RESUMEN
OBJECTIVE: Buccal myomucosal local flaps in oral cavity reconstruction are a valid option for small-to-moderate defects. Nevertheless, few articles report about functional recovery. The purpose of the present analysis is to evaluate the impact of these flaps on function and quality of life. STUDY DESIGN: The study, retrospectively conducted on 36 patients who were surgically treated for tongue cancer between 2012 and 2018 at the Unit of Maxillo-Facial Surgery, Foundation IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (Italy), evaluates functional outcomes using the following 4 questionnaires: Performance Status Scale for Head and Neck Cancer Patients, M.D. Anderson Dysphagia Inventory, Speech Handicap Index, and European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Head and Neck Module. RESULTS: All patients are able to eat soft or more solid foods, and most of them eat quietly in public. Although 50% of cases reported a certain degree of dysphagia, it does not impact self-esteem and social relationships. Only 20% of patients have severe problems with speech. However, more than half of the cases (65%) report oral problems. CONCLUSIONS: The collected data confirm the suitability of the myomucosal cheek flaps for tongue reconstruction. Most patients report a good functional recovery and satisfactory quality of life even if none of them has a recovery comparable to the presurgical state.
Asunto(s)
Trastornos de Deglución , Procedimientos de Cirugía Plástica , Neoplasias de la Lengua , Mejilla/cirugía , Humanos , Mucosa Bucal/cirugía , Calidad de Vida , Estudios Retrospectivos , Lengua/cirugía , Neoplasias de la Lengua/cirugíaRESUMEN
Many different unique functions have been attributed to lactoferrin (Lf), including DNA and RNA binding, and transport into the nucleus, where Lf binds to specific sequences and activates transcription. A pentapeptide, Gly-Arg-Arg-Arg-Arg, corresponding to a region of the N-terminal portion of human Lf rich in basic amino acids, was synthesized and its intracellular localization was investigated. Peptide internalization was assayed using the rhodaminated form of the same molecule. This N-terminal peptide sequence is able to be internalized within less than 10 min at concentration as low as 1 microM, and its intracellular localization is nuclear, mainly nucleolar. Similar behaviour was observed using peptides composed of either all l or d amino acids, the last one being a retro-inverse peptide. The internalization process does not involve an endocytotic pathway, since no inhibition of the uptake was observed at 4 degrees C. The kinetics of peptide internalization was also evaluated. The internalization properties of such a short Lf pentapeptide have been assayed for its ability to transport peptide nucleic acids (PNAs) inside cells in order to improve their efficacy. The abundant transmembrane transport and nuclear localization of the proposed peptide, deriving from hLf and, for the first time, identified as a nuclear localization signal, could be used as an alternative strategy to tackle the unsolved problem of intracellular accumulation of antisense and antigene drugs and for the development of new pharmacological tools.
Asunto(s)
Núcleo Celular/metabolismo , Lactoferrina/metabolismo , Señales de Localización Nuclear/metabolismo , Ácidos Nucleicos de Péptidos/metabolismo , Secuencias de Aminoácidos , Nucléolo Celular/metabolismo , Endocitosis , Colorantes Fluorescentes/química , Humanos , Lactoferrina/química , Imitación Molecular , Ácidos Nucleicos de Péptidos/química , Rodaminas/química , Temperatura , Células Tumorales CultivadasRESUMEN
Sponges (phylum Porifera) are the phylogenetically oldest metazoan animals, their evolution dating back to 600 million years ago. Here we demonstrate that sponges express ADP-ribosyl cyclase activity, which converts NAD(+) into cyclic ADP-ribose, a potent and universal intracellular Ca(2+) mobilizer. In Axinella polypoides (Demospongiae, Axinellidae), ADP-ribosyl cyclase was activated by temperature increases by means of an abscisic acid-induced, protein kinase A-dependent mechanism. The thermosensor triggering this signaling cascade was a heat-activated cation channel. Elucidation of the complete thermosensing pathway in sponges highlights a number of features conserved in higher organisms: (i) the cation channel thermoreceptor, sensitive to heat, mechanical stress, phosphorylation, and anesthetics, shares all of the functional characteristics of the mammalian heat-activated background K(+) channel responsible for central and peripheral thermosensing; (ii) involvement of the phytohormone abscisic acid and cyclic ADP-ribose as its second messenger is reminiscent of the drought stress signaling pathway in plants. These results suggest an ancient evolutionary origin of this stress-signaling cascade in a common precursor of modern Metazoa and Metaphyta.
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Ácido Abscísico/fisiología , Adenosina Difosfato Ribosa/fisiología , Antígenos CD , Activación del Canal Iónico , Canales Iónicos/fisiología , Poríferos/metabolismo , Transducción de Señal , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Ácido Abscísico/biosíntesis , Animales , Antígenos de Diferenciación/metabolismo , Cromatografía Líquida de Alta Presión , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Calor , Canales Iónicos/metabolismo , NAD+ Nucleosidasa/metabolismo , Poríferos/enzimología , Espectrometría de FluorescenciaRESUMEN
The presence of Ca(2+)-dependent, heat-stress-activated nitric oxide synthase (NOS) activity in peculiarly shaped, fusiform, and dendritic sponge cells is described for the first time. The NOS activity was evidenced evaluating the conversion of radioactive citrulline from [(14)C]arginine in intact cells from two different species that are phylogenetically unrelated in the class of Demospongiae: Axinella polypoides and Petrosia ficiformis. The production of nitrogen monoxide (NO) was confirmed by electron paramagnetic resonance analysis, and the histochemistry technique of NADPH diaphorase showed a specific localization of NOS activity in a particular network of dendritic cells in the sponge parenchyma. Sponges are the most primitive metazoan group; their evolution dates back 600 million years. The presence of environmental stress-activated NOS activity in these organisms may prove to be the most ancient NO-dependent signaling network in the animal kingdom.
Asunto(s)
Calcio/metabolismo , Óxido Nítrico Sintasa/metabolismo , Poríferos/enzimología , Estrés Fisiológico/metabolismo , Animales , Citrulina/metabolismo , Células Dendríticas/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Calor , Técnicas para Inmunoenzimas , NADPH Deshidrogenasa/metabolismo , Óxido Nítrico/metabolismoRESUMEN
The L-arginine uptake of resting platelets from type-2 diabetic patients and control subjects was measured and the kinetic parameters defined. The effect of platelet stimulation with agonists was also investigated. Kinetic studies showed that the K(m) value for L-arginine transport was not different in patients compared with control subjects, while V(max) was significantly decreased in patients compared with controls. Moreover, agonists able to mobilize Ca(2+) produced a further decrease in the L-arginine uptake in controls and in patients, suggesting that Ca(2+) intracellular levels down-regulate L-arginine transport in platelets from both control subjects and patients. Data suggest that drugs designed to control intracellular Ca(2+) might restore platelet function in diabetic patients.
Asunto(s)
Arginina/farmacocinética , Plaquetas/metabolismo , Diabetes Mellitus Tipo 2/sangre , Anciano , Calcimicina/farmacología , Calcio/metabolismo , Calcio/farmacología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Femenino , Humanos , Cinética , Masculino , Persona de Mediana Edad , Óxido Nítrico/metabolismo , Factores de TiempoRESUMEN
The penton base of adenovirus mediates viral attachment to integrin receptors and particle internalisation, properties that can be exploited to reengineer prokaryotic viruses for the infection of mammalian cells. We report that filamentous phage displaying either the full-length penton base gene or a central region of 107 amino acids on their surface were able to bind, internalise, and transduce mammalian cells expressing integrin receptors. Both phage bound alphavbeta3, alphavbeta5, alpha3beta1, and alpha5beta1 integrin subtypes. Cell-binding was shown by electron microscopy; internalisation was investigated by immunofluorescence and confirmed by micropanning. As it has been described for adenovirus, pharmacologic disruption of phosphoinositide-30H kinase, but not of myosin light-chain kinase, inhibited phage internalisation. Recombinant phage encoding an eukaryotic expression cassette was able to mediate gene expression in mammalian cells. Taken together, these data open insights for the exploit of recombinant phage for integrin-targeted gene delivery.
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Adenoviridae/genética , Bacteriófagos/genética , Proteínas de la Cápside , Cápside/genética , Proteínas Portadoras/metabolismo , Técnica del Anticuerpo Fluorescente , Vectores Genéticos , Células HeLa , Humanos , Integrinas/metabolismo , Microscopía Electrónica , Oligopéptidos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción GenéticaRESUMEN
BACKGROUND: The major concern for the use of adenoviral vectors for gene therapy is the viral-induced immune response that has been shown to be responsible for short-term transgene expression and inefficient viral readministration. In vivo studies and clinical trials with recombinant adenovirus have suggested a role for interleukin 6 (IL-6) in the inflammatory reaction that follows Ad-infection. IL-6 plays an important role in the acute-phase innate response, in the differentiation of B-cells and in the activation of the Th2 cell subsets. METHODS: To clarify the role of IL-6 in the immune response to Ad-vectors, we used IL-6 knock-out mice (IL-6 -/- ). E1/E3 deleted recombinant adenoviruses encoding reporter genes were administered to wild type or IL-6-/- mice; transgene expression kinetics and immune response were analyzed. RESULTS: Acute phase protein production was significantly diminished in IL-6 -/- mice after adenoviral injection. No significant difference between wild type and knock-out animals in the level or the nature of leucocyte recruitment in the liver was detectable. A minor decrease in the IgG response to Ad-recombinants was observed in knock-out mice. Gene transfer efficiency, both in terms of levels and duration of transgene expression, were comparable in IL-6+/+ and IL-6-/- mice. An increase in IL-1beta and tumor necrosis factor-alpha (TNF-alpha) levels was observed in the sera of IL-6 -/- mice as compared to wild type animals: this phenomenon represents a possible compensatory mechanism for the establishment of the immune phenotype observed in mutant mice. CONCLUSIONS: IL-6 plays a role in the acute phase response to adenoviral vectors. Nevertheless, possibly due to a compensatory mechanism exerted by other cytokines, the antibody and cellular responses to adenoviruses are very similar in wild type and IL-6 -/- mice.
Asunto(s)
Adenoviridae/genética , Formación de Anticuerpos/genética , Vectores Genéticos , Inflamación/inmunología , Interleucina-6/fisiología , Proteínas de Fase Aguda/genética , Animales , Humanos , Inmunidad Celular/genética , Inflamación/genética , Interleucina-6/genética , Ratones , Ratones NoqueadosRESUMEN
L-Arginine uptake and Ca(2+) changes in unstirred platelets activated by thrombin, collagen and Ca(2+) ionophore A23187 were evaluated. Thrombin did not affect L-arginine uptake at short incubation times (2-15 min), but at prolonged times slowed down the amino acid transport. Collagen was ineffective. A23187 decreased the L-arginine uptake in a dose-dependent manner, producing the maximal inhibition at 5 microM. In FURA 2-loaded platelets collagen did not modify Ca(2+) basal level, thrombin induced a late Ca(2+) rise and A23187 dose-dependently increased cytosolic Ca(2+), eliciting the highest increase at 5 microM. It is likely that L-arginine uptake is inversely modulated by Ca(2+) concentrations and is inhibited during platelet stimulation with agonists which induce cytosolic Ca(2+) elevation.
Asunto(s)
Arginina/farmacocinética , Plaquetas/metabolismo , Calcio/metabolismo , Calcimicina/farmacología , Colágeno/farmacología , Relación Dosis-Respuesta a Droga , Hemostáticos/farmacología , Humanos , Ionóforos/farmacología , Óxido Nítrico/metabolismo , Activación Plaquetaria/efectos de los fármacos , Transducción de Señal , Trombina/farmacología , Factores de TiempoRESUMEN
Overexpression of inducible nitric oxide synthase causes the production of high levels of nitric oxide, which, under pathological conditions, leads to immunosuppression and tissue damage. The results recently obtained using peptide nucleic acids, rather than traditional oligonucleotides as antigen and antisense molecules, prompted us to test their efficacy in the regulation of nitric oxide production, thereby overcoming the obstacle of cellular internalization. The cellular permeability of four inducible nitric oxide synthase antisense peptide nucleic acids of different lengths was evaluated. These peptide nucleic acids were covalently linked to a hydrophobic peptide moiety to increase internalization and to a tyrosine to allow selective 125I radiolabelling. Internalization experiments showed a 3-25-fold increase in the membrane permeability of the modified peptide nucleic acids with respect to controls. Inducible nitric oxide synthase inhibition experiments on intact stimulated macrophages RAW 264.7 after passive permeation of the two antisense peptide nucleic acids 3 and 4 demonstrated a significant decrease (43-44%) in protein enzymatic activity with respect to the controls. These data offer a basis for developing a good alternative to conventional drugs directed against inducible nitric oxide synthase overexpression.
Asunto(s)
Macrófagos/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Ácidos Nucleicos de Péptidos/metabolismo , Ácidos Nucleicos de Péptidos/farmacología , Animales , Elementos sin Sentido (Genética)/genética , Elementos sin Sentido (Genética)/metabolismo , Elementos sin Sentido (Genética)/farmacología , Transporte Biológico , Línea Celular , Ratones , Óxido Nítrico Sintasa de Tipo II , Ácidos Nucleicos de Péptidos/genéticaRESUMEN
Inducible nitric oxide synthase (iNOS) is modulated at the transcriptional level. Overexpression of this protein may result in high levels of nitric oxide leading to tissue damage and immunosuppression. In order to reduce the pathological effects of NO overproduction many efforts have been devoted to the identification of specific inhibitors of iNOS. The discovery of peptide nucleic acids (PNA), a novel class of molecules able to selectively interact with nucleic acids, prompted us to attempt a new way for the regulation of NO production. Here we describe the synthesis, characterization and in vitro effects of a PNA molecule bearing a homopyrimidine sequence complementary to the 5' coding region of murine iNOS mRNA. This PNA shows specific interactions with iNOS mRNA in RNase protection assays and is able to block the synthesis of iNOS protein selectively in a rabbit reticulocyte lysate system. These results strengthen the view of a possible pharmacological application of PNA as a compound able to interfere with a specific enzymatic activity even at low concentrations.
Asunto(s)
Óxido Nítrico Sintasa/genética , Oligonucleótidos Antisentido/química , Animales , Sistema Libre de Células , Ratones , Óxido Nítrico Sintasa de Tipo II , Hibridación de Ácido Nucleico , Péptidos , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/química , ConejosRESUMEN
The skeleton of the common Mediterranean demosponge Chondrosia reniformis lacks endogenous spicules; but exogenous siliceous material is selectively incorporated into its collagenous ectosome, strengthening this layer. Nevertheless, the settling of sponge buds during asexual reproduction necessitates an active incorporation of the calcareous substratum through the sponge lower ectosome. This fact suggests the presence of a polarity in the sponge, with the lower surface selecting primarily carbonates, and the upper surface selecting exclusively silicates and quartz. Our observations under experimental conditions showed that the strong selectivity of the upper ectosome is realized only when the sponge is fixed to the substratum; if detached, the sponge incorporates both quartz and carbonates. In laboratory experiments, the incapacity of both kinds of ectosome to regenerate into a new complete sponge suggests that this polarity arises early in ontogeny.
RESUMEN
The aim of this study was to investigate the circadian variability of heart rate in acute myocardial infarction (AMI) in identifying patients at high risk for malignant ventricular arrhythmias (MVA) and sudden death within 1 year of the acute event. The investigation was carried out in 43 patients, who underwent 24-hour Holter monitoring within 3 months of AMI. Besides the time domain indexes of heart rate variability (SDNN, SDNN index, pNN50, rMSSD), the circadian rhythm of hourly total beats (HTB) and hourly qualified beats (HQB) has been analyzed by the Cosinor method. The AMI patients with MVA and those with MVA who died within 1 year the acute event showed SDNN, SDNN index and pNN50 values lower than subjects without MVA and survived patients with MVA, respectively; the individuals with AMI at high risk for MVA and for sudden death had an SDNN value < 105 ms and 50 ms, respectively. The circadian rhythm of HTB and HQB was statistically validated only in the group without MVA; patients without the circadian rhythm of HTB and HQB showed a higher mortality rate within 1 year of AMI, and the majority was in the group with MVA. The contemporary evidence of an SDNN value < 105 ms and the lack of HTB and HQB circadian rhythm increased sensitivity for identifying patients with MVA to 75%. On the other hand, the contemporary evidence of an SDNN value < 50 ms and the lack of HTB and HQB circadian rhythm increased sensitivity for identifying patients who died within 1 year of AMI to 100%. In conclusion, the assayed methods seem to be both useful and complementary in identifying patients at high risk for MVA and sudden death within 1 year of AMI.
Asunto(s)
Arritmias Cardíacas/fisiopatología , Ritmo Circadiano , Frecuencia Cardíaca , Infarto del Miocardio/fisiopatología , Anciano , Arritmias Cardíacas/etiología , Arritmias Cardíacas/mortalidad , Muerte Súbita Cardíaca , Electrocardiografía Ambulatoria/estadística & datos numéricos , Femenino , Ventrículos Cardíacos , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/complicaciones , Infarto del Miocardio/mortalidad , Pronóstico , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The infectivity and replication of human (HIV-1), feline (FIV), and murine (LP-BM5) immunodeficiency viruses are all inhibited by several nucleoside analogues after intracellular conversion to their triphosphorylated derivatives. At the cellular level, the main problems in the use of these drugs concern their limited phosphorylation in some cells (e.g., macrophages) and the cytotoxic side effects of nucleoside analogue triphosphates. To overcome these limitations a new nucleoside analogue homodinucleotide, di(thymidine-3'-azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophosphat e (AZTp2AZT), was designed and synthesized. AZTp2AZT was a poor in vitro inhibitor of HIV reverse transcriptase, although it showed antiviral and cytotoxic activities comparable to those of the parent AZT when added to cultures of a HTLV-1 transformed cell line. AZTp2AZT encapsulated into erythrocytes was remarkably stable. Induction of erythrocyte-membrane protein clusterization and subsequent phagocytosis of AZTp2AZT-loaded cells allowed the targeted delivery of this impermeant drug to macrophages where its metabolic activation occurs. The addition of AZTp2AZT-loaded erythrocytes to human, feline, and murine macrophages afforded almost complete in vitro protection of these cells from infection by HIVBa-L, FIV, and LP-BM5, respectively. Therefore, AZTp2AZT, unlike the membrane-diffusing azidothymidine, acts as a very efficient antiretroviral prodrug following selective targeting to macrophages by means of loaded erythrocytes.
Asunto(s)
Antivirales/farmacología , VIH-1/efectos de los fármacos , Virus de la Inmunodeficiencia Felina/efectos de los fármacos , Macrófagos/virología , Profármacos/farmacología , Nucleótidos de Timina/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/síntesis química , Secuencia de Bases , Gatos , Células Cultivadas , Cartilla de ADN , Didesoxinucleótidos , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Eritrocitos/virología , VIH-1/patogenicidad , VIH-1/fisiología , Humanos , Virus de la Inmunodeficiencia Felina/patogenicidad , Virus de la Inmunodeficiencia Felina/fisiología , Cinética , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Estructura Molecular , Fagocitosis/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Profármacos/síntesis química , Nucleótidos de Timina/síntesis química , Zidovudina/análogos & derivadosRESUMEN
A new Azidothymidine derivative, di-(thymidine-3'- azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophosphate (AZTp2AZT), was encapsulated in human erythrocytes according to a conservative procedure of hypotonic shock-isotonic resealing and reannealing. Like in erythrocyte lysates supplemented with 1 mM ATP, intact red cells too were found to convert AZTp2AZT to 3'-Azido-3'-deoxythymidine which was then released linearly in plasma. The major metabolic pathway involved in this conversion was the symmetrical hydrolysis of AZTp2AZT to yield two 3'-Azido-3'- deoxythymidine-5'-phosphate molecules which were then dephosphorylated to 3'-Azido-3'-deoxythymidine. At late times of incubation, also a limited asymmetrical hydrolysis of AZTp2AZT became apparent in the intact erythrocytes, yielding 3'-Azido-3'-deoxythymidine-5'-diphosphate that was then converted to the triphosphorylated derivative. Therefore, erythrocytes loaded with AZTp2AZT act "in vitro" as bioreactors ensuring sustained and potentially useful release of 3'-Azido-3'-deoxythymidine.
Asunto(s)
Portadores de Fármacos , Eritrocitos/metabolismo , Profármacos/administración & dosificación , Nucleótidos de Timina/administración & dosificación , Zidovudina/administración & dosificación , Didesoxinucleótidos , Hemólisis , Humanos , Técnicas In Vitro , Profármacos/metabolismo , Nucleótidos de Timina/metabolismo , Zidovudina/metabolismoRESUMEN
Extracellular ATP potentiates, by activation of P2y-type purinergic receptors, the production of NO induced by low doses of lipopolysaccharide (LPS) in the murine macrophagic cell line RAW 264.7 (Tonetti et al. (1994) Biochem. Biophys. Res. Commun. 203, 430-434). Release of TNF-alpha, known to be an autocrine factor for iNOS expression, was enhanced, too, following exposure of either LPS-induced or uninduced cells to externally added micromolar ATP. Reverse transcription-PCR experiments showed that extracellular ATP increases mRNA levels of both inducible NO synthase (iNOS) and of TNF-alpha to extents comparable to those of enzymatic and biological activities, respectively. These data demonstrate that activation of purinergic receptors by extracellular ATP results in an enhanced expression of the iNOS and TNF-alpha genes.
Asunto(s)
Adenosina Trifosfato/fisiología , Aminoácido Oxidorreductasas/metabolismo , Macrófagos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Aminoácido Oxidorreductasas/genética , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Ratones , Datos de Secuencia Molecular , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
A new dimeric fluoropyrimidine molecule (5-fluoro-2'-deoxyuridilyl-(5'-->3')-5-fluoro-2'-deoxy-5'-uridylic acid, Compound 1) was chemically synthesized from two separately deblocked 5-fluoro-2'-deoxyuridine mononucleotide moieties. Other structurally related nucleotides, 5-fluoro-2'-deoxyuridine-5'-diphosphate (FdUDP), 5-fluoro-2'-deoxyuridine-5'-triphosphate (FdUTP) and 5-fluoro-2'-deoxyuridine-3',5'-bisphosphate were also synthesized. The structures of all synthesized molecules were verified by mass spectrometric analyses and were consistent with expected molecular mass values. The metabolic patterns of conversion of Compound 1 were investigated both in human erythrocyte lysates and in intact erythrocytes previously loaded with this molecule according to a highly conservative encapsulation procedure. In hemolysates, Compound 1 was transformed to 5-fluoro-2'-deoxyuridine (FUdR) and to 5-fluorouracil (FU) through the intermediate formation of 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP). In intact red cells, Compound 1 still generated FUdR (and to a lesser extent FU), that was then released outside. The conversion pathway involves a phosphodiesterase-catalysed hydrolysis of Compound 1 into two FdUMP molecules, followed by further dephosphorylation to FUdR and by partial conversion to FU. Unlike hemolysates, Compound 1-loaded intact erythrocytes featured transient formation of FdUDP and FdUTP, both metabolites representing storage compounds for the final and sustained production of FUdR and FU. Therefore, human erythrocytes can behave as bioreactors ensuring the time-controlled production and delivery of the two powerful antitumor drugs FUdR and FU from encapsulated Compound 1. This new molecule and other compounds as well (e.g. FdUDP and FdUTP) can be viewed as useful pre-prodrugs, exploitable for intraerythrocytic bioconversion reactions.
Asunto(s)
Eritrocitos/metabolismo , Floxuridina/metabolismo , Profármacos/síntesis química , Pirimidinas/síntesis química , Fluorodesoxiuridilato/metabolismo , Humanos , Espectrometría de Masas , Pirimidinas/metabolismoRESUMEN
Experiments were designed to investigate the influence of oxygen free radicals on the rate of conversion of the anticancer drug cis-diammine-(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA) to reactive species able to bind to DNA. A system containing the Fe-EDTA chelate and ascorbate was used to generate free radicals. The rate of drug conversion to by-products, during incubation in chloride-free phosphate buffer at 37 degrees, was determined by HPLC analysis and found to be approximately 10 times faster in the presence of the free radical generating system, compared to CBDCA alone. The hydroxyl radical scavenger, mannitol, was able to reduce the rate of CBDCA conversion significantly, while an enhancing effect was observed in the presence of superoxide dismutase. The platinum containing species, which are formed in the presence of free radicals, were demonstrated to react with isolated salmon sperm DNA. The rate of platinum binding to DNA during incubation of CBDCA in the presence of the Fe-EDTA/ascorbate system was markedly enhanced. No effect on platinum binding to DNA during incubation with cis-diamminedichloroplatinum(II) (CDDP) in the same experimental conditions was observed, thus excluding an increased susceptibility of DNA itself to binding of platinum, due to DNA damage induced by free radicals. These findings support the hypothesis that the increased conversion of CBDCA, previously observed in our laboratory, which occurs in the presence of hemoglobin could be mediated by a Fenton-like reaction resulting in oxygen free radical production, thus providing potential clues to improvements in the clinical use of this drug.
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Carboplatino/metabolismo , Oxígeno/metabolismo , Animales , Ácido Ascórbico , Carboplatino/química , Cisplatino/metabolismo , ADN/metabolismo , Estabilidad de Medicamentos , Ácido Edético , Compuestos Ferrosos , Radicales Libres , Platino (Metal)/metabolismoRESUMEN
The antineoplastic drug Carboplatin (CBDCA) was encapsulated in human erythrocytes by means of transient hypotonic hemolysis, followed by isotonic resealing. Up to 5 mg/ml of packed cells could be entrapped, with about 70% cell recovery. In vitro incubation of the CBDCA-loaded erythrocytes in autologous plasma caused a very slow release of the drug from the cells (12% approximately in 3 h). The encapsulation conditions, performed at a low hematocrit, in order to obtain high amounts of the drug inside the carriers, impaired the metabolic properties of the loaded erythrocytes significantly. In particular, an almost complete disappearance of GSH was observed. Analysis of the intraerythrocytic metabolism of CBDCA showed that, in spite of its relatively high stability in aqueous solutions, in hemolysates and in the loaded erythrocytes a significant percentage of CBDCA is rapidly converted to other species that still retain an antiproliferative activity in vitro. This fast conversion could be extensively inhibited by previous conversion of oxyhemoglobin to methemoglobin or carbomonoxyhemoglobin, suggesting an important role of heme iron in this process. Encapsulation of CBDCA in selectively targeted human erythrocytes may represent a therapeutic strategy for increasing the drug concentration in specific organs, notably liver.
Asunto(s)
Carboplatino/administración & dosificación , Eritrocitos , Carboplatino/farmacocinética , Portadores de Fármacos , Estabilidad de Medicamentos , Eritrocitos/metabolismo , Estudios de Evaluación como Asunto , Hemoglobinas/metabolismo , Humanos , Técnicas In Vitro , Hígado/metabolismoRESUMEN
The formation of DNA interstrand cross-links following exposure of target cells to the antineoplastic drugs Cisplatin and Carboplatin was measured in murine erythroleukemic cells either native or induced to differentiate and to synthesize hemoglobin by treatment with hexamethylene bisacetamide. The uptake of both drugs was identical in the uninduced and the induced cells, thus excluding differences in permeability due to differentiation. Both cell types formed comparable levels of DNA interstrand cross-links when submitted to treatment with Cisplatin. Conversely, significant differences were observed after exposure to Carboplatin. Specifically, the induced, hemoglobin containing cells showed significantly enhanced intracellular activation of the drug and subsequent binding to DNA, compared to the uninduced cells. These data suggest that, in agreement with previous results obtained with erythrocytes, the hemoglobin present in the committed cells enhances the aquation process that mediates the activation of Carboplatin.