Inductively heated plasma waste treatment for energy recovery.

An assessment of a decentralized inductively heated plasma waste treatment system for energy recovery has been done. The modular miniaturized high enthalpy plasma source IPG6 is a reference for the system and has been qualified for inert but also chemically aggressive gas compositions. An identification and review of applications were undertaken. Niches of high environmental and societal importance are considered: hospital waste (threshold countries), shipboard waste and marine litter. The wastes are reviewed deriving relevant parameter for a system analysis aiming for the derivation of energy production and efficiencies. The system analysis shows advantageous constellation due to the wastes' energy leading to self-feeding systems.

HPLC determination of brain biogenic amines following treatment with bispyridinium aldoxime K203.

Effect of a new acetylcholine-esterase reactivator, K203 as a new potential antidote in organophosphate intoxications was studied on dopamine (DA), homovanillic acid (HVA), serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels in seven brain regions (cerebellum, spinal cord, hippocampus, hypothalamus, striatum, medulla oblongata and frontal cortex) of rats by an optimized and validated HPLC method. No significant change in brain level of these neurotransmitters was found either 15 or 60 min following treatment. However, when 5-HIAA/5-HT ratios were calculated as measure of turnover, significant decreases were found in the cerebellum, hippocampus, hypothalamus and the frontal cortex 15 min following K203 administration, but after 60 min only in the frontal cortex.

Medicinal chemistry of antimigraine drugs.

Migraine is one of the most frequent neurological disorder with high impact on the quality of life. Primary headaches such as migraine are pathophysiologically complex disorders. The concept of the trigeminovascular system dysfunction in migraine has led to a number of drug discoveries dramatically changing the treatment options. Acute and prophylactic therapy targeting either the trigeminovascular system or central structures involve several groups of drugs with peculiar medicinal chemistry. In the proposed review up to date concept of treatment strategy, medicinal chemistry data of the drugs used will be summarized. The present review gives detailed information on drugs effective in aborting migraine attacks (by inhibiting prostanoid synthesis, are agonists of serotonin 5-HT1B/D receptors, on the recently introduced CGRP-receptor antagonists) and the drugs recommended for prophylactic treatment (selected beta-adrenergic receptor antagonists, Ca-channel inhibitors, antiepileptics, antidepressants). The pharmacokinetics, fate in the body (absorption, distribution, metabolism, excretion) and significant pharmacological effects as well as the recent bioanalytical methods for their determination are presented.

Effect of a single neonatal oxytocin treatment (hormonal imprinting) on the biogenic amine level of the adult rat brain: could oxytocin-induced labor cause pervasive developmental diseases?

Perinatal single-hormone treatment causes hormonal imprinting with lifelong consequences in receptor-binding capacity, hormone production as well as in social and sexual behavior. In the present experiments, newborn rats were treated with a single dose of oxytocin, and the levels of biogenic amines and their metabolites were studied in 8 different brain regions and in the sera when the male and female animals were 4 months old. Both dopaminergic and serotonergic neurotransmission was found to be significantly influenced. The levels of 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindole acetic acid metabolites decreased in the hypothalamus and striatum. Dopamine, serotonin, norepinephrine, and 5-hydroxytryptophol levels were hardly altered, and there was no difference in the epinephrine levels. The results show that dopamine and serotonin metabolism of hypothalamus and striatum are deeply and lifelong influenced by a single neonatal oxytocin treatment Oxytocin imprinting resulted in decreased dopamine turnover in the hypothalamus and decreased serotonin turnover in the hypothalamus, medulla oblongata, and striatum of females. As the disturbance of brain dopamine and serotonin system has an important role in the development of pervasive developmental diseases (eg, autism) and neuropsychiatric disorders (eg, schizophrenia), the growing number of oxytocin-induced labor as a causal factor, cannot be omitted.

Effect of perinatal stress on the biogenic amine neurotransmitter level of the adult rat's brain.

The amount of biogenic amines (dopamine and serotonin) and their metabolites (DOPAC, HVA, 5-HIAA, and 5-HTOL) in five regions of the brain (frontal cortex, hypothalamus, hippocampus, striatum, and brainstem) was studied in the male and female offspring of control and perinatally (48 h before birth or 48 h after birth) food and water deprived dams, when they were three months old, by using HPLC-EC determination. The increase of amine or metabolite level was dominant (19 values increased and 10 decreased related to control). Before-birth stress caused increase in 9 case and only 2 decreased, while in the case of after-birth stress 10 increased and 8 decreased. However, though there is no possibility to decide an exact tendency of direction, the after-birth stress (transmitted by milk) has more expressed effect. Striatum and brainstem were the most touched regions. There was a gender dependence with the dominance of males, except striatum. Blood plasma nociceptin level was also studied and there was a significant elevation in males after pre- and postnatal deprivation, while in females only after postnatal deprivation. The importance of the results in correlation with other stress effects is discussed.

The metabolism and disposition of a potent inhibitor of hepatitis C virus NS3/4A protease.

Compound A ((1aR,5S,8S,10R,22aR)-5-tert-butyl-N-{(1R,2S)-1-[(cyclopropylsulfonyl)carbamoyl]-2-ethenylcyclopropyl}-14-methoxy-3,6-dioxo-1,1a,3,4,5,6,9,10,18,19,20,21,22,22a-tetradecahydro-8H-7,10-methanocyclopropa[18,19][1,10,3,6]dioxadiazacyclononadecino[12,11-b]quinoline-8-carboxamide) is a prototype of a series of subnanomolar inhibitors of genotypes 1, 2, and 3 hepatitis C virus (HCV) NS3/4A proteases. HCV NS3/4A protease inhibitors have demonstrated high antiviral effects in patients with chronic HCV infection and are likely to form a key component of future HCV therapy. Compound A showed excellent liver exposure in rats, which is essential for compounds intended to treat HCV. The compound was mainly eliminated intact in bile and showed greater than dose proportional systemic exposure in rats. Compound A demonstrated time- and temperature-dependent uptake into rat and human hepatocytes and proved to be a substrate for rat hepatic uptake transporter Oatp1b2 and for human hepatic uptake transporters OATP1B1 and OATP1B3. The liver selectivity observed for this compound is likely to be due to transporter-mediated hepatic uptake together with moderate passive permeability. Metabolism was mainly CYP3A-mediated and generated a reactive epoxide on the vinylcyclopropyl sulfonamide moiety that could be quenched by glutathione. Similar metabolic profiles of Compound A were obtained in liver microsomes of rats and humans. The oral bioavailability at 5 mg/kg was low due to extensive hepatic first-pass effect but clearly the intestinal absorption was enough to deliver a high amount of the compound to the liver. The metabolism and disposition properties of Compound A are particularly attractive to support its evaluation as a drug candidate for the treatment of hepatitis C.

Pyridinium aldoxime analysis by HPLC: the method for studies on pharmacokinetics and stability.

Reversed-phase separation of various pyridinium aldoximes requires a certain concentration of ion-pairing agent, as their chemical structures contain two quaternary amines in the pyridinium ring. Adequate mobile phase is scouted on the basis of retention of pyridinium aldoxime (using the graph of k' versus concentration of an ion-pairing agent) compared to the chromatogram of the background peaks originated from the homogenate. Change in the ion-pairing agent concentration was more expressed for the elution of K-203 than that of the background peaks from the serum, brain and cerebrospinal fluid. Stability of K-203 was investigated using HPLC. Determination of K-203 in tissue samples requires homogenization using either trichloroacetic acid or perchloric acid. Fast degradation takes place at acidic pH. Adjusting pH to neutral in the possible shortest time frame helps to avoid degradation. Degradation of K-203 was easily followed by HPLC separation and monitoring the elution with an ultraviolet absorbance detector at 276 nm. Amperometric detection indicates only the decrease of K-203 content.

Metabolism-related liabilities of a potent histone deacetylase (HDAC) inhibitor and relevance of the route of administration on its metabolic fate.

Compound A [1-methyl-N-{(1S)-1-[5-(2-naphthyl)-1H-imidazol-2-yl]-7-oxooctyl}piperidine-4-carboxamide is a potent class I histone deacetylase (HDAC) inhibitor that demonstrated good antiproliferative activity against human tumour cell lines of different origin. This compound showed high in vivo clearance in rats (160 ml min(-1) kg(-1)) due to metabolism. The main metabolite detected in urine after intravenous dosing was characterized as a dihydrohydroxy S-mercapturic acid conjugate. Following oral dosing, however, the mercapturic acid derivative was no longer the main metabolite but the major metabolites were mono- and di-glucuronide conjugates of oxidized species having a mass shift of +34 m/z with respect to the parent. Comparison of plasma concentration after intra-arterial infusion and intravenous infusion and incubation with microsomes from different tissues (liver, kidney, small intestine and lung) in the presence of beta-nicotinamide adenine dinucleotide phosphate (NADPH) indicated that the compound was highly cleared by the lung. Oxidation of the naphthalene moiety was demonstrated to be the cause of the high in vivo clearance of compound A and the potential for bioactivation of this group was flagged.

Studies of metabolism and disposition of potent human immunodeficiency virus (HIV) integrase inhibitors using 19F-NMR spectroscopy.

(19)F-nuclear magnetic resonance (NMR) has been extensively used in a drug-discovery programme to support the selection of candidates for further development. Data on an early lead compound, N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(4-methylmorpholin-3-yl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide (compound A (+)), and MK-0518 (N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(1-methyl-1-{[(5-methyl-1,3,4-oxadiazol-2-yl)carbonyl]amino}ethyl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide), a potent inhibitor of this series currently in phase III clinical trials, are described. The metabolic fate and excretion balance of compound A (+) and MK-0518 were investigated in rats and dogs following intravenous and oral dosing using a combination of (19)F-NMR-monitored enzyme hydrolysis and solid-phase extraction chromatography and NMR spectroscopy (SPEC-NMR). Dosing with the (3)H-labelled compound A (+) enabled the comparison of standard radiochemical analysis with (19)F-NMR spectroscopy to obtain quantitative metabolism and excretion data. Both compounds were eliminated mainly by metabolism. The major metabolite identified in rat urine and bile and in dog urine was the 5-O-glucuronide.

Development of humanized mice for the study of hepatitis C virus infection.

The development of a small animal model for hepatitis C virus (HCV) infection is a critical issue for the development of novel anti-HCV drugs. To this aim, we have tried many different approaches for generating mice carrying humanized liver. Main efforts were focused on the transplantation of human hepatocytes into immunocompromised mice (SCID-/-, Bg-/-) carrying a genetic lethal liver disease (Alb-uPA). Survival of homozygotic animals should largely depend on early transplantation with healthy hepatocytes. In parallel to establishing a colony of Alb-uPA/SCID/Bg mice, we developed a microsurgical procedure for intrasplenic xenotransplantation of healthy hepatocytes in 1-week-old mice. So far, we generated several chimeras by xenotransplanting human hepatocytes in Alb-uPA+/+/SCID-/-/Bg-/- mice at 1 week after birth. In a first step, identification of successfully engrafted animals is possible by quantification of human serum albumin and human alpha 1 antitrypsin in mouse sera. Additional preliminary histomorphological analysis of liver sections from chimeric animals was also carried out. One of the mice was transiently infected with HCV, reaching viremia levels of approximately 10(5) genomes/mL. However, the efficiency of this system to generate chimeric mice is still very limited. We are currently exploring the use of more robust models of hepatic disease. Moreover, we have been also exploring novel strategies for the generation of chimeric mice by xenotransplanting human adult stem cells, instead of human hepatocytes, at preimmune stages of development.

Effects of fibrates on human organic anion-transporting polypeptide 1B1-, multidrug resistance protein 2- and P-glycoprotein-mediated transport.

The effects of different fibric acid derivatives (bezafibrate, clofibrate, clofibric acid, fenofibrate, fenofibric acid and gemfibrozil) on human organic anion transporting-polypeptide 1B1 (OATP2, OATP-C, SLC21A6), multidrug resistance protein 2 (MRP2/ABCC2) and MDR1-type P-glycoprotein (P-gp/ABCB1) were examined in vitro. Cyclosporin A (a known inhibitor of OATP1B1 and P-gp), MK-571 (a known inhibitor of MRP2) and cimetidine (an organic cation) were also tested. Bezafibrate, fenofibrate, fenofibric acid and gemfibrozil showed concentration-dependent inhibition of estradiol 17-beta-D-glucuronide uptake by OATP1B1-stably transfected HEK cells, whereas clofibrate and clofibric acid did not show any significant effects up to 100 microM. Inhibition kinetics of gemfibrozil, which exhibited the most significant inhibition on OATP1B1, was shown to be competitive with a Ki = 12.5 microM. None of the fibrates showed any significant inhibition of MRP2-mediated transport, which was evaluated by measuring the uptake of ethacrynic acid glutathione into MRP2-expressing Sf9 membrane vesicles. Only fenofibrate showed moderate P-gp inhibition as assessed by measuring cellular accumulation of vinblastine in a P-gp overexpressing cell-line. Cyclosporin A significantly inhibited OATP1B1 and P-gp, whereas only moderate inhibition was observed on MRP2. The rank order of inhibitory potency of MK-571 was determined as OATP1B1 (IC50: 0.3 microM) > MRP2 (4 microM) > P-gp (25 microM). Cimetidine did not show any effects on these transporters. In conclusion, neither MRP2- nor P-gp-mediated transport is inhibited significantly by the fibrates tested. Considering the plasma protein binding and IC50 values for OATP1B1, only gemfibrozil appeared to have a potential to cause drug-drug interactions by inhibiting OATP1B1 at clinically relevant concentrations.

Preclinical pharmacokinetics and metabolism of a potent non-nucleoside inhibitor of the hepatitis C virus NS5B polymerase.

The disposition of compound A, a potent inhibitor of the hepatitis C virus (HCV) NS5B polymerase, was characterized in animals in support of its selection for further development. Compound A exhibited marked species differences in pharmacokinetics. Plasma clearance was 44 ml min-1 kg-1 in rats, 9 ml min-1 kg-1 in dogs and 16 ml min-1 kg-1 in rhesus monkeys. Oral bioavailability was low in rats (10%) but significantly higher in dogs (52%) and monkeys (26%). Compound A was eliminated primarily by metabolism in rats, with biliary excretion accounting for 30% of its clearance. Metabolism was mainly mediated by cyclohexyl hydroxylation, with N-deethylation and acyl glucuronide formation constituting minor metabolic pathways. Qualitatively, the same metabolites were identified using in vitro systems from all species studied, including humans. The low oral bioavailability of compound A in rats was mostly due to poor intestinal absorption. This conclusion was borne out by the findings that hepatic extraction in the rat was only 30%, intraperitoneal bioavailability was good, and compound A was poorly absorbed from the rat isolated intestinal loop, with no detectable intestinal metabolism. Compound A was not an inhibitor of major human cytochrome P450 enzymes, indicating minimal potential for clinical drug-drug interactions. The metabolic clearance of compound A in rat, dog and monkey hepatocytes correlated with the systemic clearance observed in these species. Since compound A was very stable in human hepatocytes, the results suggest that it will be a low clearance drug in humans.

Enhancing B- and T-cell immune response to a hepatitis C virus E2 DNA vaccine by intramuscular electrical gene transfer.

We describe an improved genetic immunization strategy for eliciting a full spectrum of anti-hepatitis C virus (HCV) envelope 2 (E2) glycoprotein responses in mammals through electrical gene transfer (EGT) of plasmid DNA into muscle fibers. Intramuscular injection of a plasmid encoding a cross-reactive hypervariable region 1 (HVR1) peptide mimic fused at the N terminus of the E2 ectodomain, followed by electrical stimulation treatment in the form of high-frequency, low-voltage electric pulses, induced more than 10-fold-higher expression levels in the transfected mouse tissue. As a result of this substantial increment of in vivo antigen production, the humoral response induced in mice, rats, and rabbits ranged from 10- to 30-fold higher than that induced by conventional naked DNA immunization. Consequently, immune sera from EGT-treated mice displayed a broader cross-reactivity against HVR1 variants from natural isolates than sera from injected animals that were not subjected to electrical stimulation. Cellular response against E2 epitopes specific for helper and cytotoxic T cells was significantly improved by EGT. The EGT-mediated enhancement of humoral and cellular immunity is antigen independent, since comparable increases in antibody response against ciliary neurotrophic factor or in specific anti-human immunodeficiency virus type 1 gag CD8(+) T cells were obtained in rats and mice. Thus, the method described potentially provides a safe, low-cost treatment that may be scaled up to humans and may hold the key for future development of prophylactic or therapeutic vaccines against HCV and other infectious diseases.

In vivo localization and characterization of functional ciliary neurotrophic factor receptors which utilize JAK-STAT signaling.

The ciliary neurotrophic factor receptor is critically involved in embryonic motor neuron development. Postnatally, it may contribute to neuronal maintenance and regeneration. In addition, pharmacological stimulation of the receptor may slow the progression of several neurodegenerative disorders. The widespread nervous system expression of ciliary neurotrophic factor receptor components and the effects of low ciliary neurotrophic factor concentrations on a wide variety of cells in culture combine to suggest that functional ciliary neurotrophic factor receptors are expressed by many classes of neurons in vivo. However, the in vivo signaling properties and distribution of functional ciliary neurotrophic factor receptors have not been directly determined. We developed a novel in vivo assay of functional ciliary neurotrophic factor receptors which revealed that, in the adult nervous system, cranial and spinal motor neurons are very sensitive to ciliary neurotrophic factor and display a rapid, robust increase in phospho-STAT3 in their dendrites, cell bodies and nuclei, which is specifically blocked by the ciliary neurotrophic factor receptor antagonist, AADH-CNTF. In distinct contrast, several other classes of ciliary neurotrophic factor receptor expressing neurons fail to increase phospho-STAT3 levels following ciliary neurotrophic factor treatment, even when ciliary neurotrophic factor is applied at high concentrations. Leukemia inhibitory factor and epidermal growth factor elicit the same cell-type-dependent pattern of phospho-STAT3 increases. Responsive and non-responsive neurons express comparable levels of STAT3.Therefore, in vivo ciliary neurotrophic factor receptor-initiated STAT3 signal transduction is regulated in a very cell-type-dependent manner. The present data suggest that at least some of this regulation occurs at the STAT3 tyrosine phosphorylation step. These unexpected results also suggest that other forms of receptor-initiated STAT3 signal transduction may be similarly regulated.

Leptin receptor-mediated regulation of cholinergic neurotransmitter phenotype in cells of central nervous system origin.

Leptin is an adipocyte-secreted hormone that regulates body weight and exerts effects on hematopoiesis, reproduction, and immunity. The leptin receptor (OBR) shares sequence similarity and signaling capabilities with receptors for cytokines of the ciliary neurotrophic factor (CNTF) family. Our previous finding that CNTF and leptin exert similar anti-obesity effects and activate common neuronal signaling pathways, prompted us to investigate whether leptin may share with CNTF the ability to regulate the expression of specific neuronal genes. To this end, we established a cell line, derived from the murine septal cholinergic neuronal cell line SN-56, which stably expresses OBR. In this cell line, termed SN-56/OBR, leptin induces STAT transcription factor activation and STAT-dependent reporter gene expression in a manner similar to that of CNTF. Furthermore, in SN-56/OBR cells both CNTF and leptin produce changes in neurotransmitter and neuropeptide phenotype characteristic of cholinergic neurons, such as an increase in choline acetyltransferase and vasoactive intestinal polypeptide, and a decrease in neuropeptide Y expression. SN-56/OBR cells thus constitute an interesting new model system to investigate leptin action in cells of central nervous system origin. Possible physiological implications of OBR's intrinsic ability to regulate cholinergic phenotypic markers are discussed.

Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation.

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 microgram of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.

Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides.

Mutations reducing the functional activity of leptin, the leptin receptor, alpha-melanocyte stimulating hormones (alpha-MSH) and the melanocortin-4 receptor (Mc4r) all lead to obesity in mammals. Moreover, mutant mice that ectopically express either agouti (Ay/a mice) or agouti-related protein (Agrp), antagonists of melanocortin signalling, become obese. These data suggest that alpha-MSH signalling transduced by Mc4r tonically inhibits feeding; however, it is not known to what extent this pathway mediates leptin signalling. We show here that Mc4r-deficient (Mc4r-/-) mice do not respond to the anorectic actions of MTII, an MSH-like agonist, suggesting that alpha-MSH inhibits feeding primarily by activating Mc4r. Obese Mc4r-/-mice do not respond significantly to the inhibitory effects of leptin on feeding, whereas non-obese Mc4r-/- mice do. These data demonstrate that melanocortin signalling transduced by Mc4r is not an exclusive target of leptin action and that factors resulting from obesity contribute to leptin resistance. Leptin resistance of obese Mc4r-/- mice does not prevent their response to the anorectic actions of ciliary neurotrophic factor (CNTF), corticotropin releasing factor (CRF), or urocortin; or the orexigenic actions of neuropeptide Y (NPY) or peptide YY (PYY), indicating that these neuromodulators act independently or downstream of Mc4r signalling.

Agonistic and antagonistic variants of ciliary neurotrophic factor (CNTF) reveal functional differences between membrane-bound and soluble CNTF alpha-receptor.

Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR) and the signal-transducing beta-subunits gp130 and leukemia inhibitory factor receptor-beta (LIFR). CNTFR can function in either membrane-bound or soluble forms. The membrane-bound form mediates the neuronal actions of CNTF, whereas the soluble form serves to confer cytokine responsiveness to non-neuronal cells expressing gp130 and LIFR. The objective of this work was to analyze whether the two receptor isoforms differ in their ability to interact functionally with CNTF and related proteins. Two new types of CNTF variants, characterized by weakened interactions with either CNTFR or both LIFR and gp130, were developed, and the biological activities of these and other mutants were determined in non-neuronal versus neuronal cells, as well as in non-neuronal cells transfected with an expression vector for CNTFR. Membrane anchoring of CNTFR was found to render the CNTF receptor complex relatively insensitive to changes in agonist affinity for either alpha- or beta-receptor subunits and to promote a more efficient interaction with a gp130-depleting antagonistic variant of CNTF. As a result of this phenomenon, which can be rationalized in terms of the multivalent nature of CNTF receptor interaction, CNTF variants display striking changes in receptor selectivity.

Ciliary neurotrophic factor corrects obesity and diabetes associated with leptin deficiency and resistance.

Receptor subunits for the neurocytokine ciliary neurotrophic factor (CNTF) share sequence similarity with the receptor for leptin, an adipocyte-derived cytokine involved in body weight homeostasis. We report here that CNTF and leptin activate a similar pattern of STAT factors in neuronal cells, and that mRNAs for CNTF receptor subunits, similarly to the mRNA of leptin receptor, are localized in mouse hypothalamic nuclei involved in the regulation of energy balance. Systemic administration of CNTF or leptin led to rapid induction of the tis-11 primary response gene in the arcuate nucleus, suggesting that both cytokines can signal to hypothalamic satiety centers. Consistent with this idea, CNTF treatment of ob/ob mice, which lack functional leptin, was found to reduce the adiposity, hyperphagia, and hyperinsulinemia associated with leptin deficiency. Unlike leptin, CNTF also reduced obesity-related phenotypes in db/db mice, which lack functional leptin receptor, and in mice with diet-induced obesity, which are partially resistant to the actions of leptin. The identification of a cytokine-mediated anti-obesity mechanism that acts independently of the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance.