European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences

Published by Elsevier
Print ISSN: 0928-0987
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We have evaluated the chemical and microbiological stability of sufentanil citrate, levobupivacaine hydrochloride and a mixture in a 0.9% sodium chloride infusion in order to provide background information on the storage of a sufentanil-levobupivacaine mixture in polypropylene (PP) syringes. Chemical assays were performed by HPLC on days 0, 1, 2, 3, 8, 14, 23, 28 and 30 after storage at 4, 21, and 36 degrees C. Microbiological stability was evaluated under aseptic conditions using a laminar air flow station, with a grade A environment and a B background. The samples taken for microbiological analysis were collected immediately after preparation of the solutions and then after 7, 14, 21 and 28 days storage. At 4 degrees C the sufentanil citrate solution was stable for 23 days. At 21 degrees C the sufentanil citrate solution maintained chemical stability for 3 days, but thereafter the concentration of sufentanil decreased 15% from day 3 to day 8. At 36 degrees C a similar decrease was noticed from day 1 to day 3. On the contrary, the levobupivacaine hydrochloride solution maintained chemical stability for 28 days at 4 and 21 degrees C and for 23 days at 36 degrees C. The sufentanil-levobupivacaine mixture maintained chemical stability for 28 days at 4, 21 and 36 degrees C. The sufentanil and levobupivacaine solutions and the mixture studied maintained microbiological stability for 28 days. According to the chemical and microbiological stability studies, the sufentanil-levobupivacaine mixture in PP syringes could be stored for 28 days at 4 and 21 degrees C.
 
Inhibitors of dipeptidyl peptidase-4 (DPP-IV), a key regulator of the actions of incretin hormones, exert antihyperglycemic effects in type 2 diabetic patients. A major question concerns the potential ability of long term DPP-IV inhibition to have beneficial disease-modifying effects, specifically to attenuate loss of pancreatic β-cell mass due to oxidative stress induced inflammation. Here, we investigated the effects of a potent and selective DPP-4 inhibitor, an analog of vildagliptin (PKF-275-055), on glycemic control, pancreatic β-cell mass, genes and proteins expressions, tumor necrosis factor-alpha, and nitric oxide in an n2-STZ diabetic model of rat with defects in insulin sensitivity and secretion. To induce NIDDM, streptozotocin (STZ) 90 mg/kg was administered i.p. to a group of 2 days old pups. Diabetic rats were administered orally with vildagliptin analog PKF-275-055. Saline treated animals served as diabetic control. Significant and dose-dependent correction of postprandial hyperglycemia was observed in diabetic rats following 8 weeks of chronic therapy. Treatment with PKF-275-055 showed increased the number of insulin-positive β-cells in islets and improved the expressions of genes and proteins are responsible for insulin secretions. In addition, treatment of rats with PKF-275-055 significantly increased insulin content, glycogen content and total proteins content; and decreased the inflammatory markers i.e. nitric oxide and TNF-alpha. The present studies indicate that PKF-275-055 is a novel selective DPP-IV inhibitor having potential to reduce inflammation that might be a potential agent for type 2 diabetes.
 
The new anti-inflammatory agents 6-methyl-3-isopropyl-2H-1,2-benzothiazin-4(3H)-one 1,1-dioxide 6a and its analogues 6b-f were synthesized from L-valine. All compounds were characterized by physical, chemical and spectral studies. Preliminary pharmacological evaluation of the resulting products showed that compounds 6a-f (5-20 mg/kg, i.p.) are active anti-inflammatory agents in carrageenan-induced rat paw oedema assay in albino rats, and their effects are comparable to that of piroxicam (5 mg/kg, i.p.), used as a reference drug. The nature of the substituents on the sulfonamide nitrogen and those on position three had a pronounced effect on the anti-inflammatory activity. Studies of structure-activity relationships have led to selection of compound 2,6-dimethyl-3-isopropyl-1,2-benzothiazin-3,4-diol 1,1-dioxide 6 f which exhibited the most potent activity (61.7% inhibition at 5 mg/kg, i.p. and ED(50)=4.5 mg/kg, i.p.). Comparison of the gastrointestinal safety of compounds 6a-f with that of piroxicam showed a far better tolerability for our products. This comparison was based on the ulcer index and the pH of gastric content.
 
1,1-Bis(3-indolyl)-1-(p-substitutedphenyl)methane (C-DIM) compounds exhibit remarkable antitumor activity with low toxicity in various cancer cells including lung tumors. Two C-DIM analogs, DIM-C-pPhOCH3 (C-DIM-5) and DIM-C-pPhOH (C-DIM-8) while acting differentially on the orphan nuclear receptor, TR3/Nur77 inhibited cell cycle progression from G0/G1 to S-phase and induced apoptosis in A549 cells. Combinations of docetaxel (doc) with C-DIM-5 or C-DIM-8 showed synergistic anticancer activity in vitro and these results were consistent with their enhanced antitumor activities in vivo. Respirable aqueous formulations of C-DIM-5 (mass median aerodynamic diameter of 1.92 ± 0.22 um and geometric standard deviation of 2.31 ± 0.12) and C-DIM-8 (mass median aerodynamic diameter of 1.84 ± 0.31 um and geometric standard deviation of 2.11 ± 0.15) were successfully delivered by inhalation to athymic nude mice bearing A549 cells as metastatic tumors. This resulted in significant (p<0.05) lung tumor regression and an overall reduction in tumor burden. Analysis of lung tumors from mice treated with inhalational formulations of C-DIM-5 and C-DIM-8 showed decreased mRNA and protein expression of mediators of tumor initiation, metastasis, and angiogenesis including MMP2, MMP9, c-Myc, β-catenin, c-Met, c-Myc, and EGFR. Microvessel density assessment of lung tissue sections showed significant reduction (p<0.05) in angiogenesis and metastasis as evidenced by decreased distribution of immunohistochemical staining of VEGF, and CD31. Our studies demonstrate both C-DIM-5 and C-DIM-8 have similar anticancer profiles in treating metastatic lung cancer and possibly work as TR3 inactivators.
 
We characterized the morphology, structure and supramolecular organization of microparticles obtained by spray drying 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and hyaluronic acid (HA). Pure DPPC microparticles are small and strongly aggregated with phospholipids organized in a lamellar-like structure observable by scanning electron microscopy (SEM). X-ray scattering demonstrates that it corresponds to an almost dry lamellar phase with chains tilted with respect to the bilayer surface and organized according to a hexagonal lattice within the bilayer. Upon aging, DPPC reorganizes into an orthorhombic structure within the bilayer. The addition of HA leads to an increase of particle size and a decrease of aggregation and tap density associated to a morphology switch from dense spheres to hollow shells. By contrast, the supramolecular organization is not modified: HA is mostly "sandwiched" between DPPC headgroups. In addition, HA impedes phospholipids rearrangement upon aging. Altogether, for drug delivery purposes, the addition of HA is beneficial in terms of stability and physical properties.
 
5-Imino-1,2,4-thiadiazoles and quinazolines derivatives as glycogen synthase kinase 3β (GSK-3β) and phosphodiesterase 7 (PDE7) inhibitors were characterized for their ability to pass the blood-brain barrier (BBB) together with their human serum albumin (HSA) binding using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). To study the blood-brain barrier penetration, a parallel artificial membrane permeability assay (PAMPA) using a porcine brain lipid was employed. For the HPLAC investigation, HSA was previously covalently immobilized to the silica matrix of the HPLC column. This HSA-based column was used to characterize the high affinity binding sites of 5-imino-1,2,4-thiadiazoles and quinazolines derivatives to HSA. Displacement experiments in the presence of increasing concentrations of competitors known to bind selectively to the main binding sites of HSA were carried out to determine their possible binding site. The same drug-protein system was studied by CD. The analysed compounds were able to pass BBB, they present good drug-like properties and they showed a high affinity to HSA. Competition experiments showed an anticooperative interaction at sites I and II, and an independent binding at bilirubin binding site on HSA.
 
The transfer of ionic species of three beta-blockers (propranolol, sotalol and timolol) has been studied by cyclic voltammetry at a macroscopic water 1,2-dichloroethane (1,2-DCE) interface. The aqueous solution has been gellified in order to study the effect of the gel on the transport properties of the drugs. The gelling agent also stabilizes the interface overcoming mechanical instability. The standard potential and standard Gibbs energy of transfer across the interface, the partition coefficient and the diffusion coefficient of each drug were determined in the presence of a gelled interface. The diffusion coefficients were shifted relative to those obtained at normal water 1,2-DCE interfaces (free of gel).
 
4-[(1,2-Dihydro-2-oxo-3H-indol-3-ylidene) amino]-N(4,6-dimethyl-2-pyrimidinyl)-benzene sulphonamide and its derivatives were synthesized by reaction of isatin and its derivatives with sulphadimidine. Their chemical structures have been confirmed by IR, (1)H NMR data and elemental analysis. Investigation of anti-HIV activity of compounds were tested against replication of HIV-1 (IIIB) and HIV-2 (ROD) strains in acutely infected MT-4 cells and the activity compared with standard azidothymidine. Among the compounds tested, 4-[(1,2-dihydro-2 oxo-3H-indol-3-ylidene)amino]-N(4,6-dimethyl-2-pyrimidinyl)-benzene sulphonamide and its N-acetyl derivative were the most active compounds.
 
Tricyclic analogs of melatonin with alkyl and cycloalkyl moieties in the beta position of the ethylamido chain have been prepared and tested for their ability to activate pigment granule aggregation in Xenopus laevis melanophores. The introduction of two methyl groups in the beta position of the side-chain of the methoxyl-substituted ligands induces a synergistic effect in agonist potency, which, importantly, is maintained after the methoxyl substituent is removed. The presence of more bulky beta-substituents, regardless of the size of the R group, seems to lead to antagonism.
 
In spite of the development of new anticancer drugs by the pharmaceutical industry, melanoma and T lymphomas are diseases for which medical advances remain limited. Thus, there was an urgent need of new therapeutics with an original mechanism of action. Since several years, our group develops quinoxalinic compounds. In this paper, the first preclinical results concerning one lead compound, EAPB0203, are presented. This compound exhibits in vitro cytotoxic activity on A375 and M4Be human melanoma cell lines superior to that of imiquimod and fotemustine. A liquid chromatography-mass spectrometry method was first validated to simultaneously quantify EAPB0203 and its metabolite, EAPB0202, in rat plasma. Thereafter, the pharmacokinetic profiles of EAPB0203 were studied in rat after intravenous and intraperitoneal administrations. After intraperitoneal administration the absolute bioavailability remains limited (22.7%). In xenografted mouse, after intraperitoneal administration of 5 and 20mg/kg, EAPB0203 is more potent than fotemustine. The survival time was increased up to 4 and 2 weeks compared to control mice and mice treated by fotemustine, respectively. The results of this study demonstrate the relationship between the dose of EAPB0203 and its effects on tumor growth. Thus, promising efficacy, tolerance and pharmacokinetic data of EAPB0203 encourage the development towards patient benefit.
 
Four 2,3-diarylimidazo[1,2-a]pyridines (I, 1a-c) were synthesized as inhibitors of UV-induced apoptosis and showed quite different properties. First, only the pyridinyl derivative I showed protection in molt cells. From the supposed intracellular target, phospholipid membrane models were studied by (1)H, (2)H and (31)P NMR spectroscopy. All these molecules can incorporate the membrane bilayer of small unilamellar vesicles of lecithin (SUV). However, I is clearly closed to the external polar head of the lipids, and is relatively mobile in the layer. Conversely, the other molecules are strongly immobilized in the deep part of the external layer. (31)P solid-state NMR spectra recorded on phospholipid dispersions (multilayers vesicles (MLV)) completely excluded any detergent effect or any modification of temperature transition. The only structural or dynamic effect observed was a homogeneous, but limited, reduction in the chemical shift anisotropy in the presence of I, in agreement with its superficial location. (2)H NMR experiment performed on the same model using perdeuterated phospholipids showed no significant fluidity reduction at the level of terminal CD(3) groups in the presence of 1a-c, according to their deep location. Finally, their interactions with synthetic oligonucleotide, d(CGATCG)(2) was studied showing non specific interactions of 1a on the external GC pair, while no interaction was observed with the other derivatives.
 
Chloral hydrate, the oldest synthetic hypnotic, is still among the most common agents used for conscious sedation of infants and children. Chloral (Clo) spontaneously condenses with the biogenic amine tryptamine (Ta) giving rise to the endogenous formation of 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo). TaClo constitutes a mammalian alkaloid and a potent neurotoxin, which is capable of inducing a slowly developing degeneration of the dopaminergic system in rats. Due to the late onset of parkinsonian-type symptoms after TaClo administration, this agent has been postulated to be a potential natural inducer of Parkinson's disease. In order to elucidate its pharmacokinetics, its tissue distribution and excretion profile, radiolabelled [3-14C]-TaClo was prepared in a convenient four-step synthetic pathway. Studies on rats intraperitoneally treated with a single dose of 2.1 mg/kg (0.6 microCi/kg) of [3-14C]-TaClo.HCl (specific activity: 0.28 microCi/mg) revealed the radioactivity to be rapidly incorporated with the highest concentrations of 14C found in the excretory organs. [3-14C]-TaClo was poorly absorbed systemically, as indicated by the very low plasma radioactivity levels. Maximum levels of 14C were reached between 2 and 6 h postdose, with the exception of large intestine peaking at 12 h after dosing. Total mean percent recovery of the radioactive dose was about 96% within the 48-h period examined. Urinary excretion accounted for ca. 34.5%, with the majority of the applied dose being eliminated by the hepatobiliary pathway.
 
We investigated neurochemically and neuropathologically the utility of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice as a model of Parkinson's disease. The changes in dopamine D1 and D2 receptors and dopamine uptake sites were determined by quantitative autoradiography using [3H]SCH23390, [3H]raclopride and [3H]mazindol, respectively. Dopamine and 3,4-dihydroxyphenyl acetic acid (DOPAC) contents in the striatum were measured by high-performance liquid chromatography. The distribution of nigral neurons and reactive astrocytes was determined by immunohistochemical staining with antibody against tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP). The mice received four intraperitoneal injections of MPTP (10 mg/kg) at 1-h intervals and then the brains were analyzed at 3 and 7 days after the treatments. No significant change in dopamine D1 receptors was observed in the striatum and substantia nigra after acute treatment with MPTP. Dopamine D2 receptors were reduced significantly in the substantia nigra only 7 days after the MPTP treatment, whereas striatum showed no significant change in the binding throughout the experiments. In contrast, dopamine uptake sites were reduced markedly in the striatum and substantia nigra 3 and 7 days after the MPTP treatment. Dopamine and DOPAC content were also reduced in the striatum 3 and 7 days after the MPTP treatment. An immunohistochemical study indicated a loss of the number of TH-positive neurons in the substantia nigra 7 days after the MPTP treatment. In contrast, numerous GFAP-positive astrocytes were evident in the striatum 7 days after the MPTP treatment. These results provide valuable information for the pathogenesis of acute stage of Parkinson's disease.
 
A series of 3,7-dimethyl-pyrazolo[3,4-e][1,2,4]triazin-4-yl thiosemicarbazide derivatives 3-22 were prepared and evaluated in vitro against HM1:1MSS strain of Entamoeba histolytica, to identify the compounds for antiamoebic activity. They exhibited antiamoebic activity in the range (IC50=0.81-7.31microM). The results were compared to the activity of known drug metronidazole. It is inferred from the in vitro studies that the compounds 10, 11, 17 and 18 were found to be significantly better inhibitors of E. histolytica since IC50 values in the muM range elicited by these compounds are much lower than metronidazole. Besides, compounds 11 and 17 have shown the most promising antiamoebic activity (IC50=0.81microM of 11, IC50=0.84 microM of 17 versus IC50=1.81microM of metronidazole). The study suggests the possibility of developing triazine analogues as potential drug candidates for antiamoebic activity.
 
Structures I-IX based on a drug-like 7,8-dihydroimidazo[2,1-c][1,2,4]triazin-4(6H)-one scaffold (Sztanke, 2009; Sztanke et al., 2006a, 2008a, 2009; Sztanke and Tkaczyn´skiTkaczyn´Tkaczyn´ski, 1997a; Kandefer-SzerszenétSzerszenét al., 2014; Szuster-Ciesielska et al., 2012).
General structure for 8-(4-chlorophenyl)-4-oxo-4,6,7,8-tetrahydroimidazo[2,1-c][1,2,4]triazine-3-carbohydrazide (20) with an atom numbering of the bicyclic scaffold.
Terms for the linear correlation expressed by Eq. (2) (log k = log k w À Su) for the
The chromatographic behaviour and significant lipophilicity/hydrophobicity indices (log kw, S, φ0) are presented for 21 biologically active fused 1,2,4-triazinones based on the linear relationship: log k = log kw - Sφ established for the retention on LC-18 HPLC column, using as mobile phases mixtures of three organic modifiers with water. The effect of these mobile phase modifiers on the chromatographic behaviour of solutes was established and the organic modifier of choice is suggested. The complex correlation of slopes versus intercepts obtained for acetonitrile, contrary to linear ones obtained for methanol and dioxane are disclosed. The observed difference in retention mechanism for acetonitrile compared to methanol and dioxane is explained by intermolecular interactions encoded in lipophilicity. Linear correlations with statistically significant levels between log kw values determined from three different chromatographic systems were obtained. The relationships between log kw constants (derived from the linear model for methanol-water mobile phases) and predicted log P and log S values by the use of various computational methods were investigated and these were established with high correlation coefficients. The predicted log P values plotted against φ0 (MeOH) indices showed the best fit. Principal component analysis was used to compare various lipophilicity parameters of the solutes and their in silico biological descriptors relevant to optimal pharmacokinetics profile. The similarities and dissimilarities between all the variables and molecular structures of solutes are presented. Statistically significant correlations were found between the chromatographic lipophilicity indices and the calculated pharmacokinetic descriptors: fraction unbound in brain (fu, brain), oral bioavailability (%F), permeability and intestinal absorption in jejunum (Caco-2), skin permeation (log Kp) and blood/brain concentration (log BB). Copyright © 2014. Published by Elsevier B.V.
 
The inhibitory effects of a series of 5,6-dihydro-4H-1,3-selenazine derivatives, 1,3-selenazole, and 5,6-dihydro-4H-1,3-thiazine derivatives on the proliferation of human HT-1080 fibrosarcoma cells were investigated. The compounds 4-ethyl-4-hydroxy-2-p-tolyl-5, 6-dihydro-4H-1,3-selenazine (TS-2) and 4-hydroxy-4-methyl-6-propyl-2-p-tolyl-5,6-dihydro-4H-1,3-selenazine++ + (TS-6) exhibited the strongest inhibitory effect among 1, 3-selenazine derivatives, and the EC(50) of TS-2 and TS-6 was 7.76 and 8.40 microM, respectively. On the other hand, 1,3-selenazole and 5,6-dihydro-4H-1,3-thiazines had no inhibitory effects. TS-2 and TS-6 inhibited the proliferation of HT-1080 cells time- and dose-dependently. They induced dose-dependent DNA fragmentation in HT-1080 cells, revealing a typical apoptosis characteristics. The present study demonstrated that TS-2 and TS-6 inhibited HT-1080 proliferation through the induction of DNA fragmentation.
 
A series of eight 5,6-dihydro-4H-1,3-thiazine derivatives was synthesized by the BF3 x Et2O-catalyzed reaction of selected alpha,beta-unsaturated ketones with thiobenzamide at room temperature. The antimycobacterial activities of these compounds were determined against Mycobacterium tuberculosis H37Rv (ATCC 27294) using the Alamar blue susceptibility assay. Three compounds, 5-hydroxy-3-phenyl-4-aza-2-thiabicyclo[3.3.1]none-3-ene 3a, 4-hydroxy-4-methyl-6-pentyl-2-phenyl-5,6-dihydro-4H-1,3-thiazine 3b, and 4-ethyl-4-hydroxy-2-phenyl-5,6-dihydro-4H-1,3-thiazine 3c exhibited inhibitory activities of 97, 77 and 76%, respectively, at a concentration of 6.25 microg/ml. The actual MIC99 for the most active of these compounds, 3a, was also determined to be >6.25 microg/ml. These results, and especially those for 3a, suggest that 1,3-thiazines are potential lead compounds in the search for new antitubercular agents.
 
Pharmacokinetic parameters of theophylline and one of its metabolites, 1,3-dimethyluric acid (1,3-DMU), were compared after intravenous and oral administration of aminophylline, 5mg/kg as theophylline, to diabetes mellitus rats induced by alloxan (DMIA) or streptozotocin (DMIS), and their respective control rats. In DMIA and DMIS rats, expression of CYP1A2 and 2E1 increased approximately three times. Theophylline was metabolized to 1,3-DMU by CYP1A2 and 2E1 in rats. Hence, it was expected that formation of 1,3-DMU increased in DMIA or DMIS rats. This was proven by the following results. First, after intravenous administration of theophylline, the AUC of 1,3-DMU was significantly greater in DMIA (110% increase) or DMIS (47.4% increase) rats. Second, the AUC of theophylline was significantly smaller in DMIA (26.1% decrease) or DMIS (30.1% decrease) rats because of significantly faster time-averaged total body clearance in DMIA (34.8% increase) or DMIS (42.7% increase) rats. Third, based on in vitro hepatic microsomal studies, intrinsic 1,3-DMU formation clearances were significantly faster in DMIA (20.4% increase) or DMIS (30.7% increase) rats than respective control rats. Similar results (AUC values of theophylline and 1,3-DMU) were also obtained after oral administration.
 
The cannabinoid receptors type 2 (CBR2) are attractive therapeutic targets of the endocannabinoid signaling system (ECS) as they are not displaying the undesired psychotropic and cardiovascular side-effects seen with cannabinoid receptor type 1 (CB1R) agonists. In continuation of our previous work on 2,4,6-trisubstituted 1,3,5-triazines as potent CB2 agonists, we synthesized an additional series of more polar analogues (1-10), which were found to possess high CB2R agonist activity with enhanced water solubility. The most potent compound in the series was N-(adamantan-1-yl)-4-ethoxy-6-(4-(2-fluoroethyl)piperazin-1-yl)-1,3,5-triazin-2-amine (9) with EC50 value of 0.60nM. To further evaluate the biological effects of the compounds, the selected compounds were tested in vitro against four different cell lines. A human retinal pigment epithelial cell line (ARPE-19) was used to evaluate the cytotoxicity of the compounds whereas an androgen-sensitive human prostate adenocarcinoma cell line (LNCaP), a Jurkat leukemia cell line and a C8161 melanoma cell line were used to assess the antiproliferative activity of the compounds. The most interesting results were obtained for N-(adamantan-1-yl)-4-ethoxy-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (6), which induced cell viability decrease in prostate and leukemia cell lines, and diminished proliferation of C8161 melanoma cells. The results could be reversed in leukemia cells with the selective CB2R antagonist AM630, whereas in prostate cells the AM630 induced a significant cell viability decrease with a mechanism probably unlinked to CB2 cannabinoid receptor. The antiproliferative effect of 6 on the melanoma cells seemed not to be mediated via the CB1R or CB2R. No cytotoxicity was detected against ARPE-19 cell line at concentrations of 1 and 10μM for compound 6. However, at 30μM concentration the compound 6 decreased the cell viability. Finally, in order to estimate in vivo behavior of these compounds, (18)F labeled PET ligand, N-cyclopentyl-4-ethoxy-6-(4-(2-fluoro-18-ethyl)piperazin-1-yl)-1,3,5-triazin-2-amine ([(18)F]5), was synthesized and its biodistribution was determined in healthy male Sprague-Dawley rats. As a result, the tracer showed a rapid (<15min) elimination in urine accompanied by a slower excretion via the hepatobiliary route. In conclusion, we further demonstrated that 1,3,5-triazine scaffold serves as a suitable template for the design of highly potent CB2R agonists with reasonable water solubility properties. The compounds may be useful when studying the role of the endocannabinoid system in different diseases. The triazine scaffold is also a promising candidate for the development of new CB2R PET ligands. Copyright © 2014 Elsevier B.V. All rights reserved.
 
3D ligand-based virtual screening was employed to identify novel scaffolds for cannabinoid receptor ligand development. A total of 112 compounds with diverse structures were purchased from commercial vendors. Twelve CB1 receptor antagonists/inverse agonists and ten CB2 receptor agonists were identified in vitro. One of the CB2 agonists, N-cyclopentyl-4-ethoxy-6-(4-methylpiperidin-1-yl)-1,3,5-triazin-2-amine (19, -log EC(50) = 7.5, E(max) = 255%) was selected for further development. As far as we are aware, the compound's 1,3,5-triazine scaffold represents a new core structure for CB2 agonists. A library of fifty-seven 2,4,6-trisubstituted-1,3,5-triazines was created to clarify the structure-activity relationship study of the analogs.
 
Several thiourea and urea derivatives were prepared by the reaction of 4-aminopyrazoles with substituted isothiocyanates or isocyanates. The novel compounds were tested anticonvulsant activity using by pentylenetetrazole-induced seizure (PTZ) and maximal electroshock seizure (MES) tests. Among the tested compounds, thiourea derivatives of 4b were afforded 90 and 100% protection in PTZ and MES tests at 50mg/kg, respectively. Urea derivatives of 5a and 5b were afforded 82 and 100% protection both at 25 and 50mg/kg. Also synthesized compounds were screened for antituberculosis activity against Mycobacterium tuberculosis H37Rv at 6.25 microg/mL concentration but they were not found active at these concentration. In addition, some selected compounds were evaluated for in vitro anti-HIV activity and they were all negative.
 
Ketosis is a metabolic disorder closely associated with both lipid and carbohydrate metabolism. Recent studies show that angiopoietin-like protein 3 (ANGPTL3) contributes to the development of metabolic disorder. The objective of this study was to explore the inhibitory effect of 1,3,5,8-tetrahydroxyxanthone (Xan), a naturally occurring flavonoid compound, on ketosis and the mechanisms involved in this regulation. After 4weeks, Xan (10 or 30mg/kg, intragastrically) treatment decreased plasma total ketone bodies, malondialdehyde, 8-isoprostane, triglyceride, total cholesterol levels, and hepatic ANGPTL3 expression concomitantly with increased plasma glucose concentration and adipose lipoprotein lipase (LPL) expression in ketosis murine. The present results suggest that Xan regulates ANGPTL3-LPL pathway to lessen the ketosis in mice.
 
A series of 6-aryl-4-isopropyl-2-[2-(1-phenylalkylidene)hydrazino]-1,4-dihydropyrimidine hydrochlorides was prepared and tested for antibacterial and antifungal effects. The title compounds were synthesized by cyclocondensation of N(2)-(1-phenylalkylideneamino)guanidines with 1-aryl-4-methyl-2-penten-1-ones. Structure analyses of the prepared compounds were accomplished by means of 1D and 2D NMR spectroscopy. The analyses showed that the ring closure reaction took place regioselectively in all cases and generated exclusively 2-(phenylalkylidenehydrazino)dihydropyrimidines. According to the NOE experiments, the applied N(2)-(1-phenylalkylideneamino)guanidines exist in [D(6)]DMSO solution as s-trans-(E)- and the title compounds as s-trans-(E,E)-isomers. The antimicrobial activity was evaluated against representative Gram-negative and Gram-positive bacteria, and against the pathogenic yeast Candida albicans. The tested title compounds showed weak antibacterial activity against Gram-positive bacteria, and one compound was active against Candida albicans.
 
Given the fundamentally multifactorial character of Alzheimer's disease (AD), addressing more than one target for disease modification or therapy is expected to be highly advantageous. Here, following the cholinergic hypothesis, we aimed to inhibit both acetyl- and butyrylcholinesterase (AChE and BuChE) in order to increase the concentration of acetylcholine in the synaptic cleft. In addition, the formation of the amyloid fibrils should be inhibited and already preformed fibrils should be destroyed. Based on a recently identified AChE inhibitor with a 1,4-substituted 4-(1H)-pyridylene-hydrazone skeleton, a substance library has been generated and tested for inhibition of AChE, BuChE, and fibril formation. Blood-brain barrier mobility was ensured by a transwell assay. Whereas the p-nitrosubstituted compound 18C shows an anti-AChE activity in the nanomolar range of concentration (IC50 = 90 nM), the bisnaphthyl substituted compound 20L was found to be the best overall inhibitor of AChE/BuChE and enhances the fibril destruction.
 
Double-charged 1,4-dihydropyridine (1,4-DHP) amphiphiles have been shown to condense DNA and efficiently transfect it into cells in vitro [Hyvönen et al., Biochim. Biophys. Acta 1509 (2000) 451]. Alkyl chain length and buffering capacity at endosomal pH range (5.0-7.4) affected complexation and transfection activity. In this study we examined how those chemical modifications of amphiphile-DNA complexes (amphiplexes) affect their interactions with extracellular polyanions (glycosaminoglycans, albumin) and lipid bilayers, their cellular uptake and intracellular distribution. To evaluate cellular uptake, CV1-P cells were incubated with labeled DNA-amphiphile complexes and analyzed by flow cytometry. Confocal laser fluorescence microscopy was used to investigate the intracellular distribution of amphiplexes. The results showed that biophysical properties of compounds can be changed by slight structural modifications. These factors determine the intracellular kinetics and transfection efficacy of the compounds. Some extracellular glycosaminoglycans and serum interfere with 1,4-DHP-amphiphile-mediated transfection by destabilizing the amphiplexes. Neither high cellular uptake, membrane destabilizing activity nor buffering capacity alone is adequate for high transfection efficacy. The activity results from complex interplay of various factors that determine intracellular kinetics and, consequently, transfection.
 
A series of N4-alkyl-1,6,7,11b-tetrahydro-2H-pyrimido[4,3-a]isoquinolinamine hydroiodides with triazasterol-related structures was designed and synthesized to mimic, as stable analogues, native high energy intermediates (HEI) of ergosterol biosynthesis. The title compounds can be regarded as 8,13,15-triaza-13,17-secosteroids with aromatic ring A bearing the positive charge in the guanidinium moiety. Hence, these compounds present structural similarities with corresponding carbocationic intermediates occurring during the enzyme catalyzed transformation of squalene into ergosterol. The N4-alkylaminopyrimidoisoquinolinium salts were prepared by reaction of respective S-methylthiotetrahydropyrimidoisoquinoline hydroiodides with octylamine, and appropriately methyl-branched alkyl- and alkenylamines. In order to prepare (3R)-6-isopropyl-3-methyl-6-hepten-1-amine several synthetic routes were investigated. The structures of all reported compounds were proved and completely assigned on the basis of homo- and heteronuclear correlated 1D and 2D NMR spectroscopy. The in vitro antifungal susceptibility tests of the title compounds with a standard panel of eight pathogenic fungi revealed especially against the used dermatophytes and yeasts with MICs in the range of 1-32 microg/ml moderate to good antimycotic effects. Depending on the nature of the N4-alkyl substituents structure-activity relationships were found with a maximum of antifungal efficacy of the N4-3,7-dimethyloctylaminopyrimidoisoquinolinium iodide.
 
Solid dispersion formulations made up of d-alpha-tocopheryl polyethylene glycol succinate 1000 (TPGS 1000) and polyvinyl pyrrolidone co-vinyl acetate 64 (PVPVA 64) or hydroxy propyl methyl cellulose 2910 (HPMC 2910) were developed in order to improve the dissolution of UC 781. UC 781 dissolution rate was markedly improved as compared to the physical mixtures and the pure drug, attaining maximum drug releases of up to 100% after only 5 min in the case of TPGS 1000-UC 781-PVPVA 64 solid dispersions and 30 min in TPGS 1000-UC 781-HPMC 2910. The increased UC 781 dissolution rate could be maintained when formulating UC 781 in PVPVA 64 tablets. The latter disintegrated in only 4 min, reaching drug releases of up to 90% (w/w). In addition, as opposed to the corresponding solid dispersions, no decrease in drug release occurred upon dissolution of PVPVA 64 tablets when the pH was increased to 6.8. Contrary to the PVPVA 64 tablet formulations, HPMC 2910 tablets showed a slow dissolution process due to the gelling nature of the polymer. The drug was slowly released as HPMC 2910 dissolved in the medium, however also in this case 90% (w/w) of the drug was dissolved after 4 h. Both polymers formed compatible blends in combination with the drug. Thermal analysis of the ternary mixtures revealed eutectic behavior exhibiting an extremely fine dispersion of the drug in the carrier. This was confirmed by the fact that no drug crystals could be detected using X-ray diffraction (XRD). As opposed to the physical mixtures, PVPVA 64 and HPMC 2910 solid dispersions did not contain any isolated polymer-rich phases, hence showed improved homogeneity. Amorphous TPGS 1000 clusters occurred in PVPVA 64 and HPMC 2910 formulations upon addition of at least 10% (w/w) UC 781, showing extremely low glass transition temperatures depending of the thermal history of the samples.
 
Solid dispersions and physical mixtures made up of the poorly water-soluble drug UC 781, a polymer and a surfactant were prepared to contribute to the understanding of the relationship between physicochemical characteristics and dissolution behaviour. In addition, to facilitate downstream processing while still favouring drug dissolution to a maximum extent, formulation conditions were investigated to obtain a free flowing powder which contains a maximum amount of surfactant. Poloxamer 407, a polyethylene-polypropylene glycol block copolymer, was selected as a suitable polymer based on UC 781 supersaturation results. d-Alpha-tocopheryl polyethyleneglycol succinate 1000 (TPGS 1000) was preferred as a surfactant since it increased UC 781 dissolution when formulated in a self-micro emulsifying drug delivery system (SMEDDS), as compared to TPGS 400, TPGS 4000 and TPGS 6000. Based on flow properties, a TPGS 1000/Poloxamer 407 ratio of 80/20 was used to prepare solid dispersions by spray drying. Pure drugs, physical mixtures and solid dispersions were characterized by differential scanning calorimetry and X-ray powder diffraction. Eutectic phase behaviour was obtained in which the relative distribution of the polyethylene glycol folding was dependent on UC 781 concentration. Drug release was markedly increased when formulated as a solid dispersion with Poloxamer 407 and TPGS 1000. Formulation of solid dispersions did however not further improve the drug dissolution rate compared to that of physical mixtures. Nonetheless, variability of dissolution results was considerably reduced upon solid dispersion formulation.
 
The impact of biodegradable pH-sensitive surfactant (BPS)-liposomes on nucleic acid, i.e., oligonucleotide and plasmid DNA, cellular delivery was examined. Fluorescein-labeled nucleic acids complexed with 1,2-dioleoyl-3-trimethylammonium propane cationic liposomes and BPS at a charge ratio (+/-) of 10 were incubated in CV-1 cells and analyzed by flow cytometry. The fluorescence intensity of oligonucleotides but not plasmid DNA complexed with BPS-liposomes was higher than those complexed with BPS-free liposomes at early time points. However, when cells were fixed to equalize the intracellular pH since fluorescein, a pH-sensitive fluorophore, has higher fluorescence intensity in alkaline pH than acidic, no difference in intensity was observed. This indicated the incorporation of BPS in liposomes did not increase oligonucleotide cellular uptake over control liposomes, but redistributed oligonucleotides into a more basic environment, e.g., cytoplasm. An explanation consistent with the presented data is the formation of small transient membrane defects within the endosomal membrane as presented previously [Liang, E., Hughes, J.A., 1998a. Membrane fusion and rupture in liposomes: effect of biodegradable pH-sensitive surfactants. J. Membr. Biol. 166, 37-49.]. The above findings suggested that BPS may be effective agents of disrupting one of the major barriers, endosomal membrane, to enhance nucleic acid cellular transport.
 
Knockout (KO) animals are useful tools with which to assess the interplay between P-glycoprotein (P-gp; Abcb1) and the breast cancer resistance protein (Bcrp, Abcg2), two major ABC-transporters expressed at the blood-brain barrier (BBB). However, one major drawback of such deficient models is the possible involvement of compensation between transporters. In the present study, P-gp and Bcrp distribution in the brain as well as P-gp expression levels at the BBB were compared between the Bcrp TGEM KO rat model and the wild-type (WT) strain. Therefore, we used confocal microscopy of brain slices and western blot analysis of the isolated brain microvessels forming the BBB. This deficient rat model was used to assess the influence of Bcrp on the brain and peripheral kinetics of its substrate [(11)C]befloxatone using positron emission tomography (PET). The influence of additional P-gp inhibition was tested using elacridar (GF120918) 2 mg/kg in Bcrp KO rats. The distribution pattern of P-gp in the brain as well as P-gp expression levels at the BBB was similar in Bcrp-deficient and WT rats. Brain and peripheral kinetics of [(11)C]befloxatone were not influenced by the lack of Bcrp. Neither was the brain uptake of [(11)C]befloxatone in Bcrp-deficient rats influenced by the inhibition of P-gp. In conclusion, the Bcrp-deficient rat strain, in which we detected no compensatory mechanism or modification of P-gp expression as compared to WT rats, is a suitable model to study Bcrp function separately from that of P-gp at the BBB. However, although selectively transported by BCRP in vitro, our results suggest that [(11)C]befloxatone PET imaging might not be biased by impaired function of this transporter in vivo.
 
In this study five macrolide antibiotics (azithromycin, erythromycin, clarithromycin, roxithromycin and telithromycin) were compared based on their ability to interact with human MDR1 (ABCB1, P-glycoprotein), studied from two main aspects: by determining the influence of macrolide antibiotics on MDR1 function, as well as the influence of MDR1 on macrolide accumulation in MES-SA/Dx5 cells overexpressing human MDR1. At higher micromolar concentrations five tested macrolides were shown to inhibit MDR1 function in terms of rhodamine-123 efflux and verapamil-activated ATPase function, whereas at lower concentrations they activated MDR1 ATPase. They were confirmed to be substrates of MDR1 and to compete with each other, as well as with verapamil for transport via this transporter. Expression of MDR1 on cells decreased macrolide accumulation in cells from 2- to 80-fold with the most pronounced change observed for azithromycin and erythromycin. Moreover, presence of active MDR1 highly affected the relative ranking of tested macrolides according to their accumulation in cells. In conclusion, out of seven applied methods and assessed parameters, four of them gave similar rough evaluation on the strength of interaction of five macrolides with MDR1, with clarithromycin, roxithromycin and telithromycin showing stronger interaction than azithromycin and erythromycin.
 
Based on our initial results on the effects of several ATP-binding cassette (ABC) transporter inhibitors on rhodamine-123 efflux from A549, a human lung carcinoma, and MES-SA, a human uterine sarcoma cell line, the aim of this study was to identify the transporter responsible for this export. Export of two fluorescent dyes, rhodamine-123 and calcein, was investigated in both cell lines by testing five commonly used inhibitors of ABC transporters: verapamil, cyclosporin A, MK571, GF129018 and fumitremorgin C. A very high degree of correlation (R(2)=0.91-0.99) between results obtained in the two cell lines suggested that the same transporter was involved in the export of tested fluorescent substrates in both cell lines. Expression analysis and gene silencing techniques, as well as transport of additional substrate 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) on membrane vesicles revealed that the transporter was multidrug resistance related protein 2 (MRP2, ABCC2). Furthermore, it was found that the tested modulators showed very diverse effects on the export of three fluorescent substrates via MRP2, with some modulators being inhibitory in one, while having no effect or even stimulating the transport in the other fluorescent dye assay. Verapamil inhibited rhodamine-123, but stimulated CDCF transport and did not affect calcein export. GF129018 did not affect calcein and CDCF transport, but it inhibited rhodamine-123 transport. These results demonstrate the importance of studying various combinations of potential substrates and modulators of MRP2 in order to estimate possible drug-drug interactions in living organisms. In addition, A549 and MES-SA cells were shown to be good cell models for studying interactions of compounds with human MRP2.
 
Objectives: To investigate the absorption, distribution, metabolism and excretion of AFN-1252, a novel inhibitor of the essential FabI enzyme in Staphylococcus spp., in vitro and following microdosing in healthy adult male subjects following intravenous and oral administration. Methods: Three ADME studies, comprising a Caco-2 assay, a rat intestinal perfusion model and a microdosing study in healthy human volunteers, were conducted. Results: The Caco-2 assay indicated that AFN-1252 in solution is well-absorbed and undergoes insignificant efflux, and its transport across the intestinal wall is probably passive. In the rat intestinal perfusion model, AFN-1252 exhibited high permeability potential across three segments, in the rank order of jejunum=ileum>colon. Taken together with the low aqueous solubility, the data from these studies indicate that AFN-1252 is a BCS Class II molecule with solubility-limited absorption. Analysis of the [(14)C]-AFN-1252 radioactivity concentration-time data indicated similar pharmacokinetics following intravenous and oral administration in the microdosing study in healthy volunteers. These included long terminal half-lives of ∼7 h and 83% bioavailability, indicating that there was little first-pass metabolism following oral dosing. AFN-1252 exhibited good distribution to skin and skin structures where its anti-staphylococcal activity may be required. Urinary and faecal excretion are major elimination routes for [(14)C]-AFN-1252 following intravenous or oral administration. Conclusions: AFN-1252 has the potential for both intravenous and oral administration, once- or twice-daily dosing and good tissue distribution in humans. Further safety, efficacy and pharmacokinetic studies in man are required to investigate therapeutically-relevant doses for this novel agent and its targeted selectivity and high potency against Staphylococcus spp.
 
Pluronic F127 (PF127), one of the polymers which can inhibit drug efflux transporters in cancer therapy, was used to produce amphiphilic nanocarriers for doxorubicin (DOX). In order to stabilize the nanocarriers, the hydroxyl groups on both termini of PF127 were acrylated and reacted with methacrylated chondroitin sulfate (CSMA) to form CS-PF127 nanogel. The introducing CSMA has carboxylic acid groups which can be used to react with a folic acid-polyethylene glycol (FA-PEG). Folic acid, having high binding affinity to tumor-associated folate receptors (FR), provides a selective delivery of doxorubicin (DOX) to FR-positive tumor cells. DOX was loaded either in a cationic DOX.HCl form through the electrostatic interactions with the negative charges of chondroitin sulfate, or in a free DOX form by solubilization into the PPO core compartment of PF127. The loading efficiency and release behavior of DOX prepared from two different formulations are compared. The synthesis of CS-PF127 and FA-PEG grafted CS-PF127 (FA-CS-PF127) was characterized by nuclear magnetic resonance spectrometry (NMR), ultraviolet/visible spectroscopy (UV), and X-ray photoelectron spectroscopy (XPS). With a fluorescent probe technique, the critical aggregation concentrations (CAC) are 7.5 x 10(-2)mg/mL for CS-PF127 and 7.9 x 10(-2)mg/mL for FA-CS-PF127, respectively. The spherical images of nanogels were visualized with the use of the transmission electron microscope (TEM). The particle diameters measured by dynamic light scattering (DLS) are 299.6+/-8.2nm for CS-PF127 and 138.3+/-12.3 for FA-CS-PF127, neither aggregation nor change in sizes in double deionized (DD) water after 20 days. The better cellular uptake of FA-CS-PF127 in KB cells was evidenced by confocal laser scanning microscopy (CLSM) and flow cytometry upon loading Rhodamine123 as a probe.
 
13-cis-Retinoic acid (13-cis-RA) is a synthetic retinoid commonly used in the treatment of severe acne. It has also been found to possess potential chemopreventive activity. It has extremely low aqueous solubility and high photo-sensitivity. This study investigated the effects of the complexation of 13-cis-RA with alpha-cyclodextrin (alpha-CD) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on its phase solubility. HP-beta-CD was found to be more effective in increasing the aqueous solubility of 13-cis-RA compared to alpha-CD. Phase solubility studies indicated that the solubility of 13-cis-RA was increased dramatically by the formation of inclusion complex with HP-beta-CD. The solubility was further enhanced by pH adjustment. The photostability of the selected inclusion complex of 13-cis-RA:HP-beta-CD was then evaluated. Complexation with HP-beta-CD was found to delay the photo-degradation of 13-cis-RA in aqueous solution. The physicochemical properties of the solid inclusion complex were characterized by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and X-ray diffractometry (XRD). Molecular modeling with MMFF94s force field (SYBYL V6.6) was utilized to predict the preferred orientation of 13-cis-RA in the CD cavity and the main structural features responsible for the enhancement of its solubility and photostability. The energy scores estimated from the computational analysis were found capable of reflecting the stability constants of the cyclodextrin complexes obtained in the phase solubility studies. The results showed that HP-beta-CD was a proper excipient for increasing solubility and stability of 13-cis-RA.
 
Human brain chemistry is incompletely understood and better methodologies are needed. Traumatic brain injury (TBI) causes metabolic perturbations, one result of which includes increased brain lactate levels. Attention has largely focussed on glycolysis, whereby glucose is converted to pyruvate and lactate, and is proposed to act as an energy source by feeding into neurons’ tricarboxylic acid (TCA) cycle, generating ATP. Also reportedly upregulated by TBI is the pentose phosphate pathway (PPP) that does not generate ATP but produces various molecules that are putatively neuroprotective, antioxidant and reparative, in addition to lactate among the end products. We have developed a novel combination of 13C-labelled cerebral microdialysis both to deliver 13C-labelled substrates into brains of TBI patients and recover the 13C-labelled metabolites, with high-resolution 13C NMR analysis of the microdialysates. This methodology has enabled us to achieve the first direct demonstration in humans that the brain can utilise lactate via the TCA cycle. We are currently using this methodology to make the first direct comparison of glycolysis and the PPP in human brain. In this article, we consider the application of 13C-labelled cerebral microdialysis for studying brain energy metabolism in patients. We set this methodology within the context of metabolic pathways in the brain, and 13C research modalities addressing them.
 
Articaine hydrochloride, 4-methyl-3-(2-[propylamino]propionamido)-2-thiophenecarboxylic acid, methyl ester hydrochloride, is a local anaesthetic commonly used in dentistry, and is classified as an amide local anaesthetic. Solid-state (13)C and (31)P NMR were used to investigate the uncharged articaine species (sample pH 10.0) when interacting with distearoylphosphatidylcholine (DSPC) model membranes. The DSPC phospholipid bilayer was studied at four different molar ratios of articaine, 10, 25, 40, and 55 mol%, respectively. The articaine concentration-dependent decrease in the DSPC bilayer gel-to-liquid-crystalline phase-transition temperature demonstrates substantial articaine interaction with this bilayer. A DSPC bilayer contains a large hydrophobic core and the (13)C and (31)P NMR spectra of the 40 mol% articaine-containing sample demonstrate a disturbance in the molecular packing of the polar bilayer region that extends into the hydrophobic region, evidenced by carbon 2 and 3 of the stearoyl acyl chains. Observed (31)P and (13)C NMR spectral changes when articaine is increased from 40 to 55 mol%, suggest formation of articaine aggregates and decrease in DSPC bilayer perturbation at the latter articaine level.
 
Stringent control of gene expression in human gene therapy strategies is important for both therapeutic and safety reasons. Replication-defective vectors derived from adenoviruses have been shown to be capable of highly efficient in vivo gene delivery to a wide variety of dividing and nondividing human cells. Here, we review the progress in the development of regulatable adenovirus vectors that allow gene expression to be tightly controlled by low concentrations of tetracyclines. As an example of the potential clinical utility of this technology, we highlight our results obtained in an immunotherapy model for prostate cancer with a tetracycline-regulatable adenovirus vector expressing the cytokine interleukin-12. Recombinant adenovirus vectors with tetracycline-regulatable gene expression provide new opportunities and improved safety for gene therapy applications in humans.
 
This study compares the intrinsic permeability coefficients of 40 drug molecules, obtained by three popular variants of the PAMPA assay, based on: (a) n-hexadecane, (b) 2% w/v dioleyoylphosphatidylcholine in n-dodecane, and (c) 20% w/v lecithin in n-dodecane, the HDM-, DOPC-, DS-PAMPA models, respectively. It was shown that PAMPA permeability values consistently rank in magnitude according to: DS>DOPC>HDM, with molecules like metoprolol showing 1000-fold greater permeability in DS than in HDM. Abraham descriptors were used to rationalize these observations. Water-solubilized polar molecules form very strong H-bonds with the solvent. Such molecules need to break these bonds in order to enter the pure alkane phase, which, in turn, offers no compensating H-bond solvation. Thus, more energy appears to be needed for a polar molecule to penetrate a pure alkane barrier, compared to a barrier possessing some H-bond interactions. The 20% phospholipid content of the DS-PAMPA lipid may be thought to ease the permeation process, by offering a compensating source of H-bonding within the membrane phase.
 
Cisplatin (cis-diamminedichloroplatinum(II)) is used in chemotherapy and it is well established that cisplatin forms platinum-DNA adducts that initiate tumor cell death. Drawbacks are side effects such as neurotoxicity and cellular cisplatin resistance and it is possible that part of these effects are linked to cisplatin interaction with lipids and the phospholipid bilayer. 13C magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of total lipid extract from pig brain with and without cisplatin show that the phosphatidylserine (PS) carboxyl resonance disappears in presence of cisplatin and that a new resonance of similar intensity appears at 185.5 ppm. Thus, indicating cisplatin interaction with the PS head-group. Static and MAS 31P NMR spectra of lipid extract with and without cisplatin show that the phospholipids to a large extent reside in a bilayer environment in pure lipid extract, and that the presence of cisplatin promotes isotropic and/or hexagonal lipid phases.
 
For local administration of drugs or enzyme inhibitors in the human gut, a small-bore, smooth tube was introduced through the nose, retrieved from the pharynx, equipped with a firm radio-opaque capsule, and swallowed. Peristalsis moves the capsule to the desired location in the gut where it is anchored before administration via the tube. Drug uptake is followed by plasma sampling. One capsule type is used for solutions, another for solid formulations. With solutions, repeated administrations could be done with the capsule being anchored for 24h or longer or, alternatively, at several locations along the gut. This communication presents the method and an overview of 13 uptake and enzyme/transporter localization studies. Altogether, 268 intubations were undertaken in a total of 128 subjects. Plasma concentrations found with terbutaline and metoprolol are presented showing that terbutaline has its best uptake in the upper small intestine, whereas metoprolol shows the same bioavailability along the whole gut. Subjects could undertake most of their normal activities while carrying the equipment. No serious adverse events (AEs) occurred. Possibly intubation-related AEs were abdominal pain (n=8) and constipation (n=5). In conclusion, the method has been found to be safe, convenient and multifunctional for studies of drug uptake at predetermined gut locations in healthy subjects.
 
The aim of this paper is to assess the performance of a new mechanistic model for analysing (13)C-octanoic acid breath test data using simulation studies. The (13)C-octanoic acid breath test is widely used for indirect assessment of the rate of gastric emptying and it is yet to achieve universal acceptance due to inconsistencies when the results are compared with simultaneous and direct measurements using scintigraphy. The new semi-mechanistic model has five separate compartments; stomach, intestine, central and peripheral body and breath compartments. Stomach and breath profiles were simulated for 50 individuals under four conditions: variability on all parameters; no variability on the rate constant of gastric emptying and the rate constant of absorption; variability on the rate constant of gastric emptying and the rate constant of absorption only; and no variability on all parameters. A mono-exponential model was fitted to the stomach profile and the new semi-mechanistic model and three other widely used methods were fitted to the breath profiles. The gastric emptying half times from stomach profiles correlate better (R(2)=1,1,1,1 for the four conditions) with the half emptying times from the semi-mechanistic model compared with half emptying times from the modified exponential model (R(2)=0.72,0.53,0.88,1), Ghoos method (R(2)=0.72,0.54,0.88,1) and Wagner-Nelson method (R(2)=0.79,0.68,0.89,1) for the four simulation studies. The semi-mechanistic model is very effective for the assessment of GE using the (13)C-octanoic acid breath test and could be applied in the development of drugs that influence GE.
 
The purpose of this study was to investigate the formulation and delivery of a protein in a pressurized metered-dose inhaler (pMDI) containing HFA 134a as the propellant for aerosol delivery. Ethanol and surfactants, including polyoxyethylene 10 oleyl ether (Brij 97), polyoxyethylene 20 oleyl ether (Brij 98), polyoxyethylene sorbitan monooleate (Tween 80) and Aerosol OT (AOT), were investigated as formulation adjuvants to improve the dose delivery characteristics of the model protein (bovine serum albumin) containing pMDI formulations. The aqueous solution of a surfactant and protein was lyophilized to obtain a solid carrier system of the protein. Readily dispersible suspensions were obtained by suspending this solid carrier system in HFA 134a with ethanol as a dispersing aid. The formulations containing Tween 80 resulted in the highest respirable fraction. This study suggested a potential formulation containing a lyophilized complex of surfactant and protein readily dispersible in HFA 134a for delivering a therapeutic protein to the respiratory tract by inhalation.
 
In order to improve the efficacy of doxorubicin (DOXO) in the treatment of hepatocellular carcinomas, the drug was conjugated with lactosaminated human albumin (L-HSA), a hepatotropic drug carrier. Conjugation was performed using the (6-maleimidocaproyl)hydrazone derivative of the drug (DOXO-EMCH). The maleimide group of DOXO-EMCH reacts with the aminoacidic residues of the carrier forming stable bonds, whereas the hydrazone bond is rapidly hydrolysed in the acidic endosomal and lysosomal compartments of the cells allowing the intracellular release of DOXO. To identify the amino acids of L-HSA involved in the bond with DOXO-EMCH, in the present study we synthesized this compound with the 2,3 carbon atoms of the maleimide moiety enriched in the (13)C isotope and used this labelled DOXO-EMCH to prepare two types of L-HSA conjugate. Type I was prepared in analogy to those studied in the anticancer experiments using tris(2-carboxyethyl)phosphine (TCEP) to reduce l-cysteine disulfides and make the sulfhydryl groups available for the reaction with DOXO-EMCH; type II was synthesized omitting TCEP. By (13)C NMR spectroscopy we could demonstrate that in type I conjugate cysteine was the only amino acid residue that reacted with DOXO-EMCH, whereas in type II conjugate lysine was the only amino acid in the reaction with DOXO-EMCH. When hydrolysed in an acidic medium to cleave the hydrazone bond, type I conjugate released only DOXO, whereas type II conjugate also released a derivative of the drug.
 
Stability of the antihypertensive drug nifedipine (NIF) has been studied experimentally in solid state by (1)H-(14)N NMR-NQR double resonance (NQDR) and theoretically by the Density Functional Theory (DFT). Photodegradation of NIF to its metabolite in vivo nitrosonifedipine, NO-NIF (antioxidative agent) upon long term daylight exposure was detected and the changes in the molecular structure of NIF were analysed. The photoconversion of NIF to NO-NIF in solid was found to be accompanied with the electron density redistribution at nitrogen sites (NH to N and NO(2) to NO) and proved to be successfully detected with identification of photoproducts by (1)H-(14)N NQDR and DFT methods. The increase in the e(2)qQ/h and η describing EFG tendency towards non-spherical symmetry was significantly greater upon the reduction of NO(2) site than upon hydrogen abstraction from NH site. The level of sensitivity of detection of the photodegradation product was about 1% of the original sample. The Quantum Theory of Atoms in Molecules (QTAIM) analysis has been found useful in predicting photoreactive sites in the molecules and finding the explanation of differences in reactivity between parent NIF and its photoproduct NO-NIF. Using NIF as a model, this study demonstrates the suitability of NQDR supported by DFT for non-destructive determination of the photodegradation products in solid state.
 
Reducing the differentiation period for obtaining an in vitro intestinal barrier model is required to reduce the duration and cost for drug screening assays. In this frame, the Caco-2/TC7 subclone differentiation state was investigated from day 0 (D0) to day 32 (D32). As such, the expression of 45 genes (including cell junction, cell polarization, cell functionality, drug transport and metabolism genes) was followed throughout the 32 days. In parallel, the monolayer polarization and the formation of the cellular junctions were characterized by the immuno-staining of occludin, claudin-1 and actin proteins. The cell monolayer permeability was analyzed via transepithelial electric resistance measurements and paracellular transport of Lucifer Yellow. The P-gp efflux efficiency was assessed by rhodamine 123 transport. Alkaline phosphate activity was quantified to assess the cell differentiation. Three stages of differentiation were observed using the clustering of principal component analysis of the RTqPCR data and the overall assays. From D0 to D10, cells were in a proliferation stage and under-differentiated; from D14 to D21 a stable differentiation stage was reached; from D25 to D32 the epithelium seemed to enter into a post-differentiated stage. This study demonstrates that Caco-2/TC7 cells are functional and ready for use in drug screening permeability assays from 14 days in culture when compared with conventional 21 days for Caco-2 cells. In addition, this study provides a refined set of data allowing temporal and multi scale investigations, due to the intracellular kinetics and mRNA levels that can be correlated with membrane protein kinetics and functional extracellular activities. Therefore, shorter time in culture combined with a better knowledge of the cells during the time in culture will in turn help to improve the quality and cost of Caco-2/TC7 assays for drug development. Copyright © 2015. Published by Elsevier B.V.
 
Microdose study enables us to understand the pharmacokinetic profiles of drugs in humans prior to the conventional clinical trials. The advantage of microdose study is that the unexpected pharmacological/toxicological effects of drugs caused by drug interactions or genetic polymorphisms of metabolic enzymes/transporters can be avoided due to the limited dose. With a combination use of accelerator mass spectrometry (AMS) and (14)C-labaled compounds, the pharmacokinetics of both parent drug and its metabolites can be sensitively monitored. Thus, to demonstrate the usability of microdose study with AMS for the prediction of the impact of genetic polymorphisms of CYP enzyme on the pharmacokinetics of unchanged drugs and metabolites, we performed microdose pharmacogenetic study using tolbutamide as a CYP2C9 probe drug. A microdose of (14)C-tolbutamide (100 μg) was administered orally to healthy volunteers with the CYP2C9∗1/∗1 or CYP2C9∗1/∗3 diplotype. Area under the plasma concentration-time curve for the (14)C-radioactivity, determined by AMS, or that for the parent drug, determined by liquid chromatography/mass spectrometry, was about 1.6 times or 1.7 times greater in the CYP2C9∗1/∗3 than in the CYP2C9∗1/∗1 group, which was comparable to the previous reports at therapeutic dose. In the plasma and urine, tolbutamide, carboxytolbutamide, and 4-hydroxytolbutamide were detected and practically no other metabolites could be found in both diplotype groups. The fraction of metabolites in plasma radioactivity was slightly lower in the CYP2C9∗1/∗3 group. Microdose study can be used for the prediction of the effects of genetic polymorphisms of enzymes on the pharmacokinetics and metabolic profiles of drugs with minimal care of their pharmacological/toxicological effects.
 
8-(N-2-hydroxy-5-chlorobenzoyl)-amino-caprylic acid (5-CNAC), a compound lacking pharmacological activity enhances the absorption of salmon calcitonin, when co-administered. Disposition and biotransformation of 5-CNAC was studied in six healthy postmenopausal women following a single oral dose of 200mg (14)C-radiolabeled 5-CNAC (as disodium monohydrate salt). Blood, plasma, urine and feces collected over 7 days were analyzed for radioactivity. Metabolite profiles were determined in plasma and excreta and metabolite structures were elucidated by LC-MS/MS, LC-(1)H NMR, enzymatic methods and by comparison with reference compounds. Oral 5-CNAC was safe and well tolerated in this study population. 5-CNAC absorption was rapid (t(max)=0.5h; C(max)=9.00 ± 2.74 μM (mean ± SD, n=6) and almost complete. The elimination half-life (t(½)) was 1.5 ± 1.1h. The radioactive dose was excreted mainly in urine (≥ 90%) in form of metabolites and 0.071% as intact 5-CNAC. Excretion of radioactivity in feces was minor and mostly as metabolites (<3%). Radioactivity in plasma reached C(max) (35.4 ± 7.9 μM) at 0.75 h and declined with a half-life of 13.9 ± 4.3h. 5-CNAC accounted for 5.8% of the plasma radioactivity AUC(0-24h). 5-CNAC was rapidly cleared from the systemic circulation, primarily by metabolism. Biotransformation of 5-CNAC involved: (a) stepwise degradation of the octanoic acid side chain and (b) conjugation of 5-CNAC and metabolites with glucuronic acid at the 2-phenolic hydroxyl group. The metabolism of 5-CNAC in vivo could be reproduced in vitro in human hepatocytes. No metabolism of 5-CNAC was observed in human liver microsomes.
 
Opioids are the most effective analgesics for pain management, and efficient pain control is a therapeutic priority. Herein, we describe the synthesis and pharmacological activities of the 5-benzyl analogue of the mu opioid analgesic 14-methoxymetopon (14-MM). The result of the replacement of the 5-methyl in 14-MM with a benzyl group on in vitro opioid receptor binding and functional profiles, and in vivo behavioural properties, i.e. nociception and motor activity, was investigated. In rodent brain membranes, the 5-benzyl derivative showed high affinity at the micro opioid receptor and decreased interaction with delta and kappa receptors, hence displaying a similar binding profile as 14-MM. It displayed potent agonist activity in vitro and in vivo. In in vitro guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding assay, it activated G-proteins in rat brain membranes through a micro opioid receptor-mediated mechanism having significantly enhanced potency compared to DAMGO (D-Ala(2),Me-Phe(4),Gly-ol(5)]enkephalin), and to the micro opioid agonist morphinans 14-MM, 14-O-methyloxymorphone (14-OMO) and morphine. In vivo, the 5-benzyl analogue of 14-MM elicited dose-dependent and naloxone-sensitive antinociceptive effects in hot-plate and tail-flick tests in mice after subcutaneous (s.c.) administration. Its analgesic potency was comparable to 14-MM, and was 50-fold higher than that of morphine. Contrary to morphine, 14-MM and 14-OMO, no motor dysfunction was produced by the new opioid in the mouse rotarod test at any of the tested doses. In summary, the 5-benzyl analogue of 14-MM emerged as a novel potent mu opioid antinociceptive agent with reduced propensity to cause unwanted motor impairment.
 
Chemical structure of SB203580 compound.
The effects of specific p38-MAPK inhibitor, SB203580, on the resistance of L1210 / VCR cells. A-Effect of SB203580 on the cytotoxicity of vincristine (LC value) in resistant L1210 / VCR cells. R-Cytotoxicity of vincristine on L1210 / VCR cells; SB-cytotoxicity of vincristine on 50 L1210 / VCR cells in the presence of 30 mM SB203580; S-cytotoxicity of vincristine on parental sensitive L1210 cells. LC-concentration of 50 3 vincristine that is lethal for 50% of cells. B-Effect of SB203580 on intracellular accumulation of [ H]-vincristine in L1210 / VCR cells. The resistant cells were treated with 15, 30 and 60 mM SB203580 or 0.1% DMSO (negative control) and the accumulation of vincristine was determined as described in Materials and methods. R-resistant L1210 / VCR cells cultivated in the basal medium; RD-L1210 / VCR cells cultivated in the presence of 0.1% DMSO (negative control); SB1-resistant cells treated with 60 mM SB203580; SB2-resistant cells treated with 30 mM SB203580; SB3-resistant cells treated 3 with 15 mM SB203580; S-intracellular accumulation of [ H]-vincristine in sensitive L1210 cells. Results represent means6S from three independent d measurements. C-Cytotoxicity of SB203580 on sensitive L1210 (S) and resistant L1210 / VCR (R) cells. LC-Concentration of SB203580 that is lethal 50
A – Effect of SB203580 treatment on levels of p38-MAPK. The L1210 / VCR cells were treated for 72 h with 30 m M SB203580 (R ) or 0.05% SB DMSO (R ) and the p38-MAPK levels were determined by Western blot analysis using specific p38-MAPK antibody. B – Effect of SB203580 treatment D on the gene expression of P-glycoprotein. The L1210 / VCR cells were treated for 72 h with 30 m M SB203580 (R ) or 0.05% DMSO (R ) and the SB D 
Levels and phosphorylation state of p38-MAPK in parental sensitive L1210 (S) multidrug resistant L1210 / VCR cells cultivated in the absence (R) and presence of vincristine (R ). A – Western blot record showing the protein levels of p38-MAPK in sensitive and resistant cells, determined by specific V anti-p38-MAPK antibody. B – Record showing the content of a phosphorylated form of 38-MAPK. Phospho-p38-MAPK was determined by Western blot analysis using an antibody that reacts specifically only with dual phosphorylated p38-MAPK (Thr180 / Tyr182). 
P-glycoprotein (P-gp) is the plasma membrane transport pump responsible for efflux of chemotherapeutic agents from cells and is one of the systems that secures multidrug resistance (MDR) of neoplastic cells. In the present study, drug sensitive L1210 and multidrug resistant L1210/VCR (characterized by overexpression of P-gp) mouse leukemic cell lines were used as an experimental model. We have found that SB203580, a specific inhibitor of p38-MAPK pathway, significantly reduced the degree of the vincristine resistance in L1210/VCR cells. This phenomenon was accompanied by a decrease in the LC(50) value of vincristine from 3.203+/-0.521 to 0.557+/-0.082 microM. The LC(50) value of sensitive cells for vincristine was about 0.011 microM. The effect of SB203580 on L1210/VCR cells was associated with significantly increased intracellular accumulation of [3H]-vincristine in the concentration dependent manner. Prolonged exposure of resistant cells to 30 microM SB203580 did neither significantly influence the gene expression of P-gp, nor change the protein levels of p38-MAPK. Western blot analysis revealed that the MDR phenotype in L1210/VCR cells was associated with increased level and activity of cytosolic p38-MAPK. In resistant cells, the enhanced phosphorylation of both, p38-MAPK and ATF-2 (endogenous substrate for p38-MAPK) was found as well. In conclusion we could remark that SB203580, an inhibitor of p38 kinase pathway, reversed the MDR resistance of L1210/VCR cells. MDR phenotype of these cells is connected with increased levels and activities of p38-MAPK. These findings point to the possible involvement of the p38-MAPK pathway in the modulation of P-gp mediated multidrug resistance in the L1210/VCR mouse leukemic cell line. However, the mechanisms of SB203580 action should be further investigated.
 
Top-cited authors
José Manuel Sousa Lobo
  • University of Porto
Paulo Jorge Cardoso Costa
  • University of Porto
Per Artursson
  • Uppsala University
Anette Müllertz
  • University of Copenhagen
Colin W Pouton
  • Monash University (Australia)