin " Osteoblasts " Bone health " In vitro.