Ramakrishna Samala

Anticonvulsant profile of a balanced ketogenic diet in acute mouse seizure models

Anticonvulsant effects of the ketogenic diet (KD) have been reported in the mouse, although previous studies did not control for intake of vitamins, minerals and antioxidants. The aim of this study was to examine the effects of balanced ketogenic and control diets in acute mouse seizure models. The behavior in four mouse seizure models, plasma d-beta-hydroxybutyrate (d-BHB) and glucose levels were determined after feeding control diet, 4:1 and 6:1 KDs with matched vitamins, minerals and antioxidants. Feeding 4:1 and 6:1 KDs ad lib to 3-week-old (adolescent) mice resulted in 1.2-2.2mM d-BHB in plasma, but did not consistently change glucose levels. The 6:1 KD reproducibly elevated the CC50 (current that initiates seizures in 50% mice tested) in the 6-Hz model after 14 days of feeding to adolescent CD1 mice. Higher plasma d-BHB levels correlated with anticonvulsant effects. Despite ketosis, no consistent anticonvulsant effects of KDs were found in the fluorothyl or pentylenetetrazole CD1 mouse models. The 4:1 KD was neither anticonvulsant nor neuroprotective in hippocampus in the C3H mouse kainate model. Taken together, the KDs anticonvulsant effect was limited to the 6-Hz model, required chronic feeding with 6:1 fat content, and was independent from lowering plasma glucose.

Eicosapentaenoic and docosahexaenoic acids are not anticonvulsant or neuroprotective in acute mouse seizure models

Docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or vegetable oil (control) were added to standard rodent chow (6 g/kg) and fed to mice ad lib for 4 weeks to determine if polyunsaturated fatty acids (PUFA) are anticonvulsant or neuroprotective in mice. The seizure susceptibility of these mice was compared using the fluorothyl, pentylenetetrazole (PTZ), 6 Hz, and kainate models. We found that PUFA feeding significantly altered the fatty acid profile in both plasma and brain, but did not change seizure thresholds in the fluorothyl, PTZ, or 6 Hz models nor did it significantly alter seizure behavior or hippocampal damage following kainate injection. In conclusion, DHA or EPA feeding did not show anticonvulsant or neuroprotective activity in four acute seizure models. Chronic seizure models remain to be examined.

β-Hydroxybutyric acid grafted solid lipid nanoparticles: A novel strategy to improve drug delivery to brain

UNLABELLED Delivery of drugs to brain is an elusive task in the therapy of many serious neurological diseases. With the aim to create a novel formulation to enhance the drug uptake to brain, betreliesoxybutyric acid (HBA) grafted docetaxel loaded solid lipid nanoparticles (HD-SLNs) were explored. Transportation of HD-SLNs relies on the transport of novel ligand, HBA, by monocarboxylic acid transporter (MCT1). Expression of MCT1 transporter on brain endothelial cells (bEnd cells) was studied using immunocytochemistry. Stearylamine-HBA conjugate was used to modify the surface of SLNs and it was confirmed using XPS (X-Ray Photon Spectroscopy) analysis. In vitro release studies revealed the controlled release of drug from HD-SLNs. Cytotoxicity and cell uptake studies revealed the increased uptake of docetaxel with HD-SLNs. Mechanism involved in the uptake of HD-SLNs was studied in bEnd cells by saturating MCT1 with excess HBA. Pharmacokinetic and brain distribution demonstrated increased docetaxel concentrations in brain compared with Taxotere®. FROM THE CLINICAL EDITOR The authors of this study demonstrate enhanced drug delivery to the brain using a novel formulation of beta-hydroxybutyric acid grafted docetaxel loaded solid lipid nanoparticles. The results show increased uptake of docetaxel compared with Taxotere.

The ketogenic diet changes metabolite levels in hippocampal extracellular fluid

Despite successful use of the ketogenic diet (KD) for the treatment of drug-resistant epilepsy, its mechanism of action is unclear. After KD-feeding, increased plasma D-beta-hydroxybutyrate (BHB) levels appear to be important for protection against seizures. We hypothesized that the KD leads to metabolic changes in the brain, which are reflected in the hippocampal extracellular fluid (hECF). CD1 mice were fed control or KD for 2-3 weeks since weaning. In vivo microdialysis of hECF was used to measure the levels of glucose, lactate, as well as BHB under basal conditions and during 30 min stimulation with 60 mM K(+), which was retrodialysed. The hECF BHB concentration in KD-fed mice was determined as 43.4±10.1 μM using the zero-flow method and 50.7±5.5 μM based on in vitro recovery. The total BHB concentration in brain homogenate from KD-fed mice was 180 nmol/g. The intracellular BHB concentration is therefore estimated to be about 3-fold higher than the extracellular level, which suggests that BHB in adolescent mouse brains may not be quickly metabolized. The basal hECF glucose concentration was 30% lower in KD-fed mice, indicating that glucose may be less important as an energy source. Lactate levels were similar in control and KD-fed mice. High potassium stimulation elevated lactate by 3-3.5-fold and decreased glucose by 40-50% in both diet groups, consistent with similar anaerobic and aerobic metabolism in both diet groups during high hippocampal activity. Overall, these data (1) defined the BHB concentration in the hippocampal extracellular fluid in KD-fed mice and (2) showed lower glucose metabolism compared to control diet-fed mice. This work will now enable other researchers to mimic the hippocampal extracellular environment in experiments aimed at deciphering the mechanisms of the KD.

Vinorelbine Delivery and Efficacy in the MDA-MB-231BR Preclinical Model of Brain Metastases of Breast Cancer.

PurposeTo evaluate vinorelbine drug exposure and activity in brain metastases of the human MDA-MB-231BR breast cancer model using integrated imaging and analysis.MethodsBrain and systemic metastases were created by administration of cancer cells in female NuNu mice. After metastases developed, animals were administered vinorelbine at the maximal tolerated dose (12 mg/kg), and were evaluated thereafter for total and unbound drug pharmacokinetics, biomarker TUNEL staining, and barrier permeability to Texas red.ResultsMedian brain metastasis drug exposure was 4-fold greater than normal brain, yet only ~8% of non-barrier systemic metastases, which suggests restricted brain exposure. Unbound vinorelbine tissue/plasma partition coefficient, Kp,uu, equaled ~1.0 in systemic metastases, but 0.03–0.22 in brain metastases, documenting restricted equilibration. In select sub-regions of highest drug-uptake brain metastases, Kp,uu approached 1.0, indicating complete focal barrier breakdown. Most vinorelbine-treated brain metastases exhibited little or no positive early apoptosis TUNEL staining in vivo. The in vivo unbound vinorelbine IC50 for TUNEL-positive staining (56 nM) was 4-fold higher than that measured in vitro (14 nM). Consistent with this finding, P-glycoprotein expression was observed to be substantially upregulated in brain metastasis cells in vivo.ConclusionsVinorelbine exposure at maximum tolerated dose was less than one-tenth that in systemic metastases in >70% of brain metastases, and was associated with negligible biomarker effect. In small subregions of the highest uptake brain metastases, compromise of blood-tumor barrier appeared complete. The results suggest that restricted delivery accounts for 80% of the compromise in drug efficacy for vinorelbine against this model.

Highly sensitive LC–MS/MS-ESI method for determination of phenelzine in human plasma and its application to a human pharmacokinetic study

A selective, sensitive and rapid LC-MS/MS method has been developed and validated for quantification of the phenelzine (PZ) in 200μL of human plasma using hydroxyzine (HZ) as an internal standard (IS) as per regulatory guidelines. The sample preparation involved the derivatization of PZ using pentaflurobenzaldehyde followed by solid phase extraction process to extract PZ and HZ from human plasma. LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electro spray ionization technique in positive ion mode and the transitions of m/z 305.1→105.1 and m/z 375.3→201.1 were used to measure the derivative of PZ and IS, respectively. The total run time was 3.5min and the elution of PZ and HZ occurred at 2.53, and 1.92min, respectively; this was achieved with a mobile phase consisting of 10mM ammonium acetate: acetonitrile (20:80, v/v) at a flow rate of 1.0mL/min on an Ace C18 column with a split ratio of 70:30. The developed method was validated in human plasma with a lower limit of quantitation 0.51ng/mL. A linear response function was established for the range of concentrations 0.51-25.2ng/mL (r>0.995) for PZ. The intra- and inter-day precision values met the acceptance criteria. PZ was stable in the battery of stability studies viz., stock solution, bench-top, auto-sampler, long-term and freeze/thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.

In Situ and In Vivo Animal Models

An important property in any central nervous system drug is the ability to cross the blood–brain barrier (BBB) and to reach therapeutic concentrations in brain at safe and acceptable doses. Multiple parameters influence brain drug bioavailability, including solubility, membrane permeation, and affinity for influx and efflux transporters. This chapter overviews the primary in vivo methods to assess brain drug distribution in preclinical and clinical reports. In most studies, two parameters, the BBB permeability-surface area product (PS) and brain distribution volume or partition coefficient (K p,brain), are used to characterize the ability of a drug compound to gain access to and distribute in brain. Together with the time course of systemic drug exposure, these two parameters can be used to predict total drug concentration within brain. Further, because unbound drug concentration often correlates better with drug activity, a number of studies also determine the drug free fraction (f u) so that the free concentrations can be calculated. Specific methods, such as in situ brain perfusion, brain efflux index, and brain microdialysis, are valuable to dissect specific elements of BBB drug permeation or transport as well as equilibration in brain interstitial fluid and cellular elements. Overall, these approaches complement in vivo drug distribution studies and in vitro BBB permeation methods.

One-pot Synthesis and Antitumor Activity of Unsymmetrical Terphenyls.

In this paper a simple and efficient method for the unsymmetrical terphenyls via sequential one-pot Suzuki coupling reactions using Pd(OAc)2 without isolation of the intermediate is described. The prepared terphenyls were found to possess potent anticancer properties against a panel of cancer cells which includes A549, HeLa, MCF7, DU145, HT29 and BxPC-3. Structural similarity with combretastatin A4, these terphenyls disrupted the tubulin polymerization in vitro and destabilized the microtubules in cells. Flow cytometry studies indicated growth arrest of cells in the G2/M phase of the cell cycle corresponding to antimitotic action. Furthermore, compound 4c showed potent anti-mitotic activity even in zebrafish model and could likely be a potential therapeutic compound as it is active both in in vitro and in vivo.