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1.
J Exp Biol ; 222(Pt 7)2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30862703

RESUMO

Several studies show effects of yolk androgens in avian eggs on the phenotype of the offspring. Yolk hormone concentrations decline strongly within the first few days of incubation. Although early embryonic uptake of yolk androgens is suggested by the presence of radioactivity in the embryo when eggs are injected with radiolabelled androgens, these studies do not verify the chemical identity of radioactive compound(s), although it is known that these androgens can be metabolized substantially. By using stable isotope-labelled testosterone and androstenedione in combination with mass spectrometry, enabling verification of the exact molecular identity of labelled compounds in the embryo, we found that after 5 days of incubation the androgens were not taken up by the embryo. However, their concentrations in the entire yolk albumen homogenates declined strongly, even when corrected for dilution by albumen and water. Our results indicate metabolism of maternal androgens, very likely to 5ß-androstane-3α,17ß-diol, etiocholanolone and their conjugated forms. The results imply that the effects of increased exposure of the embryo to maternal androgens take place either before this early conversion or are mediated by these metabolites with an as yet unknown function, opening new avenues for understanding hormone-mediated maternal effects in vertebrates.


Assuntos
Androstenodiona/metabolismo , Embrião de Galinha/metabolismo , Testosterona/metabolismo , Animais , Isótopos de Carbono , Cromatografia Líquida , Gema de Ovo/química , Espectrometria de Massas em Tandem , Trítio
2.
Inorg Chem ; 57(8): 4510-4515, 2018 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-29601196

RESUMO

Non-heme iron(II) complexes of pentadentate N4Py ( N,N-bis(2-pyridylmethyl)- N-bis(2-pyridyl)methylamine) type ligands undergo visible light-driven oxidation to their iron(III) state in the presence of O2 without ligand degradation. Under mildly basic conditions, however, highly selective base catalyzed ligand degradation with O2, to form a well-defined pyridyl-imine iron(II) complex and an iron(III) picolinate complex, is accelerated photochemically. Specifically, a pyridyl-CH2 moiety is lost from the ligand, yielding a potentially N4 coordinating ligand containing an imine motif. The involvement of reactive oxygen species other than O2 is excluded; instead, deprotonation at the benzylic positions to generate an amine radical is proposed as the rate determining step. The selective nature of the transformation holds implications for efforts to increase catalyst robustness through ligand design.

3.
Drug Metab Dispos ; 44(5): 683-91, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26932816

RESUMO

P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) are differentially expressed along the intestine and work coordinately to reduce the intracellular concentration of xenobiotics and the absorption of orally taken drugs. Drug-drug interactions (DDIs) based on P-gp/CYP3A interplay are of clinical importance and require preclinical investigation. We investigated the P-gp/Cyp3a interplay and related DDIs with different P-gp inhibitors in the various regions of the rat intestine ex vivo using precision-cut intestinal slices (PCIS) with quinidine (Qi), a dual substrate of P-gp and Cyp3a, as the probe. The results showed that P-gp efflux was the main factor limiting the intracellular Qi content at concentrations below 5µM, whereas both efflux and metabolism were saturated at [Qi] > 50µM. The selective P-gp inhibitors CP100356 [N-(3,4-dimethoxyphenethyl)-4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2[1H]-yl)-6,7-dimethoxyquinazolin-2-amine] and PSC833 [valspodar, 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-l-valine-cyclosporin A] enhanced the Qi accumulation in slices in line with the different P-gp expression in the intestinal regions and, as a result, also enhanced metabolism in the jejunum and ileum. Dual inhibitors of both P-gp and Cyp3a (verapamil and ketoconazole) increased the concentration of Qi in the jejunum and ileum, but less 3-hydroxy-quinidine was produced due to inhibition of Cyp3a. The results indicate that the P-gp/Cyp3a interplay depends on the concentration of the drug and on the intestinal region under study. Furthermore, due to the P-gp/Cyp3a interplay, DDIs can lead to remarkable changes in the intracellular concentration of both the parent drug and the metabolite, which varies among the intestinal regions and depends on the selectivity of the inhibitors, with potentially important implications for disposition and toxicity of drugs and their metabolites.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas/fisiologia , Íleo/metabolismo , Jejuno/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Ciclosporinas/metabolismo , Absorção Intestinal , Cetoconazol/metabolismo , Masculino , Quinidina/metabolismo , Ratos , Ratos Wistar , Valina/metabolismo , Verapamil/metabolismo
4.
Drug Metab Dispos ; 41(8): 1557-65, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23733277

RESUMO

The inclusion of nanoparticles dispersed in a hydrophilic matrix is one of the formulation strategies to improve the bioavailability of orally administered Biopharmaceutics Classification System (BCS) class II and IV drugs by increasing their dissolution rate in the intestine. To confirm that the increased dissolution rate results in increased bioavailability, in vitro and in vivo animal experiments are performed, however, translation to the human situation is hazardous. In this study, we used a range of in vitro and ex vivo methods, including methods applying human tissue, to predict the in vivo oral bioavailability of a model BCS class II CB-1 antagonist, formulated as a nanoparticle solid dispersion. The enhanced dissolution rate from the nanoparticle formulation resulted in an increased metabolite formation in both rat and human precision-cut intestinal slices, suggesting increased uptake and intracellular drug concentration in the enterocytes. In Ussing chamber experiments with human tissue, both the metabolite formation and apical efflux of the metabolite were increased for the nanoparticulate solid dispersion compared with a physical mixture, in line with the results in intestinal slices. The pharmacokinetics of the different formulations was studied in rats in vivo. The nanoparticle formulation indeed improved the absorption of the cannabinoid receptor 1 (CB-1) antagonist and the delivery into the brain compared with the physical mixture. In conclusion, the combined approach provides a valuable set of tools to investigate the effects of formulation on the absorption of poorly soluble compounds in human intestine and may provide relevant information on the oral bioavailability in humans early in the development process.


Assuntos
Antagonistas de Receptores de Canabinoides/administração & dosagem , Absorção Intestinal , Nanopartículas/administração & dosagem , Receptor CB1 de Canabinoide/antagonistas & inibidores , Animais , Encéfalo/metabolismo , Antagonistas de Receptores de Canabinoides/química , Antagonistas de Receptores de Canabinoides/farmacocinética , Química Farmacêutica , Humanos , Masculino , Ratos , Ratos Wistar , Solubilidade
5.
J Leukoc Biol ; 94(1): 89-98, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23682128

RESUMO

Natural hibernation consists of torpid phases with metabolic suppression alternating with euthermic periods. Induction of torpor holds substantial promise in various medical conditions, including trauma, major surgery, and transplantation. Torpor in mice can be induced pharmacologically by 5'-AMP. Previously, we showed that during natural torpor, the reduction in body temperature results in lymphopenia via a reduction in plasma S1P. Here, we show that during torpor induced by 5'-AMP, there is a similar reduction in the number of circulating lymphocytes that is a result of their retention in secondary lymphoid organs. This lymphopenia could be mimicked by engagement of A(2B)Rs by a selective A(2B)R agonist (LUF6210) in the absence of changes in temperature and prevented by A(2B)R antagonists during 5'-AMP-induced torpor. In addition, forced cooling of mice led to peripheral blood lymphopenia, independent of A(2B)R signaling. The induction of torpor using 5'-AMP impacted the migration of lymphocytes within and between secondary lymphoid organs. During torpor, the homing into LNs was impaired, and two-photon intravital microscopy revealed that cell motility was decreased significantly and rapidly upon 5'-AMP administration. Furthermore, the S1P plasma concentration was reduced by 5'-AMP but not by LUF6210. S1P plasma levels restored upon arousal. Likely, the reduced migration in LNs combined with the reduced S1P plasma level substantially reduces lymphocyte egress after injection of 5'-AMP. In conclusion, 5'-AMP induces a state of pharmacological torpor in mice, during which, lymphopenia is governed primarily by body temperature-independent suppression of lymphocyte egress from LNs.


Assuntos
Monofosfato de Adenosina/farmacologia , Regulação da Temperatura Corporal/efeitos dos fármacos , Cálcio/metabolismo , Hibernação/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfopenia/etiologia , Receptor A2B de Adenosina/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Citometria de Fluxo , Hibernação/fisiologia , Linfopenia/patologia , Camundongos , Camundongos Endogâmicos C57BL
6.
J Pharm Sci ; 102(6): 1734-1741, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23589380

RESUMO

The aim of this study was to investigate the effect of divalent metal ions (Ca, Mg(2+) , and Zn(2+) ) on the stability of oxytocin in aspartate buffer (pH 4.5) and to determine their interaction with the peptide in aqueous solution. Reversed-phase high-performance liquid chromatography and high-performance size-exclusion chromatography measurements indicated that after 4 weeks of storage at 55°C, all tested divalent metal ions improved the stability of oxytocin in aspartate-buffered solutions (pH 4.5). However, the stabilizing effects of Zn(2+) were by far superior compared with Ca(2+) and Mg(2+) . Liquid chromatography-tandem mass spectrometry showed that the combination of aspartate and Zn(2+) in particular suppressed the formation of peptide dimers. As shown by isothermal titration calorimetry, Zn(2+) interacted with oxytocin in the presence of aspartate buffer, whereas Ca(2+) or Mg(2+) did not. In conclusion, the stability of oxytocin in the aspartate-buffered solution is strongly improved in the presence of Zn(2+) , and the stabilization effect is correlated with the ability of the divalent metal ions in aspartate buffer to interact with oxytocin. The reported results are discussed in relation to the possible mode of interactions among the peptide, Zn(2+) , and buffer components leading to the observed stabilization effects.


Assuntos
Ácido Aspártico/química , Ocitocina/química , Zinco/química , Soluções Tampão , Cátions Bivalentes/química , Cromatografia em Gel , Cromatografia de Fase Reversa , Dimerização , Estabilidade de Medicamentos
7.
Phytochemistry ; 87: 51-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23280038

RESUMO

Trichomes, especially the capitate-stalked glandular hairs, are well known as the main sites of cannabinoid and essential oil production of Cannabis sativa. In this study the distribution and density of various types of Cannabis sativa L. trichomes, have been investigated by scanning electron microscopy (SEM). Furthermore, glandular trichomes were isolated over the flowering period (8 weeks) by laser microdissection (LMD) and the cannabinoid profile analyzed by LCMS. Cannabinoids were detected in extracts of 25-143 collected cells of capitate-sessile and capitate stalked trichomes and separately in the gland (head) and the stem of the latter. Δ(9)-Tetrahydrocannabinolic acid [THCA (1)], cannabidiolic acid [CBDA (2)], and cannabigerolic acid [CBGA (3)] were identified as most-abundant compounds in all analyzed samples while their decarboxylated derivatives, Δ(9)-tetrahydrocannabinol [THC (4)], cannabidiol [CBD (5)], and cannabigerol [CBG (6)], co-detected in all samples, were present at significantly lower levels. Cannabichromene [CBC (8)] along with cannabinol (CBN (9)) were identified as minor compounds only in the samples of intact capitate-stalked trichomes and their heads harvested from 8-week old plants. Cryogenic nuclear magnetic resonance spectroscopy (NMR) was used to confirm the occurrence of major cannabinoids, THCA (1) and CBDA (2), in capitate-stalked and capitate-sessile trichomes. Cryogenic NMR enabled the additional identification of cannabichromenic acid [CBCA (7)] in the dissected trichomes, which was not possible by LCMS as standard was not available. The hereby documented detection of metabolites in the stems of capitate-stalked trichomes indicates a complex biosynthesis and localization over the trichome cells forming the glandular secretion unit.


Assuntos
Canabinoides/análise , Cannabis/metabolismo , Cromatografia Líquida/métodos , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Canabinoides/química
8.
Int J Biochem Cell Biol ; 45(2): 326-34, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23178537

RESUMO

We investigated the effect of myriocin treatment, which extensively depletes sphingolipids from cells, on multidrug resistance-related protein 1 (MRP1) efflux activity in MRP1 expressing cells and isolated plasma membrane vesicles. Our data reveal that both short term (3 days) and long term (7 days) treatment effectively reduce the cellular sphingolipid content to the same level. Intriguingly, a two-fold increase in MRP1-mediated efflux activity was observed following long term treatment, while short term treatment had no impact. Very similar data were obtained with plasma membrane vesicles isolated from myriocin-treated cells. Exploiting the cell-free vesicle system, Michaelis-Menten analysis revealed that the intrinsic MRP1 activity remained unaltered; however, the fraction of active transporter molecules increased. We demonstrate that the latter effect is due to an enhanced recruitment of MRP1 into lipid raft fractions, thereby promoting MRP1 activity.


Assuntos
Ácidos Graxos Monoinsaturados/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Caveolinas/metabolismo , Cricetinae , Humanos , Cinética , Leucotrieno C4/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Camundongos , Células NIH 3T3 , Fosfatidilserinas/metabolismo , Transporte Proteico , Esfingolipídeos/metabolismo
9.
Phytochemistry ; 72(17): 2172-9, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21889175

RESUMO

The aryltetralin lignan deoxypodophyllotoxin is much more widespread in the plant kingdom than podophyllotoxin. The latter serves as a starting compound for the production of cytostatic drugs like etoposide. A better insight into the occurrence of deoxypodophyllotoxin combined with detailed knowledge of its biosynthestic pathway(s) may help to develop alternative sources for podophyllotoxin. Using HPLC combined with electrospray tandem mass spectrometry and NMR spectroscopy techniques, we found nine lignans and five related structures in roots of Anthriscus sylvestris (L.) Hoffm. (Apiaceae), a common wild plant in temperate regions of the world. Podophyllotoxone, deoxypodophyllotoxin, yatein, anhydropodorhizol, 1-(3'-methoxy-4',5'-methylenedioxyphenyl)1-ξ-methoxy-2-propene, and 2-butenoic acid, 2-methyl-4-[[(2Z)-2-methyl-1-oxo-2-buten-1-yl]oxy]-, (2E)-3-(7-methoxy-1,3-benzodioxol-5-yl)-2-propen-1-yl ester, (2Z)- were the major compounds. α-Peltatin, podophyllotoxin, ß-peltatin, isopicropodophyllone, ß-peltatin-a-methylether, (Z)-2-angeloyloxymethyl-2-butenoic acid, anthriscinol methylether, and anthriscrusin were present in lower concentrations. α-Peltatin, ß-peltatin, isopicropodophyllone, podophyllotoxone, and ß-peltatin-a-methylether have not been previously reported to be present in A. sylvestris. Based on our findings we propose a hypothetical biosynthetic pathway of aryltetralin lignans in A. sylvestris.


Assuntos
Apiaceae/química , Extratos Vegetais/química , Podofilotoxina/análogos & derivados , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas , Espectroscopia de Ressonância Magnética , Raízes de Plantas/química , Podofilotoxina/análise , Podofilotoxina/biossíntese , Espectrometria de Massas por Ionização por Electrospray
10.
Proc Natl Acad Sci U S A ; 108(5): 2052-7, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21245336

RESUMO

Hibernation is an energy-conserving behavior consisting of periods of inhibited metabolism ('torpor') with lowered body temperature. Torpor bouts are interspersed by arousal periods, in which metabolism increases and body temperature returns to euthermia. In deep torpor, the body temperature typically decreases to 2-10 °C, and major physiological and immunological changes occur. One of these alterations constitutes an almost complete depletion of circulating lymphocytes that is reversed rapidly upon arousal. Here we show that torpor induces the storage of lymphocytes in secondary lymphoid organs in response to a temperature-dependent drop in plasma levels of sphingosine-1-phosphate (S1P). Regulation of lymphocyte numbers was mediated through the type 1 S1P receptor (S1P(1)), because administration of a specific antagonist (W146) during torpor (in a Syrian hamster at ∼8 °C) precluded restoration of lymphocyte numbers upon subsequent arousal. Furthermore, S1P release from erythrocytes via ATP-binding cassette (ABC)-transporters was significantly inhibited at low body temperature (4 °C) but was restored upon rewarming. Reversible lymphopenia also was observed during daily torpor (in a Djungarian hamster at ± 25 °C), during forced hypothermia in anesthetized (summer-active) hamsters (at ± 9 °C), and in a nonhibernator (rat at ∼19 °C). Our results demonstrate that lymphopenia during hibernation in small mammals is driven by body temperature, via altered plasma S1P levels. S1P is recognized as an important bioactive lipid involved in regulating several other physiological processes as well and may be an important factor regulating additional physiological processes in hibernation as well as in mediating the effects of therapeutic hypothermia in patients.


Assuntos
Regulação da Temperatura Corporal , Hibernação , Depleção Linfocítica , Linfócitos/citologia , Lisofosfolipídeos/fisiologia , Esfingosina/análogos & derivados , Animais , Cricetinae , Lisofosfolipídeos/sangue , Mesocricetus , Esfingosina/sangue , Esfingosina/fisiologia
11.
Biochem J ; 430(3): 519-29, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20604746

RESUMO

We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C(16) species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1.


Assuntos
Microdomínios da Membrana/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Esfingolipídeos/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Linhagem Celular Tumoral , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Fluoresceínas/metabolismo , Glicerofosfolipídeos/metabolismo , Humanos , Immunoblotting , Lipídeos/análise , Lipídeos/química , Microdomínios da Membrana/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Polietilenoglicóis/química , Espectrometria de Massas por Ionização por Electrospray , Esfingolipídeos/química
12.
Peptides ; 31(5): 893-8, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20206220

RESUMO

Instability and proteolytic degradation limit the delivery options and in vivo efficacy of many therapeutic peptides. We previously generated a thioether stabilized angiotensin-(1-7) analog, cAng-(1-7), which is resistant against proteolytic degradation in the circulation. We here investigated oral and pulmonary delivery of this compound. In a first step we investigated the in vitro stability of the peptide under conditions that mimic those that will be met after oral administration. We demonstrated that cAng-(1-7) is stable at pH 2.0, a pH value close to that of the stomach, has enhanced resistance to breakdown by proteases from pancreas at pH 7.4, and is resistant to breakdown by proteases from liver at the lysosomal pH 5.0. We subsequently demonstrated that, in the absence of any delivery system or formulation, cAng-(1-7) can be delivered orally and via the lung, with bioavailabilities of 0.28+/-0.05% and 28+/-5%, whereas drug uptake was maximal after subcutaneous administration (bioavailability of 98+/-6%). Therapeutic concentrations could be reached via all three routes of administration. The data prove that introduction of a thioether bridge in peptides opens novel delivery options for medically important peptides.


Assuntos
Administração Oral , Angiotensina I/administração & dosagem , Angiotensina I/farmacocinética , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/farmacocinética , Administração Cutânea , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Vias de Administração de Medicamentos , Masculino , Espectrometria de Massas , Ratos , Ratos Sprague-Dawley , Traqueia/metabolismo
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