RESUMEN

The World Health Organization (WHO) model "List of Essential Medicines" includes among indispensable medicines antibacterials and pain and migraine relievers. Monitoring their concentration in the environment, while challenging, is important in the context of antibiotic resistance as well as their production of highly toxic compounds via hydrolysis. Traditional detection methods such as high-performance liquid chromatography (HPLC) or LC combined with tandem mass spectrometry or UV-vis spectroscopy are time-consuming, have a high cost, require skilled operators and are difficult to adapt for field operations. In contrast, (electrochemical) sensors have elicited interest because of their rapid response, high selectivity, and sensitivity as well as potential for on-site detection. Previously, we reported a novel sensor system based on a type II photosensitizer, which combines the advantages of enzymatic sensors (high sensitivity) and photoelectrochemical sensors (easy baseline subtraction). Under red-light illumination, the photosensitizer produces singlet oxygen which oxidizes phenolic compounds present in the sample. The subsequent reduction of the oxidized phenolic compounds at the electrode surface gives rise to a quantifiable photocurrent and leads to the generation of a redox cycle. Herein we report the optimization in terms of pH and applied potential of the photoelectrochemical detection of the hydrolysis product of paracetamol, i.e., 4-aminophenol (4-AP), and two antibacterials, namely, cefadroxil (CFD, ß-lactam antibiotic) and doxycycline (DXC, tetracycline antibiotic). The optimized conditions resulted in a detection limit of 0.2 µmol L-1 for DXC, but in a 10 times higher sensitivity, 20 nmol L-1, for CFD. An even higher sensitivity, 7 nmol L-1, was noted for 4-AP.

Asunto(s)

Medicamentos Esenciales/análisis , Técnicas Electroquímicas/métodos , Luz , Fenoles/química , Acetaminofén/análisis , Acetaminofén/metabolismo , Cefadroxilo/análisis , Cefadroxilo/metabolismo , Doxiciclina/análisis , Doxiciclina/metabolismo , Medicamentos Esenciales/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Límite de Detección , Oxidación-Reducción , Fármacos Fotosensibilizantes/química

RESUMEN

Penicillin G acylase (PGA) was isolated from Providencia rettgeri PX04 (PrPGApx04) and utilized for the kinetically controlled synthesis of ß-lactam antibiotics. Site-directed mutagenesis was performed to increase the process efficiency. Molecular docking was carried out to speculate the key mutant positions corresponding with synthetic activity, which resulted in the achievement of an efficient mutant, ßF24G. It yielded higher conversions than the wild-type enzyme in the synthesis of amoxicillin (95 versus 17.2%) and cefadroxil (95.4 versus 43.2%). The reaction time for achieving the maximum conversion decreased from 14 to 16 h to 2-2.5 h. Furthermore, the secondary hydrolysis of produced antibiotics was hardly observed. Kinetic analysis showed that the (kcat/Km)AD value for the activated acyl donor D-hydroxyphenylglycine methyl ester (D-HPGME) increased up to 41 times. In contrast, the (kcat/Km)Ps values for the products amoxicillin and cefadroxil decreased 6.5 and 21 times, respectively. Consequently, the α value (kcat/Km)Ps/(kcat/Km)AD, which reflected the relative hydrolytic specificity of PGA for produced antibiotics with respect to the activated acyl donor, were only 0.028 and 0.043, respectively. The extremely low hydrolytic activity for the products of the ßF24G mutant enabled greater product accumulation to occur during synthesis, which made it a promising enzyme for industrial applications.

Asunto(s)

Antibacterianos/metabolismo , Proteínas Mutantes/metabolismo , Penicilina Amidasa/metabolismo , Providencia/enzimología , Providencia/metabolismo , beta-Lactamas/metabolismo , Amoxicilina/metabolismo , Cefadroxilo/metabolismo , Glicina/análogos & derivados , Glicina/metabolismo , Hidrólisis , Cinética , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Penicilina Amidasa/genética , Penicilina Amidasa/aislamiento & purificación , Factores de Tiempo

RESUMEN

The proton-coupled oligopeptide transporter PEPT2 (SLC15A2) plays an important role in the disposition of di/tripeptides and peptide-like drugs in kidney and brain. However, unlike PEPT1 (SLC15A1), there is little information about species differences in the transport of PEPT2-mediated substrates. The purpose of this study was to determine whether PEPT2 exhibited a species-dependent uptake of glycylsarcosine (GlySar) and cefadroxil using yeast Pichia pastoris cells expressing cDNA from human, mouse, and rat. In such a system, the functional activity of PEPT2 was evaluated with [3H]GlySar as a function of time, pH, substrate concentration, and specificity, and with [3H]cefadroxil as a function of concentration. We observed that the uptake of GlySar was pH-dependent with an optimal uptake at pH 6.5 for all three species. Moreover, GlySar showed saturable uptake kinetics, with Km values in human (150.6 µM) > mouse (42.8 µM) ≈ rat (36.0 µM). The PEPT2-mediated uptake of GlySar in yeast transformants was specific, being inhibited by di/tripeptides and peptide-like drugs, but not by amino acids and nonsubstrate compounds. Cefadroxil also showed a saturable uptake profile in all three species, with Km values in human (150.8 µM) > mouse (15.6 µM) ≈ rat (11.9 µM). These findings demonstrated that the PEPT2-mediated uptake of GlySar and cefadroxil was specific, species dependent, and saturable. Furthermore, based on the Km values, mice appeared similar to rats but both were less than optimal as animal models in evaluating the renal reabsorption and pharmacokinetics of peptides and peptide-like drugs in humans.

Asunto(s)

Cefadroxilo/metabolismo , Dipéptidos/metabolismo , Pichia/metabolismo , Simportadores/fisiología , Animales , Transporte Biológico , Técnicas de Cultivo de Célula , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Humanos , Concentración de Iones de Hidrógeno , Cinética , Ratones , Pichia/genética , Ratas , Especificidad de la Especie , Especificidad por Sustrato , Simportadores/genética

RESUMEN

PepT1 (SLC15A1) is a high-capacity low-affinity transporter that is important in the absorption of digested di/tripeptides from dietary protein in the small intestine. PepT1 is also crucial for the intestinal uptake and absorption of therapeutic agents such as the ß-lactam aminocephalosporins and antiviral prodrugs. Species differences, however, have been observed in PepT1-mediated intestinal absorption and pharmacokinetics, thereby, making it more difficult to predict systemic drug exposure. In the present study, we evaluated the in situ intestinal permeability of the PepT1 substrate cefadroxil in wildtype and humanized PepT1 (huPepT1) mice, and the in vivo absorption and disposition of drug after escalating oral doses. The in situ perfusions indicated that cefadroxil had a twofold higher affinity (i.e., twofold lower Km) for jejunal PepT1 in huPepT1 mice, lower but substantial permeability in all regions of the small intestine, and low but measureable permeability in the colon as compared to wildtype animals. The in vivo experiments indicated almost superimposable pharmacokinetic profiles between the two genotypes after intravenous bolus dosing of cefadroxil. In contrast, after oral dose escalation, the systemic exposure of cefadroxil was reduced in huPepT1 mice as compared to wildtype animals. Moreover, the AUC and Cmax versus dose relationships were nonlinear for huPepT1 but not wildtype mice, and similar to that observed from human subjects. In conclusion, our findings indicate that huPepT1 mice may provide a valuable tool in the drug discovery process by better predicting the oral pharmacokinetic profiles of PepT1 substrates in humans.

Asunto(s)

Antibacterianos/farmacocinética , Cefadroxilo/farmacocinética , Absorción Intestinal , Mucosa Intestinal/metabolismo , Yeyuno/metabolismo , Simportadores/metabolismo , Animales , Antibacterianos/administración & dosificación , Antibacterianos/sangre , Antibacterianos/metabolismo , Cefadroxilo/administración & dosificación , Cefadroxilo/sangre , Cefadroxilo/metabolismo , Colon/metabolismo , Cruzamientos Genéticos , Relación Dosis-Respuesta a Droga , Semivida , Humanos , Técnicas In Vitro , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Transportador de Péptidos 1 , Perfusión , Especificidad de la Especie , Simportadores/genética , Distribución Tisular

RESUMEN

The aim of the present study was to develop a method for water flux reabsorption measurement in Doluisio's Perfusion Technique based on the use of phenol red as a non-absorbable marker and to validate it by comparison with gravimetric procedure. The compounds selected for the study were metoprolol, atenolol, cimetidine and cefadroxil in order to include low, intermediate and high permeability drugs absorbed by passive diffusion and by carrier mediated mechanism. The intestinal permeabilities (Peff) of the drugs were obtained in male and female Wistar rats and calculated using both methods of water flux correction. The absorption rate coefficients of all the assayed compounds did not show statistically significant differences between male and female rats consequently all the individual values were combined to compare between reabsorption methods. The absorption rate coefficients and permeability values did not show statistically significant differences between the two strategies of concentration correction. The apparent zero order water absorption coefficients were also similar in both correction procedures. In conclusion gravimetric and phenol red method for water reabsorption correction are accurate and interchangeable for permeability estimation in closed loop perfusion method.

Asunto(s)

Indicadores y Reactivos , Absorción Intestinal , Intestino Delgado/metabolismo , Fenolsulfonftaleína , Agua/metabolismo , Animales , Atenolol/metabolismo , Cefadroxilo/metabolismo , Cimetidina/metabolismo , Femenino , Masculino , Metoprolol/metabolismo , Perfusión , Ratas Wistar , Reproducibilidad de los Resultados

RESUMEN

Among all the pharmaceutical drugs that contaminate the environment, antibiotics occupy an important place due to their high consumption rates in both veterinary and human medicine. The present study examined the ability of Pseudomonas putida to grow on the antibiotic wastewater, currently expanding in Tunisia, containing amoxicillin and cefadroxil. P. putida was very efficient to grow quickly in pharmaceutical wastewater (PW) and in reducing the total dissolved solids to 80.1 %. Cytotoxicity of PW, before and after biodegradation with P. putida mt-2, was evaluated in vitro, using the MTT assay, against four human tumor cell lines such as A549 (lung cell carcinoma), HCT15 (colon cell carcinoma), MCF7 (breast adenocarcinoma), and U373 (glioma cell carcinoma). The PW reduced all human cell lines viability in a dose-dependent manner. This activity was very remarkable against U373 cell line. For this reason, we have tested the genotoxicity of PW using comet assay for quantification of DNA fragmentation. In fact, PW has statistically significant (p<0.001) influence on DNA. Indeed, the percentage of genotoxicity was 66.87 and 87.5 %, after 24 and 48 h of treatment, respectively. However, cytotoxicity and genotoxicity decreased strongly when tested the PW obtained after incubation with P. putida mt-2. Our results indicate that P. putida is a promising and improved alternative to treating industrial-scale effluent compared to current chemical treatment procedures used by the industrials.

Asunto(s)

Antibacterianos/metabolismo , Daño del ADN/efectos de los fármacos , Pseudomonas putida/crecimiento & desarrollo , Aguas Residuales/química , Amoxicilina/metabolismo , Biodegradación Ambiental , Cefadroxilo/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , Fragmentación del ADN , Humanos , Residuos Industriales/análisis , Células MCF-7 , Pseudomonas putida/metabolismo , Túnez

RESUMEN

The purpose of this study was to quantitatively determine the contribution of PepT1 [peptide transporter 1 (SLC15A1)] to the intestinal permeability of valacyclovir, an ester prodrug of the antiviral drug acyclovir. In situ single-pass intestinal perfusions were employed (pH 6.5 × 90 minutes) to assess the effective permeability (P(eff)) of 100 µM [(3)H]valacyclovir in wild-type and PepT1 knockout mice. Acyclovir pharmacokinetics was also evaluated after oral administration of 25 nmol/g valacyclovir. In wild-type mice, jejunal uptake of valacyclovir was best described by both saturable (K(m) = 10.2 mM) and nonsaturable components where the saturable pathway accounted for 82% of total transport. Valacyclovir P(eff) was 2.4 × 10(-4) cm/s in duodenum, 1.7 × 10(-4) cm/s in jejunum, 2.1 × 10(-4) cm/s in ileum, and 0.27 × 10(-4) cm/s in colon. In Pept1 knockout mice, P(eff) values were about 10% of that in wild-type animals for these small intestinal segments. Valacyclovir P(eff) was similar in the colon of both genotypes. There were no differences in valacyclovir P(eff) between any of the intestinal segments of PepT1 knockout mice. Valacyclovir P(eff) was significantly reduced by the dipeptide glycylsarcosine and the aminocephalosporin cefadroxil, but not by the amino acids l-valine or l-histidine, the organic acid p-aminohippurate, or the organic base tetraethylammonium (all at 25 mM). PepT1 ablation resulted in 3- to 5-fold reductions in the in vivo rate and extent of valacyclovir absorption. Our findings conclusively demonstrate, using in situ and in vivo validations in genetically modified mice, that PepT1 has a major influence in improving the oral absorption of valacyclovir.

Asunto(s)

Aciclovir/análogos & derivados , Antivirales/farmacocinética , Mucosa Intestinal/metabolismo , Simportadores/deficiencia , Valina/análogos & derivados , Aciclovir/administración & dosificación , Aciclovir/farmacocinética , Administración Oral , Animales , Antivirales/administración & dosificación , Unión Competitiva , Biotransformación , Cefadroxilo/metabolismo , Cromatografía Líquida de Alta Presión , Colon/metabolismo , Dipéptidos/metabolismo , Duodeno/metabolismo , Íleon/metabolismo , Absorción Intestinal , Yeyuno/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Transportador de Péptidos 1 , Perfusión , Permeabilidad , Reproducibilidad de los Resultados , Simportadores/genética , Valaciclovir , Valina/administración & dosificación , Valina/farmacocinética

RESUMEN

PURPOSE: To determine the contribution of intestinal PepT1 on the permeability and oral absorption of the ß-lactam antibiotic drug cefadroxil. METHODS: The effective permeability (P eff ) of cefadroxil was evaluated in wild-type and PepT1 knockout mice following in situ single-pass intestinal perfusions. The plasma concentration-time profiles of cefadroxil were also examined after oral gavage. RESULTS: The P eff (cm/s) of cefadroxil in wild-type mice was 0.49 × 10(-4) in duodenum, 0.80 × 10(-4) in jejunum, 0.88 × 10(-4) in ileum and 0.064 × 10(-4) in colon. The P eff (cm/s) in PepT1 knockout mice was significantly reduced in small intestine, but not in colon, as shown by values of 0.003 × 10(-4), 0.090 × 10(-4), 0.042 × 10(-4) and 0.032 × 10(-4), respectively. Jejunal uptake of cefadroxil was saturable (Km = 2-4 mM) and significantly attenuated by the sodium-proton exchange inhibitor 5-(N,N-dimethyl)amiloride. Jejunal permeability of cefadroxil was not affected by L-histidine, glycine, cephalothin, p-aminohippurate or N-methylnicotinamide. In contrast, cefadroxil permeability was significantly reduced by glycylproline, glycylsarcosine, or cephalexin. Finally, PepT1 ablation resulted in 23-fold reductions in peak plasma concentrations and 14-fold reductions in systemic exposure of cefadroxil after oral dosing. CONCLUSIONS: The findings are definitive in demonstrating that PepT1 is the major transporter responsible for the small intestinal permeability of cefadroxil as well as its enhanced oral drug performance.

Asunto(s)

Antibacterianos/farmacocinética , Cefadroxilo/farmacocinética , Técnicas de Inactivación de Genes , Mucosa Intestinal/metabolismo , Simportadores/genética , Administración Oral , Animales , Antibacterianos/metabolismo , Cefadroxilo/metabolismo , Femenino , Masculino , Ratones , Ratones Noqueados , Transportador de Péptidos 1 , Permeabilidad , Simportadores/metabolismo

RESUMEN

Quantitative structure/activity relationship (QSAR) approaches have widely been applied to gain deeper understandings of the relationships between ADME parameters and molecular structure and properties. QSAR models for predicting ADME properties are required to cover structurally diverse compounds. In the present investigation, we describe application of genetic algorithm-combined partial least squares (GA-PLS) method to QSAR modelling of various ADME properties. By selecting an appropriate set of molecular descriptors automatically by the use of genetic algorithm, many ADME properties could be well-explained by simple molecular descriptors derived from 2-dimensional chemical structure.

Asunto(s)

Algoritmos , Análisis de los Mínimos Cuadrados , Conformación Molecular , Farmacocinética , Relación Estructura-Actividad Cuantitativa , Transporte Biológico , Células CACO-2 , Cefadroxilo/química , Cefadroxilo/metabolismo , Cefadroxilo/farmacocinética , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Modelos Genéticos , Modelos Moleculares , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Solubilidad

RESUMEN

Cefadroxil is a cephalosporin antibiotic used in the treatment of infection. However, cerebrospinal fluid (CSF) concentrations of cefadroxil and other aminocephalosporins are not adequate for the treatment of bacterial meningitis. To evaluate the relevance of PEPT2 in affecting the exposure of aminocephalosporins in brain, we investigated the transport properties of cefadroxil at the blood-CSF interface using primary-cultured epithelial cells and isolated whole tissues of choroid plexus. Our results indicated that cefadroxil was preferentially taken up from the apical as opposed to basal side of the monolayer (5-fold), and its apical uptake was stimulated by an inwardly directed proton gradient. The concentration-dependent apical uptake of cefadroxil was characterized by a high-affinity/low-capacity transport system (Km = 39.0 +/- 22.7 microM; Vmax = 22.9 +/- 6.6 pmol/mg/min) and a nonsaturable component (Kd = 0.15 +/- 0.01 microl/mg/min); in contrast, only a nonsaturable component was found for the basal uptake of cefadroxil (Kd = 0.14 +/- 0.01 microl/mg/min). The apical-to-basal transepithelial transport of 2 microM cefadroxil was greater than its basal-to-apical transport, but no differences were observed in directionality when 5 mM concentrations of cefadroxil were studied. Moreover, the cellular efflux of cefadroxil was not saturable in either direction (i.e., to apical or basal side). Finally, no differences were observed in the choroid plexus tissue efflux of 2 microM cefadroxil from wild-type and PEPT2 null mice. These findings demonstrate that PEPT2 has an important role in limiting the exposure of cefadroxil in CSF. Located at the apical membrane of choroid plexus epithelium, PEPT2 acts in a unidirectional (as opposed to bidirectional) manner in transporting cefadroxil from CSF into the cell.

Asunto(s)

Antibacterianos/metabolismo , Cefadroxilo/metabolismo , Líquido Cefalorraquídeo/metabolismo , Plexo Coroideo/metabolismo , Simportadores/metabolismo , Animales , Animales Recién Nacidos , Antibacterianos/farmacocinética , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/fisiología , Cefadroxilo/farmacocinética , Técnicas de Cultivo de Célula , Células Cultivadas , Plexo Coroideo/citología , Relación Dosis-Respuesta a Droga , Impedancia Eléctrica , Células Epiteliales/metabolismo , Femenino , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratones Noqueados , Modelos Biológicos , Embarazo , Ratas , Simportadores/antagonistas & inhibidores , Simportadores/genética , Factores de Tiempo

RESUMEN

Uptake of cyclic dipeptides by H+/oligopeptide cotransporter (PEPT1) was studied in monolayers of the human intestinal cell line, Caco-2. The cyclic dipeptides studied were cyclic glycylphenylalanine (cyclo(Gly-Phe)), cyclic phenylalanylserine (cyclo(Phe-Ser)), cyclic seryltyrosine (cyclo(Ser-Tyr)) and cyclic glycyltyrosine (cyclo(Gly-Tyr)). These molecules have both peptide bonds and aromatic rings, and are similar in structure to cephalexin and cephadroxil, which are transported by PEPT1. Cellular uptake of these cyclic dipeptides was pH dependent, and was inhibited by the addition of PEPT1 substrates such as glycylsarcosine, indicating PEPT1-mediated transport. Michaelis constants (Km) for these cyclic dipeptides were cyclo(Ser-Tyr) < cyclo(Phe-Ser), and cyclo(Gly-Tyr) < cyclo(Gly-Phe), indicating that tyrosine possessing phenol moiety has higher affinity for PEPT1 than phenylalanine possessing benzen moiety. The Km for cephadroxil possessing phenol moiety was reportedly lower than that for cephalexin possessing benzen moiety. Therefore, it was concluded that the phenolic hydroxyl group of the substrate may enhance affinity for PEPT1.

Asunto(s)

Proteínas Portadoras/fisiología , Cefadroxilo/metabolismo , Dipéptidos/metabolismo , Simportadores , Células CACO-2 , Cromatografía Líquida de Alta Presión , Humanos , Concentración de Iones de Hidrógeno , Transportador de Péptidos 1

RESUMEN

The nature of protein breakdown products and peptidomimetic drugs such as beta-lactams is crucial for their transmembrane transport across apical enterocyte membranes, which is accomplished by the pH-dependent high-capacity oligopeptide transporter PEPT1. To visualize oligopeptide transporter-mediated uptake of oligopeptides, an ex vivo assay using the fluorophore-conjugated dipeptide derivative D-Ala-Lys-N(epsilon)-7-amino-4-methylcoumarin-3-acetic acid (D-Ala-Lys-AMCA) was established in the murine small intestine and compared with immunohistochemistry for PEPT1 in murine and human small intestine. D-Ala-Lys-AMCA was accumulated by enterocytes throughout all segments of the murine small intestine, with decreasing intensity from the top to the base of the villi. Goblet cells did not show specific uptake. Inhibition studies revealed competitive inhibition by the beta-lactam cefadroxil, the angiotensin-converting enzyme inhibitor captopril, and the dipeptide glycyl-glutamine. Controls were performed using either the inhibitor diethylpyrocarbonate or an incubation temperature of 4 degrees C to exclude unspecific uptake. Immunohistochemistry for PEPT1 localized immunoreactivity to the enterocytes, with the highest intensity at the apical membrane. This is the first study that visualizes dipeptide transport across the mammalian intestine and indicates that uptake assays using D-Ala-Lys-AMCA might be useful for characterizing PEPT1-specific substrates or inhibitors.

Asunto(s)

Proteínas Portadoras/metabolismo , Cumarinas/metabolismo , Dipéptidos/metabolismo , Intestino Delgado/metabolismo , Oligopéptidos/metabolismo , Simportadores , Inhibidores de la Enzima Convertidora de Angiotensina/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacocinética , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Captopril/metabolismo , Captopril/farmacocinética , Cefadroxilo/metabolismo , Cefadroxilo/farmacocinética , Cumarinas/farmacocinética , Dietil Pirocarbonato/farmacología , Dipéptidos/farmacocinética , Enterocitos/metabolismo , Femenino , Colorantes Fluorescentes , Humanos , Inmunohistoquímica , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/fisiología , Intestino Delgado/citología , Intestino Delgado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/farmacocinética , Transportador de Péptidos 1

RESUMEN

This paper reports the development of new interpenetrating polymeric networks of sodium alginate with gelatin or egg albumin cross-linked with a common cross-linking agent, glutaraldehyde, for the in-vitro release of cefadroxil. The beads formed were characterized by Fourier transform infra-red spectroscopy, scanning electron microscopy and differential scanning calorimetry. Swelling/drying experiments were performed to compute the diffusion coefficients and the molecular mass between cross-links of the beads. The release results were evaluated using an empirical equation to understand the transport mechanism. The extent of cross-linking was studied in terms of the size and release characteristics of the beads. The experimental and derived quantities have been used to study their dependencies on the nature of the polymeric beads, transport mechanism, encapsulation efficiency and drug diffusion, as well as the cross-linking abilities of the polymers.

Asunto(s)

Alginatos/química , Cefadroxilo/farmacocinética , Cefalosporinas/farmacocinética , Portadores de Fármacos/química , Hidrogeles/química , Polímeros/química , Albúminas/química , Rastreo Diferencial de Calorimetría , Cefadroxilo/metabolismo , Cefalosporinas/metabolismo , Difusión , Fijadores/química , Gelatina/química , Ácido Glucurónico , Glutaral/química , Ácidos Hexurónicos , Microscopía Electrónica de Rastreo , Microesferas , Espectroscopía Infrarroja por Transformada de Fourier , Agua/química

RESUMEN

Penicillin-G acylase (EC 3.5.1.11) from Escherichia coli catalyzed the synthesis of various beta-lactam antibiotics in ice at -20 degrees C with higher yields than obtained in solution at 20 degrees C. The initial ratio between aminolysis and hydrolysis of the acyl-enzyme complex in the synthesis of cephalexin increased from 1.3 at 20 degrees C to 25 at -20 degrees C. The effect on the other antibiotics studied was less, leading us to conclude that freezing of the reaction medium influences the hydrolysis of each nucleophile-acyl-enzyme complex to a different extent. Only free penicillin-G acylase could perform transformations in frozen media: immobilized preparations showed a low, predominantly hydrolytic activity under these conditions.

Asunto(s)

Antibacterianos/síntesis química , Penicilina Amidasa/química , Amoxicilina/síntesis química , Amoxicilina/metabolismo , Ampicilina/síntesis química , Ampicilina/metabolismo , Antibacterianos/metabolismo , Cefadroxilo/síntesis química , Cefadroxilo/metabolismo , Cefalexina/síntesis química , Cefalexina/metabolismo , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Escherichia coli/enzimología , Congelación , Penicilina Amidasa/metabolismo

RESUMEN

l. Recently two genes have been identified by expression cloning that encode mammalian epithelial peptide transporters capable of translocating di- and tripeptides and selected peptidomimetics by stereoselective and rheogenic substrate-H+ cotransport. PepT1 from rabbit or human small intestine induces a transport activity with high transport capacity but rather low substrate affinity when expressed in Xenopus oocytes. In contrast, the renal carrier PepT2 is a high affinity-type transporter with a lower maximal transport capacity. In addition, both transporters show differences in pH dependence and substrate specificity. 2. As a first approach to identify structural components of the transport proteins that determine their phenotypical characteristics, we constructed a recombinant chimeric peptide transporter (CH1Pep) in which the aminoterminal region (residues 1-401) is derived from PepT2 whereas the carboxyterminal region (residues 402-707) starting at the end of transmembrane domain 9 is derived from PepT1. Expression of PepT1, PepT2 and CH1Pep in Xenopus oocytes allowed the characteristics of the transporters to be determined by flux studies employing a radiolabelled dipeptide and by the two-electrode voltage clamp technique. 3. Our studies indicate that CH1Pep conserves the characteristics of PepT2 including the high affinity for dipeptides and peptidomimetics, the substrate specificity, the pH dependence of transport activation and the electrophysiological parameters. We conclude that the phenotypical characteristics of the renal peptide transporter are determined by its amino-terminal region.

Asunto(s)

Proteínas Portadoras/fisiología , Proteínas Recombinantes de Fusión/fisiología , Simportadores , Animales , Cefadroxilo/metabolismo , Dipéptidos/metabolismo , Escherichia coli , Potenciales Evocados , Humanos , Concentración de Iones de Hidrógeno , Oocitos/metabolismo , Transportador de Péptidos 1 , Conejos , Especificidad por Sustrato , Xenopus

RESUMEN

The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences.

Asunto(s)

Proteínas Portadoras/genética , Corteza Renal/metabolismo , Simportadores , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/metabolismo , Cefadroxilo/metabolismo , Clonación Molecular , ADN Complementario/genética , Expresión Génica , Biblioteca de Genes , Intestino Delgado/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Oocitos , ARN Mensajero/genética , Conejos , Xenopus laevis

RESUMEN

This study was initiated to determine if there are differences in the recognition of beta -lactam antibiotics as substrates between intestinal and renal peptide transporters, PEPT 1 and PEPT 2. Reverse transcription-coupled polymerase chain reaction and/or Northern blot analysis have established that the human intestinal cell line Caco-2 expresses PEPT 1 but not PEPT 2, whereas the rat proximal tubule cell line SKPT expresses PEPT 2 but not PEPT 1. Detailed kinetic analysis has provided unequivocal evidence for participation of PEPT 2 in SKPT cells in the transport of the dipeptide glycylsarcosine and the aminocephalosporin cephalexin. The substrate recognition pattern of PEPT 1 and PEPT 2 was studied with cefadroxil (a cephalosporin) and cyclacillin (a penicillin) as model substrates for the peptide transporters constitutively expressed in Caco-2 cells (PEPT 1) and SKPT cells (PEPT 2). Cyclacillin was 9-fold more potent than cefadroxil in competing with glycylsacosine for uptake via PEPT 1. In contrast, cefadroxil was 13-fold more potent than cyclacillin in competing with the dipeptide for uptake via PEPT 2. The substrate recognition pattern of PEPT 1 and PEPT 2 was also investigated using cloned human peptide transporters functionally expressed in HeLa cells. Expression of PEPT 1 or PEPT 2 in HeLa cells was found to induce H(+)-coupled cephalexin uptake in these cells. As was the case with Caco-2 cells and SKPT cells, the uptake of glycylsarcosine induced in HeLa cells by PEPT 1 cDNA and PEPT 2 cDNA was inhibitable by cyclacillin and cefadroxil. Again, the PEPT 1 cDNA-induced dipeptide uptake was inhibited more potently by cyclacillin than by cefadroxil, and the PEPT 2 cDNA-induced dipeptide uptake was inhibited more potently by cefadroxil than by cyclacillin. It is concluded that there are marked differences between the intestinal and renal peptide transporters in the recognition of beta -lactam antibiotics as substrates.

Asunto(s)

Antibacterianos/metabolismo , Proteínas Portadoras/metabolismo , Intestino Delgado/metabolismo , Riñón/metabolismo , Simportadores , Animales , Antibacterianos/farmacología , Transporte Biológico/efectos de los fármacos , Northern Blotting , Proteínas Portadoras/genética , Cefadroxilo/metabolismo , Células Cultivadas , Cefalexina/metabolismo , Cefalosporinas/metabolismo , Ciclacilina/metabolismo , Dipéptidos/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Intestino Delgado/citología , Riñón/citología , Penicilinas/metabolismo , Transportador de Péptidos 1 , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Ratas , Proteínas Recombinantes/metabolismo , Virus Vaccinia/genética

RESUMEN

The apparent functional molecular mass of the kidney peptide/H(+)-symporter was determined by radiation inactivation in brush-border membrane vesicles (BBMV) of rat kidney cortex. Purified BBMV were irradiated at low temperatures with high energy electrons generated by a 10-MeV linear accelerator at doses from 0 to 30 megarads. Uptake studies were performed with [3H]cefadroxil, a beta-lactam antibiotic which serves as a substrate for the kidney peptide/H(+)-symporter. Inhibition of influx of [3H]cefadroxil into BBMV was used to determine the functional molecular mass of the transporter. Additionally, direct photoaffinity labeling of the transport- and/or binding proteins for [3H]cefadroxil in control and irradiated BBMV was performed to determine the molecular mass of the putative transporter by SDS-polyacrylamide gel electrophoresis. Initial rates of pH-gradient dependent uptake of [3H]cefadroxil decreased progressively as a function of radiation dose. The apparent radiation inactivation size (RIS) of the transport function was found to be 414 +/- 16 kDa. Direct photoaffinity labeling yielded labeled membrane proteins with apparent molecular masses of 130 kDa and 105 kDa, respectively. The proteins displayed different labeling characteristics with respect to incubation time, specificity and the response to irradiation. It appears that only a 105 kDa protein is directly involved in transport function since (a) only it showed a specific pH gradient dependent labeling pattern and (b) the covalent incorporation of [3H]cefadroxil into this protein decreased parallel to the loss of transport function in irradiated BBMV. The peptide/H(+)-symporter in kidney brush-border membranes therefore appears to have a monomer mass of 105 kDa and may function in an oligomeric arrangement.

Asunto(s)

Proteínas Portadoras/efectos de la radiación , Corteza Renal/ultraestructura , Simportadores , Marcadores de Afinidad , Animales , Transporte Biológico Activo , Proteínas Portadoras/metabolismo , Cefadroxilo/metabolismo , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Masculino , Microvellosidades , Peso Molecular , Aceleradores de Partículas , Transportador de Péptidos 1 , Fotoquímica , Ratas , Ratas Wistar

RESUMEN

Uptake of cefadroxil and its two acetyl-derivatives, N-acetyl- and O-acetyl-cefadroxil, into the brush-border membrane vesicles (BBMV) was measured at [pH]o = 5.5, 7.4 and [pH]i = 7.4. Both acetyl-derivatives showed a significantly slower uptake than cefadroxil at [pH]o = 5.5 and 7.4. Cefadroxil and the two derivatives showed a higher uptake rate in the presence of an inward H+ gradient ([pH]o = 5.5, [pH]i = 7.4). At [pH]o = 5.5, uptake of cefadroxil into BBMV was inhibited by N-acetyl-, O-acetyl-, N-BOC-, and N-BOC-O-acetyl-cefadroxil, but not by cephalothin and cefuroxime. At [pH]o = 7.4, no inhibition of cefadroxil uptake was evident for any inhibitors. There were two different transporters responsible for the uptake of cefadroxil at pH 5.5 and 7.4. One is the H(+)-coupled dipeptide transport system, and the other is the neutral pH-preferring system. The alpha-amino group may be essential for the transport of cefadroxil by both transport systems. Although the phenolic group in the side chain is not an essential functional group of beta-lactam antibiotics, an additional derivation on the phenolic group of cefadroxil also inhibited both the H(+)-coupled dipeptide transport system and the neutral pH-preferring transport system.

Asunto(s)

Cefadroxilo/farmacocinética , Microvellosidades/metabolismo , Animales , Cefadroxilo/metabolismo , Concentración de Iones de Hidrógeno , Absorción Intestinal , Concentración Osmolar , Ratas

RESUMEN

The therapeutic efficacies of cefadroxil and cephalexin were compared in a Streptococcus pyogenes-induced lung infection in rats. Although MICs, rates of in vitro killing in rat serum, and antibiotic serum levels after oral administration were similar for both drugs, cefadroxil was about eight times more effective than cephalexin in reducing the number of viable streptococci at the site of infection. This excellent in vivo bactericidal activity of cefadroxil in lung tissue and bronchial secretions was reflected in the 50% protective dose (PD50) after single or multiple oral treatments. A single treatment given 24 h after infection resulted in a PD50 of 2.8 mg of cefadroxil per kg, compared with 21 mg of cephalexin per kg. When treatment was administered three times, at 24, 27, and 30 h postinfection, the PD50s of cefadroxil and cephalexin were 0.7 and 8.0 mg/kg, respectively. In infected animals, treated 24 h postinfection, the area under the lung tissue concentration versus time curve for cefadroxil was significantly greater than that of cephalexin. This difference in pharmacokinetic behavior may account, at least in part, for the superior therapeutic results obtained with cefadroxil in this experimental pulmonary infection.

Asunto(s)

Cefadroxilo/uso terapéutico , Cefalexina/uso terapéutico , Neumonía/tratamiento farmacológico , Animales , Cefadroxilo/sangre , Cefadroxilo/metabolismo , Cefalexina/sangre , Cefalexina/metabolismo , Pulmón/metabolismo , Masculino , Pruebas de Sensibilidad Microbiana , Neumonía/etiología , Ratas , Infecciones Estreptocócicas/tratamiento farmacológico