RESUMEN
Intestinal transport and metabolism of modified kyotorphin (KTP) were studied in rats. Modified KTPs studied were C-terminally modified KTP with p-aminophenyl-beta-D-glucoside (KTP-pAPbeta glc), N-terminally modified KTP-pAPbeta glc with t-butyloxycarbonyl group (Boc-KTP-pAPbeta glc) and the N- and C-terminally modified KTP by cyclization (cyclic KTP). KTP-pAPbeta glc was metabolized at a similar rate to that of KTP, and did not appear on the serosal side. Although Boc-KTP-pAPbeta glc was also metabolized, it was more stable than KTP and appeared on the serosal side. Cyclic KTP was also quite stable and appeared on the serosal side. The modified KTPs were evaluated kinetically for absorption consisting of membrane transport and metabolism. Absorption clearance (CL(abs)) of cyclic KTP, Boc-KTP-pAPbeta glc and Boc-KTP was higher than that of KTP (0.247 microl/min/cm) (Mizuma et al., Biochim. Biophys. Acta 1335 (1997) 111-119), which is the theoretical maximum by complete inhibition of peptidase activity, indicating that derivatization of KTP increases the membrane permeability. Furthermore, the data clearly showed that the greater the metabolic clearance (CL(met)) of KTP and the KTP derivatives, the lower the absorption clearance (CL(abs)). These results and further simulation study led to the conclusion that metabolic degradation in the intestinal tissues is more critical than membrane permeability (transport) for oral delivery of peptide drugs. Based on the stability of cyclic KTP in serum, this appears to be a good candidate analgesic peptide drug.
Asunto(s)
Endorfinas/farmacocinética , Mucosa Intestinal/metabolismo , Péptidos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Acetaminofén/farmacocinética , Animales , Transporte Biológico , Cromatografía Líquida de Alta Presión , Endorfinas/sangre , Endorfinas/química , Glucosa/química , Absorción Intestinal , Masculino , Péptidos Cíclicos/metabolismo , Ratas , Ratas WistarRESUMEN
Intestinal metabolism and transport of p-nitrophenyl beta-D-thioglucoside (p-NPbetaSglc) and p-nitrophenyl beta-D-thiogalactoside (p-NPbetaSgal) by the Na+/glucose cotransporter were studied in excised small intestine of the rat. p-NPbetaSglc and p-NPbetaSgal were stable enough on the mucosal side to be transported to the serosal side. Transport of p-NPbetaSglc was inhibited in the presence of phloridzin (a Na+/glucose cotransporter inhibitor), glucose, or 3-O-methylglucose (3-OMG). p-NPbetaSglc transport was dependent on Na+ concentration in a sigmoidal manner. The activation energy for transport was 19.4 kcal mol(-1). The distribution of transport activity of p-NPbetaSglc in each region of the small intestine correlated well with that of 3-OMG. These results indicate that p-NPbetaSglc is transported by the Na+/glucose cotransporter in small intestine. The order of turnover rate for glycoside transport by Na+/glucose cotransporter was 3-OMG > p-nitrophenyl beta-O-glucoside > p-NPbetaSglc > p-NPbetaSgal, indicating that the presence of a galactose moiety and a sulphur between the monosaccharide moiety and aglycone decreases the turnover rate of the Na+/glucose cotransporter in the transport of glycosides. In an inhibition study using stable p-NPbetaSglc as a Na+/glucose cotransporter-transportable marker glucoside, it was also shown that the Na+/glucose cotransporter recognized several types of glycosides. In conclusion, displacement of the oxygen at carbon C-1 of glucose by sulphur in thioglycosides decreases the turnover rate of the Na+/glucose cotransporter, but thioglycosides are stable in the small intestine and are transported by the Na+/glucose cotransporter.
Asunto(s)
Intestino Delgado/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Tioglicósidos/farmacocinética , 2,4-Dinitrofenol/farmacología , 3-O-Metilglucosa/farmacocinética , Animales , Transporte Biológico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glicósidos/farmacología , Técnicas In Vitro , Masculino , Florizina/farmacología , Ratas , Ratas Wistar , Sodio/farmacología , Tiogalactósidos/farmacocinética , Tioglucósidos/farmacocinética , Extractos de Tejidos/químicaRESUMEN
Transport and recognition of aminopeptidase-resistant cellobiose-coupled tyrosylglycylglycine (CcpTGG) by intestinal Na+/glucose cotransporter (SGLT1) was examined in rat small intestine. Inhibitory effect of phloridzin (SGLT1 inhibitor) on the CcpTGG transport was extremely low. Concentration dependence of the CcpTGG transport was observed, but the primary component of transport was passive diffusion. However, CcpTGG significantly inhibited SGLT1-mediated transport, indicating its recognition by SGLT1. Other glucose-conjugates also inhibited SGLT1-mediated transport. These results indicate that recognition of sugar conjugates by SGLT1 is much less restricted than transport, and that it should be relatively easier to design SGLT1 inhibitors than SGLT1-transportable sugar conjugates.
Asunto(s)
Aminopeptidasas/metabolismo , Celobiosa/metabolismo , Mucosa Intestinal/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Oligopéptidos/metabolismo , Animales , Secuencia de Carbohidratos , Masculino , Ratas , Ratas Wistar , Transportador 1 de Sodio-GlucosaRESUMEN
AIMS: To study reaction of photoactivated frusemide (F) and F glucuronide (Fgnd metabolite) with human serum albumin in order to find a clue to clarify a mechanism of phototoxic blisters from high frusemide dosage. METHODS: F was exposed to light in the presence of human serum albumin (HSA). HSA treated with this method (TR-HSA) was characterized by fluorescence spectroscopic experiment, alkali treatment and reversible binding experiment. RESULTS: Less 4-hydroxyl-N-furfuryl-5-sulphamoylanthranilic acid (4HFSA, a photodegradation product of F) was formed in the presence of HSA than in the absence of HSA. A new fluorescence spectrum excited at 320 nm was observed for TR-HSA. Alkali treatment of TR-HSA released 4HFSA. Quenching of the fluorescence due to the lone tryptophan near the warfarin-binding site of HSA was observed in TR-HSA. The reversible binding of F or naproxen to the warfarin-binding site of TR-HSA was less than to that of native HSA. These results indicate the photoactivated F was covalently bound to the warfarin-binding site of HSA. The covalent binding of Fgnd, which is also reversibly bound to the warfarin-binding site of HSA, was also induced by exposure to sunlight. Fgnd was more photoactive than F, indicating that F could be activated by glucuronidation to become a more photoactive compound. CONCLUSIONS: The reactivity of photoactivated F and Fgnd to HSA and/or to other endogenous compounds may cause the phototoxic blisters that result at high F dosage.
Asunto(s)
Diuréticos/farmacología , Furosemida/análogos & derivados , Furosemida/farmacología , Albúmina Sérica/metabolismo , Unión Competitiva/efectos de la radiación , Diuréticos/química , Diuréticos/efectos de la radiación , Estabilidad de Medicamentos , Furosemida/química , Furosemida/efectos de la radiación , Humanos , Fotoquímica , Espectrometría de Fluorescencia , Luz SolarRESUMEN
Furosemide 1-O-acyl glucuronide (Fgnd) was reversibly bound to a single class of binding sites on human serum albumin (HSA), and the binding of Fgnd decreased with increasing F concentrations, suggesting that Fgnd binds to the same warfarin binding sites on HSA as F binds. The rate of Fgnd degradation (hydrolysis and acyl migration) decreased in the presence of HSA. Although the formation of acyl migration isomers of Fgnd was slower in the presence of HSA than in its absence, hydrolysis of Fgnd to F was faster in the presence of HSA. Rapid minor irreversible binding of Fgnd to HSA within 30 min was followed by slow major irreversible binding. Slow irreversible binding of Fgnd to HSA was decreased by F, though not significantly. This suggests that major irreversible binding may proceed via reversible binding. It has been reported that acyl migration is a prerequisite for irreversible binding. Therefore, these results indicate that HSA decreases irreversible binding of Fgnd to protein by suppressing acyl migration. Furthermore, these results suggest that HSA may prevent irreversible binding of Fgnd to other proteins in the body by decreasing the concentration of reactive Fgnd in the unbound form. HSA eliminates reactive Fgnd by hydrolysis to F. Therefore, it is concluded that HSA works as a scavenger to decrease reactive compounds by reversible binding or eliminates reactive compounds by irreversible binding.
Asunto(s)
Furosemida/análogos & derivados , Albúmina Sérica/metabolismo , Acilación , Sitios de Unión , Furosemida/metabolismo , Humanos , Hidrólisis , EstereoisomerismoRESUMEN
PURPOSE: To characterize the intestinal absorption of a beta-glucose conjugate of acyclovir (9-[(2-hydroxyethoxy) methyl] guanine, ACV) and compare it to ACV and its analogues in terms of stability and transport by Na+/glucose cotransporter (SGLTI). METHODS: ACVbeta(glc) was enzymatically synthesized using cellulase. Intestinal absorption experiments were performed with rat everted small intestine. Conformation of the glucose moiety was analyzed by NMR spectroscopy. RESULTS: The ACVbeta(glc) was stable on the mucosal side, and was transported to the serosal side in all regions of the small intestine. However, significant contribution of SGLTI to the transport of ACVbetaglc was not observed. NMR spectroscopic analysis indicated that the glucose conformation of ACVbeta(glc) was the 4C1 chair form, identical to beta-glucose or SGLT1-transportable beta-glucosides reported previously. Therefore, other factors such as molecular size and charge due to aglycone may cause no transport of ACVbeta(glc) by SGLT1. On the other hand, the hydrophilicity of ACVbeta(glc) was much higher than of ACV, suggesting water solubility-derived improvement of intestinal absorption of ACV. CONCLUSIONS: ACVbeta(glc) is stable and absorbable, but it is not transported by SGLT1. No involvement of SGLT1 in the ACVbeta(glc) transport is not due to glucose conformation.
Asunto(s)
Aciclovir/análogos & derivados , Adenina/análogos & derivados , Glucósidos/farmacocinética , Guanosina/farmacocinética , Absorción Intestinal , Cinetina , Aciclovir/farmacocinética , Adenina/farmacocinética , Animales , Transporte Biológico , Glucosa/metabolismo , Intestino Delgado/metabolismo , Espectroscopía de Resonancia Magnética , Masculino , Glicoproteínas de Membrana/metabolismo , Estructura Molecular , Proteínas de Transporte de Monosacáridos/metabolismo , Aceites , Ratas , Ratas Wistar , Sodio/metabolismo , Transportador 1 de Sodio-Glucosa , AguaRESUMEN
Stability of furosemide glucuronide, the major metabolite of furosemide, was studied in order to accurately assess the glucuronidation of furosemide. Furosemide glucuronide was purified by high-performance liquid chromatography, and the mass spectrum of furosemide glucuronide showed the molecular ion peaks [M-H]- at 505 and 507 (m/z). Furosemide glucuronide was photodegraded to the compound, which was shown more hydrophilic than furosemide glucuronide by high-performance liquid chromatography assay. The photodegradation product of furosemide glucuronide was hydrolyzed to one of the photodegradation products of furosemide by beta-glucuronidase, indicating that the photodegradation product of furosemide glucuronide possessed a glucuronic acid moiety. Furthermore, the mass spectrum of the photodegradation product of furosemide glucuronide exhibited molecular ion peaks [M-H]- at 487 and [M-2H+2Na]- at 509, indicating the chlorine displacement of furosemide glucuronide by a hydroxyl group. Furosemide glucuronide was unstable in an aqueous solution (pH=7.4), and presumed acyl migration isomers of furosemide glucuronide (furosemide glucuronide-isomers) were detected by high-performance liquid chromatography equipped with photodiode array UV detector. The UV spectra of seven furosemide glucuronide-isomers were closely similar to that of furosemide glucuronide but not furosemide. Exposing a mixture of furosemide glucuronide and furosemide glucuronide-isomers to light resulted in the production of new compounds. UV spectra of photodegradation products of furosemide glucuronide-isomers were closely similar to those of photodegradation product of furosemide glucuronide. These results suggested that furosemide glucuronide-isomers were also photodegraded, resulting in the displacement of chlorine by a hydroxyl group as in furosemide glucuronide.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Furosemida/análogos & derivados , Furosemida/análisis , Furosemida/metabolismo , Glucuronidasa/metabolismo , Espectrometría de Masas , Fotoquímica , Espectrofotometría UltravioletaRESUMEN
Intestinal glucuronidation and absorption of p-nitrophenol (p-NP), acetaminophen (APAP) and 1-naphthol (alpha-NA) in the presence of phloridzin (inhibitor of Na+/glucose cotransporter) and phloretin (aglycone of phloridzin) were studied. Glucuronides of p-NP, APAP and alpha-NA appeared on both the serosal and mucosal sides. The amounts of glucuronides on the serosal side were decreased in the presence of phloridzin and phloretin. p-NP, APAP and alpha-NA appeared on the serosal side as well, and the amounts of p-NP, APAP and alpha-NA on the serosal side were increased by the presence of phloridzin and phloretin. Furthermore, the intestinal glucuronidation and absorption of alpha-NA at various concentrations were studied in the presence and absence of phloretin. Metabolic clearance was decreased in the presence of phloretin, and the absorption clearance was increased. The higher concentrations of alpha-NA caused higher absorption clearance. The lower the metabolic clearance, the higher the absorption clearance. The relationship between glucuronidation metabolism and absorption in intestine was kinetically analyzed by the metabolic inhibition model. Complete inhibition of glucuronidation improved the intestinal absorption of alpha-NA, and the absorption clearance increased to 7.17 microliter/min/cm. The formation of phloretin and an unknown metabolite from phloridzin were observed. An unknown metabolite from phloretin was observed, and was suppressed by the presence of alpha-NA. This suggests that phloridzin was hydrolyzed to phloretin, which was metabolized to glucuronide, and thereby inhibited glucuronidation of p-NP, APAP and alpha-NA.
Asunto(s)
Glucuronidasa/antagonistas & inhibidores , Absorción Intestinal/efectos de los fármacos , Floretina/farmacología , Florizina/farmacología , Acetaminofén/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Glucuronatos/metabolismo , Glucuronidasa/metabolismo , Masculino , Naftoles/metabolismo , Nitrofenoles/metabolismo , Ratas , Ratas WistarRESUMEN
The intestinal transport of glucose- and galactose-conjugated acetaminophen (APAP glycoside) by Na+/glucose cotransporter (SGLT1) was studied. SGLT1-mediated transport of APAP glycosides preferred glucoside>galactoside and beta-anomer>alpha-anomer. These preferences agree with previous studies. NMR spectroscopic and molecular modeling studies indicated that the conformation of the glucose ring of alpha- and beta-glucosides of APAP, as well as glycosides in previous studies, is in the 4C1 chair form, the same form as glucose itself. Molecular dynamics analysis also indicated that the glucose ring was in the 4C1 chair form, and that there are differences between the rotational spaces of aglycones and hydroxy groups of glucose moieties between anomers. Therefore, we conclude that the beta-anomeric preference of glucose conjugate transport by SGLT1 is not due to the conformation of the glucose ring, but to the configuration of the aglycone at C-1 of the monosaccharide moiety.
Asunto(s)
Glicoconjugados/metabolismo , Mucosa Intestinal/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Monosacáridos/metabolismo , Acetaminofén/metabolismo , Animales , Transporte Biológico , Galactosa/metabolismo , Glucosa/metabolismo , Glicoconjugados/química , Cinética , Modelos Moleculares , Conformación Molecular , Monosacáridos/química , Ratas , Transportador 1 de Sodio-Glucosa , Relación Estructura-ActividadRESUMEN
Intestinal transport and metabolism of p-nitrophenyl alpha-disaccharides were studied. In the absorption of p-nitrophenyl alpha-melibioside, no compounds other than p-nitrophenyl alpha-melibioside were detected on either the mucosal or the serosal side. In the absorption of p-nitrophenyl alpha-maltoside, on the other hand, p-nitrophenyl alpha-glucoside was formed on the mucosal side to appear on the serosal side. p-Nitrophenol and p-nitrophenyl beta-glucuronide also appeared on the serosal side in the absorption of p-nitrophenyl alpha-maltoside, and the total amount transported to the serosal side was significantly decreased in the absence of Na+ (a cosubstrate of Na+/glucose cotransporter (SGLT1)). Furthermore, the total transport clearance of p-nitrophenyl alpha-glucoside formed from p-nitrophenyl alpha-maltoside on the mucosal side in the p-nitrophenyl alpha-maltoside absorption, was similar to that of the absorption of p-nitrophenyl alpha-glucoside itself. These results led to the conclusion that the intestinal absorption of disaccharide conjugate depended on disaccharidase, and the absorption of the alpha-maltose conjugate occurred sequentially by the maltase-catalyzed hydrolysis of the disaccharide conjugate and SGLT1-mediated transport of the glucose conjugate.
Asunto(s)
Disacaridasas/metabolismo , Disacáridos/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Animales , Transporte Biológico Activo/fisiología , Cromatografía Líquida de Alta Presión , Hidrólisis , Absorción Intestinal , Intestino Delgado/metabolismo , Masculino , Ratas , Ratas WistarRESUMEN
We examined the mechanism of p-nitrophenyl-beta-d-glucopyranoside (p-NP-beta-d-Glc) transport in brush-border membrane vesicles from rat small intestine. The initial uptake rate showed an overshoot phenomenon in the presence of an inwardly directed sodium-ion concentration gradient. The overshoot disappeared when the sodium-ion concentration gradient was replaced with a potassium ion concentration gradient. d-Glucose and p-NP-beta-D-Glc analogues inhibited the uptake, whereas uridine, leucine and disaccharide did not. Data on the concentration dependence of p-NP-beta-D-Glc uptake indicated that two carrier-mediated systems are involved. The uptake via the high-affinity site required an inwardly directed sodium-ion concentration gradient, while the uptake via the low-affinity site proceeded such a gradient. D-Glucose competitively inhibited the initial uptake of p-NP-beta-D-Glc via the high-affinity site with a Ki value of 301 microM. The p-NP-beta-D-Glc is transported in the small intestine via both the same carrier-mediated transport system that takes up D-glucose and a distinct low-affinity carrier-mediated transport system.
Asunto(s)
Glucósidos/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestructura , Fosfatasa Alcalina/análisis , Animales , Sitios de Unión/efectos de los fármacos , Unión Competitiva , Transporte Biológico , Glucosa/metabolismo , Glucosa/farmacología , Intestino Delgado/citología , Intestino Delgado/metabolismo , Masculino , Microvellosidades/metabolismo , Concentración Osmolar , Ratas , Ratas Wistar , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismoRESUMEN
Intestinal absorption of four cyclic dipeptides was studied in the everted small intestine of the rat. Cyclic seryltyrosine (cyclo(Ser-Tyr)) was stable enough to be transported whereas linear seryltyrosine was not. The absorption clearance of cyclo(Ser-Tyr) was concentration-dependent, and for cyclo(Ser-Tyr) at 125 microM decreased in the presence of glycylsarcosine (10 mM) or cephalexin (10 mM), which were reported to be absorbed by oligopeptide transporter. The absorption clearance was also reduced at 4 degrees C and in the presence of 1 mM dinitrophenol. Kinetic analysis of cyclo(Ser-Tyr) absorption showed that Km and Vmax were 19.8 microM and 0.295 nmol min(-1) cm(-1), respectively. It was also suggested that cyclic aspartylphenylalanine and cyclic histidylphenylalanine were absorbed by oligopeptide transporters, but cyclic histidylproline was not. The absorption clearance of cyclo(Ser-Tyr) in the control was much higher than the value of the correlation line representing a plot of passive transport (which was obtained from the absorption clearance of cyclic peptides in the presence of glycylsarcosine (10 mM)) against hydrophobicity (oil-water partition coefficient). These results indicate that cyclo(Ser-Tyr) is absorbed by the oligopeptide transporter.
Asunto(s)
Proteínas Portadoras/metabolismo , Dipéptidos/farmacocinética , Absorción Intestinal , Péptidos Cíclicos/farmacocinética , Animales , Transporte Biológico/efectos de los fármacos , Dipéptidos/química , Estabilidad de Medicamentos , Mucosa Intestinal/metabolismo , Yeyuno/metabolismo , Masculino , Péptidos Cíclicos/química , Ratas , Ratas Wistar , Membrana Serosa/metabolismo , Relación Estructura-ActividadRESUMEN
Furosemide 1-O-acyl glucuronide (Fgnd) was extracted from the urine following oral administration of furosemide. The crude Fgnd was applied to micronized Amberlite XAD-2 column (2.5 cm i.d. x 90 cm length, 75-500 microns particle size). The purified Fgnd was identified by mass spectrometry and beta-glucuronidase treatment. This method was also applicable to the purification of glucuronide of tolmetin (nonsteroidal anti-inflammatory drug, NSAID), suggesting that it was applicable to the other NSAIDs, most of which were known to be metabolized to acyl-glucuronides.
Asunto(s)
Furosemida/análogos & derivados , Glucuronatos/orina , Antiinflamatorios no Esteroideos/análisis , Bencimidazoles/análisis , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/métodos , Clofibrato/análogos & derivados , Clofibrato/análisis , Ácido Flufenámico/análogos & derivados , Ácido Flufenámico/análisis , Furosemida/orina , Glucuronidasa/metabolismo , Indometacina/análogos & derivados , Indometacina/análisis , Espectrometría de Masas , Estructura Molecular , Probenecid/análogos & derivados , Probenecid/análisis , Tolmetina/análogos & derivados , Tolmetina/análisisRESUMEN
Intestinal absorption of beta-disaccharide (cellobiose, maltose and lactose) conjugates of p-nitrophenol (p-nitrophenyl beta-disaccharide) were examined in terms of the hydrolysis of disaccharide conjugate to monosaccharide conjugate and the transport of monosaccharide conjugate by Na+/glucose transport carrier (SGLT1). beta-Cellobioside, beta-maltoside and beta-lactoside of p-nitrophenol (p-NP) were hydrolyzed to p-nitrophenyl beta-glucoside (p-NPbeta glc) on the mucosal side, and p-NPbeta glc appeared on the serosal side. Although p-NP beta-disaccharide, p-NP and p-NP glucuronide also appeared on the serosal side, their amounts were much lower than that of p-NPbeta glc. The amount of p-NPbeta glc transported to the serosal side was decreased in the presence of phloridzin (transport inhibitor of SGLT1) and in the absence of Na+ (a cosubstrate of SGLT1), indicating that p-NPbeta glc was formed from p-NP beta-disaccharide on the mucosal side and transported to the serosal side by SGLT1. Furthermore, the absorption clearance of p-NPbeta glc, which was formed from p-NP beta-cellobioside and p-NP beta-lactoside by lactase-phloridzin hydrolase (LPH), was much higher than that of p-NPbeta glc itself, although the absorption clearance of p-NPbeta glc, which was formed from p-NP beta-maltoside by maltase was similar to that of p-NPbeta glc itself. These results indicated that p-NPbeta glc was transported by the vectorial cooperation of SGLT1 with LPH from mucosal p-NP beta-cellobioside or p-NP beta-lactoside.
Asunto(s)
Disacáridos/metabolismo , Glucósidos/metabolismo , Intestino Delgado/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Animales , Transporte Biológico , Glucuronatos/análisis , Glucuronatos/metabolismo , Glicósido Hidrolasas/metabolismo , Glicósidos/metabolismo , Absorción Intestinal/fisiología , Lactasa-Florizina Hidrolasa/metabolismo , Masculino , Maltosa/análogos & derivados , Maltosa/metabolismo , Estructura Molecular , Nitrofenoles/análisis , Nitrofenoles/metabolismo , Florizina/farmacología , Ratas , Ratas Wistar , Transportador 1 de Sodio-GlucosaRESUMEN
Leucine enkephalinamide (LEamide), aminopeptidase-degradable opioid peptide, was coupled with cellobiose (cellobiose-coupled LEamide, CcpLEamide). CcpLEamide was absorbed from the rat small intestine in vitro, whereas LEamide was not. CcpLEamide on the mucosal side was more stable than aminopeptidase-resistant cellobiose-coupled leucine enkephalin (CcpLE) in the presence of inhibitors of enkephalinase and angiotensin converting enzyme, and much more stable than LEamide. The absorption rate (clearance) of CcpLEamide was comparable with that of CcpLE in the presence of these peptidase inhibitors. Analgesic activities of these enkephalins were tested by the acetic acid writhing assay and hot-plate assay after subcutaneous administration to mice. Both assays indicated CcpLEamide-induced analgesia. On the other hand, the analgesic activity of LEamide was not observed, but pretreatment with amastatin (a peptidase inhibitor) produced LEamide-induced analgesia. These results indicate that CcpLEamide is stable in the body and has analgesic effects without pretreatment with peptidase inhibitors, and was stable enough to be absorbed from the small intestine. We propose CcpLEamide as an orally active analgesic peptide candidate.
Asunto(s)
Analgésicos/farmacocinética , Encefalina Leucina/análogos & derivados , Absorción Intestinal , Aminopeptidasas/metabolismo , Analgésicos/metabolismo , Animales , Celobiosa/química , Celobiosa/farmacocinética , Portadores de Fármacos , Encefalina Leucina/química , Encefalina Leucina/metabolismo , Encefalina Leucina/farmacocinética , Masculino , Ratones , Ratones Endogámicos ICR , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The absorption, especially the stability and transportability, of the cyclic peptide cyclic glycylphenylalanine (cyclo(Gly-Phe)) and the linear peptides glycylphenylalanine, glycyl-D-phenylalanine and phenylalanylglycine have been studied in rat small intestine. Linear peptides were degraded on the mucosal side and only glycyl-D-phenylalanine appeared on the serosal side. However, cyclo(Gly-Phe) was stable on the mucosal side and appeared on the serosal side. Furthermore, the absorption clearance of cyclo(Gly-Phe) was higher than that of glycyl-D-phenylalanine. In the presence of the peptidase inhibitor bestatin, the degradation of linear peptides was reduced and linear peptides appeared on the serosal side, but only phenylalanylglycine, which is transported by the oligopeptide transporter, was absorbed faster than cyclo(Gly-Phe). The absorption clearance of cyclo(Gly-Phe) was reduced as its concentration was increased from 125 microM to 500 microM. Furthermore, the absorption clearance of cyclo(Gly-Phe) at 125 microM was reduced at 4 degrees C or in the presence of glycylsarcosine and cephalexin, which are transported by the oligopeptide transporter. These results indicated that cyclo(Gly-Phe) was stable enough to be absorbed and was transported in part by the oligopeptide transporter rather than completely by passive diffusion.
Asunto(s)
Dipéptidos/farmacocinética , Absorción Intestinal/efectos de los fármacos , Animales , Dipéptidos/química , Leucina/análogos & derivados , Leucina/farmacología , Masculino , Inhibidores de Proteasas/farmacología , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
The intestinal absorption of oligopeptides, peptidase-degradable tyrosylglycylglycine (TGG) and peptidase-resistant L-tyrosyl-D-arginine (D-kyotorphin, D-KTP) across Peyer's patches (PP) in rabbit intestine were studied using an Ussing-type chamber and in situ closed perfusion methods. The clearance for the serosal appearance of intact TGG across PP by the Ussing-type chamber method was a little higher than that across the jejunal epithelium (JE). Meanwhile, the in situ closed perfusion experiment showed that the clearance for the plasma appearance of intact TGG across PP was about 10 times that of JE. Furthermore, it was shown that the clearance for the plasma appearance of D-KTP in PP was about twice that in JE by the in situ closed perfusion method, indicating that the membrane permeability of PP was higher than that of JE. Therefore, these results indicate that PP had less metabolic peptidase activity than JE, and that the PP was a suitable site for the intestinal absorption of oligopeptides, especially peptidase-degradable peptides.
Asunto(s)
Endorfinas/farmacocinética , Absorción Intestinal/fisiología , Mucosa Intestinal/metabolismo , Yeyuno/metabolismo , Oligopéptidos/farmacocinética , Ganglios Linfáticos Agregados/metabolismo , Animales , Mucosa Intestinal/patología , Yeyuno/patología , Masculino , ConejosRESUMEN
The effect of anionic compounds on the hepatic uptake of paracetamol sulphate, a conjugative metabolite of paracetamol, has been studied. Hepatic uptake of paracetamol sulphate by isolated hepatocytes was inhibited by bromosulphophthalein, dibromosulphophthalein and p-nitrophenyl sulphate, but not by probenecid or cholic acid. Bromosulphophthalein and dibromosulphophthalein also inhibited the uptake of paracetamol sulphate in the rat isolated perfused liver. Saturable uptake of paracetamol sulphate was also observed in the absence of inorganic sulphate. The uptake of paracetamol sulphate was reduced by 1 and 10 mM inorganic sulphate. Bromosulphophthalein and p-nitrophenyl sulphate inhibited the uptake of paracetamol sulphate in the absence of inorganic sulphate. These results indicate that paracetamol sulphate shares a transporter with bromosulphophthalein, dibromosulphophthalein and p-nitrophenyl sulphate, all of which contain the sulphate or sulphonate group. Therefore, the sulphate or sulphonate moiety might be crucial for interaction with the transporter, and mutual inhibition of hepatic uptake among these compounds is likely.
Asunto(s)
Acetaminofén/análogos & derivados , Hígado/efectos de los fármacos , Hígado/metabolismo , Acetaminofén/farmacocinética , Animales , Aniones , Ácido Cólico , Ácidos Cólicos/farmacología , Colorantes/farmacología , Interacciones Farmacológicas , Masculino , Nitrobencenos/farmacología , Perfusión , Probenecid/farmacología , Ratas , Ratas Wistar , Sulfatos/farmacología , Sulfobromoftaleína/farmacología , Uricosúricos/farmacologíaRESUMEN
Hepatic uptake of the sulfate conjugate of p-nitrophenol (p-NPsul) has been studied. Uptake clearance of p-NPsul by isolated rat hepatocytes was dependent on p-NPsul concentration, suggesting carrier-mediated transport. The uptake of p-NPsul by isolated rat hepatocytes was inhibited by acetaminophen sulfate (APAPsul), the uptake of which had been previously reported to be inhibited by p-NPsul. The hepatic uptake of p-NPsul was also inhibited by bromosulfophthalein (BSP) or dibromosulfophthalein (DBSP), which had been reported to inhibit hepatic uptake of APAPsul in the previous study. These inhibitory effects were observed in isolated perfused liver experiments as well as in isolated hepatocyte experiments. Therefore, it was concluded that hepatic uptake of p-NPsul was mediated by a transporter, which mediates hepatic uptake of APAPsul as well as BSP or DBSP. It is suggested that sulfate moiety may have a key role as a vector for the hepatic transport of sulfate conjugate metabolites. Interaction between sulfate conjugate metabolites in hepatic uptake may also be expected.
Asunto(s)
Acetaminofén/farmacología , Analgésicos no Narcóticos/farmacología , Hígado/metabolismo , Nitrobencenos/metabolismo , Sulfatos/farmacología , Análisis de Varianza , Animales , Transporte Biológico Activo , Células Cultivadas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hígado/efectos de los fármacos , Masculino , Nitrobencenos/farmacocinética , Probenecid/farmacología , Ratas , Ratas Wistar , Sulfobromoftaleína/farmacologíaRESUMEN
Intestinal transport and metabolism of kyotorphin (KTP) were studied in rat everted small intestine. KTP on the mucosal side was metabolized completely within 60 min, and any amounts of KTP were not detected on the serosal side. On the other hand, [D-Arg2]-KTP (D-KTP) was stable on the mucosal side to appear on the serosal side. However, N-t-butoxycarbonyl-KTP (Boc-KTP), which was metabolized on the mucosal side faster than KTP, appeared on the serosal side. In intestinal homogenate, KTP was metabolized, and the metabolic clearance (CL(met)) was decreased by peptidase inhibitors, bestatin, o-phenanthrolin and tryptophan hydroxamate. In the presence of these peptidase inhibitors, the absorption clearance (CL(abs)) of KTP was increased. The less the CL(met) of KTP was, the more the CL(abs) of KTP was. Meanwhile, Boc-KTP in intestinal homogenate was stable even in the absence of peptidase inhibitors. The CL(abs) of Boc-KTP was constant irrespective of the stability on the mucosal side. Kinetic analysis by the metabolic inhibition model indicated that the stabilization of KTP in the intestinal tissue could increase the CL(abs) up to 0.247 microl/min per cm, which was as much as the CL(abs) of stable D-KTP. These results led to the conclusion that rate-limiting process in intestinal absorption of KTP is metabolic degradation in intestinal tissue during the absorption.