Pharmacokinetics of d- and l-norfenfluramine following their administration as individual enantiomers in rats.

The effect of fenfluramine and norfenfluramine enantiomers in rodent seizure models and their correlation with the pharmacokinetics of d- and l-fenfluramine in rats have been reported recently. To complement these findings, we investigated the pharmacokinetics of d- and l- norfenfluramine in rat plasma and brain. Sprague-Dawley rats were injected intraperitoneally with 20 mg/kg and 1 mg/kg l- norfenfluramine. A 1 mg/kg dose of d-norfenfluramine was used because higher doses caused severe toxicity. The concentration of each enantiomer in plasma and brain was determined at different time points by liquid chromatography/mass spectrometry. Pharmacokinetic parameters were compared between norfenfluramine enantiomers, and with those reported previously for fenfluramine enantiomers after a 20 mg/kg, i.p., dose. All enantiomers were absorbed rapidly and eliminated, with half-lives ranging from 0.9 h (l-fenfluramine) to 6.1 h (l- norfenfluramine, 20 mg/kg) in plasma, and from 3.6 h (d-fenfluramine) to 8.0 h (l-fenfluramine) in brain. Brain-to-plasma concentration ratios ranged from 15.4 (d-fenfluramine) to 27.6 (d-norfenfluramine), indicating extensive brain penetration. The fraction of d- and l-fenfluramine metabolized to norfenfluramine was estimated to be close to unity. This work is part of ongoing investigations to determine the potential value of developing enantiomerically pure l-fenfluramine or l-norfenfluramine as follow-up compounds to the marketed racemic fenfluramine.

Lung dendritic-cell metabolism underlies susceptibility to viral infection in diabetes.

People with diabetes feature a life-risking susceptibility to respiratory viral infection, including influenza and SARS-CoV-2 (ref. 1), whose mechanism remains unknown. In acquired and genetic mouse models of diabetes, induced with an acute pulmonary viral infection, we demonstrate that hyperglycaemia leads to impaired costimulatory molecule expression, antigen transport and T cell priming in distinct lung dendritic cell (DC) subsets, driving a defective antiviral adaptive immune response, delayed viral clearance and enhanced mortality. Mechanistically, hyperglycaemia induces an altered metabolic DC circuitry characterized by increased glucose-to-acetyl-CoA shunting and downstream histone acetylation, leading to global chromatin alterations. These, in turn, drive impaired expression of key DC effectors including central antigen presentation-related genes. Either glucose-lowering treatment or pharmacological modulation of histone acetylation rescues DC function and antiviral immunity. Collectively, we highlight a hyperglycaemia-driven metabolic-immune axis orchestrating DC dysfunction during pulmonary viral infection and identify metabolic checkpoints that may be therapeutically exploited in mitigating exacerbated disease in infected diabetics.

Therapeutic efficacy and pharmacokinetics of liposomal-cannabidiol injection: a pilot clinical study in dogs with naturally-occurring osteoarthritis.

Introduction: Osteoarthritis is a common disease in dogs resulting in chronic pain and decreased wellbeing. Common analgesics such as non-steroidal anti-inflammatories may fail to control pain and can produce major adverse effects. Study objectives were to evaluate pharmacokinetics, therapeutic efficacy, and safety of subcutaneous liposomal-cannabidiol (CBD) as an additional analgesic therapy in dogs suffering from naturally-occurring osteoarthritis. Methods: Six such dogs were recruited following ethics approval and owner consent. Dogs were administered a single subcutaneous injection of 5 mg/kg liposomal-CBD. Plasma concentrations of CBD, blood work, activity monitoring collar data, wellbeing questionnaire (owners) and pain scoring (veterinarian) were performed at baseline and monitored up to six weeks following intervention. Data overtime were compared with baseline using linear-regression mixed-effects. P-value was set at 0.05. Results: CBD plasma concentrations were observed for 6 weeks; median (range) peak plasma concentration (Cmax) was 45.2 (17.8-72.5) ng/mL, time to Cmax was 4 (2-14) days and half-life was 12.4 (7.7-42.6) days. Median (range) collar activity score was significantly increased on weeks 5-6; from 29 (17-34) to 34 (21-38). Scores of wellbeing and pain evaluations were significantly improved at 2-3 weeks; from 69 (52-78) to 53.5 (41-68), and from 7.5 (6-8) to 5.5 (5-7), respectively. The main adverse effect was minor local swelling for several days in 5/6 dogs. Conclusion: Liposomal-CBD administered subcutaneously produced detectable CBD plasma concentrations for 6 weeks with minimal side effects and demonstrated reduced pain and increased wellbeing as part of multimodal pain management in dogs suffering from osteoarthritis. Further placebo-controlled studies are of interest.

The Beneficial Effect of Mitochondrial Transfer Therapy in 5XFAD Mice via Liver-Serum-Brain Response.

We recently reported the benefit of the IV transferring of active exogenous mitochondria in a short-term pharmacological AD (Alzheimer's disease) model. We have now explored the efficacy of mitochondrial transfer in 5XFAD transgenic mice, aiming to explore the underlying mechanism by which the IV-injected mitochondria affect the diseased brain. Mitochondrial transfer in 5XFAD ameliorated cognitive impairment, amyloid burden, and mitochondrial dysfunction. Exogenously injected mitochondria were detected in the liver but not in the brain. We detected alterations in brain proteome, implicating synapse-related processes, ubiquitination/proteasome-related processes, phagocytosis, and mitochondria-related factors, which may lead to the amelioration of disease. These changes were accompanied by proteome/metabolome alterations in the liver, including pathways of glucose, glutathione, amino acids, biogenic amines, and sphingolipids. Altered liver metabolites were also detected in the serum of the treated mice, particularly metabolites that are known to affect neurodegenerative processes, such as carnosine, putrescine, C24:1-OH sphingomyelin, and amino acids, which serve as neurotransmitters or their precursors. Our results suggest that the beneficial effect of mitochondrial transfer in the 5XFAD mice is mediated by metabolic signaling from the liver via the serum to the brain, where it induces protective effects. The high efficacy of the mitochondrial transfer may offer a novel AD therapy.

Metabolomic profiling of triple negative breast cancer cells suggests that valproic acid can enhance the anticancer effect of cisplatin.

Cisplatin is an effective chemotherapeutic agent for treating triple negative breast cancer (TNBC). Nevertheless, cisplatin-resistance might develop during the course of treatment, allegedly by metabolic reprograming, which might influence epigenetic regulation. We hypothesized that the histone deacetylase inhibitor (HDACi) valproic acid (VPA) can counter the cisplatin-induced metabolic changes leading to its resistance. We performed targeted metabolomic and real time PCR analyses on MDA-MB-231 TNBC cells treated with cisplatin, VPA or their combination. 22 (88%) out of the 25 metabolites most significantly modified by the treatments, were acylcarnitines (AC) and three (12%) were phosphatidylcholines (PCs). The most discernible effects were up-modulation of AC by cisplatin and, contrarily, their down-modulation by VPA, which was partial in the VPA-cisplatin combination. Furthermore, the VPA-cisplatin combination increased PCs, sphingomyelins (SM) and hexose levels, as compared to the other treatments. These changes predicted modulation of different metabolic pathways, notably fatty acid degradation, by VPA. Lastly, we also show that the VPA-cisplatin combination increased mRNA levels of the fatty acid oxidation (FAO) promoting enzymes acyl-CoA synthetase long chain family member 1 (ACSL1) and decreased mRNA levels of fatty acid synthase (FASN), which is the rate limiting enzyme of long-chain fatty acid synthesis. In conclusion, VPA supplementation altered lipid metabolism, especially fatty acid oxidation and lipid synthesis, in cisplatin-treated MDA-MB-231 TNBC cells. This metabolic reprogramming might reduce cisplatin resistance. This finding may lead to the discovery of new therapeutic targets, which might reduce side effects and counter drug tolerance in TNBC patients.

A Case Report of Subcutaneously Injected Liposomal Cannabidiol Formulation Used as a Compassion Therapy for Pain Management in a Dog.

A 14-year-old intact mixed breed dog (26 kg) was submitted for a novel cannabidiol (CBD) analgesic treatment. The dog was cachectic and had a testicular neoplasia, hip and elbow osteoarthritis and severe cervical pain. Analgesic treatment included canine osteoarthritic supplement, robencoxib and gabapentin. An additional liposomal CBD injectable formulation at 5 mg/kg was administered subcutaneously between the shoulder blades. The dog was monitored using an activity monitoring collar (PetPace), owner wellbeing questionnaire (Canine Brief Pain Inventory; CBPI), pain interactive visual analog scale (iVAS), blood work and CBD plasma concentrations. A week from the injection and up to 3 weeks afterwards the dog had improved CBPI and iVAS pain scores, and increased collar activity scores. CBD was quantified in plasma for 28 days. Due to disease progression, further difficulty to rise and walk, and relapse to pain after 3 weeks, the owners requested a second liposomal CBD injection, which was performed 4 weeks following the first injection using 3 mg/kg dose. Two days later, the dog was found dead in the yard under direct sun, while environmental temperature was 37°C. Major findings on necropsy revealed evidence of heat stroke and severe cervical disc protrusion with spinal hematoma, none related to liposomal CBD. In conclusion, subcutaneous liposomal CBD produced quantifiable CBD plasma concentrations for 28 days and may be an effective additional treatment as part of multimodal pain management in dogs.

Multifaceted Analyses of Isolated Mitochondria Establish the Anticancer Drug 2-Hydroxyoleic Acid as an Inhibitor of Substrate Oxidation and an Activator of Complex IV-Dependent State 3 Respiration.

The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) has been extensively investigated as a cancer therapy mainly based on its regulation of membrane lipid composition and structure, activating various cell fate pathways. We discovered, additionally, that 2OHOA can uncouple oxidative phosphorylation, but this has never been demonstrated mechanistically. Here, we explored the effect of 2OHOA on mitochondria isolated by ultracentrifugation from U118MG glioblastoma cells. Mitochondria were analyzed by shotgun lipidomics, molecular dynamic simulations, spectrophotometric assays for determining respiratory complex activity, mass spectrometry for assessing beta oxidation and Seahorse technology for bioenergetic profiling. We showed that the main impact of 2OHOA on mitochondrial lipids is their hydroxylation, demonstrated by simulations to decrease co-enzyme Q diffusion in the liquid disordered membranes embedding respiratory complexes. This decreased co-enzyme Q diffusion can explain the inhibition of disjointly measured complexes I-III activity. However, it doesn't explain how 2OHOA increases complex IV and state 3 respiration in intact mitochondria. This increased respiration probably allows mitochondrial oxidative phosphorylation to maintain ATP production against the 2OHOA-mediated inhibition of glycolytic ATP production. This work correlates 2OHOA function with its modulation of mitochondrial lipid composition, reflecting both 2OHOA anticancer activity and adaptation to it by enhancement of state 3 respiration.

Therapeutic Potential of Injectable Nano-Mupirocin Liposomes for Infections Involving Multidrug-Resistant Bacteria.

Antibiotic resistance is a global health threat. There are a few antibiotics under development, and even fewer with new modes of action and no cross-resistance to established antibiotics. Accordingly, reformulation of old antibiotics to overcome resistance is attractive. Nano-mupirocin is a PEGylated nano-liposomal formulation of mupirocin, potentially enabling parenteral use in deep infections, as previously demonstrated in several animal models. Here, we describe extensive in vitro profiling of mupirocin and Nano-mupirocin and correlate the resulting MIC data with the pharmacokinetic profiles seen for Nano-mupirocin in a rat model. Nano-mupirocin showed no cross-resistance with other antibiotics and retained full activity against vancomycin-, daptomycin-, linezolid- and methicillin- resistant Staphylococcus aureus, against vancomycin-resistant Enterococcus faecium, and cephalosporin-resistant Neisseria gonorrhoeae. Following Nano-mupirocin injection to rats, plasma levels greatly exceeded relevant MICs for >24 h, and a biodistribution study in mice showed that mupirocin concentrations in vaginal secretions greatly exceeded the MIC90 for N. gonorrhoeae (0.03 µg/mL) for >24 h. In summary, Nano-mupirocin has excellent potential for treatment of several infection types involving multiresistant bacteria. It has the concomitant benefits from utilizing an established antibiotic and liposomes of the same size and lipid composition as Doxil®, an anticancer drug product now used for the treatment of over 700,000 patients globally.

Dragon Fruits as a Reservoir of Natural Polyphenolics with Chemopreventive Properties.

Dragon fruits are a valued source of bioactive compounds with high potential to become a functional food. The aim of the study was to evaluate and compare the chemopreventive potential and chemical composition of fruits harvested in Thailand and Israel. The amount of different compounds in water and methanol extracts and antioxidant activity was investigated. Moreover, cytotoxic activity against cancer and normal cells of skin, prostate, and gastrointestinal origin was performed, accompanied by anti-inflammatory assay based on NO production in RAW 264.7 macrophage model. Additionally, the quenching properties of polyphenols from fruits were determined by the interaction of the main drug carrier in blood human serum (HSA). The chemometric analysis was used to reveal the relationships between the determined parameters. Dragon fruits harvested in Israel revealed higher antioxidant properties and total content of polyphenols and betacyanins when compared to those from Thailand. The examined fruits of both origins showed significant cytotoxic activity toward colon and prostate cancer cells, with no toxic effect on normal cells, but also no anti-inflammatory effect. Moreover, a high binding ability to HSA was observed for water extracts of dragon fruits. All these predestine dragon fruits are the candidates for the attractive and chemopreventive elements of daily diet.

Prolonged Delivery of Apomorphine Through the Buccal Mucosa, Towards a Noninvasive Sustained Administration Method in Parkinson's Disease: In Vivo Investigations in Pigs.

In the current work, prolonged systemic delivery of apomorphine via buccal mucosa was shown to be a promising treatment for Parkinson's disease as a substitute for clinically utilized subcutaneous infusions. Due to extensive 'first-pass' metabolism, apomorphine is administered parenterally to bypass liver metabolism. Drawbacks of parenteral administration cause low patient compliance and adherence to treatment. On the other hand, while also bypassing the liver, delivery through buccal mucosa has a superior safety profile, is less costly, lacks pain and discomfort, and possesses excellent accessibility, overall augmenting patient compliance. Current in vivo study in pigs showed: (1) steady plateau levels of apomorphine in plasma were obtained 30 min following administration and remained constant for 8 h until a delivery device was removed, (2) bioavailability of apomorphine was 55%-80% as opposed to <2% peroral and (3) simulation of the pharmacokinetic profile obtained in pigs predicted therapeutically relevant levels of apomorphine in human. Furthermore, antipyrine was incorporated as a permeation marker to enable mechanistic investigation of apomorphine release from the delivery device and its permeation through the buccal mucosa. In addition, limitations of an Ussing diffusion chamber as an ex vivo research tool were also discussed.

Saturated and aromatic aldehydes originating from skin and cutaneous bacteria activate the Nrf2-keap1 pathway in human keratinocytes.

Skin homeostasis is constantly challenged by environmental factors, affecting its delicate redox balance. The skin is also home to a wide variety of bacterial species, including Staphylococci. The cutaneous redox state is governed by the Nrf2-keap1 pathway, which is responsible for the induction of phase II cytoprotective enzymes, thus sustaining a healthy oxidative state. As part of normal metabolism, both bacteria and cutaneous tissue emit copious amounts of volatile organic compounds (VOCs), one subgroup of which are aldehydes. α,ß-unsaturated aldehydes are known activators of Nrf2-keap1 pathway by direct oxidation of the keap1 protein. However, we did not encounter reports of Nrf2 activation by saturated or aromatic aldehydes, neither bacteria nor skin-derived. We hypothesized that non-α,ß-unsaturated aldehydes derived from skin or cutaneous bacteria may act as Nrf2-keap1 pathway activators and therefore afford protection against environmental insults. The saturated aldehydes nonanal and decanal (known skin metabolites) and the aromatic aldehyde benzaldehyde (known skin and Staphylococcus epidermidis metabolite) were shown to induce the Nrf2-keap1 pathway in human keratinocytes. We also identified a newly described aromatic aldehyde, 3-furaldehyde (3-FA), emitted from S. aureus and S. epidermidis cultures, which also activated the pathway. Moreover, Nrf2-keap1 induction led to a significant protection against UVB-induced apoptosis. The mechanism involved in this activation has been partially elucidated. This work emphasizes the importance of cutaneous bacteria, as well as healthy skin lipid peroxidation processes in the maintenance and regulation of the cellular antioxidant response, namely with regard to coping with environmental stressors.

Pharmacokinetic investigation of synthetic cannabidiol oral formulations in healthy volunteers.

Recent advances in the research of medicinal cannabis has placed the non-intoxicating cannabinoid cannabidiol (CBD) at the front of scientific research. The reasons behind this popularity is the compound's therapeutic properties, alongside a safe profile of administration lacking addictive properties such as euphoric state of mind and a wide dosing range. Oral administration of CBD is challenging due to poor solubility in the gastro-intestinal system and susceptibility to extensive first pass metabolism. As a result, the practice in clinic and investigational trials is to administer cannabinoids in edible oils or oil-based solutions. Nonetheless, reported pharmacokinetics of cannabinoids and CBD in particular are not uniform among research groups and are affected by the vehicle of administration. The purpose of the work presented here is to investigate oral absorption processes of synthetic CBD when given in different oral formulations in healthy volunteers. The study design was a three way, blind, cross-over single administration study of 12 healthy male volunteers. CBD was administered in powder form, dissolved in sesame oil and in self-nano-emulsifying drug delivery system (SNEDDS). Administration of CBD in lipid-based vehicles resulted in a significant increase in Cmax and AUC of CBD, as compared to powder form. Overall plasma exposure of CBD did not differ between sesame oil vehicle and the SNEDDS formulation. However, administration of CBD in pure oil resulted in two absorption behaviors of early and delayed absorption among subjects, as opposed to SNEDDS platform that resulted in a uniform early absorption profile. Results of this trial demonstrate the importance of solubilization process of lipophilic drugs such as CBD and demonstrated the ability of the nano formulation to achieve a reliable, predictable PK profile of the drug. These findings offer a standardized oral formulation for the delivery of cannabinoids and contribute data for the growing field of cannabinoid pharmacokinetics.

Prolonged oral transmucosal delivery of highly lipophilic drug cannabidiol.

Delivery of drugs through oral mucosa enables bypass of the gastrointestinal tract and "first pass" metabolism in the liver and the gut. Thus, a higher and less variable bioavailability can be obtained. Mechanisms of this administration route for cannabidiol were investigated in the current research in pigs. Results show that cannabidiol has substantially low permeability rate over 8 h through oral mucosa and accumulates significantly within it. Furthermore, following the removal of the delivery device, residual prolongation of release from the oral mucosa into systemic blood circulation continues for several hours. This method of delivery enabled acquisition of clinically relevant plasma levels of cannabidiol. The absorption profile indicates that cannabidiol, as well as other lipophilic molecules, should be delivered through oral mucosa for systemic absorption from a device that conceals the drug and prevents its washout by the saliva flow and subsequent ingestion into gastrointestinal tract.

Nanocapsules embedded in microparticles for enhanced oral bioavailability and efficacy of Lopinavir as an anti-AIDS drug.

Lopinavir (LPV), an efficient drug for HIV infection treatment, was incorporated into biodegradable PLGA nanocapsules (NCs) embedded in microparticles (MCPs) using the spray-drying technique in an attempt to bypass the P-gp efflux and protect the drug from CYP3A pre-systemic metabolism without ritonavir (RTV). SEM observations confirmed the formation of NCs and their entrapment in the MCPs. LPV-loaded NCs and free LPV were released from the MCPs at pH of 7.4 as evidenced by in vitro release studies. Results obtained from rat studies showed a two-fold higher bioavailability of LPV following oral administration of the optimal formulation than Kaletra®, the marketed drug, showing that when properly entrapped, LPV can be effectively protected from CYP degradation in the gut as well as from the liver following systemic absorption. It was also shown that serum derived from rats following LPV oral administration in two formulations and Kaletra® significantly decreased the multiplication of HIV-1 in cultured SupT1 cells. Furthermore, the LPV formulations markedly restricted the titre of infectious HIV-1 production compared with Kaletra® confirming the improved antiviral activity of LPV delivered in the rat blood circulation by the nanocapsules embedded in microparticle formulations.

Piperine-pro-nanolipospheres as a novel oral delivery system of cannabinoids: Pharmacokinetic evaluation in healthy volunteers in comparison to buccal spray administration.

Nowadays, therapeutic indications for cannabinoids, specifically Δ9-tetrahydrocannabinol (THC) and Cannabidiol (CBD) are widening. However, the oral consumption of the molecules is very limited due to their highly lipophilic nature that leads to poor solubility at the aqueous environment. Additionally, THC and CBD are prone to extensive first pass mechanisms. These absorption obstacles render the molecules with low and variable oral bioavailability. To overcome these limitations we designed and developed the advanced pro-nanolipospheres (PNL) formulation. The PNL delivery system is comprised of a medium chain triglyceride, surfactants, a co-solvent and the unique addition of a natural absorption enhancer: piperine. Piperine was selected due to its distinctive inhibitory properties affecting both Phase I and Phase II metabolism. This constellation self emulsifies into nano particles that entrap the cannabinoids and the piperine in their core and thus improve their solubility while piperine and the other PNL excipients inhibit their intestinal metabolism. Another clear advantage of the formulation is that its composition of materials is approved for human consumption. The safe nature of the excipients enabled their direct evaluation in humans. In order to evaluate the pharmacokinetic profile of the THC-CBD-piperine-PNL formulation, a two-way crossover, single administration clinical study was conducted. The trial comprised of 9 healthy volunteers under fasted conditions. Each subject received a THC-CBD (10.8mg, 10mg respectively) piperine (20mg)-PNL filled capsule and an equivalent dose of the oromucosal spray Sativex® with a washout period in between treatments. Single oral administration of the piperine-PNL formulation resulted in a 3-fold increase in Cmax and a 1.5-fold increase in AUC for THC when compared to Sativex®. For CBD, a 4-fold increase in Cmax and a 2.2-fold increase in AUC was observed. These findings demonstrate the potential this formulation has in serving as a standardized oral cannabinoid formulation. Moreover, the concept of improving oral bioavailability described here, can pave the way for other potential lipophilic active compounds requiring enhancement of their oral bioavailability.

Bioactivity and nutritional properties of hardy kiwi fruit Actinidia arguta in comparison with Actinidia deliciosa 'Hayward' and Actinidia eriantha 'Bidan'.

The aim of this research is to identify and compare the bioactive compounds, antioxidant capacities and binding potentials to human protein in different varieties of hardy kiwi (Actinidia (A.) arguta), 'Hayward' (Actinidia deliciosa) and less - known 'Bidan' (Actinidia eriantha). Polyphenols, flavonoids, flavanols, tannins, vitamin C, lutein, zeaxanthin and dietary fibers were significantly higher in cultivar 'M1' among the A. arguta than in 'Hayward'. The binding properties of studied kiwi fruits were determined by interaction of polyphenols with human serum albumin (HSA). An internal standard FTIR technique allowed the quantitative comparison of specific IR absorption bands (Amides I, II, III) of different kiwi fruit samples after interaction with HSA. It was shown that the antioxidant and binding capacities and FTIR quantitative estimations of A. arguta fruits were significantly higher than in 'Hayward', but lower than the 'Bidan'. In MS spectra were found some slight differences in A. arguta kiwis in comparison with 'Hayward' and 'Bidan'. Two A. arguta cultivars were similar to 'Bidan'. The interaction of polyphenols with HSA, evaluated by fluorometry/FTIR, made it possible to compare the bioactivity of different cultivars and families. In conclusion, for the first time fruits A. arguta, cultivated in Poland, were compared with widely consumed kiwi fruits, using advanced analytical methods. The high bioactivity and nutritional value of A. arguta fruits from Polish ecological plantation enables us to recommend them for marketing and consumption.

Rapana venosa consumption improves the lipid profiles and antioxidant capacities in serum of rats fed an atherogenic diet.

In the recent years, the consumption of seafood has increased. There are no results on the studies of Rapana venosa (Rv) as a supplementation to the diets. We hypothesized that Rv would increase antioxidant capacity and reduce blood lipids, based on the composition of bioactive compounds and fatty acids. Therefore, the aim of this investigation was to evaluate in vitro and in vivo actions of Rv from contaminated (C) and non-C (NC) regions of collection on lipid profiles, antioxidant capacity, and enzyme activities in serum of rats fed an atherogenic diet. Twenty-four male Wistar rats were divided into 4 groups of 6 each and named control, cholesterol (Chol), Chol/RvC and Chol/RvNC. Rats of all 4 groups were fed the basal diet, which included wheat starch, casein, soybean oil, cellulose, vitamin (American Institute of Nutrition for laboratory animals vitamin mixtures), and mineral mixtures (American Institute of Nutrition for laboratory animals mineral mixtures). During 28 days of the experiment, the rats of the control group received the basal diet only, and the diets of the other 3 groups were supplemented with 1% of Chol, 1% of Chol, and 5% of Rv dry matter from C and NC areas. Dry matter from C and NC areas supplemented diets slightly hindered the rise in serum lipids vs. Chol group: total Chol, 13.18% and 11.63% and low-density lipoprotein Chol, 13.57% and 15.08%, respectively. Cholesterol significantly decreased the value of total antioxidant capacity. The supplementation of Rv to the Chol diet significantly affected the increase of antioxidant capacity in serum of rats, expressed by the 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) method. The water extracts of Rv exhibited high binding properties with bovine serum albumin in comparison with quercetin. In conclusion, atherogenic diets supplemented with Rv from C and NC areas hindered both the rise in serum lipids levels and the decrease in the antioxidant capacity. Based on fluorescence and electrospray-ionization mass spectrometry profiles and in vivo studies, changes in the intensity of the found peaks were estimated in the serum samples after supplemented diets. These findings indicate that the supplementation of Rv to the atherogenic diets improve the lipid profiles and the antioxidant status in serum of rats.

Glycolysis-mediated changes in acetyl-CoA and histone acetylation control the early differentiation of embryonic stem cells.

Loss of pluripotency is a gradual event whose initiating factors are largely unknown. Here we report the earliest metabolic changes induced during the first hours of differentiation. High-resolution NMR identified 44 metabolites and a distinct metabolic transition occurring during early differentiation. Metabolic and transcriptional analyses showed that pluripotent cells produced acetyl-CoA through glycolysis and rapidly lost this function during differentiation. Importantly, modulation of glycolysis blocked histone deacetylation and differentiation in human and mouse embryonic stem cells. Acetate, a precursor of acetyl-CoA, delayed differentiation and blocked early histone deacetylation in a dose-dependent manner. Inhibitors upstream of acetyl-CoA caused differentiation of pluripotent cells, while those downstream delayed differentiation. Our results show a metabolic switch causing a loss of histone acetylation and pluripotent state during the first hours of differentiation. Our data highlight the important role metabolism plays in pluripotency and suggest that a glycolytic switch controlling histone acetylation can release stem cells from pluripotency.

Levels of sirolimus in saliva and blood following oral topical sustained-release varnish delivery system application.

PURPOSE: Sirolimus (rapamycin) is a mammalian target of rapamycin pathway blocker. The efficacy of sirolimus is currently studied for its antiproliferative properties in various malignancies and particularly in squamous cell carcinoma and other oral disorders. Topical application at the oral cavity can augment sirolimus availability at the site of action by increasing sirolimus levels in saliva and hence efficacy, along with improved safety (low levels in the blood to avoid side effects) and compliance. Our purpose was to evaluate the release profile and safety of a topical sirolimus sustained-release varnish drug delivery system. SUBJECTS AND METHODS: Sirolimus sustained-release varnish drug delivery system containing a total of 0.5 mg of the drug was applied to nine healthy male volunteers. Saliva and blood levels were determined utilizing mass spectrometry and chemiluminescent microparticle immunoassay, respectively. The prolonged release profile and safety were evaluated for the oral topical delivery system. RESULTS: After the application of the drug delivery system, a sustained-release profile was observed in the oral cavity. We have measured moderate sirolimus levels for up to 12 h. The safety was confirmed, and systemic sirolimus blood levels were negligible. CONCLUSIONS: After an application of sirolimus sustained-release varnish drug delivery system, prolonged drug levels can be achieved in the saliva. The oral topical sirolimus concentrations were potentially therapeutic along with minimal systemic exposure. These results broaden the potential clinical use of sustained-release oral topical rapalogs.

Dose-limiting inhibition of acetylcholinesterase by ladostigil results from the rapid formation and fast hydrolysis of the drug-enzyme complex formed by its major metabolite, R-MCPAI.

Ladostigil is a pseudo reversible inhibitor of acetylcholinesterase (AChE) that differs from other carbamates in that the maximal enzyme inhibition obtainable does not exceed 50-55%. This could explain the low incidence of cholinergic adverse effects induced by ladostigil in rats and human subjects. The major metabolite, R-MCPAI is believed to be responsible for AChE inhibition by ladostigil in vivo. Therefore we determined whether the ceiling in AChE inhibition resulted from a limit in the metabolism of ladostigil to R-MCPAI by liver microsomal enzymes, or from the kinetics of enzyme inhibition by R-MCPAI. Ladostigil reduces TNF-α in lipopolysaccharide-activated microglia. In vivo, it may also reduce pro-inflammatory cytokines by inhibiting AChE and increasing the action of ACh on macrophages and splenic lymphocytes. We also assessed the contribution of AChE inhibition in the spleen of LPS-injected mice to the anti-inflammatory effect of ladostigil. As in other species, AChE inhibition by ladostigil in spleen, brain and plasma did not exceed 50-55%. Since levels of R-MCPAI increased with increasing doses of ladostigil we concluded that there was no dose or rate limitation of metabolism. The kinetics of enzyme inhibition by R-MCPAI are characterized by a rapid formation of the drug-enzyme complex and fast hydrolysis which limits the attainable degree of AChE inhibition. Ladostigil and its metabolites (1-100 nM) decreased TNF-α in lipopolysaccharide-activated macrophages. Ladostigil (5 and 10mg/kg) also reduced TNF-α in the spleen after injection of lipopolysaccharide in mice. However, AChE inhibition contributed to the anti-inflammatory effect only at a dose of 10mg/kg.