Metformin hydrolase is a recently evolved nickel-dependent heteromeric ureohydrolase.

The anti-diabetic drug metformin is one of the most widely prescribed medicines in the world. Together with its degradation product guanylurea, it is a major pharmaceutical pollutant in wastewater treatment plants and surface waters. An operon comprising two genes of the ureohydrolase family in Pseudomonas and Aminobacter species has recently been implicated in metformin degradation. However, the corresponding proteins have not been characterized. Here we show that these genes encode a Ni2+-dependent enzyme that efficiently and specifically hydrolyzes metformin to guanylurea and dimethylamine. The active enzyme is a heteromeric complex of α- and ß- subunits in which only the α-subunits contain the conserved His and Asp residues for the coordination of two Ni2+ ions in the active site. A crystal structure of metformin hydrolase reveals an α2ß4 stoichiometry of the hexameric complex, which is unprecedented in the ureohydrolase family. By studying a closely related but more widely distributed enzyme, we find that the putative predecessor specifically hydrolyzes dimethylguanidine instead of metformin. Our findings establish the molecular basis for metformin hydrolysis to guanylurea as the primary pathway for metformin biodegradation and provide insight into the recent evolution of ureohydrolase family proteins in response to an anthropogenic compound.

Dual-Locked Probe with Activatable Sonoafterglow Luminescence for Precise Imaging of MET-Induced Liver Injury.

Metformin (MET) is currently the first-line treatment for type 2 diabetes mellitus (T2DM). However, overdose and long-term use of MET may induce a serious liver injury. What's worse, diagnosis of MET-induced liver injury remains challenging in clinic. Although several probes have been reported for imaging MET-induced liver injury utilizing upregulated hepatic H2S as a biomarker, they are still at risk of nonspecific activation in complex physiological environments and rely on light excitation with limited imaging depth. Herein, we rationally designed and developed a dual-locked probe, DPA-H2S, for precise imaging of MET-induced liver injury by H2S-activated sonoafterglow luminescence. DPA-H2S is a small molecule consisting of a sonosensitizer protoporphyrin IX (PpIX) and an afterglow substrate that is dual-locked with a H2S-responsive 2,4-dinitrobenzene group and a 1O2-responsive electron-rich double bond. When employing DPA-H2S for imaging of MET-induced liver injury in vivo, since the PpIX moiety can produce 1O2 in situ at the liver site under focused ultrasound (US) irradiation, the two locks of DPA-H2S can be specifically activated by the highly upregulated H2S at the liver injury sites and the in situ generated 1O2, respectively. Thus, the sonoafterglow signal of DPA-H2S is significantly turned on, enabling precise imaging of the MET-induced liver injury. In vitro results showed that, through H2S-activated sonoafterglow luminescence, DPA-H2S was capable of imaging H2S with good sensitivity and high selectivity and realized deep tissue imaging (∼20 mm, signal-to-background ratio (SBR) = 3.4). Furthermore, we successfully applied DPA-H2S for precise in vivo imaging of MET-induced liver injury. We anticipate that our dual-locked probe, DPA-H2S, may serve as a promising tool in assisting the diagnosis of MET-induced liver injury in clinics and informing the clinical utilization of MET in the near future.

Efficacy of pegylated Graphene oxide quantum dots as a nanoconjugate sustained release metformin delivery system in in vitro insulin resistance model.

Metformin, the primary therapy for type 2 diabetes mellitus (T2DM), showed limitations such as varying absorption, rapid system clearance, required large amount, resistance, longstanding side effects. Use of Nano formulations for pharmaceuticals is emerging as a viable technique to reduce negative consequences of drug, while simultaneously attaining precise release and targeted distribution. This study developed a Polyethylene Glycol conjugated Graphene Oxide Quantum dots (GOQD-PEG) nanocomposite for the sustained release of metformin. Herein, we evaluated the effectiveness of metformin-loaded nanoconjugate in in vitro insulin resistance model. Results demonstrated drug loaded nanoconjugate successfully restored glucose uptake and reversed insulin resistance in in vitro conditions at reduced dosage compared to free metformin.

A Semi-Mechanistic Physiologically Based Biopharmaceutics Model to Describe Complex and Saturable Absorption of Metformin: Justification of Dissolution Specifications for Extended Release Formulation.

Physiologically based pharmacokinetic (PBPK) or physiologically based biopharmaceutics models (PBBM) demonstrated plethora of applications in both new drugs and generic product development. Justification of dissolution specifications and establishment of dissolution safe space is an important application of such modeling approaches. In case of molecules exhibiting saturable absorption behavior, justification of dissolution specifications requires development of a model that incorporates effects of transporters is critical to simulate in vivo scenario. In the present case, we have developed a semi-mechanistic PBBM to describe the non-linearity of BCS class III molecule metformin for justification of dissolution specifications of extended release formulation at strengths 500 mg and 1000 mg. Semi-mechanistic PBBM was built using physicochemical properties, dissolution and non-linearity was accounted through incorporation of multiple transporter kinetics at absorption level. The model was extensively validated using literature reported intravenous, oral (immediate & extended release) formulations and further validated using in-house bioequivalence data in fasting and fed conditions. Virtual dissolution profiles at lower and upper specifications were generated to justify the dissolution specifications. The model predicted literature as well as in-house clinical study data with acceptable prediction errors. Further, virtual bioequivalence trials predicted the bioequivalence outcome that matched with clinical study data. The model predicted bioequivalence when lower and upper specifications were compared against pivotal test formulations thereby justifying dissolution specifications. Overall, complex and saturable absorption pathway of metformin was successfully simulated and this work resulted in regulatory acceptance of dissolution specifications which has ability to reduce multiple dissolution testing.

Development of poly (lactic-co-glycolic acid) (PLGA) based implants using hot melt extrusion (HME) for sustained release of drugs: The impacts of PLGA's material characteristics.

Hot melt extrusion (HME) processed Poly (lactic-co-glycolic acid) (PLGA) implant is one of the commercialized drug delivery products, which has solid, well-designed shape and rigid structures that afford efficient locoregional drug delivery on the spot of interest for months. In general, there are a variety of material, processing, and physiological factors that impact the degradation rates of PLGA-based implants and concurrent drug release kinetics. The objective of this study was to investigate the impacts of PLGA's material characteristics on PLGA degradation and subsequent drug release behavior from the implants. Three model drugs (Dexamethasone, Carbamazepine, and Metformin hydrochloride) with different water solubility and property were formulated with different grades of PLGAs possessing distinct co-polymer ratios, molecular weights, end groups, and levels of residual monomer (high/ViatelTM and low/ ViatelTM Ultrapure). Physicochemical characterizations revealed that the plasticity of PLGA was inversely proportional to its molecular weight; moreover, the residual monomer could impose a plasticizing effect on PLGA, which increased its thermal plasticity and enhanced its thermal processability. Although the morphology and microstructure of the implants were affected by many factors, such as processing parameters, polymer and drug particle size and distribution, polymer properties and polymer-drug interactions, implants prepared with ViatelTM PLGA showed a smoother surface and a stronger PLGA-drug intimacy than the implants with ViatelTM Ultrapure PLGA, due to the higher plasticity of the ViatelTM PLGA. Subsequently, the implants with ViatelTM PLGA exhibited less burst release than implants with ViatelTM Ultrapure PLGA, however, their onset and progress of the lag and substantial release phases were shorter and faster than the ViatelTM Ultrapure PLGA-based implants, owing to the residual monomer accelerated the water diffusion and autocatalyzed PLGA hydrolysis. Even though the drug release profiles were also influenced by other factors, such as composition, drug properties and polymer-drug interaction, all three cases revealed that the residual monomer accelerated the swelling and degradation of PLGA and impaired the implant's integrity, which could negatively affect the subsequent drug release behavior and performance of the implants. These results provided insights to formulators on rational PLGA implant design and polymer selection.

Gaining Insight into Mitochondrial Targeting: AUTAC-Biguanide as an Anticancer Agent.

AUTAC-Biguanide is a hybrid compound designed to target mitochondria, inducing their degradation by mitophagy. This study unveils the potential of biguanides as cancer cell-targeting agents, emphasizing AUTAC-Biguanide's superior antiproliferative properties compared to metformin and its selectivity for cancer cells. The mechanism behind this heightened effect includes the ability of AUTAC-Biguanide to trigger mitophagy. By providing a comprehensive analysis of these findings, this study adds valuable insights to the field of mitochondrial-targeting anticancer agents.

Innovative berberine nanoethosomal vaginal in situ gel: Unraveling polycystic ovary syndrome treatment on female Wistar rats.

PURPOSE: The present work seeks to develop, assess and refine a nanoethosomal vaginal in situ gel containing Berberine, aimed at enhancing its efficacy in treating Poly Cystic Ovary Syndrome (PCOS). This formulation aims to augment drug permeation, enable controlled release kinetics, and mitigate oral adverse effects commonly associated with Berberine administration. METHOD: Nanoethosomes formulated using diverse soya lecithin-ethanol concentrations within a 32 full-factorial-design, sought optimal formulations based on particle size and %entrapment-efficiency. Subsequent scrutiny involved PDI, Zeta potential and drug-content evaluation. TEM analysis authenticated morphology, while in vitro drug release from Nanoethosomes was examined. Pluronic F-127 concentrations (16%-21%w/v) were explored for the in situ gel, analyzing pH, gelation time and gelation temperature. The refined gel underwent evaluations for viscosity and in vitro diffusion. In vivo assessment covered pharmacokinetics, vaginal irritancy and Mifepristone-induced PCOS management, validated through histopathological and biochemical analysis, juxtaposing findings across normal, diseased, plain Berberine gel and standard metformin administered groups. RESULTS: Optimized Nanoethosomal Formulation(F3) displayed particle size of 183.5 nm, 82.58 % as %entrapment-efficiency, PDI of 0.137, -50.34 mV as zeta potential and 81.64 ± 1.57 % drug-content. TEM analysis confirmed spherical, nano-sized particles. In vitro studies exhibited 80.45 % drug release over 24 h. The formulated gel with 18 % Pluronic F-127 showed viscosity ranging from 193.01 ± 0.16cps to 1817.08 ± 1.67cps with temperature changes from 25 ± 2.0 °C to 38 ± 2.0 °C. In vitro diffusion revealed 85.99 %drug release from optimized gel. In vivo animal studies demonstrated increased plasma drug concentration, non-irritating properties in vaginal tests, and efficacy in managing Mifepristone-induced PCOS compared to other treatments. Short-term stability evaluations confirmed thermodynamic stability at room-temperature.

Co-Delivery of Valsartan and Metformin from a Thermosensitive Hydrogel-Nanoparticle System Promotes Collagen Production in Proliferating and Senescent Primary Human Dermal Fibroblasts.

Aging negatively impacts skin health, notably through the senescent cell phenotype, which reduces collagen production and leads to thinner, more fragile skin prone to injuries and chronic wounds. We designed a drug delivery system that addresses these age-related issues using a hybrid hydrogel-nanoparticle system that utilizes a poly(δ-valerolactone-co-lactide)-b-poly(ethylene-glycol)-b-poly(δ-valerolactone-co-lactide) (PVLA-PEG-PVLA) hydrogel. This hydrogel allows for the local, extended release of therapeutics targeting both proliferating and senescent cells. The PVLA-PEG-PVLA hydrogel entrapped valsartan, and metformin-loaded liposomes functionalized with a fibronectin-mimetic peptide, PR_b. Metformin acts as a senomorphic, reversing aspects of cellular senescence, and valsartan, an angiotensin receptor blocker, promotes collagen production. This combination treatment partially reversed the senescent phenotype and improved collagen production in senescent dermal fibroblasts from both young and old adults. Our codelivery hydrogel-nanoparticle system offers a promising treatment for improving age-related dermal pathologies.

A multifunctional injectable, self-healing, and adhesive hydrogel-based wound dressing stimulated diabetic wound healing with combined reactive oxygen species scavenging, hyperglycemia reducing, and bacteria-killing abilities.

The proficient handling of diabetic wounds, a rising issue coinciding with the global escalation of diabetes cases, poses significant clinical difficulties. A range of biofunctional dressings have been engineered and produced to expedite the healing process of diabetic wounds. This study proposes a multifunctional hydrogel dressing for diabetic wound healing, which is composed of Polyvinyl Alcohol (PVA) and N1-(4-boronobenzyl)-N3-(4-boronophenyl)-N1, N1, N3, N3-teramethylpropane-1, 3-diaminium (TSPBA), and a dual-drug loaded Gelatin methacryloyl (GM) microgel. The GM microgel is loaded with sodium fusidate (SF) and nanoliposomes (LP) that contain metformin hydrochloride (MH). Notably, adhesive and self-healing properties the hydrogel enhance their therapeutic potential and ease of application. In vitro assessments indicate that SF-infused hydrogel can eliminate more than 98% of bacteria within 24 h and maintain a sustained release over 15 days. Additionally, MH incorporated within the hydrogel has demonstrated effective glucose level regulation for a duration exceeding 15 days. The hydrogel demonstrates a sustained ability to neutralize ROS throughout the entire healing process, predominantly by electron donation and sequestration. This multifunctional hydrogel dressing, which integrated biological functions of efficient bactericidal activity against both MSSA and MRSA strains, blood glucose modulation, and control of active oxygen levels, has successfully promoted the healing of diabetic wounds in rats in 14 days. The hydrogel dressing exhibited significant effectiveness in facilitating the healing process of diabetic wounds, highlighting its considerable promise for clinical translation.

Enhanced anticancer effect of lysozyme-functionalized metformin-loaded shellac nanoparticles on a 3D cell model: role of the nanoparticle and payload concentrations.

Here we used a 3D human hepatic tumour cell culture model to assess the in vitro efficacy of "active" metformin-loaded nanoparticles (NPs) as anticancer therapeutics. The metformin nanocarrier design was repurposed from previous studies targeting bacterial and fungal biofilms with antimicrobials loaded in protease-coated nanoparticles. These active nanocarriers were constructed with shellac cores loaded with metformin as the anticancer agent and featured a surface coating of the cationic protease lysozyme. The lysozyme's role as a nanocarrier surface coating is to partially digest the extracellular matrix (ECM) of the 3D tumour cell culture which increases its porosity and the nanocarrier penetration. Hep-G2 hepatic 3D clusteroids were formed using a water-in-water (w/w) Pickering emulsion based on an aqueous two-phase system (ATPS). Our specific metformin nano-formulation, comprising 0.25 wt% lysozyme-coated, 0.4 wt% metformin-loaded, 0.2 wt% shellac NPs sterically stabilized with 0.25 wt% Poloxamer 407, demonstrated significantly enhanced anticancer efficiency on 3D hepatic tumour cell clusteroids. We examined the role of the lysozyme surface functionality of the metformin nanocarriers in their ability to kill both 2D and 3D hepatic tumour cell cultures. The anticancer efficiency at high metformin payloads was compared with that at a high concentration of nanocarriers with a lower metformin payload. It was discovered that the high metformin payload NPs were more efficient than the lower metformin payload NPs with a higher nanocarrier concentration. This study introduces a reliable in vitro model for potential targeting of solid tumours with smart nano-therapeutics, presenting a viable alternative to animal testing for evaluating anticancer nanotechnologies.

Bacterial valorization of agricultural-waste into a nano-sized cellulosic matrix for mitigating emerging pharmaceutical pollutants: An eco-benign approach.

For Bacterial Nanocellulose (BNC) production, standard methods are well-established, but there is a pressing need to explore cost-effective alternatives for BNC commercialization. This study investigates the feasibility of using syrup prepared from maize stalk as a valuable nutrient and sustainable carbon source for BNC production. Our study achieved a remarkable BNC production yield of 19.457 g L-1 by utilizing Komagataeibacter saccharivorans NUWB1 in combination with components from the Hestrin-Schramm (HS) medium. Physicochemical properties revealed that the obtained BNC exhibited a crystallinity index of 60.5 %, tensile strength of 43.5 MPa along with enhanced thermostability reaching up to 360 °C. N2 adsorption-desorption isotherm of the BNC displayed characteristics of type IV, indicating the presence of a mesoporous structure. The produced BNC underwent thorough investigation, focusing on its efficacy in addressing environmental concerns, particularly in removing emerging pharmaceutical pollutants like Metformin and Paracetamol. Remarkably, the BNC exhibited strong adsorption capabilities, aligning with the Langmuir isotherm and pseudo-second-order model. Thermodynamic analysis confirmed a spontaneous and endothermic adsorption process. Furthermore, the BNC showed potential for regeneration, enabling up to five recycling cycles. Cytotoxicity and oxidative stress assays validated the biocompatibility of BNC. Lastly, the BNC films displayed an impressive 88.73 % biodegradation within 21 days.

Intravenous injectable metformin-Cu(II)-EGCG coordination polymer nanoparticles for electrothermally enhanced dual-drug synergistic tumor therapy.

Intravenous injectable metformin-Cu(II)-EGCG infinite coordination polymer nanoparticles (metformin-Cu(II)-EGCG ICP NPs) have been synthesized, and an efficient strategy for synergistic tumor therapy by utilizing these nanoparticles in conjunction with micro-electrothermal needles (MENs) was proposed. These nanoparticles display exceptional uniformity with a diameter of 117.5 ± 53.3 nm, exhibit an extraordinary drug loading capacity of 90% and allow for precise control over the drug ratio within the range of 1 : 1 to 1 : 20 while maintaining excellent thermal stability. Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and X-ray diffraction were employed to determine their chemical structure and coordination state. The combination index (CI) value of the metformin-Cu(II)-EGCG ICP NPs was calculated to be 0.19, surpassing that of the two individual drugs and metformin mixed with EGCG (0.98). Importantly, upon intravenous injection, metformin in nanoparticles demonstrated exceptional stability in the bloodstream, while both drugs were rapidly released within the acidic tumor microenvironment. Animal experiments showcased an impressive tumor inhibition rate of 100% within a mere 20-day time frame after the synergistic therapy with a lower dosage (5.0 mg kg-1 of nanoparticles), coupled with a minimal tumor recurrence rate of only 18.75% over a 60-day observation period. These findings indicate the promising prospects of these nanoparticles in tumor treatment.

Metformin-Induced Invertase Unfolding: Enzyme Kinetics and Activity Regulation.

The effects of metformin on invertase activity and its inhibition on sucrose digestion were studied. The rapid unfolding kinetics of invertases, followed a two-state model with an inactive intermediate formation. The dynamic interaction between metformin and invertase caused the secondary structure of the enzyme to become less ß-sheet, more α-helix, and random coiling oriented, which weakened the binding force between enzyme and its substrate. Metformin acted as a chaotrope and disrupted the hydrogen bonds of water, which facilitated the unfolding of invertase. However, some sugar alcohols, which promoted the H-bond formation of water, could repair the secondary structure of metformin-denatured invertase and therefore regulate the enzyme activity. This research enriches our understanding of the mechanism of enzyme unfolding induced by guanidine compounds. Moreover, because metformin and sugar substitutes are of concern to diabetes, this research also provides useful information for understanding the activity of the digestive enzyme that coexists with metformin and sugar alcohols.

Discovery of a Ni2+-dependent heterohexameric metformin hydrolase.

The biguanide drug metformin is a first-line blood glucose-lowering medication for type 2 diabetes, leading to its presence in the global environment. However, little is known about the fate of metformin by microbial catabolism. Here, we characterize a Ni2+-dependent heterohexameric enzyme (MetCaCb) from the ureohydrolase superfamily, catalyzing the hydrolysis of metformin into guanylurea and dimethylamine. Either subunit alone is catalytically inactive, but together they work as an active enzyme highly specific for metformin. The crystal structure of the MetCaCb complex shows the coordination of the binuclear metal cluster only in MetCa, with MetCb as a protein binder of its active cognate. An in-silico search and functional assay discover a group of MetCaCb-like protein pairs exhibiting metformin hydrolase activity in the environment. Our findings not only establish the genetic and biochemical foundation for metformin catabolism but also provide additional insights into the adaption of the ancient enzymes toward newly occurred substrate.

A doxycycline-loaded microfiber of poly-metformin/PCL for eradicating melanoma stem cells.

Nowadays, electrospun fibrous mats are used as drug delivery systems for loading of potential drugs in order to kill cancer cells. In the study, a skin patch for treating melanoma cancer after surgery was made using polycaprolactone and polymetformin microfibers that were loaded with doxycycline (PolyMet/PCL@DOX), an anti-cancer stem cell agent. The morphology, structure, mechanical characteristics, swelling, and porosity of the electrospun microfibers were examined. Drug release andanticancereffectiveness of PolyMet/PCL@DOXwas evaluated against A375 melanoma cancer stem cells using the MTS, Flow cytometry, colony formation and CD44 expression assays. Scanning electron microscopy (SEM) verified the micro fibrous structure with a diameter of about 2.31 µm. The porosity and swelling percentages for microfibers was 73.5 % and 2.9 %, respectively. The tensile strength at the breaking point was equal to 3.84 MPa. The IC50 of PolyMet/PCL@DOX was 7.4 µg/mL. The survival rate of A375 cells after 72 h of PolyMet/PCL@DOX treatment was 43.9 %. The colony formation capacity of A375 cells decreased after PolyMet/PCL@DOX treatment. The level of CD44 expression in the PolyMet/PCL@DOX group decreased compared to the control group. Generally, PolyMet/PCL@DOX microfibers can be a promising candidate as a patch after surgery to eradicate cancer stem cells, effectively.

Application of deep UV resonance Raman spectroscopy to column liquid chromatography: Development of a low-flow method for the identification of active pharmaceutical ingredients.

In this study, deep UV resonance Raman spectroscopy (DUV-RRS) was coupled with high performance liquid chromatography (HPLC) to be applied in the field of pharmaceutical analysis. Naproxen, Metformin and Epirubicin were employed as active pharmaceutical ingredients (APIs) covering different areas of the pharmacological spectrum. Raman signals were successfully generated and attributed to the test substances, even in the presence of the dominant solvent bands of the mobile phase. To increase sensitivity, a low-flow method was developed to extend the exposure time of the sample. This approach enabled the use of a deep UV pulse laser with a low average power of 0.5 mW. Compared to previous studies, where energy-intensive argon ion lasers were commonly used, we were able to achieve similar detection limits with our setup. Using affordable lasers with low operating costs may facilitate the transfer of the results of this study into practical applications.

Research into how carvacrol and metformin affect several human proteins in a hyperglycemic condition: A comparative study in silico and in vitro.

Carvacrol (CV) is an organic compound found in the essential oils of many aromatic herbs. It is nearly unfeasible to analyze all the current human proteins for a query ligand using in vitro and in vivo methods. This study aimed to clarify whether CV possesses an anti-diabetic feature via Docking-based inverse docking and molecular dynamic (MD) simulation and in vitro characterization against a set of novel human protein targets. Herein, the best poses of CV docking simulations according to binding energy ranged from -7.9 to -3.5 (kcal/mol). After pathway analysis of the protein list through GeneMANIA and WebGestalt, eight interacting proteins (DPP4, FBP1, GCK, HSD11ß1, INSR, PYGL, PPARA, and PPARG) with CV were determined, and these proteins exhibited stable structures during the MD process with CV. In vitro application, statistically significant results were achieved only in combined doses with CV or metformin. Considering all these findings, PPARG and INSR, among these target proteins of CV, are FDA-approved targets for treating diabetes. Therefore, CV may be on its way to becoming a promising therapeutic compound for treating Diabetes Mellitus (DM). Our outcomes expose formerly unexplored potential target human proteins, whose association with diabetic disorders might guide new potential treatments for DM.

In vitro and ex vivo evaluation of chitosan gel containing metformin-loaded polymeric nanoparticles for topical treatment of melanoma.

OBJECTIVE: The purpose of this study was to prepare and evaluate chitosan (CS) gel containing metformin hydrochloride (MET)-loaded polycaprolactone (PCL) nanoparticles (NPs) for topical treatment of melanoma. SIGNIFICANCE: Topical administration of MET-PCL NPs-CS gel improves penetration of drug, decreases side effects, and increases efficacy of treatment. METHODS: MET-PCL NPs were prepared by double emulsion method. Particle size, charge, encapsulation efficiency (EE), release, and morphology were evaluated. MET-PCL NPs-CS gel formulation was characterized in terms of organoleptic properties, pH, gelling time, viscosity, spreadability, release, and morphology. Cytotoxicity was performed on B16F10 cells. Ex vivo permeability was done with pig skin. RESULTS: The size, charge, and EE were found to be 180 ± 10 nm, -11.4 mV, and 93%. SEM images showed that NPs were spherical and smooth. An initial burst release followed by a slower release was observed. MET-PCL NPs-CS gel was found to be transparent. The pH was 4.9 ± 0.05. The gelation time was 1.6 ± 0.2 min. The viscosity results confirm pseudoplastic behavior of gel. The spreadability by % area was 392 ± 6.4 cm. The images showed that gelling network of CS gel was composed of suspended NPs. The viscosity was between 554 and 3503 cP. MET-PCL NPs-CS gel showed prolonged release up to 72 h. On B16F10 cells, gel showed higher cytotoxicity compared to MET solution. MET-PCL NPs-CS gel had twofold higher permeability in pig skin compared with MET-CS gel. CONCLUSION: Topical administration of MET-PCL NPs-CS gel into the skin resulted in improved dermal penetration and this promising approach may be of value in effective treatment of melanoma and other skin cancers.

Metformin drugs under simulated gastric conditions can generate high nitrite-dependent levels of N-nitrosodimethylamine.

N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA), group 2A carcinogens, were detected in finished drug products, including metformin, ranitidine, sartans and other drugs which caused multiple recalls in the USA and Europe. Important studies also reported the formation of NDMA when ranitidine and nitrite were added to simulated gastric fluid. Our objective was to screen finished drug products from Europe and USA for nitrosamine impurities and investigate the formation of NDMA in metformin finished drug products when added to simulated gastric fluid. One dosage unit of 30 different commercially available drugs, including metformin, sartans, and ranitidine were tested for NDMA, NDEA, and dimethylformamide (DMF) impurities, using a liquid chromatography-mass spectrometry (LC-MS) method. Then, 6 metformin finished drug products were tested in stomach conditions for 2 h at 37 °C in a 100 mL solution with a pH of 2.5 and different nitrite concentrations (40, 10, 1, 0.1 mM) and tested for NDMA, and DMF using LC-MS. We measured NDMA, NDEA, and DMF in 30 finished drug products. NDMA and DMF were quantified for metformin drug products in simulated gastric fluid with different nitrite concentrations. None of the 30 drugs showed concerning levels of NDMA, NDEA, or DMF when tested as single tablets. However, when metformin tablets are added to simulated gastric fluid solutions with high nitrite concentrations (40 mM and 10 mM), NDMA can reach amounts of thousands of nanograms per tablet. At the closest concentration to physiologic conditions we used, 1 mM, NDMA is still present in the hundreds of nanograms in some metformin products. In this in vitro study, nitrite concentration had a very important effect on NDMA quantification in metformin tablets added to simulated gastric fluid. 1 mM nitrite caused an increase above the acceptable daily intake set by the U.S. Food and Drug Administration (FDA) for some of the metformin drugs. 10 mM, 40 mM nitrite solutions generated NDMA amounts exceeding by more than a hundred times the acceptable daily intake set by the FDA of 96 nanograms. These findings suggest that metformin can react with nitrite in gastric-like conditions and generate NDMA. Thus, patients taking metformin could be exposed to NDMA when high nitrite levels are present in their stomach, and we recommend including a statement within the Patient Package Inserts/Instructions for use.

Synergistic Effect of Flavonoids and Metformin on Protection of the Methylglyoxal-Induced Damage in PC-12 Neuroblastoma Cells: Structure-Activity Relationship and Potential Target.

Methylglyoxal-induced ROS elevation is the primary cause of neuronal damage. Metformin is a traditional hypoglycemic drug that has been reported to be beneficial to the nervous system. In this study, flavonoids were found to enhance the protective effect of metformin when added at a molar concentration of 0.5%. The structure-activity relationship (SAR) analysis indicated that ortho- substitution in the B ring, and the absence of double bonds between the 2 and 3 position combined with the gallate substitution with R configuration at the 3 position in the C ring played crucial roles in the synergistic effects, which could be beneficial for designing a combination of the compounds. Additionally, the mechanism study revealed that a typical flavonoid, EGCG, enhanced ROS scavenging and anti-apoptotic ability via the BCL2/Bax/Cyto C/Caspase-3 pathway, and synergistically inhibited the expression of GSK-3ß, BACE-1, and APP in PC-12 cells when used in combination with metformin. The dose of metformin used in the combination was only 1/4 of the conventional dose when used alone. These results suggested that ROS-mediated apoptosis and the pathways related to amyloid plaques (Aß) formation can be the targets for the synergistic neuroprotective effects of flavonoids and metformin.