Antidiabetic Biguanide Research Articles

To Formulate and Evaluate the Metformin Hydrochloride Floating Tablet

The aim of the research was to create a gastro retentive system for prolonged release of metformin HCl in the proximal part of the gastrointestinal tract (GIT) in the form of an oral floating tablet. Metformin HCl is an antidiabetic biguanide with poor bioavailability and an absorption window in the upper GIT. Floating tablets were prepared using a wet granulation method containing the natural polymers guar gum and kcarrageenan and the synthetic polymer HPMC K100 (HPMC) either alone or in combination. Sodium bicarbonate and citric acid were used as gas generators. Floating tablets were evaluated for weight variation, hardness and friability, drug content, swelling index, in vitro buoyancy and in vitro drug release. The formulation is optimized based on buoyancy, matrix integrity and in vitro drug release in simulated gastric fluid at pH 1.2. A formulation formulated with a combination of 6 wt. % k-carrageenan and 11 wt. % guar gum showed good gel strength, stable and continuous buoyancy for 12 hours, minimal buoyancy delay of 58 seconds, and good matrix integrity during dissolution period. The drug release of the optimized formulation followed the Korsmeyer- Peppas model and the mechanism was non-Fickian/divergent. PXRD and DSC studies showed partial amorphization of the drug. The mechanism of drug release appeared to be a diffusion mechanism. Stability studies have shown that the drug does not degrade when stored for 3 months at 40˚C.

Metronomic cyclophosphamide and metformin inhibited tumor growth and repopulated tumor-infiltrating lymphocytes in an experimental carcinoma model

Metformin is a widely used antidiabetic biguanide. Retrospective data demonstrated the association of metformin use with survival benefit in multiple tumor types. Interest in repurposing metformin to treat cancer has not been translated into encouraging clinical benefit. In animal models, metformin activated cytotoxic T cells and exerted an immune-mediated anticancer effect. The current research was conducted to investigate the possible therapeutic benefit of metformin in combination with metronomic cyclophosphamide in an experimental cancer model. Ehrlich ascites carcinoma was injected into the subcutaneous tissue to induce solid tumors in syngeneic mice. Exponential solid tumor growth ensued and was effectively arrested with the administration of a cytotoxic dose of parenteral cyclophosphamide. Alternatively, oral metformin and continuous, low-dose cyclophosphamide significantly inhibited tumor growth relative to untreated mice. The drug combination was well tolerated. Histopathological examination of the tumor showed an increased number of tumor-infiltrating lymphocytes and enhanced expression of granzyme B by this drug combination. The current data suggests a potential role of metformin and metronomic chemotherapy that warrants further investigation.

Regulatory Effects of Metformin, an Antidiabetic Biguanide Drug, on the Metabolism of Primary Rat Adipocytes.

Metformin is a biguanide compound commonly applied in humans with type 2 diabetes. The drug affects different tissues, including fat tissue. The direct influence of metformin on cells of fat tissue, i.e., adipocytes, is poorly elucidated. In the present study, the short-term (4-h) effects of metformin on lipogenesis, glucose transport, lipolysis, and lactate release in primary rat adipocytes were explored. It was demonstrated that metformin reduced insulin-induced lipogenesis and increased glucose transport into adipocytes. The tested compound also decreased lactate release from fat cells. It was shown that metformin substantially limited lipolysis stimulated by epinephrine (adrenergic receptor agonist) and dibutyryl-cAMP (direct activator of protein kinase A). Moreover, metformin decreased the lipolytic process triggered by DPCPX (adenosine A1 receptor antagonist). In the case of each lipolytic stimulator, the drug evoked a similar inhibitory effect in the presence of 3 and 12 mM glucose. The lipolytic response of adipocytes to epinephrine was also found to be reduced by metformin when glucose was replaced by alanine. It was demonstrated that the tested compound limits the release of both glycerol and fatty acids from fat cells. The results of the present study provided evidence that metformin significantly affects the metabolism of primary rat adipocytes. Its action covers processes related to lipid accumulation and release and occurs after relatively short-term exposure.

Decavanadate and metformin-decavanadate effects in human melanoma cells

Decavanadate is a polyoxometalate (POMs) that has shown extensive biological activities, including antidiabetic and anticancer activity. Importantly, vanadium-based compounds as well as antidiabetic biguanide drugs, such as metformin, have shown to exert therapeutic effects in melanoma. A combination of these agents, the metformin-decavanadate complex, was also recognized for its antidiabetic effects and recently described as a better treatment than the monotherapy with metformin enabling lower dosage in rodent models of diabetes. Herein, we compare the effects of decavanadate and metformin-decavanadate on Ca2+-ATPase activity in sarcoplasmic reticulum vesicles from rabbit skeletal muscles and on cell signaling events and viability in human melanoma cells. We show that unlike the decavanadate-mediated non-competitive mechanism, metformin-decavanadate inhibits Ca2+-ATPase by a mixed-type competitive–non-competitive inhibition with an IC50 value about 6 times higher (87 μM) than the previously described for decavanadate (15 μM). We also found that both decavanadate and metformin-decavanadate exert antiproliferative effects on melanoma cells at 10 times lower concentrations than monomeric vanadate. Western blot analysis revealed that both, decavanadate and metformin-decavanadate increased phosphorylation of extracellular signal-regulated kinase (ERK) and serine/threonine protein kinase AKT signaling proteins upon 24 h drug exposure, suggesting that the anti-proliferative activities of these compounds act independent of growth-factor signaling pathways.

Preliminary Evidence for IL-10-Induced ACE2 mRNA Expression in Lung-Derived and Endothelial Cells: Implications for SARS-Cov-2 ARDS Pathogenesis.

Angiotensin-converting enzyme 2 (ACE2) is a receptor for the spike protein of SARS-COV-2 that allows viral binding and entry and is expressed on the surface of several pulmonary and non-pulmonary cell types, with induction of a “cytokine storm” upon binding. Other cell types present the receptor and can be infected, including cardiac, renal, intestinal, and endothelial cells. High ACE2 levels protect from inflammation. Despite the relevance of ACE2 levels in COVID-19 pathogenesis, experimental studies to comprehensively address the question of ACE2 regulations are still limited. A relevant observation from the clinic is that, besides the pro-inflammatory cytokines, such as IL-6 and IL-1β, the anti-inflammatory cytokine IL-10 is also elevated in worse prognosis patients. This could represent somehow a “danger signal”, an alarmin from the host organism, given the immuno-regulatory properties of the cytokine. Here, we investigated whether IL-10 could increase ACE2 expression in the lung-derived Calu-3 cell line. We provided preliminary evidence of ACE2 mRNA increase in cells of lung origin in vitro, following IL-10 treatment. Endothelial cell infection by SARS-COV-2 is associated with vasculitis, thromboembolism, and disseminated intravascular coagulation. We confirmed ACE2 expression enhancement by IL-10 treatment also on endothelial cells. The sartans (olmesartan and losartan) showed non-statistically significant ACE2 modulation in Calu-3 and endothelial cells, as compared to untreated control cells. We observed that the antidiabetic biguanide metformin, a putative anti-inflammatory agent, also upregulates ACE2 expression in Calu-3 and endothelial cells. We hypothesized that IL-10 could be a danger signal, and its elevation could possibly represent a feedback mechanism fighting inflammation. Although further confirmatory studies are required, inducing IL-10 upregulation could be clinically relevant in COVID-19-associated acute respiratory distress syndrome (ARDS) and vasculitis, by reinforcing ACE2 levels.

Inhibition of glycolysis and mitochondrial respiration promotes radiosensitisation of neuroblastoma and glioma cells

BackgroundNeuroblastoma accounts for 7% of paediatric malignancies but is responsible for 15% of all childhood cancer deaths. Despite rigorous treatment involving chemotherapy, surgery, radiotherapy and immunotherapy, the 5-year overall survival rate of high-risk disease remains < 40%, highlighting the need for improved therapy. Since neuroblastoma cells exhibit aberrant metabolism, we determined whether their sensitivity to radiotherapy could be enhanced by drugs affecting cancer cell metabolism.MethodsUsing a panel of neuroblastoma and glioma cells, we determined the radiosensitising effects of inhibitors of glycolysis (2-DG) and mitochondrial function (metformin). Mechanisms underlying radiosensitisation were determined by metabolomic and bioenergetic profiling, flow cytometry and live cell imaging and by evaluating different treatment schedules.ResultsThe radiosensitising effects of 2-DG were greatly enhanced by combination with the antidiabetic biguanide, metformin. Metabolomic analysis and cellular bioenergetic profiling revealed this combination to elicit severe disruption of key glycolytic and mitochondrial metabolites, causing significant reductions in ATP generation and enhancing radiosensitivity. Combination treatment induced G2/M arrest that persisted for at least 24 h post-irradiation, promoting apoptotic cell death in a large proportion of cells.ConclusionOur findings demonstrate that the radiosensitising effect of 2-DG was significantly enhanced by its combination with metformin. This clearly demonstrates that dual metabolic targeting has potential to improve clinical outcomes in children with high-risk neuroblastoma by overcoming radioresistance.

Metformin: Targeting the Metabolo-Epigenetic Link in Cancer Biology.

Metabolism can directly drive or indirectly enable an aberrant chromatin state of cancer cells. The physiological and molecular principles of the metabolic link to epigenetics provide a basis for pharmacological modulation with the anti-diabetic biguanide metformin. Here, we briefly review how metabolite-derived chromatin modifications and the metabolo-epigenetic machinery itself are both amenable to modification by metformin in a local and a systemic manner. First, we consider the capacity of metformin to target global metabolic pathways or specific metabolic enzymes producing chromatin-modifying metabolites. Second, we examine its ability to directly or indirectly fine-tune the activation status of chromatin-modifying enzymes. Third, we envision how the interaction between metformin, diet and gut microbiota might systemically regulate the metabolic inputs to chromatin. Experimental and clinical validation of metformin’s capacity to change the functional outcomes of the metabolo-epigenetic link could offer a proof-of-concept to therapeutically test the metabolic adjustability of the epigenomic landscape of cancer.

Antineoplastic Activity of an Old Natural Antidiabetic Biguanide on the Human Thyroid Carcinoma Cell Line.

In the last decades, metformin (Met), an herbal anti-diabetic medicine, has been proposed as an anti-cancer agent. Thyroid cancers are the most common malignancy of the endocrine system. Therefore, the current study was performed to assess the effects of Met on cell proliferation and activation of the Phosphoinositide 3- Kinase (PI3K)/Protein kinase B (AKT)/Forkhead Box O1 (FOXO1) signaling pathway in the Medullary Thyroid Carcinoma (MTC) cells. The effects of Met on the expression of REarranged during Transfection (RET) proto-oncogene were also investigated. MTC cell line (TT) was treated with 0, 2.5, 5, 10, 20, 30, 40, 50, and 60 mM concentrations of Met for 24, 48, and 72h. The viability and apoptosis of the treated cells were measured by the 3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V- Propidium Iodide (PI) assays. The expression level of PI3K, AKT, FOXO1, and RET genes was investigated by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), and phosphorylation of their proteins was determined by the Enzyme-Linked Immunosorbent Assay (ELISA). Results showed that Met significantly decreased the viability of the MTC cells. Met also reduced the expression level of PI3K, AKT, and FOXO1 genes (P<0.05), whereas it elevated the expression level of RET proto-oncogene (P<0.05). It seems that the Met has a cytostatic effect on the TT cells. Our results showed that anti-tumoral effects of Met may be cell type-specific, and according to the induction of RET (as a proto-oncogene) and inhibition of FOXO1 (as a tumor suppressor gene), Met could not be an appropriate agent in the treatment of MTC. The antineoplastic activity of Met has been confirmed against several malignancies in "in vitro" and "in vivo" studies. However, its molecular mechanisms in the treatment of different carcinomas particularly in thyroid cancers are not clearly understood and more studies are required to confirm its exact effect on the MTC.

Abstract P3-06-16: Synergistic effect of biguanides and fatty acid β-oxidation inhibitor in triple-negative breast cancers

Abstract Triple negative breast cancer (TNBC) is one of non-targetable cancers which are still poorly understood for the driver pathways and therapeutic targets. Over the past decade, cancer metabolism researches have a huge advance. Thus, it has enhanced our understanding on metabolic reprogramming in cancer therapy. We have previously shown that metabolic reprogramming to fatty acid β-oxidation (FAO) is a key energy metabolic pathway in TNBC. Moreover, we reported that FAO regulates c-Src, one of the frequently upregulated oncopathways in TNBC via autophosphorylation of Src at Y419. However, single treatment by FAO inhibitor alone cannot effectively control the tumor progression in TNBC because of metabolic reprogramming and flexibility of tumors. Therefore, proper combination therapies with other metabolic targets are necessary. Recently increasing studies demonstrate that anti-diabetic biguanides have attractive anti-cancer effect in various cancer types including breast cancer (BC). However, its significance as an anti-cancer drug is not well established due to parallel metabolic pathways that support tumor growth. Biguanide like metformin is the most widely administered anti-diabetic agent worldwide. Phenformin, a biguanide derivative similar to metformin, has a greater potency than metformin. Like metformin, phenformin also inhibits mitochondrial electron transport chain (ETC) through complex I inhibition. In addition, biguanides lead to the activation of AMPK, which plays a important role in insulin signaling and energy sensing. Importantly, AMPK is an upstream regulator of FAO pathway because it can phosphorylate ACC to activate FAO. Considering the dependency of TNBC to FAO, we evaluated the therapeutic significance of the combination of biguanides (ETC inhibitors) and FAO inhibitors in TNBC progression and metastasis. And we investigated that targeting both ‘arms’ of the pathway can provide more effective and durable responses in TNBC treatments. Our various in vitro and in vivo studies using TNBC cell lines and PDX models suggest that the combination of both inhibitors can provide better therapeutic benefits in TNBCs. This is a rationale and cost-effective metabolic approach to overcome the currently non-targetable TNBCs. Further study toward the clinical effectiveness of this combination may offer better treatment opportunities for TNBC patients. Citation Format: Jun Hyoung Park, Kwang Hwa Jung, Dongya Jia, Benny A Kaipparettu. Synergistic effect of biguanides and fatty acid β-oxidation inhibitor in triple-negative breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-16.

Aging and cancer: Is glucose a mediator between them?

Aging can increase cancer incidence because of accumulated mutations that initiate cancer and via compromised body control of premalignant lesions development into cancer. Relative contributions of these two factors are debated. Recent evidence suggests that the latter is rate limiting. In particular, hyperglycemia caused by compromised body control of blood glucose may be a factor of selection of somatic mutation-bearing cells for the ability to use glucose for proliferation. High glucose utilization in aerobic glycolysis is a long known characteristic of cancer. The new evidence adds to the concepts that have been being developed starting from mid-1970ies to suggest that age-related shifts in glucose and lipid metabolism increase the risk of cancer and compromise prognoses for cancer patients and to propose antidiabetic biguanides, including metformin, for cancer prevention and as an adjuvant means of cancer treatment aimed at the metabolic rehabilitation of patients. The new evidence is consistent with several effects of glucose contributing to aging and acting synergistically to enhance carcinogenesis. Glucose can affect (i) separate cells (via promoting somatic mutagenesis and epigenetic instability), (ii) cell populations (via being a factor of selection of phenotypic variants in cell populations for higher glucose consumption and, ultimately, for high aerobic glycolysis); (iii) cell microenvironment (via modification of extracellular matrix proteins), and (iv) the systemic levels (via shifting the endocrine regulation of metabolism toward increasing blood lipids and body fat, which compromise immunological surveillance and promote inflammation). Thus, maintenance of youthful metabolic characteristics must be important for cancer prevention and treatment.

Abstract 361: NT1195, a novel biguanide, exhibits superior uterine penetration and anti-tumorigenic efficacy as compared to metformin/phenformin in endometrial cancer

Abstract Objectives: Obesity and diabetes are associated with increased risk and worse outcomes in endometrial cancer (EC). Anti-diabetic biguanide drugs such as metformin/phenformin may have anti-tumorigenic effects by behaving as AMPK activators. Metformin requires organic cation transporters (OCTs) for entry into cells whereas phenformin is not as reliant on OCTs for cell entry. NT1195 (NovaTarg) is a novel biguanide designed to have greater affinity for the OCT1/3, PMAT and MATE1 transporters. Thus, we compared head-to-head the pharmacokinetic properties and anti-tumorigenic potential of metformin vs phenformin vs NT1195 in a genetically engineered mouse model of endometrioid EC (LKB1fl/flp53fl/fl). Methods: Pharmacokinetic parameters, including half-life, AUC, bioavailability and uterine concentration were measured in female control mice after oral dosing of metformin, phenformin and NT1195 (5 mg/kg oral for all drugs). AdCre was injected at 6 wks of age to induce invasive EC in LKB1fl/flp53fl/fl mice. Following tumor onset, mice were treated with placebo vs metformin vs phenformin vs NT1195 for 4 wks (50 mg/kg, oral for all drugs). Results: NT1195 (&amp;gt; 24 hrs) had a longer half-life when compared to metformin (3.7 hrs) and phenformin (7.2 hrs). Drug bioavailability was heightened in the NT1195-treated mice when compared to metformin and phenformin (149 vs 83.5 vs 8.31%, respectively). The AUC (h*kg*ng/ml/mg) for NT1195 was significantly higher at 1143 than that of metformin (26) and phenformin (184). Drug accumulation in uterine tissue at 24 hrs post dose demonstrated markedly increased presence of NT1195 (45.4 ng/g), compared to metformin and phenformin, which yielded levels below the limits of quantification. NT1195 had increased anti-tumorigenic efficacy in LKB1fl/flp53fl/fl mice, inhibiting tumor growth by 88% compared to 78% for metformin and 80% for phenformin (p&amp;lt;0.01). Conclusions: When compared to metformin/phenformin, NT1196 had superior bioavailability, a longer half-life and greater uterine tissue penetration, culminating in improved efficacy in an EC mouse model. Thus, NT1195 may be a promising novel biguanide therapy in EC, particularly in women who want to avoid hysterectomy and preserve fertility or are poor surgical candidates. Citation Format: Katherine Tucker, Allison Staley, Yali Fan, Xiaoling Zhao, Yajie Yin, Ziwei Fang, Wenchuan Sun, Kenneth Batchelor, Nick Livingston, Chunziao Zhou, Victoria Bae-Jump. NT1195, a novel biguanide, exhibits superior uterine penetration and anti-tumorigenic efficacy as compared to metformin/phenformin in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 361.

PV-0539 Antidiabetic biguanides radiosensitize hypoxic cancer cells through a decrease in oxygen consumption

PV-0539 Antidiabetic biguanides radiosensitize hypoxic cancer cells through a decrease in oxygen consumption

Abstract P2-02-11: Combinational treatment of biguanides and fatty acid β-oxidation inhibitor in triple-negative breast cancers

Abstract Among breast cancers (BCs), the driver pathways and therapeutic targets are still poorly understood for triple negative (TN) BCs. Advances in cancer metabolism research over the last decade have enhanced our understanding on metabolic reprogramming in cancer therapy. We have previously shown that metabolic reprogramming to fatty acid β-oxidation (FAO) is a major energy pathway in metastatic TNBC. Moreover, we reported that FAO regulates c-Src, one of the frequently upregulated oncopathways in TNBC via autophosphorylation of Src at Y419. Since FAO inhibitors alone cannot effectively control the tumor progression in TNBC, suitable combination therapies with other metabolic targets are necessary. Recently increasing evidences show that anti-diabetic biguanides have attractive anticancer effect in various cancer types including BC. However, its significance as an anticancer drug is not well established due to parallel metabolic pathways that support tumor growth. Phenformin, a biguanide derivative similar to metformin, has a greater potency than metformin. Like metformin, phenformin also inhibits mitochondrial electron transport chain (ETC) through complex I inhibition. In addition, biguanides lead to the activation of AMPK, which plays a key role in insulin signaling and energy sensing. Importantly, AMPK is an upstream regulator of FAO pathway because it can phosphorylate ACC to activate FAO. Considering the dependency of TNBC to FAO, we evaluated the therapeutic significance of the combination of biguanides(ETC inhibitors) and FAO inhibitors in TNBC progression and metastasis. We hypothesize that blocking both 'arms' of the pathway can provide more pronounced and durable responses in TNBCs. Our different in vitro and in vivo studies using TNBC cell line and PDX models suggest that the combination of both inhibitors can provide better therapeutic significance in metastatic TNBCs. This is a rationale and cost-effective metabolic approach to manage the currently non-targetable metastatic TNBCs. Further investigation into the clinical effectiveness of this combination may provide better treatment opportunities for TNBC patients. Citation Format: Park JH, Jung KH, Vithayathil S, Jia D, Kaipparettu BA. Combinational treatment of biguanides and fatty acid β-oxidation inhibitor in triple-negative breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-02-11.

Metformin administration prevents memory impairment induced by hypobaric hypoxia in rats

Metformin, an antidiabetic biguanide, reduces hyperglycemia by improving glucose utilization and reducing gluconeogenesis. Recently, an increasing number of studies have shown that metformin also led to a significant clinical improvement in memory and cognition in different clinical settings. In the present study, we investigated whether metformin administration protects against memory impairment and neuron damage caused by acute exposure to hypobaric hypoxia and screened the possible molecular mechanisms with a focused gene array. We found that metformin treatment obviously attenuated spatial memory and recognition memory impairment resulting from acute hypobaric hypoxia exposure but had no effect on general locomotor and behavioral activity. Moreover, the results of Nissl and TUNEL staining showed that neuron damage and cell apoptosis caused by hypobaric hypoxia exposure was also inhibited by metformin pretreatment. At the molecular level, we found that metformin pretreatment not only prevented the changes of FOS, JUNB and BDNF at both mRNA and protein levels, but also increased the expression of the postsynaptic scaffold genes HOMER and PSD95 after exposure to hypobaric hypoxia. These data suggested that metformin pretreatment is a feasible strategy for preventing memory impairment under hypobaric hypoxia.

Antidiabetic Biguanides Radiosensitize Hypoxic Colorectal Cancer Cells Through a Decrease in Oxygen Consumption.

Background and Purpose: The anti-diabetic biguanide drugs metformin and phenformin exhibit antitumor activity in various models. However, their radiomodulatory effect under hypoxic conditions, particularly for phenformin, is largely unknown. This study therefore examines whether metformin and phenformin as mitochondrial complex I blockades could overcome hypoxic radioresistance through inhibition of oxygen consumption.Materials and Methods: A panel of colorectal cancer cells (HCT116, DLD-1, HT29, SW480, and CT26) was exposed to metformin or phenformin for 16 h at indicated concentrations. Afterward, cell viability was measured by MTT and colony formation assays. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry. Phosphorylation of AMP-activated protein kinase (AMPK) was examined by western blot. Mitochondria complexes activity and oxygen consumption rate (OCR) were measured by seahorse analyzer. The radiosensitivity of tumor cells was assessed by colony formation assay under aerobic and hypoxic conditions. The in vitro findings were further validated in colorectal CT26 tumor model.Results: Metformin and phenformin inhibited mitochondrial complex I activity and subsequently reduced OCR in a dose-dependent manner starting at 3 mM and 30 μM, respectively. As a result, the hypoxic radioresistance of tumor cells was counteracted by metformin and phenformin with an enhancement ratio about 2 at 9 mM and 100 μM, respectively. Regarding intrinsic radioresistance, both of them did not exhibit any effect although there was an increase of phosphorylation of AMPK and ROS production. In tumor-bearing mice, metformin or phenformin alone did not show any anti-tumor effect. While in combination with radiation, both of them substantially delayed tumor growth and enhanced radioresponse, respectively, by 1.3 and 1.5-fold.Conclusion: Our results demonstrate that metformin and phenformin overcome hypoxic radioresistance through inhibition of mitochondrial respiration, and provide a rationale to explore metformin and phenformin as hypoxic radiosensitizers.

Inhibition of Chloride Intracellular Channel 1 (CLIC1) as Biguanide Class-Effect to Impair Human Glioblastoma Stem Cell Viability.

The antidiabetic biguanide metformin exerts antiproliferative effects in different solid tumors. However, during preclinical studies, metformin concentrations required to induce cell growth arrest were invariably within the mM range, thus difficult to translate in a clinical setting. Consequently, the search for more potent metformin derivatives is a current goal for new drug development. Although several cell-specific intracellular mechanisms contribute to the anti-tumor activity of metformin, the inhibition of the chloride intracellular channel 1 activity (CLIC1) at G1/S transition is a key events in metformin antiproliferative effect in glioblastoma stem cells (GSCs). Here we tested several known biguanide-related drugs for the ability to affect glioblastoma (but not normal) stem cell viability, and in particular: phenformin, a withdrawn antidiabetic drug; moroxydine, a former antiviral agent; and proguanil, an antimalarial compound, all of them possessing a linear biguanide structure as metformin; moreover, we evaluated cycloguanil, the active form of proguanil, characterized by a cyclized biguanide moiety. All these drugs caused a significant impairment of GSC proliferation, invasiveness, and self-renewal reaching IC50 values significantly lower than metformin, (range 0.054–0.53 mM vs. 9.4 mM of metformin). All biguanides inhibited CLIC1-mediated ion current, showing the same potency observed in the antiproliferative effects, with the exception of proguanil which was ineffective. These effects were specific for GSCs, since no (or little) cytotoxicity was observed in normal umbilical cord mesenchymal stem cells, whose viability was not affected by metformin and moroxydine, while cycloguanil and phenformin induced toxicity only at much higher concentrations than required to reduce GSC proliferation or invasiveness. Conversely, proguanil was highly cytotoxic also for normal mesenchymal stem cells. In conclusion, the inhibition of CLIC1 activity represents a biguanide class-effect to impair GSC viability, invasiveness, and self-renewal, although dissimilarities among different drugs were observed as far as potency, efficacy and selectivity as CLIC1 inhibitors. Being CLIC1 constitutively active in GSCs, this feature is relevant to grant the molecules with high specificity toward GSCs while sparing normal cells. These results could represent the basis for the development of novel biguanide-structured molecules, characterized by high antitumor efficacy and safe toxicological profile.

Treatment with antidiabetic biguanide drugs directly impacts the function of multiple pituitary cell types from two non-human primate models

Treatment with antidiabetic biguanide drugs directly impacts the function of multiple pituitary cell types from two non-human primate models

On Choosing Control Objects in Experimental Gerontological Research

Recently, a large number of papers have appeared that describe the successful use of various biologically active compounds (short peptides, mitochondrial antioxidants, antidiabetic biguanides, mimetics of dietary restriction, autophagy modulators, etc.) as geroprotectors. However, in our opinion, in most cases, the positive results of such studies are determined by a “successful” selection of control objects. Animals with certain abnormalities are often used for this purpose, so that any favorable effect on the corresponding pathological processes leads to an increase in their lifespan. In addition, control animals can be normal (i.e., wildtype) but placed under certain extreme conditions that can be overcome just by using certain biologically active compounds. Thus, in this case, the treatment of pathologies rather than the effect on fundamental processes of aging is observed. There is a point of view that the results of Clive McCay’s well-known experiments, which have significantly prolonged the life of rats by limiting caloric intake, were determined by the facts that, firstly, the control animals fed ad libitum (which is absolutely untypical for animals in the wild) and, secondly, Fisher-344 rats, which were used in these experiments, are short-lived. The above considerations, apparently, also apply to the gerontological experiments on cultured cells. In particular, we sometimes hear remarks from our colleagues regarding the model of “stationary phase aging” of cell cultures, which is used in our laboratory, due to the fact that most of the experiments are performed on transformed rather than normal cells. However, this approach seems to us quite justified, because the phenomenon of “stationary phase”/chronological aging is common to a wide variety of cells, including bacteria, yeasts, cyanobacteria, mycoplasmas, as well as animal and plant cells. Cells with an unlimited mitotic potential do not change either from experiment to experiment or during long-term cultivation both with and without subcultivation (within the framework of the stationary phase aging model), which cannot be said of the normal diploid fibroblasts, whose telomeres are shortened with each division. In the period from seeding to entering the stationary phase of growth, the cells divide up to ten times! We believe that, to search for effective geroprotectors that affect the fundamental mechanisms of aging, it is necessary to perform studies on “maximally healthy” animals or on “maximally stable” model systems.

Protective effect of metformin on D-galactose-induced aging model in mice.

Objective(s):Metformin (Met), an antidiabetic biguanide, reduces hyperglycemia via improving glucose utilization and reducing the gluconeogenesis. Met has been shown to exert neuroprotective, antioxidant and anti-inflammatory properties. The present study investigated the possible effect of Met on the D-galactose (D-gal)-induced aging in mice.Materials and Methods:Met (1 and 10 mg/kg/p.o.), was administrated daily in D-gal-received (500 mg/kg/p.o.) mice model of aging for six weeks. Anxiety-like behavior, cognitive function, and physical power were evaluated by the elevated plus-maze, novel object recognition task (NORT), and forced swimming capacity test, respectively. The brains were analyzed for the level of superoxide dismutase (SOD) and brain-derived neurotrophic factor (BDNF).Results:Met decreased the anxiety-like behavior in D-gal-treated mice. Also, Met treated mice showed significantly improved learning and memory ability in NORT compared to the D-gal-treated mice. Furthermore, Met increased the physical power as well as the activity of SOD and BDNF level in D-gal-treated mice.Conclusion:Our results suggest that the use of Met can be an effective strategy for prevention and treatment of D-gal-induced aging in animal models. This effect seems to be mediated by attenuation of oxidative stress and enhancement of the neurotrophic factors.

Salting-out assisted liquid-liquid extraction coupled with hydrophilic interaction chromatography for the determination of biguanides in biological and environmental samples

A new salting-out assisted liquid-liquid extraction (SALLE) sample preparation method for the determination of the polar anti-diabetic biguanide drugs (metformin, buformin and phenformin) in blood plasma, urine and lake water samples were developed. The SALLE was performed by mixing samples (plasma (0.2mL), urine or lake water (1.0mL)) with acetonitrile (0.4mL for plasma, 0.5mL for urine or lake water), sodium hydroxide powder was then added for the phase separation. The effects of type of salting-out reagent, type of extraction solvent, volumes of acetonitrile and sample, amount of sodium hydroxide, vortexing and centrifugation times on the extraction efficiency were investigated. The upper layer, containing the biguanides, was directly injected into a HPLC unit using ZIC-HILIC column (150mm×2.1mm×3.5μm) and was detected at 236nm. The method was validated and calibration curves were linear with r2>0.99 over the range of 20–2000μgL−1 for plasma and 5–2000μgL−1 for urine and lake water samples. The limits of detection were in the range (3.8–5.6)μgL−1, (0.8–1.5)μgL−1 and (0.3–0.8)μgL−1 for plasma, urine and lake water, respectively. The accuracies in the three matrices were within 87.3–103%, 87.4–109%, 82.2–109% of the nominal concentration for metformin, buformin and phenformin, respectively. The relative standard deviation for inter- and intra −day precision were in the range of 1.0–17% for all analytes in the three matrices.