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The growing interest in the composition and effects of microbiota raised the question how drug pharmacokinetics could be influenced by concomitant application of probiotics. The aim of this study was to find whether probiotic E. coli strain Nissle 1917 (EcN) influences the pharmacokinetics of concomitantly taken antiarrhythmic drug amiodarone (AMI). Live bacterial suspension of probiotic EcN (or non-probiotic E. coli strain ATCC 25922) was applied orally to male Wistar rats for seven days, while a control group of rats was treated with a saline solution. On the eighth day, the amiodarone hydrochloride was administered as one single oral dose (50 mg/kg) to all rats (N = 60). After 0, 1, 2, 3, 4, 5.5, 7, 9, 14, 22, and 30 hours, blood samples were taken from the rat abdominal aorta. The plasma level of AMI and its metabolite N-desethylamiodarone (DEA) was determined using the HPLC with UV detection. Administration of EcN led to a 43% increase of AMI AUC0-30 in comparison with control samples. However, this effect was not observed if EcN was replaced by a reference non-probiotic E. coli strain. Thus, EcN administration was most probably responsible for better drug absorption from the gastrointestinal tract. Plasma levels of DEA were also increased in plasma samples from animals treated with EcN. This change was again not found in the experiment with the reference non-probiotic strain. Higher DEA levels in samples from EcN-treated rats may be explained either by better absorption of AMI and/or by an increased activity of CYP2C forms, known to participate in metabolism of this drug, after EcN administration. In this paper, it is documented that concomitantly taken probiotic EcN may modulate pharmacokinetics of a drug; in this case, it led to an increased bioavailability of AMI. [ABSTRACT FROM AUTHOR]

Triple reuptake inhibitors (TRIs) are currently being developed as a new class of promising antidepressants that block serotonin (5-HT), dopamine (DA) and norepinephrine (NE) transporters, thereby increasing extracellular monoamine concentrations. The purpose of this study was to investigate the effects of LPM570065, a novel TRI and a desvenlafaxine prodrug, on extracellular 5-HT, DA and NE levels in the rat striatum after acute and chronic administration relative to desvenlafaxine, using High Performance Liquid Chromatography (HPLC) and microdialysis. Acute administration was performed by providing rodents with oral solutions (0.06 mmol·kg−1 p.o.), oral suspensions (0.06 mmol·kg−1 p.o.) and intravenous solutions (0.04 mmol·kg−1 i.v.) of LPM570065 and desvenlafaxine. Oral suspensions (0.06 mmol·kg−1·day−1) of the two drugs were also administered for a 14-day chronic period. HPLC analysis revealed that LPM570065 rapidly penetrated the rat striatum, converted into desvenlafaxine and exhibited larger total exposure compared with the administration of desvenlafaxine. Microdialysis revealed that acute and chronic administration of oral suspension of LPM570065 increased the 5-HT, DA and NE levels more than the relative administration of desvenlafaxine. Unlike desvenlafaxine, acute administration of an intravenous LPM570065 solution did not induce the undesirable 90% decrease in extracellular 5-HT levels. In contrast to the fully dose-dependent elevation of 5-HT induced by desvenlafaxine, the acute administration of LPM570065 showed a capped increase in extracellular 5-HT levels when combined with WAY-100635. Additionally, forced swim test demonstrated that acute and chronic administration of LPM570065 reduced the immobility time more than the relative administration of desvenlafaxine. These data suggest that LPM570065 may have greater efficacy and/or a more rapid onset of antidepressant action than desvenlafaxine and also counterbalance the harmful effects of desvenlafaxine on 5-HT neurotransmission related to 5-HT1A autoreceptors. Thus, this new class of drugs, TRIs has the potential to provide a new therapeutic mechanism for treating depression. [ABSTRACT FROM AUTHOR]

Bisphosphonates (BPs) have been widely used in clinical treatment of bone diseases with increased bone resorption because of their strong affinity for bone and their inhibition of bone resorption. Recently, there has been growing interest in their improvement of bone formation. However, the effect of local controlled delivery of BPs is unclear. We used polylactide acid-glycolic acid copolymer (PLGA) as a drug carrier to deliver various doses of the bisphosphonate zoledronate (Zol) into the distal femur of 8-week-old Sprague-Dawley rats. After 6 weeks, samples were harvested and analyzed by micro-CT and histology. The average bone mineral density and mineralized bone volume fraction were higher with medium- and high-dose PLGA-Zol (30 and 300 µg Zol, respectively) than control and low-dose Zol (3 µg PLGA-Zol; p<0.05). Local controlled delivery of Zol decreased the numbers of osteoclast and increased the numbers of osteoblast. Moreover, local controlled delivery of medium- and high-dose Zol accelerated the expression of bone-formation markers. PLGA used as a drug carrier for controlled delivery of Zol may promote local bone formation. [ABSTRACT FROM AUTHOR]

Background: Fentanyl is widely used off-label in NICU. Our aim was to investigate its cerebral, cardiovascular and pulmonary effects as well as pharmacokinetics in an experimental model for neonates. Methods: Fentanyl (5 µg/kg bolus immediately followed by a 90 minute infusion of 3 µg/kg/h) was administered to six mechanically ventilated newborn piglets. Cardiovascular, ventilation, pulmonary and oxygenation indexes as well as brain activity were monitored from T = 0 up to the end of experiments (T = 225–300 min). Also plasma samples for quantification of fentanyl were drawn. Results: A “reliable degree of sedation” was observed up to T = 210–240 min, consistent with the selected dosing regimen and the observed fentanyl plasma levels. Unlike cardiovascular parameters, which were unmodified except for an increasing trend in heart rate, some of the ventilation and oxygenation indexes as well as brain activity were significantly altered. The pulmonary and brain effects of fentanyl were mostly recovered from T = 210 min to the end of experiment. Conclusion: The newborn piglet was shown to be a suitable experimental model for studying fentanyl disposition as well as respiratory and cardiovascular effects in human neonates. Therefore, it could be extremely useful for further investigating the drug behaviour under pathophysiological conditions. [ABSTRACT FROM AUTHOR]

This study investigated the histological tissue reaction to long-term implanted cerebral open flow microperfusion (cOFM) probes in the frontal lobe of the rat brain. Most probe-based cerebral fluid sampling techniques are limited in application time due to the formation of a glial scar that hinders substance exchange between brain tissue and the probe. A glial scar not only functions as a diffusion barrier but also alters metabolism and signaling in extracellular brain fluid. cOFM is a recently developed probe-based technique to continuously sample extracellular brain fluid with an intact blood-brain barrier. After probe implantation, a 2 week healing period is needed for blood-brain barrier reestablishment. Therefore, cOFM probes need to stay in place and functional for at least 15 days after implantation to ensure functionality. Probe design and probe materials are optimized to evoke minimal tissue reaction even after a long implantation period. Qualitative and quantitative histological tissue analysis revealed no continuous glial scar formation around the cOFM probe 30 days after implantation and only a minor tissue reaction regardless of perfusion of the probe. [ABSTRACT FROM AUTHOR]

Background: The poor bioavailability of Berberine (BBR) and Betulinic acid (BA) limits the development of these promising anticancer agents for clinical use. In the current study, BBR and BA in spray dried (SD) mucoadhesive microparticle formulations were prepared. Methods: A patented dual channel spray gun technology established in our laboratory was used for both formulations. Gastrointestinal (GI) permeability studies were carried out using Caco-2 cell monolayer grown in in-vitro system. The oral bioavailability and pharmacokinetic profile of SD formulations were studied in Sprague Dawley rats. A549 orthotopic and H1650 metastatic NSCLC models were utilized for the anticancer evaluations. Results: Pharmacokinetic studies demonstrated that BBR and BA SD formulations resulted in 3.46 and 3.90 fold respectively, significant increase in plasma Cmax concentrations. AUC levels were increased by 6.98 and 7.41 fold in BBR and BA SD formulations, respectively. Compared to untreated controls groups, 49.8 & 53.4% decrease in the tumor volumes was observed in SD formulation groups of BBR and BA, respectively. Molecular studies done on excised tumor (A549) tissue suggested that BBR in SD form resulted in a significant decrease in the survivin, Bcl-2, cyclin D1, MMP-9, HIF-1α, VEGF and CD31 expressions. Cleaved caspase 3, p53 and TUNEL expressions were increased in SD formulations. The RT-PCR analysis on H1650 tumor tissue suggested that p38, Phospho-JNK, Bax, BAD, cleaved caspase 3&8 mRNA expressions were significantly increased in BA SD formulations. Chronic administration of BBR and BA SD formulations did not show any toxicity. Conclusions: Due to significant increase in oral bioavailability and superior anticancer effects, our results suggest that spray drying is a superior alternative formulation approach for oral delivery of BBR and BA. [ABSTRACT FROM AUTHOR]

Metronidazole (MTZ) and Cotrimoxazole (CTX) are used in HIV/AIDS patients eligible for antiretroviral treatment. The objective of this animal study was to determine whether pre-treatment with antibiotics affects the intestinal bioavailability of Atazanavir (ATV) and Ritonavir (RTV). After oral administration of 1 mg MTZ and CTX for 7 days, the rat colonic mucosa were analyzed for mucus thickness or placed in Ussing chambers to measure ATV and RTV net transepithelial fluxes (Jnet). 1. In control rats, the mucus thickness was 43.3±7.6 µm and 40.7±6.9 µm, in proximal and distal colon, respectively. In proximal colon, the thickness was 57.2±8.8 and 58.2±6.9 µm after MTZ and CTX, respectively whereas in distal colon, the thickness was 121.1±38.4 and 170.5±35.0 µm (P<0.05) respectively. 2. Transepithelial conductance was reduced after MTZ or CTX in the proximal and distal colon. 3. In control, net ATV secretion was observed both in proximal (−0.36±0.02 µg.hr−1 cm−2) and distal colon (−0.30±0.08 µg.hr−1 cm−2). After MTZ and CTX, it was increased in the proximal colon by two 2 fold and 4 fold, respectively and in the distal colon by 3 fold and 5 fold, respectively. 4. In control, there was no net active RTV transport either in proximal (+0.01±0.01 µg.hr−1 cm−2) or distal colon (+0.04±0.01 µg.hr−1 cm−2). After MTZ and CTX, secretion was increased 5 fold and 10 fold, respectively, in the proximal colon and two fold and 5 fold, respectively in the distal colon (p<0.001). In conclusion, after MTZ and CTX therapy, the mucus layer was enlarged, passive permeability was decreased and ATV and RTV were actively secreted by the colonic epithelium suggesting that, in rat, the intestinal bioavailability of ATV and RTV is impaired after antibiotic therapy. [ABSTRACT FROM AUTHOR]

Clobazam (CLB), a 1,5-benzodiazepine (BZD), was FDA-approved in October 2011 for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome (LGS) in patients 2 years and older. BZDs exert various CNS effects through allosteric modulation of GABAA receptors. The structurally distinct, 1,4-BZD clonazepam (CLN) is also approved to treat LGS. The precise mechanisms of action and clinical efficacy of both are unknown. Data show that the GABAA α1-subunit–selective compound zolpidem [ZOL] exhibits hypnotic/sedative effects. Conversely, data from knock-in mice carrying BZD binding site mutations suggest that the α2 subunit mediates anticonvulsant effects, without sedative actions. Hence, the specific pattern of interactions across the GABAA receptor complexes of BZDs might be reflected in their clinical efficacies and adverse effect profiles. In this study, GABAA-receptor binding affinities of CLB, N-desmethylclobazam (N-CLB, the major metabolite of CLB), CLN, and ZOL were characterized with native receptors from rat-brain homogenates and on cloned receptors from HEK293 cells transfected with combinations of α (α1, α2, α3, or α5), β2, and γ2 subtypes. Our results demonstrate that CLB and N-CLB have significantly greater binding affinities for α2- vs. α1-receptor complexes, a difference not observed for CLN, for which no distinction between α2 and α1 receptors was observed. Our experiments with ZOL confirmed the high preference for α1 receptors. These results provide potential clues to a new understanding of the pharmacologic modes of action of CLB and N-CLB. [ABSTRACT FROM AUTHOR]

The reductively activated nitroaromatic class of antimicrobials, which include nitroimidazole and the more metabolically labile nitrofuran antitubercular agents, have demonstrated some potential for development as therapeutics against dormant TB bacilli. In previous studies, the pharmacokinetic properties of nitrofuranyl isoxazolines were improved by incorporation of the outer ring elements of the antitubercular nitroimidazole OPC-67683. This successfully increased stability of the resulting pentacyclic nitrofuran lead compound Lee1106 (referred to herein as 9a). In the current study, we report the synthesis and antimicrobial properties of 9a and panel of 9a analogs, which were developed to increase oral bioavailability. These hybrid nitrofurans remained potent inhibitors of Mycobacterium tuberculosis with favorable selectivity indices (>150) and a narrow spectrum of activity. In vivo, the pentacyclic nitrofuran compounds showed long half-lives and high volumes of distribution. Based on pharmacokinetic testing and lack of toxicity in vivo,9a remained the series lead. 9a exerted a lengthy post antibiotic effect and was highly active against nonreplicating M. tuberculosis grown under hypoxia. 9a showed a low potential for cross resistance to current antitubercular agents, and a mechanism of activation distinct from pre-clinical tuberculosis candidates PA-824 and OPC-67683. Together these studies show that 9a is a nanomolar inhibitor of actively growing as well as nonreplicating M. tuberculosis. [ABSTRACT FROM AUTHOR]

The aim of this study was to achieve real-time imaging of the in vivo behavior of a green tea polyphenol, catechin, by positron emission tomography (PET). Positron-labeled 4″ -[11C]methyl-epigallocatechin gallate ([11C]Me-EGCG) was orally administered to rats, and its biodistribution was imaged for 60 min by using a small animal PET system. As the result, images of [11C]Me-EGCG passing through the stomach into the small intestines were observed; and a portion of it was quantitatively detected in the liver. On the other hand, intravenous injection of [11C]Me-EGCG resulted in a temporal accumulation of the labeled catechin in the liver, after which almost all of it was transferred to the small intestines within 60 min. In the present study, we succeeded in obtaining real-time imaging of the absorption and biodistribution of [11C]Me-EGCG with a PET system. [ABSTRACT FROM AUTHOR]

P-glycoprotein (P-gp) expression determines the absorption, distribution, metabolism and excretion of many drugs in the body. Also, up-regulation of P-gp acts as a defense mechanism against acute inflammation. This study examined expression levels of abcb1 mRNA and localization of P-gp protein in the liver, kidney, duodenum, jejunum and ileum in healthy and E. coli infected broilers by real time RT-PCR and immunohistochemistry. Meanwhile, pharmacokinetics of orally administered enrofloxacin was also investigated in healthy and infected broilers by HPLC. The results indicated that E. coli infection up-regulated expression of abcb1 mRNA levels significantly in the kidney, jejunum and ileum (P<0.05), but not significantly in the liver and duodenum (P>0.05). However, the expression level of CYP 3A37 mRNA were observed significantly decreased only in liver and kidney of E. coli infected broilers (P<0.05) compared with healthy birds. Furthermore, the infection reduced absorption of orally administered enrofloxacin, significantly decreased Cmax (0.34 vs 0.98 µg mL−1, P = 0.000) and AUC0-12h (4.37 vs 8.88 µg mL−1 h, P = 0.042) of enrofloxacin, but increased Tmax (8.32 vs 3.28 h, P = 0.040), T1/2a(2.66 vs 1.64 h−1, P = 0.050) and V/F (26.7 vs 5.2 L, P = 0.040). Treatment with verapamil, an inhibitor of P-gp, significantly improved the absorption of enrofloxacin in both healthy and infected broilers. The results suggest that the E. coli infection induces intestine P-gp expression, altering the absorption of orally administered enrofloxacin in broilers. [ABSTRACT FROM AUTHOR]

Tuberculosis remains a global threat due in part to the long treatment regimen and the increased prevalence of drug resistant M. tuberculosis strains. Therefore, new drug regimens are urgently required to combat this deadly disease. We previously synthesized and evaluated a series of new anti-tuberculosis compounds which belong to the family of imidazo[1,2-a]pyridines. This family of compounds showed low nM MIC (minimal inhibitory concentration) values against M. tuberculosis in vitro. In this study, a derivative of imidazo[1,2-a]pyridines, (N-(4-(4-chlorophenoxy)benzyl)-2,7-dimethylimidazo[1,2-a]pyridine-3-carboxamide) (ND-09759), was selected as a promising lead compound to determine its protective efficacy using a mouse infection model. Pharmacokinetic analysis of ND-09759 determined that at a dosage of 30 mg/kg mouse body weight (PO) gave a maximum serum drug concentration (Cmax) of 2.9 µg/ml and a half-life of 20.1 h. M. tuberculosis burden in the lungs and spleens was significantly decreased in mice treated once daily 6 days per week for 4-weeks with ND-09759 compared to untreated mice and this antibiotic activity was equivalent to isoniazid (INH) and rifampicin (RMP), two first-line anti-TB drugs. We observed slightly higher efficacy when using a combination of ND-09759 with either INH or RMP. Finally, the histopathological analysis revealed that infected mice treated with ND-09759 had significantly reduced inflammation relative to untreated mice. In conclusion, our findings indicate ND-09759 might be a potent candidate for the treatment of active TB in combination with current standard anti-TB drugs. [ABSTRACT FROM AUTHOR]

Baicalin purified from the root of Radix scutellariae is widely used in clinical practices. This study aimed to evaluate the effect of baicalin on the pharmacokinetics of nifedipine, a CYP3A probe substrate, in rats in vivo and in vitro. In a randomised, three-period crossover study, significant changes in the pharmacokinetics of nifedipine (2 mg/kg) were observed after treatment with a low (0.225 g/kg) or high (0.45 g/kg) dose of baicalin in rats. In the low- and high-dose groups of baicalin-treated rats, Cmax of total nifedipine decreased by 40%±14% (P<0.01) and 65%±14% (P<0.01), AUC0–∞ decreased by 41%±8% (P<0.01) and 63%±7% (P<0.01), Vd increased by 85%±43% (P<0.01) and 224%±231% (P<0.01), and CL increased by 97%±78% (P<0.01) and 242%±135% (P<0.01), respectively. Plasma protein binding experiments in vivo showed that Cmax of unbound nifedipine significantly increased by 25%±19% (P<0.01) and 44%±29% (P<0.01), respectively, and there was a good correlation between the unbound nifedipine (%) and baicalin concentrations (P<0.01). Furthermore, in vitro results revealed that baicalin was a competitive displacer of nifedipine from plasma proteins. In vitro incubation experiments demonstrated that baicalin could also competitively inhibit CYP3A activity in rat liver microsomes in a concentration-dependent manner. In conclusion, the pharmacokinetic changes of nifedipine may be modulated by the inhibitory effects of baicalin on plasma protein binding and CYP3A–mediated metabolism. [ABSTRACT FROM AUTHOR]

The aim of this work was to conduct a comprehensive study about the transport properties of NSAIDs across the blood-brain barrier (BBB) in vitro. Transport studies with celecoxib, diclofenac, ibuprofen, meloxicam, piroxicam and tenoxicam were accomplished across Transwell models based on cell line PBMEC/C1-2, ECV304 or primary rat brain endothelial cells. Single as well as group substance studies were carried out. In group studies substance group compositions, transport medium and serum content were varied, transport inhibitors verapamil and probenecid were added. Resulted permeability coefficients were compared and normalized to internal standards diazepam and carboxyfluorescein. Transport rankings of NSAIDs across each model were obtained. Single substance studies showed similar rankings as corresponding group studies across PBMEC/C1-2 or ECV304 cell layers. Serum content, glioma conditioned medium and inhibitors probenecid and verapamil influenced resulted permeability significantly. Basic differences of transport properties of the investigated NSAIDs were similar comparing all three in vitro BBB models. Different substance combinations in the group studies and addition of probenecid and verapamil suggested that transporter proteins are involved in the transport of every tested NSAID. Results especially underlined the importance of same experimental conditions (transport medium, serum content, species origin, cell line) for proper data comparison. [ABSTRACT FROM AUTHOR]

Background and Purpose: Mycophenolate mofetil (MMF) per se is not known to have negative effects on the kidney. MMF alone or in combination with sirolimus, can be the basis of calcineurin inhibitor (CNI)-free, kidney sparing drug protocols. However, long-term outcomes in patients on MMF/SRL seem to be inferior to those treated with regimens that include the CNI tacrolimus (TAC) due to an increased risk of allo-immune reactions. Interestingly, potential enhancement of the negative effects of SRL and TAC on the kidney by MMF has never been considered. Experimental Approach: It was our aim to study the effects of TAC, SRL and MMF alone and evaluate their interactions when combined on the rat kidney. For this purpose we used a comprehensive molecular marker approach including measurements of urinary 8-isoprostane concentrations (oxidative stress marker) and changes of urinary metabolite patterns (1H-NMR spectroscopy) and comparing these markers to renal function (glomerular filtration rate (GFR)) and morphologic alterations (histology). Key Results: While MMF alone did not impact GFR, its interaction with SRL and TAC led to a significant decrease of rats’ renal function. The decline went in parallel with a significant increase in urinary isoprostane concentrations and an enhancement of negative effects on urinary metabolite patterns. Conclusions: In broad summary, the present study showed that MMF may enhance the negative effects of TAC on kidney function and may even display nephrotoxic properties when combined with SRL. [ABSTRACT FROM AUTHOR]

Overexpression or/and activating mutation of FLT3 kinase play a major driving role in the pathogenesis of acute myeloid leukemia (AML). Hence, pharmacologic inhibitors of FLT3 are of therapeutic potential for AML treatment. In this study, BPR1J-340 was identified as a novel potent FLT3 inhibitor by biochemical kinase activity (IC50 approximately 25 nM) and cellular proliferation (GC50 approximately 5 nM) assays. BPR1J-340 inhibited the phosphorylation of FLT3 and STAT5 and triggered apoptosis in FLT3-ITD+ AML cells. The pharmacokinetic parameters of BPR1J-340 in rats were determined. BPR1J-340 also demonstrated pronounced tumor growth inhibition and regression in FLT3-ITD+ AML murine xenograft models. The combination treatment of the HDAC inhibitor vorinostat (SAHA) with BPR1J-340 synergistically induced apoptosis via Mcl-1 down-regulation in MOLM-13 AML cells, indicating that the combination of selective FLT3 kinase inhibitors and HDAC inhibitors could exhibit clinical benefit in AML therapy. Our results suggest that BPR1J-340 may be further developed in the preclinical and clinical studies as therapeutics in AML treatments. [ABSTRACT FROM AUTHOR]

A novel supersaturated self-emulsifying drug delivery system (Super-SEDDS) loaded with scutellarin-phospholipid complex (SPC) was developed. The system aimed to address the limitations presented by conventional SEDDS as delivery carriers for drugs with poor water-solubility, low liposolubility and high dose. As an intermediate, SPC was first prepared based on the response surface design. The presence of amorphous scutellarin was demonstrated through differential scanning calorimetry (DSC) and X-ray diffraction (XRD), while enhanced liposolubility was confirmed through comparison with scutellarin powder via an octanol/water distribution test. On the basis of the solubility study and ternary phase diagram, Super-SEDDS containing SPC of up to 200% equilibrium solubility (Seq) was designed, which composed of ethyl oleate, Cremophor RH40 and Transcutol HP with a ratio of 60∶25∶15 (w/w%). The subsequent in vitro lipolysis study and ex vivo intestinal absorption test indicated that Super-SEDDS enhanced the cumulative dissolution from 70% to 100% and improved the intestinal absorption from 0.04 to 0.12 µg/cm2 compared with scutellarin powder. Furthermore, an in vivo study demonstrated that Super-SEDDS achieved the AUC0-t of scutellarin up to approximate 1.7-fold as scutellarin powder. It was also proved superior to SPC and the conventional SEDDS. Super-SEDDS showed great potential for expanding the usage of SEDDS and could act as an alternative to conventional SEDDS. [ABSTRACT FROM AUTHOR]

Lung cancer ranks among the most common malignancies, and is the leading cause of cancer-related mortality worldwide. Chemotherapy for lung cancer can be made more specific to tumor cells, and less toxic to normal tissues, through the use of ligand-mediated drug delivery systems. In this study, we investigated the targeting mechanism of the ligand-mediated drug delivery system using a peptide, SP5-2, which specifically binds to non-small cell lung cancer (NSCLC) cells. Conjugation of SP5-2 to liposomes enhanced the amount of drug delivered directly into NSCLC cells, through receptor-mediated endocytosis. Functional SP5-2 improved the therapeutic index of Lipo-Dox by enhancing therapeutic efficacy, reducing side effects, and increasing the survival rate of tumor-bearing mice in syngenic, metastatic and orthotopic animal models. Accumulation of SP5-2-conjugated liposomal doxorubicin (SP5-2-LD) in tumor tissues was 11.2-fold higher than that of free doxorubicin, and the area under the concentration-time curve (AUC0–72 hours) was increased 159.2-fold. Furthermore, the experiment of bioavailability was assessed to confirm that SP5-2 elevates the uptake of the liposomal drugs by the tumor cells in vivo. In conclusion, the use of SP5-2-conjugated liposomes enhances pharmacokinetic properties, improves efficacy and safety profiles, and allows for controlled biodistribution and drug release. [ABSTRACT FROM AUTHOR]

In this study, the in vivo pharmacokinetics and pharmacodynamics of a novel recombinant human erythropoietin (rhEPO) Fc fusion protein, rhEPO-Fc, were studied in both rodents and rhesus monkeys. Animal models of anemia induced by irradiation, cyclophosphamide and partial renal ablation were used to evaluate therapeutic effects of rhEPO-Fc. We have demonstrated that serum half-life of rhEPO-Fc was 29.5 to 38.9 h at doses of 8, 25, 80 µg/kg in rhesus monkeys and 35.5 to 43.5 h at doses of 16, 50, 160 µg/kg in rats. In anemia animal models, rhEPO-Fc dose-dependently (7.5–30.0 µg/kg in mice, 5.4–21.4 µg/kg in rats and 5.0–10.0 µg/kg in rhesus monkeys) increased reticulocyte level, followed by an increase of RBC count, hemoglobin and hematocrit levels. At reduced intervention frequency of weekly treatments, rhEPO-Fc showed similar hematopoietic effects as compared with rhEPO given three times a week. These results indicated that rhEPO-Fc could potentially be used in treatment of anemia and warrants future clinical trials. [ABSTRACT FROM AUTHOR]

A sensitive, simple and reproducible protocol for nanoparticle-assisted laser desorption/ionization mass spectrometry imaging technique is described. The use of commercially available TiO2 nanoparticles abolishes heterogeneous crystallization, matrix background interferences and enhances signal detection, especially in the low mass range. Molecular image normalization was based on internal standard deposition on tissues, allowing direct comparison of drug penetration and distribution between different organs and tissues. The method was applied to analyze the distribution of the anticancer drug paclitaxel, inside normal and neoplastic mouse tissue sections. Spatial resolution was good, with a linear response between different in vivo treatments and molecular imaging intensity using therapeutic drug doses. This technique distinguishes the different intensity of paclitaxel distribution in control organs of mice, such as liver and kidney, in relation to the dose. Animals treated with 30 mg/kg of paclitaxel had half of the concentration of those treated with 60 mg/kg. We investigated the spatial distribution of paclitaxel in human melanoma mouse xenografts, following different dosage schedules and found a more homogeneous drug distribution in tumors of mice given repeated doses (5×8 mg/kg) plus a 60 mg/kg dose than in those assigned only a single 60 mg/kg dose. The protocol can be readily applied to investigate anticancer drug distribution in neoplastic lesions and to develop strategies to optimize and enhance drug penetration through different tumor tissues. [ABSTRACT FROM AUTHOR]

Isoniazid (INH) and Rifampicin (RFP) are widely used in the world for the treatment of tuberculosis, but the hepatotoxicity is a major concern during clinical therapy. Previous studies showed that these drugs induced oxidative stress in liver, and several antioxidants abated this effect. Metallothionein (MT), a member of cysteine-rich protein, has been proposed as a potent antioxidant. This study attempts to determine whether endogenous expression of MT protects against INH and RFP-induced hepatic oxidative stress in mice. Wild type (MT+/+) and MT-null (MT−/−) mice were treated intragastrically with INH (150 mg/kg), RFP (300 mg/kg), or the combination (150 mg/kg INH +300 mg/kg RFP) for 21 days. The results showed that MT−/− mice were more sensitive than MT+/+ mice to INH and RFP-induced hepatic injuries as evidenced by hepatic histopathological alterations, increased serum AST levels and liver index, and hepatic oxidative stress as evidenced by the increase of MDA production and the change of liver antioxidant status. Furthermore, INH increased the protein expression of hepatic CYP2E1 and INH/RFP (alone or in combination) decreased the expression of hepatic CYP1A2. These findings clearly demonstrate that basal MT provides protection against INH and RFP-induced toxicity in hepatocytes. The CYP2E1 and CYP1A2 were involved in the pathogenesis of INH and RFP-induced hepatotoxicity. [ABSTRACT FROM AUTHOR]

The discovery that paracetamol is metabolized to the potent TRPV1 activator N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404) and that this metabolite contributes to paracetamol’s antinociceptive effect in rodents via activation of TRPV1 in the central nervous system (CNS) has provided a potential strategy for developing novel analgesics. Here we validated this strategy by examining the metabolism and antinociceptive activity of the de-acetylated paracetamol metabolite 4-aminophenol and 4-hydroxy-3-methoxybenzylamine (HMBA), both of which may undergo a fatty acid amide hydrolase (FAAH)-dependent biotransformation to potent TRPV1 activators in the brain. Systemic administration of 4-aminophenol and HMBA led to a dose-dependent formation of AM404 plus N-(4-hydroxyphenyl)-9Z-octadecenamide (HPODA) and arvanil plus olvanil in the mouse brain, respectively. The order of potency of these lipid metabolites as TRPV1 activators was arvanil = olvanil>>AM404> HPODA. Both 4-aminophenol and HMBA displayed antinociceptive activity in various rodent pain tests. The formation of AM404, arvanil and olvanil, but not HPODA, and the antinociceptive effects of 4-aminophenol and HMBA were substantially reduced or disappeared in FAAH null mice. The activity of 4-aminophenol in the mouse formalin, von Frey and tail immersion tests was also lost in TRPV1 null mice. Intracerebroventricular injection of the TRPV1 blocker capsazepine eliminated the antinociceptive effects of 4-aminophenol and HMBA in the mouse formalin test. In the rat, pharmacological inhibition of FAAH, TRPV1, cannabinoid CB1 receptors and spinal 5-HT3 or 5-HT1A receptors, and chemical deletion of bulbospinal serotonergic pathways prevented the antinociceptive action of 4-aminophenol. Thus, the pharmacological profile of 4-aminophenol was identical to that previously reported for paracetamol, supporting our suggestion that this drug metabolite contributes to paracetamol’s analgesic activity via activation of bulbospinal pathways. Our findings demonstrate that it is possible to construct novel antinociceptive drugs based on fatty acid conjugation as a metabolic pathway for the generation of TRPV1 modulators in the CNS. [ABSTRACT FROM AUTHOR]

Background: The current study was carried out to examine the gastroprotective effects of Parkia speciosa against ethanol-induced gastric mucosa injury in rats. Methodology/Principal Findings: Sprague Dawley rats were separated into 7 groups. Groups 1–2 were orally challenged with carboxymethylcellulose (CMC); group 3 received 20 mg/kg omeprazole and groups 4–7 received 50, 100, 200 and 400 mg/kg of ethanolic leaf extract, respectively. After 1 h, CMC or absolute ethanol was given orally to groups 2–7. The rats were sacrificed after 1 h. Then, the injuries to the gastric mucosa were estimated through assessment of the gastric wall mucus, the gross appearance of ulcer areas, histology, immunohistochemistry and enzymatic assays. Group 2 exhibited significant mucosal injuries, with reduced gastric wall mucus and severe damage to the gastric mucosa, whereas reductions in mucosal injury were observed for groups 4–7. Groups 3–7 demonstrated a reversal in the decrease in Periodic acid-Schiff (PAS) staining induced by ethanol. No symptoms of toxicity or death were observed during the acute toxicity tests. Conclusion: Treatment with the extract led to the upregulation of heat-shock protein 70 (HSP70) and the downregulation of the pro-apoptotic protein BAX. Significant increases in the levels of the antioxidant defense enzymes glutathione (GSH) and superoxide dismutase (SOD) in the gastric mucosal homogenate were observed, whereas that of a lipid peroxidation marker (MDA) was significantly decreased. Significance was defined as p<0.05 compared to the ulcer control group (Group 2). [ABSTRACT FROM AUTHOR]

Withaferin A (WFA) is a natural product that binds to soluble forms of the type III intermediate filament (IF) vimentin. Currently, it is unknown under what pathophysiological contexts vimentin is druggable, as cytoskeltal vimentin-IFs are abundantly expressed. To investigate druggability of vimentin, we exploited rabbit Tenon's capsule fibroblast (RbTCF) cell cultures and the rabbit glaucoma filtration surgical (GFS) model of fibrosis. WFA potently caused G0/G1 cell cycle inhibition (IC50 25 nM) in RbTCFs, downregulating ubiquitin E3 ligase skp2 and inducing p27Kip1 expression. Transforming growth factor (TGF)-ß-induced myofibroblast transformation caused development of cell spheroids with numerous elongated invadopodia, which WFA blocked potently by downregulating soluble vimentin and α-smooth muscle actin (SMA) expression. In the pilot proof-of-concept study using the GFS model, subconjunctival injections of a low WFA dose reduced skp2 expression in Tenon's capsule and increased p27Kip1 expression without significant alteration to vimentin-IFs. This treatment maintains significant nanomolar WFA concentrations in anterior segment tissues that correspond to WFA's cell cycle targeting activity. A ten-fold higher WFA dose caused potent downregulation of soluble vimentin and skp2 expression, but as found in cell cultures, no further increase in p27Kip1 expression was observed. Instead, this high WFA dose potently induced vimentin-IF disruption and downregulated α-SMA expression that mimicked WFA activity in TGF-ß-treated RbTCFs that blocked cell contractile activity at submicromolar concentrations. These findings illuminate that localized WFA injection to ocular tissues exerts pharmacological control over the skp2-p27Kip1 pathway by targeting of soluble vimentin in a model of surgical fibrosis. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the use of liposomal irinotecan (Irinophore C™) plus or minus 5-fluorouracil (5-FU) for the treatment of colorectal cancer. Experimental Design: The effect of irinotecan (IRI) and/or 5-FU exposure times on cytotoxicity was assessed in vitro against HT-29 or LS174T human colon carcinoma cells. The pharmacokinetics and biodistribution of Irinophore C™ (IrC™) and 5-FU, administered alone or in combination, were compared in vivo. A subcutaneous model of HT-29 human colorectal cancer in Rag2-M mice was utilized to assess the efficacy of IrC™ alone, and in combination with 5-FU. Results: The cytotoxicity of IRI and 5-FU were strongly dependent on exposure time. Synergistic interactions were observed following prolonged exposure to IRI/5-FU combinations. Pharmacokinetics/biodistribution studies demonstrated that the 5-FU elimination rate was decreased significantly when 5-FU was co-administered intravenously with IrC™, versus alone. Significant decreases in 5-FU elimination were also observed in plasma, with an associated increase of 5-FU in some tissues when 5-FU was given by intraperitoneal injection and IrC™ was given intravenously. The elimination of IrC™ was not significantly different when administered alone or in combination with 5-FU. Therapeutic studies demonstrated that single agent IrC™ was significantly more effective than the combination of IRI/5-FU; surprisingly, IrC™/5-FU combinations were no more effective than IrC™ alone. The administration of combinations of 5-FU (16 mg/kg) and IrC™ (60 mg IRI/kg) showed increased toxicity when compared to IrC™ alone. Treatment with IrC™ alone (60 mg IRI/kg) delayed the time required for a 5-fold increase in initial tumor volume to day 49, compared to day 23 for controls. When IrC™ (40 mg IRI/kg) was used in combination with 5-FU (16 mg/kg), the time to increase tumor volume 5-fold was 43 days, which was comparable to that achieved when using IrC™ alone (40 mg IRI/kg). Conclusions: Single agent IrC™ was well tolerated and has significant therapeutic potential. IrC™ may be a suitable replacement for IRI treatment, but its use with free 5-FU is complicated by IrC™-engendered changes in 5-FU pharmacokinetics/biodistribution which are associated with increased toxicity when using the combination. [ABSTRACT FROM AUTHOR]

We have demonstrated that simple formulations composed of the parent drug in combination with generally regarded as safe (GRAS) permeability enhancers are capable of dramatically increasing the absolute bioavailability of zanamivir. This has the advantage of not requiring modification of the drug structure to promote absorption, thus reducing the regulatory challenges involved in conversion of an inhaled to oral route of administration of an approved drug. Absolute bioavailability increases of up to 24-fold were observed when Capmul MCM L8 (composed of mono- and diglycerides of caprylic/capric acids in glycerol) was mixed with 1.5 mg of zanamivir and administered intraduodenally to rats. Rapid uptake (tmax of 5 min) and a Cmax of over 7200 ng/mL was achieved. Variation of the drug load or amount of enhancer demonstrated a generally linear variation in absorption, indicating an ability to optimize a formulation for a desired outcome such as a targeted Cmax for enzyme saturation. No absorption enhancement was observed when the enhancer was given 2 hr prior to drug administration, indicating, in combination with the observed tmax, that absorption enhancement is temporary. This property is significant and aligns well with therapeutic applications to limit undesirable drug-drug interactions, potentially due to the presence of other poorly absorbed polar drugs. These results suggest that optimal human oral dosage forms of zanamivir should be enteric-coated gelcaps or softgels for intraduodenal release. There continues to be a strong need and market for multiple neuraminidase inhibitors for influenza treatment. Creation of orally available formulations of inhibitor drugs that are currently administered intravenously or by inhalation would provide a significant improvement in treatment of influenza. The very simple GRAS formulation components and anticipated dosage forms would require low manufacturing costs and yield enhanced convenience. These results are being utilized to design prototype dosage forms for initial human pharmacokinetic studies. [ABSTRACT FROM AUTHOR]

Plasma pharmacokinetics of ST-246, smallpox therapeutic, was evaluated in mice, rabbits, monkeys and dogs following repeat oral administrations by gavage. The dog showed the lowest Tmax of 0.83 h and the monkey, the highest value of 3.25 h. A 2- to 4-fold greater dose-normalized Cmax was observed for the dog compared to the other species. The mouse showed the highest dose-normalized AUC, which was 2-fold greater than that for the rabbit and monkey both of which by approximation, recorded the lowest value. The Cl/F increased across species from 0.05 L/h for mouse to 42.52 L/h for dog. The mouse showed the lowest VD/F of 0.41 L and the monkey, the highest VD/F of 392.95 L. The calculated extraction ratios were 0.104, 0.363, 0.231 and 0.591 for mouse, rabbit, monkey and dog, respectively. The dog showed the lowest terminal half-life of 3.10 h and the monkey, the highest value of 9.94 h. The simple allometric human VD/F and MLP-corrected Cl/F were 2311.51 L and 51.35 L/h, respectively, with calculated human extraction ratio of 0.153 and terminal half-life of 31.20 h. Overall, a species-specific difference was observed for Cl/F with this parameter increasing across species from mouse to dog. The human MLP-corrected Cl/F, terminal half-life, extraction ratios were in close proximity to the observed estimates. In addition, the first-in-humans (FIH) dose of 485 mg, determined from the MLP-corrected allometry Cl/F, was well within the dose range of 400 mg and 600 mg administered in healthy adult human volunteers. [ABSTRACT FROM AUTHOR]

The Hedgehog (Hh) pathway inhibitors have shown great promise in cancer therapeutics. SANT75, a novel compound we previously designed to specially inhibit the Smoothened (SMO) protein in the Hh pathway, has greater inhibitory potency than many of commonly used Hh inhibitors. However, preclinical studies of SANT75 revealed water insolubility and acute toxicity. To overcome these limitations, we developed a liposomal formulation of SANT75 and investigated its antitumor efficacy in vitro and in vivo. We encapsulated SANT75 into PEGylated liposome and the mean particle size distribution and zeta-potential (ZP) of liposomes were optimized. Using the Shh-light2 cell and Gli-GFP or Flk-GFP transgenic reporter zebrafish, we confirmed that liposome-encapsulated SANT75 inhibited Hh activity with similar potency as the original SANT75. SANT75 encapsulated into liposome exerted strong tumor growth-inhibiting effects in vitro and in vivo. In addition, the liposomal SANT75 therapy efficiently improved the survival time of tumor-bearing mice without obvious systemic toxicity. The pathological morphology and immunohistochemistry staining revealed that liposomal SANT75 induced tumor cell apoptosis, inhibited tumor angiogenesis as assessed by CD31 and down-regulated the expression of Hh target protein Gli-1 in tumor tissues. Our findings suggest that liposomal formulated SANT75 has improved solubility and bioavailability and should be further developed as a drug candidate for treating tumors with abnormally high Hh activity. [ABSTRACT FROM AUTHOR]

BYZX, [(E)-2-(4-((diethylamino)methyl)benzylidene)-5,6-dimethoxy-2,3-dihydroinden-one], belongs to a series of novel acetylcholinesterase inhibitors and has been synthesized as a new chemical entity for the treatment of Alzheimer’s disease symptoms. When incubated with human liver microsomes (HLMs), BYZX was rapidly transformed into its metabolites M1, M2, and M3. The chemical structures of these metabolites were identified using liquid chromatography tandem mass spectrometry and nuclear magnetic resonance, which indicated that M1 was an N-desethylated and C = C hydrogenation metabolite of BYZX. M2 and M3 were 2 precursor metabolites, which resulted from the hydrogenation and desethylation of BYZX, respectively. Further studies with chemical inhibitors and human recombinant cytochrome P450s (CYPs), and correlation studies were performed. The results indicated that the N-desethylation of BYZX and M2 was mediated by CYP3A4 and CYP2C8. The reduced form of β-nicotinamide adenine dinucleotide 2′-phosphate was involved in the hydrogenation of BYZX and M3, and this reaction occurred in the HLMs and in the human liver cytosol. The hydrogenation reaction was not inhibited by any chemical inhibitors of CYPs, but it was significantly inhibited by some substrates of α,β-ketoalkene C = C reductases and their inhibitors such as benzylideneacetone, dicoumarol, and indomethacin. Our results suggest that α,β-ketoalkene C = C reductases may play a role in the hydrogenation reaction, but this issue requires further clarification. [ABSTRACT FROM AUTHOR]

Heat-stress cognate 70 (Hsc70) is a host factor that helps hepatitis C virus (HCV) to complete its life cycle in infected hepatocytes. Using Hsc70 as a target for HCV inhibition, a series of novel N-substituted benzyl matrinic/sophoridinic acid derivatives was synthesized and evaluated for their anti-HCV activity in vitro. Among these analogues, compound 7c possessing N-p-methylbenzyl afforded an appealing ability to inhibit HCV replication with SI value over 53. Furthermore, it showed a good oral pharmacokinetic profile with area-under-curve (AUC) of 13.4 µM·h, and a considerably good safety in oral administration in mice (LD50>1000 mg/kg). As 7c suppresses HCV replication via an action mode distinctly different from that of the marketed anti-HCV drugs, it has been selected as a new mechanism anti-HCV candidate for further investigation, with an advantage of no or decreased chance to induce drug-resistant mutations. [ABSTRACT FROM AUTHOR]

Purpose: The PI3K/AKT/mTOR pathway is frequently dysregulated in cancers and inhibition of mTOR has demonstrated the ability to modulate pro-survival pathways. As such, we sought to determine the ability of the mTOR inhibitor everolimus to potentiate the antitumor effects of irinotecan in colorectal cancer (CRC). Experimental Design: The combinatorial effects of everolimus and irinotecan were evaluated in vitro and in vivo in CRC cell lines harboring commonly found mutations in PIK3CA, KRAS and/or BRAF. Pharmacokinetically-directed dosing protocols of everolimus and irinotecan were established and used to assess the in vivo antitumor effects of the agents. At the end of treatment, 3–6 tumors per treatment arm were harvested for biomarker analysis by NMR metabolomics. Results: Everolimus and irinotecan/SN38 demonstrated synergistic anti-proliferative effects in multiple CRC cell lines in vitro. Combination effects of everolimus and irinotecan were determined in CRC xenograft models using clinically-relevant dosing protocols. Everolimus demonstrated significant tumor growth inhibition alone and when combined with irinotecan in HT29 and HCT116 tumor xenografts. Metabolomic analysis showed that HT29 tumors were more metabolically responsive than HCT116 tumors. Everolimus caused a decrease in glycolysis in both tumor types whilst irinotecan treatment resulted in a profound accumulation of lipids in HT29 tumors indicating a cytotoxic effect. Conclusions: Quantitative analysis of tumor growth and metabolomic data showed that the combination of everolimus and irinotecan was more beneficial in the BRAF/PIK3CA mutant HT29 tumor xenografts, which had an additive effect, than the KRAS/PIK3CA mutant HCT116 tumor xenografts, which had a less than additive effect. [ABSTRACT FROM AUTHOR]

Background: Pancreatic cancer is one of the deadliest of all human malignancies with limited options for therapy. Here, we report the development of an optimized targeted drug delivery system to inhibit advanced stage pancreatic tumor growth in an orthotopic mouse model. Method/Principal Findings: Targeting specificity in vitro was confirmed by preincubation of the pancreatic cancer cells with C225 as well as Nitrobenzylthioinosine (NBMPR - nucleoside transporter (NT) inhibitor). Upon nanoconjugation functional activity of gemcitabine was retained as tested using a thymidine incorporation assay. Significant stability of the nanoconjugates was maintained, with only 12% release of gemcitabine over a 24-hour period in mouse plasma. Finally, an in vivo study demonstrated the inhibition of tumor growth through targeted delivery of a low dose of gemcitabine in an orthotopic model of pancreatic cancer, mimicking an advanced stage of the disease. Conclusion: We demonstrated in this study that the gold nanoparticle-based therapeutic containing gemcitabine inhibited tumor growth in an advanced stage of the disease in an orthotopic model of pancreatic cancer. Future work would focus on understanding the pharmacokinetics and combining active targeting with passive targeting to further improve the therapeutic efficacy and increase survival. [ABSTRACT FROM AUTHOR]

Background: Despite promising early results, clinical trials involving the continuous delivery of recombinant methionyl human glial cell line-derived neurotrophic factor (r-metHuGDNF) into the putamen for the treatment of Parkinson's disease have shown evidence of poor distribution and toxicity due to point-source accumulation. Convection-enhanced delivery (CED) has the potential to facilitate more widespread and clinically effective drug distribution. Aims: We investigated acute CED of r-metHuGDNF into the striatum of normal rats in order to assess tissue clearance, toxicity (neuron loss, gliosis, microglial activation, and decreases in synaptophysin), synaptogenesis and neurite-outgrowth. We investigated a range of clinically relevant infused concentrations (0.1, 0.2, 0.6 and 1.0 µg/µL) and time points (2 and 4 weeks) in order to rationalise a dosing regimen suitable for clinical translation. Results: Two weeks after single dose CED, r-metHuGDNF was below the limit of detection by ELISA but detectable by immunohistochemistry when infused at low concentrations (0.1 and 0.2 µg/µL). At these concentrations, there was no associated neuronal loss (neuronal nuclei, NeuN, immunohistochemistry) or synaptic toxicity (synaptophysin ELISA). CED at an infused concentration of 0.2 µg/µL was associated with a significant increase in synaptogenesis (p<0.01). In contrast, high concentrations of r-metHuGDNF (above 0.6 µg/µL) were associated with neuronal and synaptic toxicity (p<0.01). Markers for gliosis (glial fibrillary acidic protein, GFAP) and microglia (ionized calcium-binding adapter molecule 1, Iba1) were restricted to the needle track and the presence of microglia had diminished by 4 weeks post-infusion. No change in neurite outgrowth (Growth associated protein 43, GAP43, mRNA) compared to artificial cerebral spinal fluid (aCSF) control was observed with any infused concentration. Conclusion: The results of this study suggest that acute CED of low concentrations of GDNF, with dosing intervals determined by tissue clearance, has most potential for effective clinical translation by optimising distribution and minimising the risk of toxic accumulation. [ABSTRACT FROM AUTHOR]

Gastric cancer is the second common cause of cancer related death worldwide and lacks highly effective treatment for advanced disease. Nab-paclitaxel is a novel microtubule-inhibitory cytotoxic agent that has not been tested in gastric cancer as of yet. In this study, human gastric cancer cell lines AGS, NCI-N87 and SNU16 were studied. Nab-paclitaxel inhibited cell proliferation with an IC50 of 5 nM in SNU16, 23 nM in AGS and 49 nM in NCI-N87 cells after 72-hour treatment, which was lower than that of oxaliplatin (1.05 μM to 1.51 μM) and epirubicin (0.12 μM to 0.25 μM). Nab-paclitaxel treatment increased expression of the mitotic-spindle associated phospho-stathmin irrespective of the baseline total or phosphorylated stathmin level, and induced mitotic cell death as confirmed through increased expression of cleaved-PARP and caspase-3. After a two-week nab-paclitaxel, oxaliplatin or epirubicin treatment, the average in vivo local tumor growth inhibition rate was 77, 17.2 and 21.4 percent, respectively (p = 0.002). Effects of therapy on tumoral proliferative and apoptotic indices corresponded with tumor growth inhibition data, while expression of phospho-stathmin also increased in tissues. There was an increase in median animal survival after nab-paclitaxel treatment (93 days) compared to controls (31 days, p = 0.0007), oxaliplatin (40 days, p = 0.0007) or to docetaxel therapy (81 days, p = 0.0416). The strong antitumor activity of nab-paclitaxel in experimental gastric cancer supports such microtubule-inhibitory strategy for clinical application. Nab-paclitaxel benefits were observed independent from phosphorylated stathmin expression at baseline, putting into question the consideration of nab-paclitaxel use in gastric cancer based on this putative biomarker. [ABSTRACT FROM AUTHOR]

Interferon alpha (IFNα) is used for the treatment of hepatitis C infection and whilst efficacious it is associated with multiple adverse events including reduced leukocyte, erythrocyte, and platelet counts, fatigue, and depression. These events are most likely caused by systemic exposure to interferon. We therefore hypothesise that targeting the therapeutic directly to the intended site of action in the liver would reduce exposure in blood and peripheral tissue and hence improve the safety and tolerability of IFNα therapy. We genetically fused IFN to a domain antibody (dAb) specific to a hepatocyte restricted antigen, asialoglycoprotein receptor (ASGPR). Our results show that the murine IFNα2 homolog (mIFNα2) fused to an ASGPR specific dAb, termed DOM26h-196-61, could be expressed in mammalian tissue culture systems and retains the desirable biophysical properties and activity of both fusion partners when measured in vitro. Furthermore a clear increase in in vivo targeting of the liver by mIFNα2-ASGPR dAb fusion protein, compared to that observed with either unfused mIFNα2 or mIFNα2 fused to an isotype control dAb VHD2 (which does not bind ASGPR) was demonstrated using microSPECT imaging. We suggest that these findings may be applicable in the development of a liver-targeted human IFN molecule with improved safety and patient compliance in comparison to the current standard of care, which could ultimately be used as a treatment for human hepatitis virus infections. [ABSTRACT FROM AUTHOR]

1,5-Diphenyl pyrroles were previously identified as a class of compounds endowed with high in vitro efficacy against M. tuberculosis. To improve the physical chemical properties and drug-like parameters of this class of compounds, a medicinal chemistry effort was undertaken. By selecting the optimal substitution patterns for the phenyl rings at N1 and C5 and by replacing the thiomorpholine moiety with a morpholine one, a new series of compounds was produced. The replacement of the sulfur with oxygen gave compounds with lower lipophilicity and improved invitro microsomal stability. Moreover, since the parent compound of this family has been shown to target MmpL3, mycobacterial mutants resistant to two compounds have been isolated and characterized by sequencing the mmpL3 gene; all the mutants showed point mutations in this gene. The best compound identified to date was progressed to dose-response studies in an acute murine TB infection model. The resulting ED99 of 49 mg/Kg is within the range of commonly employed tuberculosis drugs, demonstrating the potential of this chemical series. The in vitro and in vivo target validation evidence presented here adds further weight to MmpL3 as a druggable target of interest for anti-tubercular drug discovery. [ABSTRACT FROM AUTHOR]

Background: Caenorhbditis elegans has being vigorously used as a model organism in many research fields and often accompanied by administrating with various drugs. The methods of delivering drugs to worms are varied from one study to another, which make difficult in comparing results between studies. Methodology/Principal Findings: We evaluated the drug absorption efficiency in C. elegans using five frequently used methods with resveratrol with low aqueous solubility and water-soluble 5-Fluoro-2′-deoxyuridine (FUDR) as positive compounds. The drugs were either applied to the LB medium with bacteria OP50, before spreading onto Nematode Growth Medium (NGM) plates (LB medium method), or to the NGM with live (NGM live method) or dead bacteria (NGM dead method), or spotting the drug solution to the surface of plates directly (spot dead method), or growing the worms in liquid medium (liquid growing method). The concentration of resveratrol and FUDR increased gradually within C. elegans and reached the highest during 12 hours to one day and then decreased slowly. At the same time point, the higher the drug concentration, the higher the metabolism rate. The drug concentrations in worms fed with dead bacteria were higher than with live bacteria at the same time point. Consistently, the drug concentration in medium with live bacteria decreased much faster than in medium with dead bacteria, reach to about half of the original concentration within 12 hours. Conclusion: Resveratrol with low aqueous solubility and water-soluble FUDR have the same absorption and metabolism pattern. The drug metabolism rate in worms was both dosage and time dependent. NGM dead method and liquid growing method achieved the best absorption efficiency in worms. The drug concentration within worms was comparable with that in mice, providing a bridge for dose translation from worms to mammals. [ABSTRACT FROM AUTHOR]

Background and Purpose: Retention of substances from systemic circulation in the brain and testes are limited due to high levels of P-glycoprotein (P-gp) in the luminal membranes of brain and testes capillary endothelial cells. From a clinical perspective, P-gp rapidly extrudes lipophilic therapeutic agents, which then fail to reach efficacious levels. Recent studies have demonstrated that acute administration of selective serotonin reuptake inhibitors (SSRI) can affect P-gp function, in vitro and in vivo. However, little is known concerning the time-course of these effects or the effects of different SSRI in vivo. Experimental Approach: The P-gp substrate, tritiated digoxin ([3H] digoxin), was co-administered with fluoxetine or sertraline to determine if either compound increased drug accumulation within the brains and testes of mice due to inhibition of P-gp activity. We undertook parallel studies in endothelial cells derived from brain microvessels to determine the dose-response and time-course of effects. Key Results: In vitro, sertraline resulted in rapid and potent inhibition of P-gp function in brain endothelial cells, as determined by cellular calcein accumulation. In vivo, a biphasic effect was demonstrated. Brain accumulation of [3H] digoxin was increased 5 minutes after treatment with sertraline, but by 60 minutes after sertraline treatment, brain accumulation of digoxin was reduced compared to control. By 240 minutes after sertraline treatment brain digoxin accumulation was elevated compared to control. A similar pattern of results was obtained in the testes. There was no significant effect of fluoxetine on P-gp function, in vitro or in vivo. Conclusions and Implications: Acute sertraline administration can modulate P-gp activity in the blood-brain barrier and blood-testes barrier. This clearly has implications for the ability of therapeutic agents that are P-gp substrates, to enter the brain when co-administered with SSRI. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β (TGF-β) is a major pro-fibrotic cytokine, causing the overproduction of extracellular matrix molecules in many fibrotic diseases. Inhibition of its type-I receptor (ALK5) has been shown to effectively inhibit fibrosis in animal models. However, apart from its pro-fibrotic effects, TGF-β also has a regulatory role in the immune system and influences tumorigenesis, which limits the use of inhibitors. We therefore explored the cell-specific delivery of an ALK5-inhibitor to hepatic stellate cells, a key cell in the development of liver fibrosis. We synthesized a conjugate of the ALK5-inhibitor LY-364947 coupled to mannose-6-phosphate human serum albumin (M6PHSA), which binds to the insulin-like growth factor II receptor on activated HSC. The effectivity of the conjugate was evaluated in primary HSC and in an acute liver injury model in mice. In vitro, the free drug and the conjugate significantly inhibited fibrotic markers in HSC. In hepatocytes, TGF-β-dependent signaling was inhibited by free drug, but not by the conjugate, thus showing its cell-specificity. In vivo, the conjugate localized in desmin-positive cells in the liver and not in hepatocytes or immune cells. In the acute liver injury model in mice, the conjugate reduced fibrogenic markers and collagen deposition more effectively than free drug. We conclude that we can specifically deliver an ALK5-inhibitor to HSC using the M6PHSA carrier and that this targeted drug reduces fibrogenic parameters in vivo, without affecting other cell-types. [ABSTRACT FROM AUTHOR]

Interleukin-1beta (IL-1β) is a major cause for induction of various inflammatory mechanisms that are decisively involved to provoke pathogenesis of type 2 diabetes mellitus (T2DM). Interleukin-1 receptor antagonist (IL-1Ra) a naturally occurring anti-inflammatory antagonist of IL-1β has been recently approved for treatment of T2DM but due to its short half-life, higher doses and frequent dosing intervals are required. Pluronic F-127 (PF127) has previously shown to prolong the release of various proteinous drugs and their serum half-lives. Subsequently, in our previous work, we developed a new dosage form of IL-1Ra using PF127 and investigated its in-vitro and in-vivo effects. Here in present work, we have extended this approach using diabetic Goto-kakizaki (GK) rats. We administered IL-1Ra loaded in PF127 gel subcutaneously for one month into GK rats. IL-1Ra loaded in PF127 gel exhibited a sustained and prolonged hypoglycemic effects on treated animals. Intraperitoneal glucose tolerance test (IPGTT) results showed that IL-1Ra loaded in PF127 gel increased glucose tolerance along with increased insulin sensitivity and β-cell’s secretory function in treated rats. Moreover, significant reduction in pro-insulin/insulin ratio, lipid profiles and interleukin 6 (IL-6) were also observed. Immunohistochemical analysis showed slight macrophages infiltration in pancreatic islets. Histochemical analysis revealed no PF127-induced alteration in the normal physiology of skin and kidney of treated animals. Hence, we concluded that IL-1Ra loaded in PF127 gel has potential to exhibit broad spectrum anti-inflammatory effects alleviating the symptoms of T2DM. [ABSTRACT FROM AUTHOR]

Although the pyrazolone derivative sulpyrine is widely used as an antipyretic analgesic drug, side effects, including fatal shock, have been reported. However, the molecular mechanism underlying such a severe side effect is largely unclear. Here, we report that the transcription factor CREBH that is highly expressed in the liver plays an important role in fatal shock induced by sulpyrine in mice. CREBH-deficient mice were resistant to experimental fatal sulpyrine shock. We found that sulpyrine-induced expression of cytochrome P450 2B (CYP2B) family genes, which are involved in sulpyrine metabolism, in the liver was severely impaired in CREBH-deficient mice. Moreover, introduction of CYP2B in CREBH-deficient liver restored susceptibility to sulpyrine. Furthermore, ectopic expression of CREBH up-regulated CYP2B10 promoter activity, and in vivo knockdown of CREBH in wild-type mice conferred a significant resistance to fatal sulpyrine shock. These data demonstrate that CREBH is a positive regulator of CYP2B in response to sulpyrine administration, which possibly results in fatal shock. [ABSTRACT FROM AUTHOR]

This study aimed to compare the anti-neoplastic effects of an Uncaria tomentosa (UT) brute hydroethanolic (BHE) extract with those of two fractions derived from it. These fractions are choroformic (CHCl3) and n-butanolic (BuOH), rich in pentacyclic oxindole alkaloids (POA) and antioxidant substances, respectively. The cancer model was the subcutaneous inoculation of Walker-256 tumour cells in the pelvic limb of male Wistar rat. Subsequently to the inoculation, gavage with BHE extract (50 mg.kg−1) or its fractions (as per the yield of the fractioning process) or vehicle (Control) was performed during 14 days. Baseline values, corresponding to individuals without tumour or treatment with UT, were also included. After treatment, tumour volume and mass, plasma biochemistry, oxidative stress in liver and tumour, TNF-α level in liver and tumour homogenates, and survival rates were analysed. Both the BHE extract and its BuOH fraction successfully reduced tumour weight and volume, and modulated anti-oxidant systems. The hepatic TNF-α level indicated a greater effect from the BHE extract as compared to its BuOH fraction. Importantly, both the BHE extract and its BuOH fraction increased the survival time of the tumour-bearing animals. Inversely, the CHCl3 fraction was ineffective. These data represent an in vivo demonstration of the importance of the modulation of oxidative stress as part of the anti-neoplastic activity of UT, as well as constitute evidence of the lack of activity of isolated POAs in the primary tumour of this tumour lineage. These effects are possibly resulting from a synergic combination of substances, most of them with antioxidant properties. [ABSTRACT FROM AUTHOR]