TP300 is a novel topoisomerase I inhibitor with neutropenia as a significant toxicity. We developed and evaluated a pharmacokinetic–pharmacodynamic (PK-PD) model, using data from Phase I and II trials to predict neutrophil decrease in patients treated with TP300.

Plasma drug concentrations of TP300, its active form TP3076 and active metabolite TP3011 and absolute neutrophil counts (ANCs) from a Phase I trial were analysed as a training dataset. A two-plus-two-compartment model was applied to the pharmacokinetics of TP3076 and TP3011. A semi-mechanistic model was used to describe the PK-PD relationship between the plasma concentration of TP3076 and TP3011, and changes in ANC.

 The model fitted well to plasma concentrations of TP3076 and TP3011. Model appropriateness was confirmed in a Phase II trial validation dataset. Body weight and liver biochemistry values were identified as covariates. A semi-mechanistic PK-PD model was applied and the longitudinal decrease in ANC was simulated. Neutrophil counts reached their nadir approximately 2 weeks after administration of TP300, and the proportion of subjects affected increased with dose.

This PK-PD model to predict neutropenia following treatment with TP300 fitted well the decrease in ANC with total concentration of TP3076 and TP3011.

Management of blood coagulation-related diseases is currently limited by the inability to provide an adequate drug concentration in blood circulation for a long term. As a promising way to overcome this problem, the long-acting forms of these drugs have attracted many interests in recent years.

In this study, chitosan-heparin nanoparticles were prepared as a polymeric delivery system intended for the prolonged intravenous delivery of heparin where the drug was used as both the therapeutic agent and a gel-forming counter-ion. The nanoparticle preparation method was optimized using a Taguchi orthogonal array. Critical formulation variables were optimized in this study in terms of their corresponding effects on the target response of particle size. Nanoparticles were characterized by the Fourier transform infrared spectroscopy, transmission electron microscopy and zeta potential.

The size, polydispersity index, zeta potential and encapsulation efficiency for the optimized formulation were found to be 61.33 ± 1.53 nm, 0.06, +15.7 mv and 74.16 ± 1.27%, respectively. The sizes of the prepared drug-loaded nanoparticles were stable at least 1 week at room temperature and 3 months in refrigerator.

The ex-vivo and in-vivo tests on the heparin-chitosan nanoparticles using activated partial thromboplastin time (aPTT) as the biological index were indicative of a smoother and longer elevation in aPTT in the presence of nanoparticulate drug.

The chemical study of a dichloromethane extract from Azorella compacta was directed to the isolation of characteristic mulinane and azorellane diterpenoids in order to determine their gastroprotective activity.

Usual chromatographic techniques on the extract led to the isolation of 12 compounds, which were identified by their spectroscopic properties. The HCl/ethanol-induced gastric lesions model in mice was used to determine the gastroprotective activity.

The new diterpenoids, 13β-hydroxymulinane (1), mulin-11,13-dien-20-ol (2), 13α-methoxyazorellanol (3) and mulin-11,13-dien-18-acetoxy-16,20-dioic acid (12) were isolated from A. compacta. The known diterpenoids mulin-11,13-dien-20-oic acid (4), 13α-hydroxyazorellane (5), 13β-hydroxyazorellane (6), mulinic acid (7), mulinolic acid (8) and azorellanol (9), and the aromatic compounds 5,7-dihydroxychromone (10) and isoflavonoid biochanin A (11), were also obtained from the extract. Compounds 6, 9 and 12 at 20 mg/kg reduced gastric lesions by 69%, 71% and 73%, respectively, being statistically similar to lansoprazole at the same dose.

The results corroborate the intraspecific chemical variations detected previously in specimens of A. compacta collected at different Chilean latitudes. A high concentration of azorellanol (9) could account in part for some of the therapeutic properties attributed to this species, in particular in ulcer treatment. Most of the mulinane and azorellane diterpenoids isolated in this study showed relevant gastroprotective activity at a low dose in the bioassay.

Whether and how synthetic cannabinoids affect inflammation and carcinogenesis has not been well studied. The present study was thus conducted to assess effects of synthetic cannabinoids on inflammation and carcinogenesis in vivo in mice.

Twenty-three analogues of synthetic cannabinoids were isolated from, and identified as adulterants in, illegal drugs distributed in the Tokyo metropolitan area, and were examined for their inhibitory effects on the induction of oedema in mouse ears by 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, selected cannabinoids, JWH-018, -122 and -210, were studied for their effects on carcinogenesis induced in mouse skin initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by TPA.

Among cannabinoids, naphthoylindoles mostly exhibited superior inhibitory effects against TPA-induced ear oedema and, especially, JWH-018, -122 and -210 showed potent activity with 50% inhibitory dose (ID50) values of 168, 346 and 542 nm, respectively (an activity corresponding to that of indometacin (ID50 = 908 nm)). Furthermore these three compounds also markedly suppressed the tumour-promoting activity of TPA.

This is the first report indicating the structure–activity relationships for the anti-inflammatory activity of synthetic cannabinoids on TPA-induced inflammation in mice. Naphthoylindoles, JWH-018, -122 and -210, had the most potent anti-inflammatory activity and also markedly inhibited tumour promotion by TPA in the two-stage mouse skin carcinogenesis model. The present results suggest that synthetic cannabinoids, such as JWH-018, -122 and -210, may be used as cancer chemopreventive agents in the future.

Due to the low stability of lipid emulsions, a lipid emulsion of prostaglandin E₁ (Lipo-PGE₁) necessitates daily intravenous drip infusions. To overcome this issue, we developed nanoparticles containing PGE₁ (Nano-PGE₁). Nano-PGE₁ showed a good sustained-release profile of PGE₁ from the nanoparticles in vitro, which may permit a longer-lasting therapeutic effect to be achieved. We here examined the pharmacological activity of Nano-PGE₁ in a rat experimental model of intermittent claudication induced by femoral artery ligation.

The walking activity of the rat was tested on a rodent treadmill. Tissue levels of PGE₁ were determined by enzyme immunoassay, and skeletal muscle angiogenesis (capillary growth) was monitored by immunohistochemical analysis.

PGE₁ could be detected in the lesion site one day after the intravenous administration of Nano-PGE₁ but not of Lipo-PGE₁. An increased accumulation of Nano-PGE₁ in the lesion site compared with control (unlesioned) site was also observed. The ligation procedure reduced the walking activity, which in turn was improved by a single administration of Nano-PGE₁ but not of Lipo-PGE₁. The single administration of Nano-PGE₁ also stimulated angiogenesis in the skeletal muscle around the ligated artery.

The findings of this study suggest that Nano-PGE₁ improves the walking activity of femoral artery-ligated rats through the accumulation and sustained release of PGE₁.

Alzheimer's disease (AD) is the most common cause of dementia, being responsible for high healthcare costs and familial hardships. Despite the efforts of researchers, no treatment able to delay or stop AD progress exists. Currently, the available treatments are only symptomatic, cholinesterase inhibitors being the most widely used drugs. Here we describe several natural compounds with anticholinesterase (acetylcholinesterase and butyrylcholinesterase) activity and also some synthetic compounds whose structures are based on those of natural compounds.

Galantamine and rivastigmine are two cholinesterase inhibitors used in therapeutics: galantamine is a natural alkaloid that was extracted for the first time from Galanthus nivalis L., while rivastigmine is a synthetic alkaloid, the structure of which is modelled on that of natural physostigmine. Alkaloids include a high number of compounds with anticholinesterases activity at the submicromolar range. Quinones and stilbenes are less well studied regarding cholinesterase inhibition, although some of them, such as sargaquinoic acid or (+)-α-viniferin, show promising activity. Among flavonoids, flavones and isoflavones are the most potent compounds. Xanthones and monoterpenes are generally weak cholinesterase inhibitors.

Nature is an almost endless source of bioactive compounds. Several natural compounds have anticholinesterase activity and others can be used as leader compounds for the synthesis of new drugs.

The development of amorphous solid dispersions is of increasing interest in the delivery of bioactive compounds; however, there is a need for a methodology that enables the rational selection of polymers for solid dispersion formulations with optimal stability to crystallization. The objective of this study was to evaluate the use of mid-infrared (IR) spectroscopy for this purpose.

Polymers evaluated included poly(vinylpyrrolidone) (PVP), Eudragit E100 (E100), carboxymethylcellulose acetate butyrate (CMCAB), hydroxypropylmethylcellulose (HPMC), HPMC acetate succinate (HPMCAS) and poly(acrylic acid) (PAA). Model crystalline bioactive polyphenols included quercetin and naringenin. Amorphous solid dispersions were prepared by dissolving both polyphenol and polymer in a common solvent followed by solvent evaporation. Mid-IR spectroscopy was used to determine and quantify the extent of polyphenol–polymer interactions, and powder X-ray diffraction was used to monitor physical stability following storage at different environmental conditions.

The mid-IR analysis suggested the following rank order for the crystallization-inhibiting performance of the different polymers: E100 > PVP > HPMCAS > HPMC ≥ CMCAB > PAA. The initial performance of the different polymers was evaluated using the highest concentration of polyphenol for which x-ray amorphous solid dispersions could be prepared via rotary evaporation. The observed stability followed that predicted from the mid-infrared spectroscopy evaluation of intermolecular interactions. The dispersions with better polyphenol–polymer interactions were stable against crystallization when exposed to high relative humidity and high temperatures; on the other hand, systems that had weak interactions were not stable to crystallization when stored at moderate environmental conditions.

Based on the observed ability of mid-IR analysis to enable the characterization of intermolecular polyphenol–polymer interactions and based on the correlation between the extent of intermolecular interactions and the crystallization-inhibiting performance of polymers, it can be concluded that this technique is an important tool for the rational formulation of solid dispersions with optimized physical stability.

The lipid excipients of the self-emulsifying drug delivery systems (SEDDS) could play a role in interfering with the drug precipitation to maintain its supersaturation, a step with possible major significance on the SEDDS. Thus, the effect of lipid chain length on indomethacin precipitation rate from SEDDS upon dilution was studied.

Indomethacin SEDDS were prepared using medium and long chain lipids at 5% and 13% (w/w) drug load. Two medium chain lipids Lauroglycol and Capryol, and two long chain lipids Labrafil and castor oil, were studied. The 13% w/w SEDDS were evaluated for drug release, and the physicochemical properties of the precipitated drug were characterized by PXRD, DSC, IR and Raman.

The final optimized SEDDS consisted of Lauroglycol (lipid): Transcutol (co-solvent): Labrasol (surfactant). No precipitate was observed with long chain lipids SEDDS, whereas medium chain lipids SEDDS showed precipitation within 30 min of drug release from 13% w/w formulations. Precipitation studies showed that the medium chain lipids resulted in a modified indomethacin form possibly an ester. The ester formation signifies the interaction between indomethacin and medium chain length lipids.

The study emphasizes the importance of lipids chain length of excipients in successful SEDDS formulations. The study provides insight into the underlying drug lipid interactions in SEDDS formulations.

The aim of this study was to investigate the in-vitro effect of rutin on glucose uptake in an insulin target (soleus muscle) and the mechanism of action involved.

Isolated soleus muscles from rats were treated with rutin (500 μm) with or without the following inhibitors; hydroxy-2-naphthalenylmethylphosphonic acid trisacetoxymethyl ester (HNMPA(AM)₃), an insulin receptor tyrosine kinase activity inhibitor, wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), RO318220, an inhibitor of protein kinase C, colchicine, a microtubule-depolymerizing agent, PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK), and cycloheximide, an inhibitor of protein synthesis on fresh Krebs Ringer-bicarbonate plus [U-¹⁴C]-2-deoxy-d-glucose (0.1 μCi/ml). Samples of tissue medium were used for the radioactivity measurements.

Rutin increased the glucose uptake in rat soleus muscle. In addition, the effect of rutin on glucose uptake was completely inhibited by pretreatment with HNMPA(AM)₃, wortmannin, RO318220, colchicine, PD98059, and cycloheximide. These results suggested that rutin stimulated glucose uptake in the rat soleus muscle via the PI3K, atypical protein kinase C and mitogen-activated protein kinase (MAPK) pathways. Also, rutin may have influenced glucose transporter translocation and may have directly activated the synthesis of the transporter GLUT-4.

The similarities of rutin action on glucose uptake compared with the signalling pathways of insulin constitute strong evidence for the insulin-mimetic role of rutin in glucose homeostasis.

The aim of this study was to investigate Pluronic F127-modified liposome-containing cyclodextrin (CD) inclusion complex (FLIC) for improving the solubility, cellular uptake and intestinal penetration of tacrolimus (FK 506) in the gastrointestinal (GI) tract.

Molecular modelling was performed to screen the optimal CD for the solubilization of FK 506. FLIC was prepared by thin-lipid film hydration with the inclusion complex solutions followed by membrane extrusion. Dilution tests were conducted in simulated gastric fluids and phosphate-buffered solution of sodium taurocholate to investigate the solubility improvement of FK506. The cellular uptake of nanocarriers was studied in Caco-2 cells, and intestinal mucous membrane penetration in the GI tract was evaluated in Sprague–Dawley rats.

The results showed that β-CD had the strongest binding energy with the guest molecule among the CDs. The prepared FLIC has an average diameter of 180.8 ± 8.1 nm with a spherical shape. The solubility and cellular uptake of FK 506 was greatly improved by the incorporation of CD complexes in the Pluronic F127-modified liposomes. Intestinal mucous membrane penetration was also significantly improved by the preparation of FLIC.

With improved drug solubility and intestinal mucous membrane penetration, FLIC shows a promising oral delivery system for FK 506.

Vesicare tablets, whose main component is solifenacin succinate, are known to be extremely bitter. The purpose of this study was to evaluate the effect of crushing on the bitterness of the Vesicare orally disintegrating tablets (ODTs).

Vesicare ODTs and conventional Vesicare tablets (CTs) were crushed either heavily or lightly. The bitterness scores and release rates of sample solutions obtained 5, 10, 30, 60, 90 or 120 s after placing a crushed CT or ODT containing 5 mg solifenacin in 40 ml of water were predicted using two taste sensors (SA402B and a-ASTREE) and HPLC, respectively. The particle size and the surfaces of the crushed tablets were observed microscopically.

The predicted bitterness scores and the drug release rates of CTs were high, irrespective of the degree of crushing. The lightly crushed ODT was predicted to be less bitter than the heavily crushed ODT. In lightly crushed ODTs, spherical particles were observed, about 200 mm in diameter.

The degree of crushing was a critical factor in determining the expression of bitterness by crushed ODTs. When intact tablets of Vesicare must be crushed to adjust the dosage, it is strongly recommended that ODTs be crushed gently.

The objective of the present investigation was to study the effect of pure gallic acid (GA) and its Tween 80 coated chitosan nanoparticles (cGANP) on the maximum tolerated dose (MTD) using Wistar rats.

The animals were administered with increasing doses (dose increasing rate = 10%) of GA and cGANP orally for 28 consecutive days until the MTD was found. The animals were monitored for bodyweight, weight indices, behavioural, biochemical and histopathological changes.

The MTD was found to be 750 mg/kg for GA and was increased to 825 mg/kg for cGANP, which indicated a significantly greater tolerance of cGANP. Slight behavioural and biochemical changes were observed above the MTD. At the MTD no significant behavioural, biochemical, histopathological changes were observed.

The brain targeted nanoparticles displayed considerable improvement in the tolerance profile of the drug as compared with the free drug.

Curculigoside, a natural compound isolated from the medicinal plant Curculigo orchioides has been reported to prevent bone loss in ovariectomized rats. However, the underlying molecular mechanisms are largely unknown. This study investigated the effects of curculigoside on proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs).

The toxicity, proliferation and osteogenic differentiation of BMSCs cultured with various concentrations (0 as control, 10, 100 and 500 µm) of curculigoside were measured by viability assay, MTT analysis, alkaline phosphatase (ALP) activity assay, alizarin red staining and mineralization assay, real-time PCR analysis on osteogenic genes including ALP, type I collagen (Col I), osteocalcin (OCN) and osteoprotegerin (OPG), runt-related transcription factor 2 (Runx2), as well as OPG enzyme-linked immunosorbent assay.

No significant cytotoxicity was observed for BMSCs after supplementation with curculigoside. The proliferation of BMSCs was enhanced after administration of curculigoside, especially 100 µm curculigoside. Moreover, the osteogenic gene expression was significantly enhanced with 100 µm curculigoside treatment. Importantly, curculigoside significantly increased OPG secretion.

The data indicate that curculigoside could promote BMSC proliferation and induce osteogenic differentiation of BMSCs. The most profound response was observed with 100 µm curculigoside. These findings may be valuable for understanding the mechanism of the effect of curculigoside on bone, especially in relation to osteoporosis.

A tacrine-silibinin codrug showed promising results in pharmacological and toxicity testing, superior to an equimolar mixture of tacrine and silibinin. The aim of this study was to get more information about its stability, possible degradation products, metabolites, and especially its active principle in vitro and in vivo.

The stability of the codrug was analysed under in-vitro assay conditions. Additionally, its metabolism was investigated using pooled human liver microsomes. Metabolites were identified via liquid chromatography-high resolution electrospray ionization mass spectrometry. Furthermore, the influence of one of the main cleavage products, tacrine hemi succinamide, on viability and mitochondria of hepatic stellate cells was analysed.

The codrug remained stable in culture medium (Dulbecco's modified Eagle's medium) over an incubation period of 24 h, whereas exposition to microsomal enzymes led to rapid cleavage of the ester bond to form silibinin and a tacrine hemi succinamide. In addition, glucuronidated metabolites of both silibinin and the codrug were detected. For the tacrine hemi succinamide, no effects were observed with regard to cell viability and mitochondrial impairment.

This study helps understand and interpret previous results concerning the effects and the absence of toxicity of the tacrine–silibinin codrug and supplies important information for further identification of the active principles of the codrug in vivo.

Mango (Mangifera indica L.) stem bark extract (MSBE) is a natural product with biological properties and mangiferin is the major component. This paper reported the evaluation of the protective effects of MSBE and mangiferin against the toxicity induced in HepG2 cells by tert-butyl hydroperoxide or amiodarone.

Nuclear morphology, cell viability, intracellular calcium concentration and reactive oxygen species (ROS) production were measured by using a high-content screening multiparametric assay.

MSBE and mangiferin produced no toxicity below 500 mg/ml doses. A marked recovery in cell viability, which was reduced by the toxicants, was observed in cells pre-exposed to MSBE or mangiferin at 5–100 mg/ml doses. We also explored the possible interaction of both products over P-glycoprotein (P-gp). MSBE and mangiferin above 100 mg/ml inhibited the activity of P-gp in HepG2 cells.

MSBE and mangiferin showed cytoprotective effects of against oxidative damage and mitochondrial toxicity induced by xenobiotics to human hepatic cells but it seemed that other constituents of the extract could contribute to MSBE protective properties. In addition, the drug efflux should be taken into account because of the inhibition of the P-gp function observed in those cells exposed to both natural products.

Suramin is a polysulfonated naphthylurea with antiparasitic and potential antineoplastic activity. Suramin's pharmacological actions, which have not yet been fully elucidated, include antagonism of the action of platelet-derived growth factor (PDGF) at its receptor. We investigated the effects of suramin on PDGF-stimulated proteoglycan synthesis.

Human vascular smooth muscle cells (VSMCs) were incubated in the presence and absence of PDGF and suramin with [³H]thymidine or ³⁵SO₄ as radiolabels. Mitogenic response was determined by [³H]thymidine incorporation. PDGFβ receptor phosphorylation was assessed by western blotting. Proteoglycan size and glycosaminoglycan chain synthesis and size were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The Alphascreen phosphotyrosine assay kit was used to investigate PDGFβ receptor tyrosine kinase inhibition by suramin.

Suramin decreased PDGF-stimulated proliferation, proteoglycan synthesis and GAG chain hyperelongation. Suramin also directly inhibited PDGFβ receptor kinase activity as well as PDGFβ receptor phosphorylation in intact VSMCs.

These data show that inhibition of PDGFβ receptor phosphorylation in intact cells is necessary to define a fully active PDGF antagonist. They also confirm that PDGFβ receptor kinase activity is necessary for PDGF-mediated atherogenic changes in proteoglycan synthesis and support efforts to develop PDGFβ receptor antagonists as potential anti-atherosclerotic agents.

Carfilzomib is a new agent for the treatment of relapsed and refractory multiple myeloma (MM). This article presents a comprehensive overview of the pharmacokinetics, pharmacodynamics, dosing schedule, safety, efficacy, preparation and administration of carfilzomib, and its role in treating MM patients.

Carfilzomib is a selective proteasome inhibitor that differs structurally and mechanistically from bortezomib. In patients' whole-blood and peripheral-blood mononuclear cells, carfilzomib inhibited proteasomal and immunoproteasomal activity by 70–80%. Approved carfilzomib dosing is based on body surface area, and is given on days 1, 2, 8, 9, 15 and 16 of a 28-day cycle (20 mg/m² in cycle 1; 27 mg/m² in cycle 2+). Premedication with dexamethasone and adequate hydration are recommended to reduce the risk of adverse events. The median t_1/2 of carfilzomib is short (0.29–0.48 h), with no accumulation detected between doses. In clinical studies in relapsed and refractory MM. and in combinations in newly diagnosed MM, single-agent carfilzomib demonstrated significant durable activity, good tolerability and a favourable safety profile, supporting its extended use.

Carfilzomib represents an important addition to the treatment armamentarium for patients with relapsed and/or refractory MM, and studies are underway evaluating the role of single-agent carfilzomib in additional clinical settings as well as in different combinations.

To investigate the herb–drug pharmacokinetic interaction of artificial calculus bovis (ACB) with diclofenac sodium (DS) and chlorpheniramine maleate (CPM) in rats.

A sensitive high-performance liquid chromatography coupled with tandem mass spectrometry method was developed and validated for the simultaneous determination of DS and CPM in rat plasma. The proposed method was successfully applied to compare the herb–drug pharmacokinetic interaction of ACB with DS and CPM in rats following intragastric administration.

The proposed method had good linearity and no endogenous material interfered with the analytes and internal standard peaks. The lower limit of quantification of DS and CPM was 1 and 0.1 ng/ml, respectively. There was no apparent pharmacokinetic interaction between DS and CPM. Co-administration of ACB with DS noticeably increased the area under the concentration–time curve (AUC_0-∞) and peak plasma concentration (C_max) of DS, while the parameters time of peak concentration (T_max), clearance (Cl_Z/F) and apparent volume of distribution (V_Z/F) of DS significantly decreased. Meanwhile, co-administration of ACB with CPM noticeably increased the T_max, Cl_Z/F and V_Z/F of CPM. A marked decline in AUC_0-∞ and C_max of CPM occurred in the presence of ACB.

This study indicated that co-administration of ACB with DS and CPM can result in an apparent herb–drug pharmacokinetic interaction in rats.

Exposure to toxicants like doxorubicin (Dox) damages cellular components by generating reactive oxygen species (ROS). This can be attenuated using free radical scavengers and/or antioxidants.

Dox-exposed cardiac myoblasts (H9c2 cells) were treated with sesamol (12.5, 25 and 50 μm), a natural phenolic compound. Intracellular ROS inhibition, cell viability and analysis of antioxidant and biochemical markers such as superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase, reduced/oxidized glutathione, lipid peroxidation and protein carbonyl content were performed. The effect of sesamol treatment on the cytotoxic and genotoxic parameters was studied by monitoring the signalling proteins involved in the apoptotic pathway.

Dox triggered cellular and genetic damage by increasing levels of intracellular ROS, thereby decreasing cell viability and increasing apoptosis. Sesamol reversed the cytotoxic and genotoxic effects of Dox. In addition, sesamol attenuated the pro-apoptotic proteins and improved the anti-apoptotic status. Sesamol pre-treatment also alleviated the disturbed antioxidant milieu by preventing ROS production and improving endogenous enzyme levels.

Among the different doses tested, 50 μm of sesamol showed maximum protection against Dox-induced oxidative damage. This reflects the significance of sesamol in ameliorating the deleterious effects associated with cancer chemotherapy.

This study aimed to investigate whether activation of haeme oxygenase (HO)-1 enzyme by haemin would have beneficial effects on the functional and histological outcome against gentamicin-induced renal damage in rats and sought to elucidate the underlying mechanisms of the therapeutic action.

Nephrotoxicity was induced by injection of gentamicin (80 mg/kg, i.p.) once daily for seven days. Haemin (50 μmol/kg, i.p.) was given to the control and gentamicin-treated rats in the presence or absence of a HO-1 inhibitor, zinc protoporphyrin IX (ZnPP, 50 μmol/kg per day, i.p.).

Haemin treatment prevented gentamicin-induced elevated serum creatinine, urinary protein levels and ameliorated the impaired creatinine clearance. Haemin compensated the deficits in antioxidant enzyme activity and attenuated lipid peroxidation along with decreased reactive oxygen species (ROS) production in renal tissues due to gentamicin. Moreover, haemin pre-administration evoked increased renal HO-1 activity. Additionally, haemin significantly attenuated elevated renal tumour necrosis factor-α (TNF-α), nuclear factor-kappaB (NF-κB) levels and caspase-3 activity alongside ameliorating glomerular pathology. These therapeutic effects were abolished by ZnPP pretreatment.

Here is the first evidence demonstrating the protective effect of HO-1 against gentamicin-associated nephrotoxicity. Suppression of oxidative/inflammatory insults alongside the corresponding decline of apoptosis were presumably responsible for this renoprotection.

The aim of this work was to determine the effect of standardized aqueous extracts from Epilobium angustifolium L., E. parviflorum Schreb. and E. hirsutum L. herbs on the apoptosis of hormone-dependent prostate cancer cells (LNCaP).

The extracts were characterized using high-performance liquid chromatography−diode array detector coupled with mass spectrometry (HPLC-DAD-MS/MS). Apoptosis in the cells was analysed using Annexin V–fluorescein isothiocyanate, and mitochondrial potential, Δψ_m, using JC-1 by flow cytometry. Caspase-3 activity was determined by enzyme-linked immunosorbent assay.

Using the HPLC-DAD-MS/MS method, 38 constituents were characterized. Extracts contained significant amounts of oenothein B as well as flavonoids and phenolic acids. Exposure of LNCaP cells to the extracts (20, 50 and 70 μg/ml) resulted in a significant increase in the level apoptotic cells, from 2.86 ± 0.5% (for untreated cells) up to 86.6 ± 1.0%. All extracts significantly decreased the mitochondrial potential, Δψ_m, resulting in an increase in the activity of caspase-3 from 0.3 ± 0.07 ng/mg of protein (for untreated cells) up to 1.26 ± 0.32 ng/mg of protein.

This study demonstrated that Epilobium extracts are active against LNCaP prostate cancer cells and that their apoptotic activity is related to activation of the mitochondrial pathway. The high oenothein B content may influence the biological activity of these plant materials.

The aim of this study was to investigate the transporter-mediated transport of N-acetyl 5-aminosalicylic acid (Ac-5-ASA) and the effect of quercetin on Ac-5-ASA transport.

Caco-2 cell monolayers grown in Transwells were used to study the transport of Ac-5-ASA in the absence or presence of quercetin, and apical-to-basolateral and basolateral-to-apical apparent permeability (PappAB and PappBA values, respectively) was determined. The effect of transporter inhibitors, such as MK571, quinidine and mitoxantrone, on the transport of Ac-5-ASA was investigated.

In the absence of transporter mediators, the transport of Ac-5-ASA was much higher in the basolateral-to-apical direction than in the opposite direction. The PappBA/PappAB ratio of Ac-5-ASA was 4.89. Quercetin inhibited the apical efflux of Ac-5-ASA and decreased the PappBA/PappAB ratio to 1.05. Of the transporter inhibitors, MK571 decreased the PappBA/PappAB ratio to 1.07; however, neither quinidine nor mitoxantrone had an effect on Ac-5-ASA transport.

Ac-5-ASA was excreted by multidrug resistance-associated protein 2 from Caco-2 cells, and its transport was inhibited by quercetin. Our findings suggest that dose levels of sulfasalazine or 5-aminosalicylic acid can be decreased by coadministration of quercetin, leading to improved pharmaceutical care for inflammatory bowel diseases.

Transepithelial di/tripeptide transport in enterocytes occurs via the apical proton-coupled peptide transporter, hPEPT1 (SLC15A1) and a basolateral peptide transporter, which has only been characterized functionally. In this study we examined the pH dependency, substrate uptake kinetics and substrate specificity of the transporter.

We studied the uptake of [¹⁴C]Gly-Sar from basolateral solution into Caco-2 cell monolayers grown for 17–22 days on permeable supports, at a range of basolateral pH values.

Basolateral Gly-Sar uptake was pH dependent, with a maximal uptake rate at a basolateral pH of 5.5. Uptake of Gly-Sar decreased in the presence of the protonophore nigericin, indicating that the uptake was proton-coupled. The uptake was saturable, with a maximal flux (V_max) of 408 ± 71, 307 ± 25 and 188 ± 19 pmol/cm²/min (mean ± S.E., n = 3) at basolateral pH 5.0, 6.0 and 7.4, respectively. The compounds Gly-Asp, Glu-Phe-Tyr, Gly-Glu-Gly, Gly-Phe-Gly, lidocaine and, to a smaller degree, para-aminohippuric acid were all shown to inhibit the basolateral uptake of Gly-Sar.

The study showed that basolateral Gly-Sar transport in the intestinal cell line Caco-2 is proton-coupled. The inhibitor profile indicated that the transporter has broad substrate specificity.

(S)-[6]-Gingerol is under investigation for a variety of therapeutic uses. Transforming growth factor (TGF)-β stimulates proteoglycan synthesis, leading to increased binding of low-density lipoproteins, which is the initiating step in atherosclerosis. We evaluated the effects of (S)-[6]-gingerol on these TGF-β-mediated proteoglycan changes to explore its potential as an anti-atherosclerotic agent.

Purified (S)-[6]-gingerol was assessed for its effects on proteoglycan synthesis by [³⁵S]-sulfate incorporation into glycosaminoglycan chains and [³⁵S]-Met/Cys incorporation into proteoglycans and total proteins in human vascular smooth muscle cells. Biglycan level was assessed by real-time quantitative polymerase chain reactions and the effects of (S)-[6]-gingerol on TGF-β signalling by assessment of the phosphorylation of Smads and Akt by western blotting.

(S)-[6]-Gingerol concentration-dependently inhibited TGF-β-stimulated proteoglycan core protein synthesis, and this was not secondary to inhibition of total protein synthesis. (S)-[6]-Gingerol inhibited biglycan mRNA expression. (S)-[6]-Gingerol did not inhibit TGF-β-stimulated glycosaminoglycan hyperelongation or phosphorylation of Smad 2, in either the carboxy terminal or linker region, or Akt phosphorylation.

The activity of (S)-[6]-gingerol to inhibit TGF-β-stimulated biglycan synthesis suggests a potential role for ginger in the prevention of atherosclerosis or other lipid-binding diseases. The signalling studies indicate a novel site of action of (S)-[6]-gingerol in inhibiting TGF-β responses.

To summarize the basis for and progress with the development of in-vitro–in-silico–in-vivo (IV-IS-IV) relationships for oral dosage forms using physiologically based pharmacokinetic (PBPK) modelling, with the focus on predicting the performance of solid oral dosage forms in humans.

Various approaches to forecasting oral absorption have been reported to date. These range from simple dissolution tests, through biorelevant dissolution testing and laboratory simulations of the gastrointestinal (GI) tract, to the use of PBPK modelling to predict oral drug absorption based on the physicochemical parameters of the drug substance. Although each of these approaches can be useful for qualitative predictions, forecasting oral absorption on a quantitative basis with an individual approach is only possible for selected drug/dosage form combinations. By integrating biorelevant dissolution test results with the PBPK models, it has become possible to achieve quantitatively accurate as well as qualitative predictions of plasma profiles after oral dosing for both immediate and modified release formulations.

With further refinement of both the biorelevant dissolution testing methods and the PBPK models, it should be possible to expedite the development and regulatory approval of optimized dosage forms and dosing conditions.

Rigosertib (ON 01910.Na, Estybon) is a novel, anticancer agent undergoing phase 3 clinical trials for a lead indication against myelodysplastic syndromes (MDS). In this research, the permeability of rigosertib was evaluated using the in-situ perfused rat intestine (IPRI) model to support development of an oral formulation for rigosertib for treating cancer patients.

Experiments (n = 6 per group) were conducted using male Sprague-Dawley rats. Studies evaluated permeability across various intestinal segments and assessed the dose-linearity of absorption over the entire intestinal length. Drug concentrations in the portal and jugular vein were collected to correlate permeability parameters with presystemic and systemic exposure.

Rigosertib permeability was highest in the jejunum, although parameter estimates indicated that rigosertib was a medium permeability compound. The compound displayed nonlinear absorption in the IPRI model, suggesting a saturable transport process. Transport inhibition studies using Caco-2 cells demonstrated that rigosertib was a P-glycoprotein (P-gp) substrate. Absolute bioavailability of rigosertib (10 and 20 mg/kg, 1-h infusion) in rats was estimated to be 10–15%. However, the fraction absorbed in humans predicted from IPRI data (52%) was consistent with published clinical data for rigosertib (35% oral bioavailability).

The results of this research indicated that rigosertib is a promising candidate for oral delivery. Further studies are needed to evaluate the potential impact of P-gp and other intestinal transporters on the oral absorption of this promising anticancer agent.

The aim of this study was to investigate the impact of commercially available, over-the-counter herbal supplements (St John's wort, black cohosh and ginger root extract) on the metabolic activation of tamoxifen and irinotecan.

Co-incubation of each drug and supplement combination over a range of concentrations was conducted in human liver microsomes and the decrease in the rate of active metabolite formation was monitored using high-performance liquid chromatography tandem mass spectrometry. Data was analysed using non-linear regression analysis and Dixon plots to determine the dominant mechanism of inhibition and to estimate the K_i and IC50 values of the commercial supplements.

The data suggest that black cohosh was the strongest inhibitor tested in this study for both CYP450 and carboxyesterase mediated biotransformation of tamoxifen and irinotecan, respectively, to their active metabolites. St John's wort was a stronger inhibitor compared with ginger root extract for tamoxifen (CYP mediated pathway), while ginger root extract was a stronger inhibitor compared with St John's wort for the carboxyesterase mediated pathway.

Commercially available supplements are widely used by patients and their potential impact on the efficacy of the chemotherapy is often unknown. The clinical significance of these results needs to be evaluated in a comprehensive clinical trial.

Drug-induced kidney injury is a serious adverse event which needs to be monitored during aminoglycoside therapy. Urine cystatin C is considered an early and sensitive marker of nephrotoxicity. Cystatin C, a low-molecular-weight serum protein, and basic drugs have a common transport system expressed in the apical membrane of renal proximal tubular cells. The aim of this study was to investigate whether aminoglycoside antibiotics influenced cystatin C binding to the renal brush-border membrane.

The binding study was performed using a rapid filtration technique and affinity column displacement method.

Concentration-dependent inhibition of chicken cystatin binding to brush-border membranes by gentamicin was observed. The gentamicin interaction with brush-border membranes was of relatively low affinity (Ki = 32 μm) in comparison with the chicken cystatin affinity to the binding sites (Kd = 3.6 μm). Amikacin and gentamicin were only able to displace chicken cystatin from the chromatographic affinity column in concentrations several times higher than normally found in the tubular fluid during standard aminoglycoside therapy.

Cystatin reabsorption in the proximal tubule cannot be significantly affected by aminoglycoside antibiotics because of their relatively low affinity to common binding sites on the brush-border membrane.

To calculate the fractal dimension values of felodipine osmotic pump tablets during drug dissolution and to characterize the mechanism of the controlled drug release kinetics through three-dimensional fractal data.

Three-dimensional fractal values of volume (D_f,volume) and surface (D_f,surface) of the tablet core were calculated based on the box counting method.

During the process of release of felodipine, both D_f,volume and D_f,surface were within the range of 2–3 and then changed markedly after a period of 3.0 h release, corresponding to extensive changes in entire shape, interior porous channels and surface structure of the tablet core. The curve of D_f,volume mirrored that for tablet volume, however the curve of D_f,surface was quite different from that of the surface area. Results showed that values of D_f,surface correlated well with the drug release rate. D_f,surface was found to be an efficient fractal parameter that could be used to characterize the complex changes to the tablet core that take place during drug release.

The fractal dimension can be used as a quantitative indicator reflecting the drug release performance and be regarded as a key indicator for the quality control of oral controlled drug delivery systems.

Autophagy is the catabolic process that facilitates the degradation of proteins and organelles into recyclable nutrients for use by the cell. This article will review current literature to support the hypothesis that autophagy is pivotal in cancer progression and survival and provides some rationale behind the notion that autophagy can be a target for future cancer therapy.

For the most part, autophagy is pro-cancerous in that it enables the affected cell to meet its nutritional requirements in hypoxic and cytotoxic environments (mainly due to chemotherapy), thus facilitating continued growth and proliferation of tumour cells. As such, it is reasonable to perceive autophagy as a mechanistic target for cancer therapy. However, the challenge to date has been the complexity of the mechanisms involved and the identification of key regulators of autophagy. This has been further complicated by the inherent variation between different cancer cell lines.

Better understanding of the role and mechanisms of autophagy in cancer, with a prelude to ways of exploiting this knowledge, may lead to better chemotherapeutic management of patients suffering from this currently incurable disease.

The aim of this research was to study and evaluate the antimicrobial activity of a novel 2-methylsulfanyl-[1,2,4]triazolo[1,5-a]quinazoline and its derivatives. Antibacterial activity of the target compounds was tested against a variety of species of Gram-positive bacteria such as Staphylococcus aureus ATCC 29213, Bacillus subtilis ATCC6633, and Gram-negative bacteria such as Pseudomonas aeruginosa ATCC27953 and Escherichia coli ATCC 25922. In addition some yeast and fungi, Candida albicans NRRL Y-477 and Aspergillus niger, respectively, were screened.

Antimicrobial tests were carried out by the agar well diffusion method, using 100 μl of suspension containing 1 × 10⁸ CFU/ml of pathological tested bacteria, 1 × 10⁶ CFU/ml of yeast, and 1 × 10⁴ spore/ml of fungi spread on nutrient agar (NA), Sabourand dextrose agar (SDA), and potato dextrose agar (PDA), respectively.

The minimum inhibitory concentration (MIC) of the tested compounds was determined using the broth double dilution method (serially diluted technique) in proper nutrient. For comparison, ciprofloxacin and ketoconazole were used as antibacterial and antifungal reference drugs, respectively. Compounds 6, 9, 13, 14, and 11 were found to have the highest broad-spectrum antibacterial activity against S. aureus ATCC 29213, B. subtilis ATCC6633 and Gram-negative bacteria such as P. aeruginosa ATCC27953 and E. coli ATCC 25922 with MIC values of 6.25 and 12.50 μg/ml.

It was clear that many of the synthesized compounds exhibited good antimicrobial activity. This study has revealed that compounds 6, 9, 13, 14, and 11 have been disclosed as moderate antimicrobial agents. These compounds could be useful as templates for further development through modification or derivatization to design more potent antimicrobial agents.

In this work, the effects of several technological factors on the stability of β-lapachone (βLAP) in solution and in the solid state were investigated.

The effects of relative humidity and light on the stability of βLAP in the solid state were studied. Samples were characterized by liquid chromatography, thermal analysis, X-ray powder diffraction and optical microscopy. In solution, the effects of light conditions and additives (cyclodextrins) were also evaluated. Molecular modelling was used to support the degradation mechanism involved. Additionally, the pH stability profile of βLAP was established.

The synergism of relative humidity and light promoted degradation of βLAP in the solid state, with important consequences for the physical and chemical characteristics of the drug after storage. Random methyl-β-cyclodextrin was able to protect the drug against the hydrolytic process in darkness. However, it accelerated the drug decomposition by photolysis in light conditions. According to the pH stability profile, βLAP undergoes an alkaline hydrolysis, its maximum stability pH being over the range 2–4.

These studies provide useful information regarding the optimal storage conditions and formulations of βLAP.

The aim of this study was to develop a curcumin intranasal thermosensitive hydrogel and to improve its brain targeting efficiency.

The hydrogel gelation temperature, gelation time, drug release and mucociliary toxicity characteristics as well as the nose-to-brain transport in the rat model were evaluated.

The developed nasal hydrogel, composed of Pluronic F127 and Poloxamer 188, had shorter gelation time, longer mucociliary transport time and produced prolonged curcumin retention in the rat nasal cavity at body temperature. The hydrogel release mechanism was diffusion-controlled drug release, evaluated by the dialysis membrane method, but dissolution-controlled release when evaluated by the membraneless method. A mucociliary toxicity study revealed that the hydrogel maintained nasal mucosal integrity until 14 days after application. The drug-targeting efficiencies for the drug in the cerebrum, cerebellum, hippocampus and olfactory bulb after intranasal administration of the curcumin hydrogel were 1.82, 2.05, 2.07 and 1.51 times that after intravenous administration of the curcumin solution injection, respectively, indicating that the hydrogel significantly increased the distribution of curcumin into the rat brain tissue, especially into the cerebellum and hippocampus.

A thermosensitive curcumin nasal gel was developed with favourable gelation, release properties, biological safety and enhanced brain-uptake efficiency.

To assess whether the composition and charge of microemulsions affect their ability to simultaneously deliver α-tocopherol and lipoic acid into viable skin layers.

α-Tocopherol and lipoic acid were added (1.1 and 0.5% w/w, respectively) to decylglucoside-based microemulsions containing mono-dicaprylin. Microemulsions containing surfactant : oil : water (w/w/w) at 60 : 30 : 10 (ME-O) and 46 : 23 : 31 (ME-W), as well as a cationic form of ME-W containing 1% phytosphingosine (ME-Wphy) were characterized, and their ability to disrupt the skin barrier and deliver the antioxidants in vitro in the skin was evaluated. Antioxidant activity in ME-Wphy-treated skin was assessed using the thiobarbituric acid-reactive substances (TBARS) assay.

The internal phase diameters of microemulsions ranged between 42 and 55 nm; phytosphingosine addition and pH adjustment to 5.0 increased zeta potential from −4.3 to +29.1 mV. ME-O displayed w/o structure, whereas ME-W and ME-Wphy were consistent with o/w. Microemulsions affected skin electrical resistance and transepidermal water loss, but did not affect lipoic acid penetration. α-Tocopherol delivery increased following the order ME-O < ME-W < ME-Wphy. ME-Wphy presented suitable short-term stability. The antioxidants delivered by ME-Wphy decreased TBARS cutaneous levels.

Even though microemulsion structure only affected tocopherol penetration, delivered levels of both antioxidants were sufficient for a decrease in TBARS, supporting their use for enhanced protection.

This work aimed to evaluate the performance of nanoparticle-loaded films based on matrices of polymethacrylates and hydroxypropylmethylcellulose (HPMC) intended for delivery of macromolecules.

Lysozyme (Lys)-loaded nanoparticles were manufactured by antisolvent co-precipitation. After size, loading efficiency and stability characterization, the selected batch of particles was further formulated into films. Films were characterized for mechanical properties, mucoadhesion, Lys release and activity after manufacture.

We found that protein-coated nanoparticles could be obtained in USP phosphate buffer pH 6.8. Particles obtained at pH 6.8 had a z-average of 347.2 nm, a zeta-potential of 21.9 mV and 99.2% remaining activity after manufacture. This formulation was further studied for its application in films for buccal delivery. Films loaded with nanoparticles that contained Eudragit RLPO (ERL) exhibited excellent mechanical and mucoadhesive properties. Due to its higher water-swelling and solubility compared with ERL, the use of HPMC allowed us to tailor the release of Lys from films. The formulation composed of equal amounts of ERL and HPMC revealed a sustained release over 4 h, with Lys remaining fully active at the end of the study.

Mucoadhesive films containing protein-coated nanoparticles are promising carriers for the buccal delivery of proteins and peptides in a stable form.

The aim of this study is to improve the dissolution and oral bioavailability of tanshinone IIA (TAN).

Solid dispersions of TAN with low-molecular-weight chitosan (LMC) were prepared and the in-vitro dissolution and in-vivo performance were evaluated.

At 1 h, the extent of dissolution of TAN from the LMC–TAN system (weight ratio 9 : 1) increased about 368.2% compared with the pure drug. Increasing the LMC content from 9 : 1 to 12 : 1 in this system did not significantly increase the rate and the extent of dissolution. Differential scanning calorimetry, X-ray diffraction and scanning electron microscopy demonstrated the formation of amorphous tanshinone IIA and the absence of crystallinity in the solid dispersion. Fourier transform infrared spectroscopy revealed that there was no interaction between drug and carrier. In-vivo test showed that LMC-TAN solid dispersion system presented significantly larger AUC_0-t, which was 0.67 times that of physical mixtures and 1.17 times that of TAN. Additionally, the solid dispersion generated obviously higher C_max and shortened T_max compared with TAN and physical mixtures.

In conclusion, the LMC -based solid dispersions could achieve complete dissolution, accelerated absorption rate and superior oral bioavailability.

Bitter perception has a particularly important role in host defence. However, to date, direct effects of bitter compounds on small intestinal motility have not been shown. This study investigated the effects of bitter compounds on the spontaneous contraction of longitudinal smooth muscle strips of rat ileum.

Isolated longitudinal smooth muscle strips of rat ileum were used for tension recording in vitro. Immunofluorescence staining was used to identify the localization of TAS2R10 receptors.

The spontaneous contraction of rat ileum was decreased after chloroquine administration. Other bitter compounds, such as quinine, denatonium and saccharin, exhibited similar effects. Chloroquine-induced relaxation was not blocked by tetrodotoxin, but was partially reversed by the nitric oxide synthase inhibitor L-NAME or the large conductance Ca²⁺-activated K⁺ (BKCa) channel antagonist iberiotoxin. By surgically removing the small intestinal mucosa or bathing in Ca²⁺-free Krebs solution, the chloroquine-induced relaxation was largely attenuated. The immunofluorescence staining showed that TAS2R10 receptors were expressed in rat ileum.

The results indicate that bitter receptor agonists induce relaxation of longitudinal smooth muscle strips of rat ileum, which is mediated by nitric oxide and BKCa channels.

The effect of anthrapyridone compound CO1 retaining cytotoxic activity against multidrug resistant (MDR) tumour cells on inducing cell death of the sensitive leukaemia HL60 cell line and its MDR sublines (HL60/VINC and HL60/DOX) was examined.

The effects of CO1 and the reference compound doxorubicin (DOX) on examined cells were analysed by studying their cytotoxicity, drug intracellular accumulation, cell cycle distribution, caspase-3 and caspase-8 activity, Fas expression and lysosomal integrity.

CO1 was much less effective at influencing the cell cycle of examined cells than DOX a well-known antitumour drug targeting cellular DNA and causing G2/M checkpoint arrest. CO1 caused much less pronounced appearance of the sub-G1 population and oligonucleosomal DNA fragmentation, characteristic of apoptosis, compared with DOX. Significantly lower caspase-3 and caspase-8 activity was also observed in the response of these cells to CO1 compared with DOX treatment. CO1 did not change the expression of the Fas death receptor, characteristic of apoptotic pathways, on the surface of studied cells. Interestingly, the results showed that CO1 caused lysosomal membrane permeability (LMP) of the cells, whereas DOX did not perturb the lysosomal integrity of the studied cells.

The results suggest that CO1 could induce LMP-mediated cell death as a main lethal effect in a caspase-independent fashion.

The purpose of this study was to demonstrate the effect of methyl-3-O-methyl gallate (M3OMG), a rare polyphenolic natural product with a potent in-vitro antioxidant effect, against sodium fluoride (NaF)-induced oxidative stress in rat erythrocytes in vivo.

Male Wistar rats were treated daily with either M3OMG (10 and 20 mg/kg) obtained through synthesis, vitamin C (10 mg/kg) or vehicle intraperitoneally for 7 days. Oxidative stress was then induced by exposing animals to NaF (600 ppm) through drinking water for 7 days. At the end of intoxication period, rats were killed and erythrocytes isolated. The activity of antioxidant enzymes (catalase and superoxide dismutase) and levels of reduced glutathione and thiobarbituric acid reactive substances were measured in erythrocyte haemolysates.

NaF intoxication resulted in a 1.9-fold increase in erythrocyte lipid peroxidation associated with significant (P < 0.001) depletion of reduced glutathione level. Superoxide dismutase and catalase activity was suppressed by NaF treatment by 3.069 and 2.3 fold when compared with untreated control groups. Pretreatment of rats with M3OMG or vitamin C afforded protection against NaF-induced oxidative stress as assessed through the measured oxidant/antioxidant markers.

This finding provided in-vivo evidence for the therapeutic potential of M3OMG in combating fluoride-induced oxidative damage in cellular systems.

Interindividual variability in glucuronidation of bilirubin and drugs by UDP-glucuronosyltransferase 1A1 (UGT1A1) is considerable and only partially explained by genetic polymorphisms and enzyme inducers. Here we determined whether a well-known epigenetic modification, cytosine methylation, explains a proportion of this variability in human liver.

UGT1A1 phenotypes, including UGT1A1 protein and bilirubin glucuronidation, and UGT1A1*28 genotype were determined using a human liver bank (n = 46). Methylation levels were quantified at 5 CpG sites associated with known transcription factor response elements in the UGT1A1 promoter and distal enhancer, as well as a CpG-rich island 1.5 kb further upstream.

Individual CpG sites showed considerable methylation variability between livers, ranging from 10- to 29-fold variation with average methylation levels from 25 to 41%. Multivariate regression analysis identified *28/*28 genotype, −4 CpG site methylation and alcohol history as significant predictors of UGT1A1 protein content. Exclusion of livers with *28/*28 genotype or alcohol history revealed positive correlations of −4 CpG methylation with bilirubin glucuronidation (R = 0.73, P < 0.00001) and UGT1A1 protein content (R = 0.54, P = 0.008).

These results suggest that differential methylation of the −4 CpG site located within a known USF response element may explain a proportion of interindividual variability in hepatic glucuronidation by UGT1A1.

To study the antiglycating, antidiabetic and antioxidant properties of Aegle marmelos Correa leaf extract and identify the bioactive constituent.

The effect of the chloroform extract of Aegle marmelos Correa was studied in streptozotocin-induced diabetic rats through evaluation of biochemical parameters. Antiglycation activity was assessed in vitro through measurement of total and specific advanced glycation end products, protein carbonyl formation and collagen solubility tests. Antioxidant potential was evaluated using the ferric-reducing antioxidant power assay and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assays. Identification of the bioactive component was attempted through silica gel column chromatography and GC-MS analysis.

In-vivo studies for 60 days revealed that the extract prevented kidney damage and other secondary complications. The chloroform extract at 16 μg could inhibit protein glycation by 44.33% and pentosidine formation by 59.31%, and could effectively inhibit protein carbonyl formation. It could scavenge DPPH radicals up to 85.26% (IC50: 26 μg). Bio-guided fractionation revealed limonene as the bioactive component, which could account for the antiglycating activity shown by the chloroform extract.

The chloroform extract of Aegle marmelos demonstrated antidiabetic antiglycating and antioxidant activity, effectively preventing kidney damage and establishment of cataracts. Limonene is reported for the first time as possessing potent antiglycating activity and is non-toxic at the concentration used.

Microglial activation has been implicated in neurological disorders for its inflammatory and neurotrophic effects. We investigated the anti-inflammatory effect of the hexane fraction from Myagropsis myagroides (Mertens ex Turner) Fensholt ethanolic extract and its underlying molecular mechanism in lipopolysaccharide-stimulated microglia.

Various solvent fractions prepared from the ethanolic extract of M. myagroides were analysed for total phenolic content, 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity and inhibitory effect on nitric oxide (NO) production in activated BV-2 microglia. We measured prostaglandin E₂ (PGE₂) and pro-inflammatory cytokine levels by enzyme-linked immunosorbent assay. Expression of inflammatory enzymes was analysed by Western blot. Nuclear translocation and activation of nuclear factor-kappaB (NF-κB) were determined by immunofluorescence and reporter gene assay, respectively.

Among the fractions, the hexane fraction (MMH), rich in fatty acid, showed the highest inhibitory activity on NO generation. Pretreatment with MMH decreased mRNA and protein levels of inducible NO synthase and cyclooxygenase-2, resulting in a decrease in NO and PGE₂ in LPS-stimulated BV-2 cells. Furthermore, MMH inhibited the production of inducible pro-inflammatory cytokines at their transcriptional level via inactivation of NF-κB. MMH inhibited the activation of extracellular signal-regulated kinase and c-Jun N-terminal kinase.

These results indicate that MMH has a strong anti-inflammatory activity in LPS-stimulated microglia, suggesting that MMH can be used as a therapeutic agent against neuroinflammatory diseases.

While the use of St John's wort extracts as treatment for mild to moderate depression is well established the mode of action is still under investiation. Individual constituents of St John's wort extract were tested for possible effects on the β₁AR density and a subsequent change in downstream signalling in rat C6 glioblastoma cells.

The effect of compounds from St John's wort extract on the downregulation of β₁-adrenergic receptor-GFP fusion proteins (β₁AR-green fluorescent protein (GFP)) of transfected rat C6 gliobastoma cells (C6-β₁AR-GFP) was investigated by means of confocal laser scanning microscopy (LSM). The influence on the lateral mobility of β₁AR-GFP in C6-β₁AR-GFP was investigated by fluorescence correlation spectroscopy. The formation of second messenger was determined by c-AMP-assay.

Confocal LSM revealed that pretreatment of cells with 1 μm of hyperforin and hyperoside for 6 days, respectively, led to an internalization of β₁AR-GFP under non-stimulating conditions. Observation by fluorescence correlation spectroscopy showed two diffusion time constants for control cells, with τ_diff1 = 0.78 ± 0.18 ms and τ_diff2 = 122.53 ± 69.41 ms, similarly distributed. Pretreatment with 1 μm hyperforin or 1 μm hyperoside for 3 days did not alter the τ_diff values but decreased the fraction of τ_diff1 whereas the fraction of τ_diff2 increased significantly. An elevated level of β₁AR-GFP with hindered lateral mobility was in line with β₁AR-GFP internalization induced by hyperforin and hyperoside, respectively. A reduced β₁-adrenergic responsiveness was assumed for C6 gliobastoma cells after pretreatment for 6 days with 1 μm of both hyperforin and hyperoside, which was confirmed by decreased cAMP formation of about 10% and 5% under non-stimulating conditions. Decrease in cAMP formation by 23% for hyperforin and 15% for hyperoside was more pronounced after stimulation with 10 μm dobutamine for 30 min.

The treatment of C6 gliobastoma cells with hyperforin and hyperoside results in a reduced β₁AR density in the plasma membrane and a subsequent reduced downstream signalling.

Insulin resistance represents a mechanism underlying defect metabolism of carbohydrate and lipid linked to inflammatory reactions in diabetic liver. We hypothesized that the changes may be secondary to endoplasmic reticulum (ER) stress, which could be alleviated by either argirein or valsartan.

Hepatosteatosis in diabetic liver was induced in rats fed with a high-fat diet (HFD) for 12 weeks combined with a single low dose of streptozotocin (STZ 35 mg/kg, ip). Interventions (mg/kg/d, po)with either argirein (50, 100 and 200) or valsartan (12) were conducted in the last 4 weeks.

In diabetic liver fat was significantly accumulated in association with elevated hepatic glucose, serum insulin and homeostasis model assessment of insulin resistance value. Downregulated glucose transporter 4, insulin receptor substrate-1 and leptin receptor (P < 0.01) were found relative to normal, where DNA ladder, downregulated B cell lymphoma/leukemia-2, upregulated B cell lymphoma/leukemia-2 Associated X protein and upregulated ER stress chaperones such as Bip/GRP78 (also known as Binding Protein, BiP), PKR-like ER kinase (PERK), p-PERK/PERK and C/EBP homologous protein were significant. These abnormalities were significantly ameliorated by argirein and valsartan.

Hepatosteatosis induced by HFD/low STZ manifests insulin resistance and apoptosis, linked to an entity of low-grade inflammation due to activated ER stress sensors. With anti-inflammatory activity either argirein or valsartan blunts hepatosteatosis through normalizing ER stress and apoptosis in the diabetic liver.

We studied the effects of ethyl acetate fraction (EAcF) obtained from Erythrina velutina leaves on mammalian myocardium.

The effect of EAcF on the contractility was studied using guinea-pig left atria mounted in a tissue bath (Tyrode's solution, 29°C, 95% CO₂, 5% O₂) and electrically stimulated (1 Hz). Concentration-response curves of EAcF were obtained in the presence of propranolol (1 μm), nifedipine (1 μm) and in reserpinized animals (5 mg/kg). The involvement of l-type calcium current (I_Ca,L) on the EAcF effect was observed in cardiomyocytes of mice assessed using patch-clamp technique.

EAcF (550 μg/ml) had a positive inotropic effect, increasing the atrial force by 164% (EC₅₀ = 157 ± 44 μg/ml, n = 6), but it was less potent than isoproterenol (EC₅₀ = 0.0036 ± 0.0019 μg/ml, n = 8). The response evoked by EAcF was abolished by propranolol or nifedipine. Reserpine did not alter the inotropic response of EAcF. Furthermore, an enhancement of the I_Ca,L peak (31.2%) with EAcF was observed. Chemical analysis of EAcF revealed the presence of at least 10 different flavonoid glycoside derivatives. Two were identified as vicenin II and isorhoifolin.

We conclude that EAcF increases the cardiac contractile force by increasing the l-type calcium current and activating the adrenergic receptor pathway.