Evaluation of Systemic and Brain Pharmacokinetic Parameters for Repurposing Metformin Using Intravenous Bolus Administration.

Metformin's potential in treating ischemic stroke and neurodegenerative conditions is of growing interest. Yet, the absence of established systemic and brain pharmacokinetic (PK) parameters at relevant pre-clinical doses presents a significant knowledge gap. This study highlights these PK parameters and the importance of using pharmacologically relevant pre-clinical doses to study pharmacodynamics (PD) in stroke and related neurodegenerative diseases. An LC-MS/MS method to measure metformin levels in plasma, brain, and cerebrospinal fluid (CSF) was developed and validated. In vitro assays examined brain tissue binding and metabolic stability. Intravenous (IV) bolus administration of metformin to C57BL6 mice covered low to high dose range maintaining pharmacological relevance. Quantification of metformin in the brain was used to assess brain pharmacokinetic parameters, such as unidirectional blood-to-brain constant (Kin) and unbound brain-to-plasma ratio (Kp, uu, brain). Metformin exhibited no binding in the mouse plasma and brain and remained metabolically stable. It rapidly entered the brain, reaching detectable levels in as little as 5 minutes. A Kin value of 1.87 {plus minus} 0.27 µl/g/min was obtained. As the dose increased, Kp, uu, brain showed decreased value, implying saturation, but this did not affect an increase in absolute brain concentrations. Metformin was quantifiable in the CSF at 30 minutes but decreased over time, with concentrations lower than those in the brain across all doses. Our findings emphasize the importance of metformin dose selection based on pharmacokinetic parameters for pre-clinical pharmacological studies. We anticipate further investigations focusing on pharmacokinetics and pharmacodynamics (PKPD) in disease conditions, such as stroke. Significance Statement The study establishes crucial pharmacokinetic parameters of metformin for treating ischemic stroke and neurodegenerative diseases, addressing a significant knowledge gap. It further emphasizes the importance of selecting pharmacologically relevant pre-clinical doses. The findings highlight metformin's rapid brain entry, minimal binding, and metabolic stability. The necessity of considering pharmacokinetic parameters in pre-clinical studies provides a foundation for future investigations into metformin's efficacy for neurodegenerative disease (s).

FAM98A is localized to stress granules and associates with multiple stress granule-localized proteins.

Stress granules are evolutionally conserved ribonucleoprotein structures that are formed in response to various stress stimuli. Recent studies have demonstrated that proteins with low complexity (LC) regions play a critical role for the formation of stress granules. In this study, we report that FAM98A, whose biological functions are unknown, is a novel component of stress granules. FAM98A is localized to stress granules, but not to P-bodies, after various stress stimuli. Analysis with deletion mutants revealed that C-terminal region that contains LC region was essential for FAM98A accumulation to stress granules. Depletion of FAM98A using two different siRNAs decreased the number of stress granules formed per cell. Finally, we show that FAM98A associates with stress granule-localized proteins, such as DDX1, ATXN2, ATXN2L, and NUFIP2. Our results show a partial role of FAM98A for the organization of stress granules.

FAM98A is a novel substrate of PRMT1 required for tumor cell migration, invasion, and colony formation.

Protein arginine methylation, which is mediated by a family of protein arginine methyltransferases (PRMTs), is associated with numerous fundamental cellular processes. Accumulating studies have revealed that the expression of multiple PRMTs promotes cancer progression. In this study, we examined the role of PRMT1 in ovarian cancer cells. PRMT1 is expressed in multiple ovarian cancer cells, and the depletion of its expression suppressed colony formation, in vivo proliferation, migration, and invasion. To gain insight into PRMT1-mediated cancer progression, we searched for novel substrates of PRMT1. We found that FAM98A, whose physiological function is unknown, was arginine-methylated by PRMT1. FAM98A is expressed in numerous ovarian cancer cell lines and is important for the malignant characteristics of ovarian cancer cells. Our results indicate the possible role of the PRMT1-FAM98A pathway in cancer progression.

Permeability of Metformin across an In Vitro Blood-Brain Barrier Model during Normoxia and Oxygen-Glucose Deprivation Conditions: Role of Organic Cation Transporters (Octs).

Our lab previously established that metformin, a first-line type two diabetes treatment, activates the Nrf2 pathway and improves post-stroke recovery. Metformin's brain permeability value and potential interaction with blood-brain barrier (BBB) uptake and efflux transporters are currently unknown. Metformin has been shown to be a substrate of organic cationic transporters (Octs) in the liver and kidneys. Brain endothelial cells at the BBB have been shown to express Octs; thus, we hypothesize that metformin uses Octs for its transport across the BBB. We used a co-culture model of brain endothelial cells and primary astrocytes as an in vitro BBB model to conduct permeability studies during normoxia and hypoxia using oxygen-glucose deprivation (OGD) conditions. Metformin was quantified using a highly sensitive LC-MS/MS method. We further checked Octs protein expression using Western blot analysis. Lastly, we completed a plasma glycoprotein (P-GP) efflux assay. Our results showed that metformin is a highly permeable molecule, uses Oct1 for its transport, and does not interact with P-GP. During OGD, we found alterations in Oct1 expression and increased permeability for metformin. Additionally, we showed that selective transport is a key determinant of metformin's permeability during OGD, thus, providing a novel target for improving ischemic drug delivery.

Impact of in-utero electronic cigarette exposure on neonatal neuroinflammation, oxidative stress and mitochondrial function.

Introduction: Despite the prevalence of the perception that electronic cigarettes (e-cig) are a safer alternative to tobacco smoke, growing concern about their potential toxic impact warrants adequate investigation focusing on special populations like maternal and pediatric groups. This study evaluated the consequences of maternal e-cig use on neonatal neuroinflammation, oxidative stress, and mitochondrial function in primary cultured neurons and postnatal day (PD) 7 and 90 brain. Methodology: Pregnant CD1 mice were exposed to e-cig vapor (2.4% nicotine) from gestational day 5 (E5) till PD7, and the primary neurons were isolated from pups at E16/17. Cellular total reactive oxygen species (ROS) and mitochondrial superoxide were measured in primary neurons using CM-H2DCFDA and Mitosox red, respectively. Mitochondrial function was assessed by Seahorse XF Cell Mitostress analysis. The level of pro-inflammatory cytokines was measured in primary neurons and PD7 and PD90 brains by RT-PCR and immunobead assay. Western blot analysis evaluated the expression of antioxidative markers (SOD-2, HO-1, NRF2, NQO1) and that of the proinflammatory modulator NF-κB. Results: Significantly higher level of total cellular ROS (p < 0.05) and mitochondrial superoxide (p < 0.01) was observed in prenatally e-cig-exposed primary neurons. We also observed significantly reduced antioxidative marker expression and increased proinflammatory modulator and cytokines expression in primary neurons and PD7 (p < 0.05) but not in PD90 postnatal brain. Conclusion: Our findings suggest that prenatal e-cig exposure induces postnatal neuroinflammation by promoting oxidative stress (OS), increasing cytokines' levels, and disrupting mitochondrial function. These damaging events can alter the fetal brain's immune functions, making such offspring more vulnerable to brain insults.

Etoricoxib treatment prevented body weight gain and ameliorated oxidative stress in the liver of high-fat diet-fed rats.

The main focus of this study was to determine the role of etoricoxib in counterbalancing the oxidative stress, metabolic disturbances, and inflammation in high-fat (HF) diet-induced obese rats. To conduct this study, 28 male Wistar rats (weighing 190-210 g) were distributed randomly into four groups: control, control + etoricoxib, HF, and HF + etoricoxib. After 8 weeks of treatment with etoricoxib (200 mg/kg), all the animals were sacrificed followed by the collection of blood and tissue samples in order to perform biochemical tests along with histological staining on hepatic tissues. According to this study, etoricoxib treatment prevented the body weight gain in HF diet-fed rats. Furthermore, rats of HF + etoricoxib group exhibited better blood glucose tolerance than the rats of HF diet-fed group. In addition, etoricoxib also markedly normalized HF diet-mediated rise of hepatic enzyme activity. Etoricoxib treatment lowered the level of oxidative stress indicators significantly with a parallel augmentation of antioxidant enzyme activities. Furthermore, etoricoxib administration helped in preventing inflammatory cell invasion, collagen accumulation, and fibrotic catastrophe in HF diet-fed rats. The findings of the present work are suggestive of the helpful role of etoricoxib in deterring the metabolic syndrome as well as other deleterious pathological changes afflicting the HF diet-fed rats.

FAM98A associates with DDX1-C14orf166-FAM98B in a novel complex involved in colorectal cancer progression.

Protein Arginine Methyl Transferase 1 (PRMT1) is deemed to be a potential oncogenic protein considering its overexpression in several malignancies including colorectal cancer. However, the molecular pathogenesis regarding PRMT1 overexpression and overall poor patient survival involved in this devastating and life threatening cancer remains obscured. In our previous study, we have identified FAM98A as a novel substrate of PRMT1 and also identified its role in ovarian cancer progression. Here, we showed that the two structural homologs FAM98A and FAM98B included in a novel complex with DDX1 and C14orf166 are required for PRMT1 expression. Analysis of the data from The Cancer Genome Atlas (TCGA) database and clinical colorectal cancer specimens also demonstrated a strong positive correlation and co-occurrence of PRMT1, FAM98A and FAM98B. These findings provide a mechanistic insight into how knockdown of FAM98A or FAM98B can suppress the malignant characteristics of cancer cells. Besides, we showed that FAM98A and FAM98B are working in the same axis as knockdown of both proteins together does not cause additional reduction in the cellular proliferation and colony formation of colorectal cancer cells.

SATB1 and SATB2 play opposing roles in c-Myc expression and progression of colorectal cancer.

Special AT-rich sequence-binding protein 1 and 2 (SATB1/2) are nuclear matrix-associated proteins involved in chromatin remodeling and regulation of gene expression. SATB2 acts as a tumor suppressor in laryngeal squamous cell carcinoma and colon cancer, whereas SATB1 promotes the progression of numerous types of cancers. In this study, we examined the effects of SATB1 and SATB2 on the malignant characteristics of colorectal cancer cells. SATB1 and SATB2 expression were negatively correlated in colorectal cancer specimens. SATB1 expression was increased, whereas SATB2 expression was reduced, in colorectal cancer tissues compared to control tissues. Exogenous expression of SATB2 in colorectal cancer cells suppressed cell proliferation, colony formation and tumor proliferation in mice. c-Myc was reduced by SATB2 expression, and exogenous expression of c-Myc in SATB2-expressing cells restored proliferation, colony formation and in vivo tumor growth of colorectal cancer cells. We also showed that c-Myc reduction by SATB2 was mediated by the inactivation of ERK5. In contrast, SATB1 promoted c-Myc expression. The expression of SATB1 in colorectal cancer tissues was positively correlated with c-Myc expression, and SATB1 knockdown reduced c-Myc expression in colorectal cancer cells. Finally, we showed that SATB1 knockdown in colorectal cancer cells suppressed cell proliferation, colony formation and cell invasion. Our results reveal interesting features of how the structural homologs SATB1 and SATB2 exert opposing functions in colorectal tumorigenesis.

Relative bioavailability and pharmacokinetic study of two trimetazidine modified release formulations in healthy Bangladeshi male volunteers.

Trimetazidine (CAS 5011-34-7) is an effective and well-tolerated antianginal drug that possesses protective properties against ischemia-induced heart injury. The relative bioavailability and pharmacokinetic characteristics of two modified release formulations of 35 mg trimetazidine, one as the test product (Metacard MR) and one as the reference product, were compared in healthy Bangladeshi male volunteers. The randomized, two-way crossover study was conducted in 24 healthy male volunteers after administration of a single 35 mg dose of each modified release formulation after 12-h overnight fasting, with a washout period of two weeks. Blood samples were collected at various time intervals following oral administration and analyzed for trimetazidine concentrations using a validated HPLC method. The pharmacokinetic parameters were determined by a non-compartmental method. After administering a single dose of 35 mg of each trimetazidine formulation, the obtained mean (SD) values for the test and reference products were 104.78 (29.3) and 98.57 (28.7) ng/ml for Cmax; 4.00 (1.1) and 3.54 (1.32) h for t(max); 423.81 (173.9) and 410.01 (195.87) ng x h/ml for AUC0-12; and 472.51 (195.2) and 462.78 (225.13) ng x h/ml for AUC0-infinity respectively. The mean t1/2 was found 3.69 (1.1) h and 3.45 (0.72) h for test and reference products respectively. From paired t-test, no significant differences were observed (p > 0.05) for any pharmacokinetic parameters. The 90% confidence intervals of the test/reference mean ratios of the In-transformed AUC0-12, AUC0-infinity, and Cmax mean values were 106.19% (97.16%-116.06%), 104.74% (95.04%-115.42%) and 106.30% (95.23%-118.66%), respectively. The two formulations demonstrated similar bioavailability with respect to both the rate and extent of trimetazidine absorption.