Metabolism and disposition of zamicastat in rats.

The metabolism and disposition of zamicastat, a reversible dopamine ß-hydroxylase (DßH) inhibitor, developed for treatment of Pulmonary Arterial Hypertension (PAH), were investigated in rats after oral and intravenous administration of [14C]-zamicastat.Zamicastat was rapidly absorbed and widely distributed to peripheral tissues, with total radioactivity almost completely recovered 168 h post-dose. Its main route of excretion was via faeces, whilst urine and expired air had minor roles.Maximum plasma concentration of zamicastat-related radioactivity occurred in the first hours, remaining quantifiable up to 144 h. The unchanged zamicastat plasma peak was 2 h post-dose and declined to low levels over 24 h.Zamicastatmetabolism occurs largely during the first 8 h with only one metabolite identified in the latest time-point (96 h), the isocyanic acid/thiocyanic acid (tautomeric forms). Zamicastat metabolic pathway involved multiple reactions comprising desulphurisation, oxidative desulphurisation, N-debenzylation followed by further oxidation or N-acetylation, and the unexpected multistep metabolic pathway leading to isocyanic acid/thiocyanic acid.

Absorption, metabolism and excretion of opicapone in human healthy volunteers.

AIMS: The absorption, metabolism and excretion of opicapone (2,5-dichloro-3-(5-[3,4-dihydroxy-5-nitrophenyl]-1,2,4-oxadiazol-3-yl)-4,6-dimethylpyridine 1-oxide), a selective catechol-O-methyltransferase inhibitor, were investigated. METHODS: Plasma, urine and faeces were collected from healthy male subjects following a single oral dose of 100 mg [14 C]-opicapone. The mass balance of [14 C]-opicapone and metabolic profile were evaluated. RESULTS: The recovery of total administered radioactivity averaged >90% after 144 hours. Faeces were the major route of elimination, representing 70% of the administered dose; 5% and 20% were excreted in urine and expired air, respectively. The Cmax of total radioactivity matched that of unchanged opicapone, whereas the total radioactivity remained quantifiable for a longer period, attributed to the contribution of opicapone metabolites, involving primarily 3-O-sulfate conjugation (58.6% of total circulating radioactivity) at the nitrocatechol ring. Other circulating metabolites, accounting for <10% of the radioactivity exposure, were formed by glucuronidation, methylation, N-oxide reduction and gluthatione conjugation. Additionally, various other metabolites resulting from combinations with the opicapone N-oxide reduced form at the 2,5-dichloro-4,6-dimethylpyridine 1-oxide moiety, including nitro reduction and N-acetylation, reductive opening and cleavage of the 1,2,4-oxadiazole ring and the subsequent hydrolysis products were identified, but only in faeces, suggesting the involvement of gut bacteria. CONCLUSION: [14 C]-opicapone was fully excreted through multiple metabolic pathways. The main route of excretion was in faeces, where opicapone may be further metabolized via reductive metabolism involving the 1,2,4-oxadiazole ring-opening and subsequent hydrolysis.

Hidden behind the mask: An authentication study on the Aztec mask of the Museum of Ethnography, Budapest, Hungary.

Turquoise covered mosaic objects - especially masks - were attractive components of treasures transported to Europe from Mexico after the fall of the Aztec Empire in the 1500s. According to our present knowledge, the mosaic masks were manufactured for ritual purpose. The main material of mosaics, the turquoise was a high-prestige semi-precious stone among Mexican native people. During the 20th century, such objects derived both from illegal treasure hunting and documented archaeological excavations. The aim of our research was the authentication of a turquoise covered Aztec wooden mask, which presumably originates from the Tehuacán Valley, Mexico and exchanged by the Museum of Ethnography, Budapest, in 1973. The detailed and complex analytical investigation of the mask is a curiosity. To reveal the origin of the object, UV photographs were taken, the wooden base was subjected to biological studies and C-14 dating, the organic glue fixing the tesserae and the inorganic mosaic tesserae were investigated by non-destructive chemical, FT-IR and Raman spectroscopic methods. Our investigations determined that the mask of the Museum of Ethnography was made of an alder species of tree and its age is AD 1492-1653. The light-coloured covering mosaic lamellae were identified as alabaster and claystone. Comparing the turquoise tesserae cover with reference materials, their chemical composition has been clearly differentiated from most of the well-known turquoise sources of the US Southwest. Based on our results, the Aztec mask of the Museum of Ethnography proved to be an original piece of art from the 15th-17th century.

Metabolism and disposition of opicapone in the rat and metabolic enzymes phenotyping.

Opicapone (2,5-dichloro-3-(5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-4,6-dimethylpyridine 1-oxide) is a selective catechol-O-methyltransferase inhibitor that has been granted marketing authorization in Europe, Japan, and United States. The present work describes the metabolism and disposition of opicapone in the rat obtained in support to its development and regulatory filling. Plasma levels and elimination of total radioactivity were determined after oral and intravenous administration of [14 C]-opicapone. The maximum plasma concentrations of opicapone-related radioactivity were reached at early time points followed by a gradual return to baseline with a biphasic elimination. Fecal excretion was the primary route of elimination of total radioactivity. Quantitative distribution of drug-related radioactivity demonstrated that opicapone and related metabolites did not distribute to the central nervous system. Opicapone was extensively metabolized in rats resulting in more than 20 phase I and phase II metabolites. Although O-glucuronidation, -sulfation, and -methylation of the nitrocatechol moiety were the principal metabolic pathways, small amount of the N-acetyl derivative was detected, as a result of reduction of the nitro group and subsequent conjugation. Other metabolic transformations included N-oxide reduction to the pyridine derivative and reductive cleavage of 1,2,4-oxadiazole ring followed by further conjugative reactions. Reaction phenotyping studies suggested that SULT 1A1*1 and *2 and UGT1A7, UGT1A8, UGT1A9, and UGT1A10 may be involved in opicapone sulfation and glucuronidation, respectively. However, the reductive metabolic pathways mediated by gut microflora cannot be excluded. Opicapone, in the rat, was found to be rapidly absorbed, widely distributed to peripheric tissues, metabolized mainly via conjugative pathways at the nitro catechol ring, and primarily excreted via feces.

Massive methane fluxing from magma-sediment interaction in the end-Triassic Central Atlantic Magmatic Province.

Exceptional magmatic events coincided with the largest mass extinctions throughout Earth's history. Extensive degassing from organic-rich sediments intruded by magmas is a possible driver of the catastrophic environmental changes, which triggered the biotic crises. One of Earth's largest magmatic events is represented by the Central Atlantic Magmatic Province, which was synchronous with the end-Triassic mass extinction. Here, we show direct evidence for the presence in basaltic magmas of methane, generated or remobilized from the host sedimentary sequence during the emplacement of this Large Igneous Province. Abundant methane-rich fluid inclusions were entrapped within quartz at the end of magmatic crystallization in voluminous (about 1.0 × 106 km3) intrusions in Brazilian Amazonia, indicating a massive (about 7.2 × 103 Gt) fluxing of methane. These micrometre-sized imperfections in quartz crystals attest an extensive release of methane from magma-sediment interaction, which likely contributed to the global climate changes responsible for the end-Triassic mass extinction.

Deep CO2 in the end-Triassic Central Atlantic Magmatic Province.

Large Igneous Province eruptions coincide with many major Phanerozoic mass extinctions, suggesting a cause-effect relationship where volcanic degassing triggers global climatic changes. In order to fully understand this relationship, it is necessary to constrain the quantity and type of degassed magmatic volatiles, and to determine the depth of their source and the timing of eruption. Here we present direct evidence of abundant CO2 in basaltic rocks from the end-Triassic Central Atlantic Magmatic Province (CAMP), through investigation of gas exsolution bubbles preserved by melt inclusions. Our results indicate abundance of CO2 and a mantle and/or lower-middle crustal origin for at least part of the degassed carbon. The presence of deep carbon is a key control on the emplacement mode of CAMP magmas, favouring rapid eruption pulses (a few centuries each). Our estimates suggest that the amount of CO2 that each CAMP magmatic pulse injected into the end-Triassic atmosphere is comparable to the amount of anthropogenic emissions projected for the 21st century. Such large volumes of volcanic CO2 likely contributed to end-Triassic global warming and ocean acidification.

Discovery of a Potent, Long-Acting, and CNS-Active Inhibitor (BIA 10-2474) of Fatty Acid Amide Hydrolase.

Fatty acid amide hydrolase (FAAH) can be targeted for the treatment of pain associated with various medical conditions. Herein we report the design and synthesis of a novel series of heterocyclic-N-carboxamide FAAH inhibitors that have a good alignment of potency, metabolic stability and selectivity for FAAH over monoacylglycerol lipase (MAGL) and carboxylesterases (CEs). Lead optimization efforts carried out with benzotriazolyl- and imidazolyl-N-carboxamide series led to the discovery of clinical candidate 8 l (3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide; BIA 10-2474) as a potent and long-acting inhibitor of FAAH. However, during a Phase I clinical trial with compound 8 l, unexpected and unpredictable serious neurological adverse events occurred, affecting five healthy volunteers, including the death of one subject.

Ischemia/reperfusion-induced Kidney Injury in Heterozygous PACAP-deficient Mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with very diverse distribution and functions. Among others, PACAP is a potent cytoprotective peptide due to its antiapoptotic, anti-inflammatory, and antioxidant actions. This also has been shown in different kidney pathologies, including ischemia/reperfusion-induced kidney injury. Similar protective effects of the endogenous PACAP are confirmed by the increased vulnerability of PACAP-deficient mice to different harmful stimuli. Kidneys of homozygous PACAP-deficient mice have more severe damages in renal ischemia/reperfusion and kidney cell cultures isolated from these mice show increased sensitivity to renal oxidative stress. In our present study we raised the question of whether the partial lack of the PACAP gene is also deleterious, i.e. whether heterozygous PACAP-deficient mice also display more severe damage after renal ischemia/reperfusion. Mice underwent 45 or 60 minutes of ischemia followed by 2 weeks reperfusion. Histological evaluation of the kidneys was performed and individual histopathological parameters were graded. Furthermore, we investigated apoptotic markers, cytokine expression, and the activity of superoxide dismutase (SOD) enzyme 24 hours after 60 minutes of renal ischemia/reperfusion. We found no difference between the intact kidneys of wild-type and heterozygous mice, but marked differences could be observed following ischemia/reperfusion. Heterozygous PACAP-deficient mice had more severe histological alterations, with significantly higher histopathological scores for most of the tested parameters. Higher level of the proapoptotic pp38 MAPK and of some proinflammatory cytokines, as well as lower activity of the antioxidant SOD could be found in these mice. In conclusion, the partial lack of the PACAP gene results in worse outcomes in cases of renal ischemia/reperfusion, confirming that PACAP functions as an endogenous protective factor in the kidney.

Medicinal chemistry of catechol O-methyltransferase (COMT) inhibitors and their therapeutic utility.

Catechol O-methyltransferase (COMT) is the enzyme responsible for the O-methylation of endogenous neurotransmitters and of xenobiotic substances and hormones incorporating catecholic structures. COMT is a druggable biological target for the treatment of various central and peripheral nervous system disorders, including Parkinson's disease, depression, schizophrenia, and other dopamine deficiency-related diseases. The purpose of this perspective is fourfold: (i) to summarize the physiological role of COMT inhibitors in central and peripheral nervous system disorders; (ii) to provide the history and perspective of the medicinal chemistry behind the discovery and development of COMT inhibitors; (iii) to discuss how the physicochemical properties of recognized COMT inhibitors are understood to exert influence over their pharmacological properties; and (iv) to evaluate the clinical benefits of the most relevant COMT inhibitors.

PACAP is an endogenous protective factor-insights from PACAP-deficient mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a widespread neuropeptide with a diverse array of biological functions. Not surprisingly, the lack of endogenous PACAP therefore results in a variety of abnormalities. One of the important effects of PACAP is its neuroprotective and general cytoprotective role. PACAP protects neurons and other tissues against ischemic, toxic, and traumatic lesions. Data obtained from PACAP-deficient mice provide evidence that endogenous PACAP also has protective functions. Mice lacking PACAP are more vulnerable to different in vitro and in vivo insults. The present review summarizes data on the increased sensitivity of PACAP-deficient mice against harmful stimuli. Mice lacking PACAP respond with a higher degree of injury in cerebral ischemia, autoimmune encephalomyelitis, and axonal lesion. Retinal ischemic and excitotoxic injuries also produce increased cell loss in PACAP-deficient mice. In peripheral organs, kidney cell cultures from PACAP-deficient mice are more sensitive to oxidative stress and in vitro hypoxia. In vivo, PACAP-deficient mice have a negative histological outcome and altered cytokine response in kidney and small intestine ischemia/reperfusion injury. Large intestinal inflammation, toxic lesion of the pancreas, and doxorubicin-induced cardiomyopathy are also more severe with a lack of endogenous PACAP. Finally, an increased inflammatory response has been described in subacute endotoxin-induced airway inflammation and in an oxazolone-induced allergic contact dermatitis model. In summary, lack of endogenous PACAP leads to higher vulnerability in a number of injuries in the nervous system and peripheral organs, supporting the hypothesis that PACAP is part of the endogenous cytoprotective machinery.

Presence of pituitary adenylate cyclase activating polypeptide and its type I receptor in the rat kidney.

Pituitary adenylate cyclase activating polypeptide (PACAP), a multifunctional neuropeptide, has 2 active forms, PACAP38 and PACAP27. It is now well-established that PACAP has several actions also in peripheral organs, including renoprotective effects. The peptide itself has not been previously identified in the rat kidney. The first aim of our study was to identify PACAP in the rat kidney using mass spectrometry and radioimmunoassay (RIA). Receptor mRNA and binding studies revealed the existence of all 3 PACAP receptors (PAC1, VPAC1, and VPAC2) in the kidney, but their exact localization in histologic sections was not evident. Because most of the cytoprotective effects of PACAP relate to its specific PAC1 receptor, our second aim was to identify the cell types wherein the PAC1 receptor is expressed in the rat kidney. Mass spectrometry revealed the presence of PACAP38 in the kidney. RIA measurements showed both PACAP38- and PACAP27-like immunoreactivities in kidney homogenates, with PACAP38 being dominant. Immunohistochemistry revealed PAC1 receptor-like immunoreactivity in kidney sections, mainly expressed in cortical tubular epithelial cells. These results showed PACAP to be endogenously present in the kidney. The tubular localization of the PAC1 receptor provides the basis for the renal effects of the peptide under physiologic and pathologic conditions.

The chemistry of catechol-O-methyltransferase inhibitors.

Despite several drawbacks, levodopa (L-dopa) remains the gold standard drug for treatment of the symptoms of Parkinson's disease (PD). L-dopa is a pro-drug of dopamine and is used to elevate striatal levels of the neurotransmitter. One approach to provide a more continuous and sustained delivery of dopamine has targeted one of the principal enzymes responsible for metabolic deactivation of L-dopa, namely catechol-O-methyltransferase (COMT). The chapter will provide a perspective of the medicinal chemistry behind the discovery of several COMT inhibitors and discuss how certain physicochemical parameters, including aqueous solubility and lipophilicity, are thought to influence pharmacokinetic properties such as absorption, distribution, and bioavailability.

Changes in pituitary adenylate cyclase-activating polypeptide following renal ischemia-reperfusion in rats.

PACAP (pituitary adenylate cyclase-activating polypeptide) occurs in two biologically active forms, with 38 and 37 amino acid residues (PACAP38 and PACAP27). In mammalian tissues, PACAP38 is the dominant form. Diverse effects have been described in the cardiovascular, respiratory, gastrointestinal, and urogenital systems. PACAP is known for its strong cytoprotective effects, which are present endogenously as well, as proven by knockout studies and results showing that PACAP is up-regulated following diverse injuries. Little is known about such effects in the kidney. We have previously shown that PACAP is protective in renal ischemia-reperfusion injury. Therefore, the aim of the present study was to investigate the changes of endogenous PACAP following 60-minute renal ischemia using radioimmunoassay. Changes were observed within 24 hours following renal vessel clamping. In the cortex, an acute decrease was followed by an increase on the intact side, and levels returned to original ones on the operated side. In the medulla, changes were only observed on the clamped side: a marked up-regulation was detected in PACAP38-like immunoreactivity within the first 24 hours. The present study showed that PACAP38- and PACAP27-like immunoreactivities sensitively react to renal ischemia-reperfusion, the physiological importance of which awaits further investigation.

Mice deficient in neuropeptide PACAP demonstrate increased sensitivity to in vitro kidney hypoxia.

One of the well-known effects of pituitary adenylate cyclase activating polypeptide (PACAP) is its neuroprotective and cytoprotective actions including renoprotective effects. Mice deficient in endogenous PACAP exhibit several behavioral, metabolic, and developmental alterations. Furthermore, PACAP-deficient mice have larger infarct volume in a model of cerebral ischemia, delayed axonal regeneration, and increased cell death in cerebellar oxidative stress. We have previously demonstrated that PACAP-deficient mice have increased susceptibility to in vitro oxidative stress, which can be counteracted by exogenous PACAP treatment. These results demonstrate that endogenous PACAP has a protective role against various stressors. The objective of the present study was to investigate whether endogenous PACAP has a protective effect in the kidney against in vitro hypoxia. Kidney cell cultures were isolated from wild-type and PACAP-deficient mice, and cell viability was assessed after in vitro hypoxia induced using CoCl(2). The sensitivity of cells from PACAP-deficient mice was increased to hypoxia: both after 24 and 48 hours of exposure, cell viability was significantly reduced compared with that in control wild-type mice. These results show that endogenous PACAP protects against noxious stimuli in the kidney and that PACAP may act as a stress sensor in renal cells.

Discovery of a long-acting, peripherally selective inhibitor of catechol-O-methyltransferase.

Novel nitrocatechol-substituted heterocycles were designed and evaluated for their ability to inhibit catechol-O-methyltransferase (COMT). Replacement of the pyrazole core of the initial hit 4 with a 1,2,4-oxadiazole ring resulted in a series of compounds endowed with longer duration of COMT inhibition. Incorporation of a pyridine N-oxide residue at position 3 of the 1,2,4-oxadiazole ring led to analogue 37f, which was found to possess activity comparable to entacapone and lower toxicity in comparison to tolcapone. Lead structure 37f was systematically modified in order to improve selectivity and duration of COMT inhibition as well as to minimize toxicity. Oxadiazole 37d (2,5-dichloro-3-(5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-4,6-dimethylpyridine 1-oxide (BIA 9-1067)) was identified as a long-acting, purely peripheral inhibitor, which is currently under clinical evaluation as an adjunct to L-Dopa therapy of Parkinson's disease.

Efficient synthesis of 2-(trifluoromethyl)nicotinic acid derivatives from simple fluorinated precursors.

Novel routes to 2-trifluoromethyl-nicotinic acid derivatives have been developed involving synthesis of the pyridine ring. These pyridyl compounds serve as key intermediates in the manufacture of the recently discovered COMT inhibitor, 3-(5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-2-(trifluoromethyl)pyridine 1-oxide.

Effects of the plant alkaloid thaliblastine on non-cross-resistant and sensitive human leukemia cells in relation with reversal of acquired anthracycline resistance.

Thaliblastine exhibits dose dependent cytotoxic effect on HL-60, HL-60/DOX, RHE and HD-MY-2 leukemia cells. Additionally, typical for apoptosis oligonucleosomal DNA fragmentation could be detected in leukemia cells treated with thaliblastine. Moreover, an MDR-phenotype reversing effect of thaliblastine was also identified.

Blast colony-forming cell binding from CML bone marrow, or blood, on stromal layers pretreated with G-CSF or SCF.

Blast colony-forming cells (CFU-BL) represent a specific subpopulation of special primitive progenitors characterized by colony formation only in close contact with a preformed stromal layer. CFU-BL derived from bone marrow of chronic myeloid leukaemia (CML) patients have been proved to adhere poorly to bone marrow derived stromal layers suggesting that the appearance of progenitors and precursors in the circulation is due to a defective adhesion of these cells to the bone marrow microenvironment. In the present experiments the effect of short-term incubation of preformed normal bone marrow stroma on the adherence of CML derived CFU-BL was studied. For stroma cultures bone marrow cells were cultured in microplates in the presence of hydrocortisone. Cultures were used when stromal layers became confluent and no sign of haemopoiesis could be observed. CFU-BL were studied by panning plastic non-adherent mononuclear (PNAMNC) bone marrow or blood cells. 8.9 +/- 2.4 colonies/103 PNAMNC (six experiments) were formed from normal bone marrow on stromal layers and 4.8 +/- 2.1 colonies/103 PNAMNC (five experiments) from CML bone marrow. Colony formation from normal bone marrow was not increased if stromal layers were incubated with 100 ng/mL granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF). Incubation of stroma with G-CSF or SCF, however, increased the colony formation of PNAMNC from CML bone marrow or blood significantly. These findings suggest that local concentration of haemopoietic growth factors at the time of panning may influence the attachment of CML progenitors to the stroma.

[Immunophenotyping in acute leukemia: detection of minimal residual disease]. / Immunfenotípus vizsgálata akut leukaemiákban: a minimális reziduális betegség kimutatási lehetósége.

Immunophenotyping improves both accuracy and reproducibility of the acute leukaemia classification and is considered particularly useful for identifying poorly differentiated subtypes of acute myeloid leukaemia and lineage association of acute lymphoid leukaemia. Immunological studies of leukaemic blasts has become critical also identifying biphenotypic leukaemias and acute myeloid leukaemia expressing lymphoid associated markers. At present, while the prognostic value of individual antigen expressions is still controversial, the immunologic detection of minimal residual disease seems to be important in monitoring the acute leukaemia patients in remission. In the present study immunophenotyping of bone marrow samples of 42 patients with acute myeloid leukaemia and 13 patients with acute lymphoid leukaemia was analysed. Patients were assessed both before and after treatment by immunophenotyping, cytogenetics and polymerase chain reaction amplification. The immunophenotyping have allowed more sensitive definition of acute leukaemia relapse, but molecular genetic methods are recommended for detection of elimination of residual disease.

Human health risk assessment and management in hazardous waste site contaminated by polychlorinated benzenes.

The improper management of large quantity of chemical waste of a chemical plant caused considerable soil contamination on a temporary hazardous waste disposal site. Initial and action-oriented exposure and health risk assessment was carried out in early 90's. The dominant contaminants were tetrachlorobenzenes (TeCBs), pentachlorobenzene and hexachlorobenzene (HCB). Concentration of TeCBs in top soil layer and vadoze zone exceeded several g/kg. After taking appropriate technical measures quantitative health risk assessment was performed for the target population living in the surrounding settlements. Health risk estimates to HCB ingested or inhaled with suspended dust and soil particulates showed that carcinogenic risk values lower than 10(-6) i.e. below the acceptable risk. For other chlorobenzenes that cause non-cancer toxic effects risk quotients showed marked health hazard inside the dump and small potential risk in vicinity. However, this assessment is based on limited monitoring data, additional information is needed to substantiate this conclusion.