RESUMO
Investigation of acetaminophen (APAP)-induced liver damage recently indicated the significance of phagocytic NADPH oxidase (NOX)-derived reactive oxygen species (ROS) and ferroptosis in the liver. Here, we focused on phagocytosis by iron-containing erythrocyte-devouring splenic macrophages and explored upstream factors of known APAP hepatotoxic mechanisms in vivo. Splenectomy did not alter hepatic cytochrome P450 (CYP) 2E1 activity or hepatic glutathione (GSH) content. APAP injection into splenectomized mice almost completely suppressed increases in plasma alanine aminotransferase levels and centrilobular hepatic necrosis showing the spleen to be a critical tissue in APAP-induced liver damage. Hepatic GSH was recovered to approximately 50 % content at 8 h. In non-splenectomized mice, liver damage was dramatically suppressed by a sensitive redox probe (DCFH-DA), macrophage-depleting clodronate (CL), and a NOX2 inhibitor. APAP treatment resulted in markedly stronger fluorescence intensity from DCFH-DA due to excessive ROS around splenic macrophages, which was lost upon co-treatment with a CYP inhibitor and CL. Deformed erythrocytes disappeared in mice co-treated with DCFH-DA, CL, the NOX2 inhibitor, and the CYP inhibitor. Simultaneously, these four compounds significantly improved APAP-depleted GSH levels. The CYP inhibitor also prevented the formation of APAP-cell adducts in the blood and spleen. In the spleen, CL co-treatment markedly reduced the number of adducts. Splenic ferrous iron levels were significantly elevated by APAP. Therefore, we demonstrated that splenic macrophages devoured APAP metabolite-erythrocyte adducts and subsequently splenic macrophage-related ROS caused sustained hepatic GSH depletion and excessive erythrocyte deformation around 7 h. Our data indicate in vivo upstream factors of known APAP hepatotoxic mechanisms.
Assuntos
Acetaminofen , Doença Hepática Induzida por Substâncias e Drogas , Eritrócitos , Glutationa , Fígado , Macrófagos , Espécies Reativas de Oxigênio , Baço , Animais , Acetaminofen/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Glutationa/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Eritrócitos/metabolismo , Eritrócitos/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Camundongos Endogâmicos C57BL , Esplenectomia , Fagocitose/efeitos dos fármacos , NADPH Oxidase 2/metabolismo , Ácido Clodrônico/farmacologiaRESUMO
Eye drops containing Tranilast (TL), N-(3,4-dimethoxycinnamoyl) anthramilic acid, are used as an anti-allergic conjunctivitis drug in the ophthalmic field. Traditional eye drops are very patient compliant, although the bioavailability (BA) of most eye drops is low since eye drops cannot be instilled beyond the capacity of the conjunctival sac due to its limited volume. Thus, traditional eye drops have low BA and a short duration of the drug on the ocular surface, so solutions to these problems are highly anticipated. In this study, we designed a sustained-release drug-delivery system (DDS) for TL nanoparticles. TL nanoparticles were prepared by bead mill treatment, and the gel formulations containing TL nanoparticles (TL-NPs-Gel, particle size 50 nm-100 nm) were provided by carboxypolymethylene. The crystal structure of TL with and without bead mill treatment is the same, but the TL solubility in formulations containing nanoparticles was 5.3-fold higher compared with gel formulations containing TL microparticles (TL-MPs-Gel). The photo and thermal stabilities of TL-NPs-Gel are also higher than those of dissolved TL. Moreover, when TL-NPs-Gel is applied to the upper eyelid skin (outside), the TL is released as nanoparticles, and delivered to the lacrimal fluid through the meibomian glands. In addition, the TL release profile for TL-NPs-Gel was sustained over 180 min after the treatment. These findings can be used to develop a sustained-release DDS in the ophthalmic field.
RESUMO
Indomethacin (IMC)-induced gastrointestinal (GI) injuries are more common in rheumatoid arthritis (RA) patients than in other IMC users, and the overexpression of nitric oxide (NO) via inducible NO synthase (iNOS) is related to the seriousness of IMC-induced GI injuries. However, sufficient strategies to prevent IMC-induced GI injuries have not yet been established. In this study, we designed dispersions of rebamipide (RBM) solid nanocrystals (particle size: 30-190 nm) by a bead mill method (RBM-NDs), and investigated whether the oral administration of RBM-NDs is useful to prevent IMC-induced GI injuries. The RBM nanocrystals were spherical and had a solubility 4.71-fold greater than dispersions of traditional RBM powder (RBM-TDs). In addition, the RBM-NDs were stable for 1 month after preparation. The RBM contents in the stomach, jejunum, and ileum of rats orally administered RBM-NDs were significantly higher than in rats administered RBM-TDs. Moreover, the oral administration of RBM-NDs decreased the NO levels via iNOS and area of the GI lesions in IMC-stimulated RA (adjuvant-induced arthritis rat) rats in comparison with the oral administration of RBM-TDs. Thus, we show that the oral administration of RBM-NDs provides a high drug supply to the GI mucosa, resulting in a therapeutic effect on IMC-induced GI injuries. Solid nanocrystalline RBM preparations may offer effective therapy for RA patients.
Assuntos
Alanina/análogos & derivados , Anti-Inflamatórios não Esteroides/efeitos adversos , Hemorragia Gastrointestinal/etiologia , Indometacina/efeitos adversos , Nanopartículas , Quinolonas/administração & dosagem , Alanina/administração & dosagem , Animais , Hemorragia Gastrointestinal/tratamento farmacológico , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Nanopartículas/química , Nanopartículas/ultraestrutura , Óxido Nítrico/metabolismo , Ratos , Difração de Raios XRESUMO
We previously reported that oral formulations containing indomethacin nanoparticles (IND-NPs) showed high bioavailability, and, consequently, improved therapeutic effects and reduced injury to the small intestine. However, the pathway for the transintestinal penetration of nanoparticles remained unclear. Thus, in this study, we investigated whether endocytosis was related to the penetration of IND-NPs (72.1 nm) using a transcell set with Caco-2 cells or rat intestine. Four inhibitors of various endocytosis pathways were used [nystatin, caveolae-dependent endocytosis (CavME); dynasore, clathrin-dependent endocytosis (CME); rottlerin, macropinocytosis; and cytochalasin D, phagocytosis inhibitor], and all energy-dependent endocytosis was inhibited at temperatures under 4 °C in this study. Although IND-NPs showed high transintestinal penetration, no particles were detected in the basolateral side. IND-NPs penetration was strongly prevented at temperatures under 4 °C. In experiments using pharmacological inhibitors, only CME inhibited penetration in the jejunum, while in the ileum, both CavME and CME significantly attenuated penetration. In conclusion, we found a novel pathway for the transintestinal penetration of drug nanoparticles. Our hypothesis was that nanoparticles would be taken up into the intestinal epithelium by endocytosis (CME in jejunum, CavME and CME in ileum), and dissolved and diffused in the intestine. Our findings are likely to be of significant use for the development of nanomedicines.
Assuntos
Endocitose/fisiologia , Indometacina/metabolismo , Intestino Delgado/metabolismo , Nanopartículas/química , Animais , Células CACO-2 , Sistemas de Liberação de Medicamentos/métodos , Humanos , Masculino , Ratos , Ratos WistarRESUMO
BACKGROUND AND OBJECTIVES: Mast cell-mediated allergic diseases are a significant global health problem. Nitric oxide (NO) produced by acute type 1 allergies greatly suppresses hepatic cytochrome P450 (CYP) metabolism. A recent in vitro study demonstrated that repeated FcεRI-mediated activation intrinsically modulates mast cell function. We investigated the effect of ovalbumin (OVA) challenges on CYP activity and NO production under real immune responses. METHODS: After repeated sensitization with OVA once a week, serum nitrate plus nitrite (NOx) and total plasma immunoglobulin E concentrations were measured using commercially available kits. Hepatic microsomal CYP-specific activities and protein expression were determined using typical substrates and by western blot, respectively. In the liver, the levels of inducible NO synthase (iNOS), F4/80, and c-kit mRNA were determined by real-time polymerase chain reaction. Hepatic total NOS activity was measured using a colorimetric assay kit. RESULTS: When mice received multiple OVA challenges, the 11th sensitization elevated NOx concentrations in serum and suppressed the activities of five major CYPs without altering protein expression levels. After the 7th, 11th, and 15th sensitizations, F4/80-positive Kupffer cell and hepatic c-kit-dependent mast cell mRNA levels were similar to those of the control. The 7th and 11th sensitizations increased hepatic iNOS mRNA expression to 15-fold and threefold above control levels, respectively, but did not enhance the total NOS activity in the liver. CONCLUSIONS: Multiple OVA challenges, unlike acute sensitization, greatly reduced serum NOx levels. The challenge-suppressed hepatic CYP metabolism was likely related to the increased serum NOx. Serum NOx may be an endogenous marker for CYP metabolism inhibition in type 1 allergic diseases.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Hipersensibilidade/enzimologia , Fígado/efeitos dos fármacos , Óxido Nítrico/biossíntese , Ovalbumina/imunologia , Animais , Sistema Enzimático do Citocromo P-450/imunologia , Feminino , Hipersensibilidade/sangue , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Fígado/enzimologia , Fígado/imunologia , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Camundongos Endogâmicos ICR , Óxido Nítrico/sangue , Óxido Nítrico/imunologia , Óxido Nítrico Sintase Tipo II/metabolismo , Ovalbumina/administração & dosagem , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Mast cells and Kupffer cells secrete interleukin (IL)-1ß, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, which stimulate excess nitric oxide (NO) producing-inducible NO synthase (iNOS). Unlike Kupffer cells, immunoglobulin E-sensitized mast cells elicit sustained NO production. We investigated the participation of mast cell-released NO and cytokine-derived iNOS activation in type 1 allergy-suppressed hepatic cytochrome P450 (CYP) metabolism. Aminoguanidine, a selective iNOS inhibitor, completely suppressed serum nitrate plus nitrite (NOx) concentrations after primary and secondary sensitization of ICR mice and markedly attenuated allergy-suppressed hepatic CYP1A2, CYP2C, CYP2E1, and CYP3A activities. In the liver, primary and secondary sensitization enhanced iNOS-stimulating IFN-γ (5-15-fold) and TNF-α (3-5-fold) mRNA levels more than IL-1ß (2-fold) and F4/80-positive Kupffer cell (2-fold) mRNA levels. When mast cell-deficient (-/-) mice were sensitized, hepatic CYP activities were not suppressed. Serum NOx levels in the sensitized -/- mice were similar with those in saline-treated ICR and -/- mice. In the liver of -/- mice, secondary sensitization markedly enhanced mRNA expression of iNOS (20-fold), IFN-γ (15-fold), and TNF-α (3-fold). However, hepatic total NOS activities in -/- mice were not significantly different between saline treatment and sensitization. Similarly, primary and secondary ICR mice did not significantly enhance total NOS activities in the liver and hepatocytes. The total NOS activities observed did not relate to the high levels of iNOS, IFN-γ, and TNF-α mRNA in the liver. Hepatic c-kit-positive mast cells in sensitized ICR mice were maintained at control levels. Therefore, our data suggest that mast cell-released NO participates in type 1 allergy-suppressed CYP1A2, CYP2C, CYP2E1, and CYP3A metabolism.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Hipersensibilidade/metabolismo , Mastócitos/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/imunologia , Feminino , Hipersensibilidade/imunologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos ICR , Microssomos Hepáticos/imunologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
Since Cav3.2â¯T-type Ca2+ channels (T-channels) expressed in the primary afferents and CNS contribute to intractable pain, we explored T-channel-blocking components in distinct herbal extracts using a whole-cell patch-clamp technique in HEK293â¯cells stably expressing Cav3.2 or Cav3.1, and purified and identified sophoraflavanone G (SG) as an active compound from SOPHORAE RADIX (SR). Interestingly, hop-derived SG analogues, (2S)-6-prenylnaringenin (6-PNG) and (2S)-8-PNG, but not naringenin, also blocked T-channels; IC50 (µM) of SG, (2S)-6-PNG and (2S)-8-PNG was 0.68-0.75 for Cav3.2 and 0.99-1.41 for Cav3.1. (2S)-6-PNG and (2S)-8-PNG, but not SG, exhibited reversible inhibition. The racemic (2R/S)-6-PNG as well as (2S)-6-PNG potently blocked Cav3.2, but exhibited minor effect on high-voltage-activated Ca2+ channels and voltage-gated Na+ channels in differentiated NG108-15â¯cells. In mice, the mechanical allodynia following intraplantar (i.pl.) administration of an H2S donor was abolished by oral or i.p. SR extract and by i.pl. SG, (2S)-6-PNG or (2S)-8-PNG, but not naringenin. Intraperitoneal (2R/S)-6-PNG strongly suppressed visceral pain and spinal ERK phosphorylation following intracolonic administration of an H2S donor in mice. (2R/S)-6-PNG, administered i.pl. or i.p., suppressed the neuropathic allodynia induced by partial sciatic nerve ligation or oxaliplatin, an anti-cancer agent, in mice. (2R/S)-6-PNG had little or no effect on open-field behavior, motor performance or cardiovascular function in mice, and on the contractility of isolated rat aorta. (2R/S)-6-PNG, but not SG, was detectable in the brain after their i.p. administration in mice. Our data suggest that 6-PNG, a hop component, blocks T-channels, and alleviates neuropathic and visceral pain with little side effects.
Assuntos
Analgésicos não Narcóticos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Flavonoides/farmacologia , Neuralgia/tratamento farmacológico , Dor Visceral/tratamento farmacológico , Analgésicos não Narcóticos/química , Analgésicos não Narcóticos/isolamento & purificação , Animais , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/isolamento & purificação , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Modelos Animais de Doenças , Flavonoides/química , Flavonoides/isolamento & purificação , Células HEK293 , Humanos , Humulus , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuralgia/metabolismo , Compostos Fitoquímicos/química , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Distribuição Aleatória , Ratos Wistar , Dor Visceral/metabolismoRESUMO
Flavin-containing monooxygenases (FMOs) are major mammalian non-cytochrome P450 oxidative enzymes. T helper 2 cell-activated allergic diseases produce excess levels of nitric oxide (NO) that modify the functions of proteins. However, it remains unclear whether allergy-induced NO affects the pharmacokinetics of drugs metabolized by FMOs. This study investigated alterations of hepatic microsomal FMO1 and FMO3 activities in type 1 allergic mice and further examined the interaction of FMO1 and FMO3 with allergy-induced NO. Imipramine (IMP; FMO1 substrate) N-oxidation activity was not altered in allergic mice with high serum NO and immunoglobulin E levels. At 7 days after primary sensitization (PS7) or secondary sensitization (SS7), benzydamine (BDZ; FMO1 and FMO3 substrate) N-oxygenation was significantly decreased to 70% of individual controls. The expression levels of FMO1 and FMO3 proteins were not significantly changed in the sensitized mice. Hepatic inducible NO synthase (iNOS) mRNA level increased 5-fold and 15-fold in PS7 and SS7 mice, respectively, and hepatic tumor necrosis factor-α levels were greatly enhanced. When a selective iNOS inhibitor was injected into allergic mice, serum NO levels and BDZ N-oxygenation activity returned to control levels. NO directly suppressed BDZ N-oxygenation, which was probably related to FMO3-dependent metabolism in comparison with IMP N-oxidation. In hepatic microsomes from PS7 and SS7 mice, the suppression of BDZ N-oxygenation was restored by ascorbate. Therefore, type 1 allergic mice had differentially suppressed FMO3-dependent BDZ N-oxygenation. The suppression of FMO3 metabolism related to reversible S-nitrosyl modifications of iNOS-derived NO. NO is expected to alter FMO3-metabolic capacity-limited drug pharmacokinetics in humans.
Assuntos
Hipersensibilidade Imediata/metabolismo , Fígado/enzimologia , Microssomos Hepáticos/enzimologia , Óxido Nítrico/metabolismo , Oxigenases/metabolismo , Animais , Benzidamina/farmacocinética , Modelos Animais de Doenças , Feminino , Humanos , Hipersensibilidade Imediata/sangue , Hipersensibilidade Imediata/imunologia , Imipramina/farmacocinética , Imunoglobulina E/sangue , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Oxirredução , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In a study to find ways to prevent the side effects of indomethacin (IMC), we previously reported that magnesium ion (Mg2+) can prevent the onset of IMC-induced gastric mucosa in adjuvant-induced arthritis (AA) rats, a model for rheumatoid arthritis (RA). In this study we investigated whether the co-administration of IMC and Mg2+ prevents the formation and aggravation of intestinal ulcerogenic lesions in AA rats. The single oral administration of an excessive dose of IMC (40 mg/kg) induces hemorrhagic lesions and nitric oxide (NO) production via inducible nitric oxide synthase (iNOS) in the jejunal and ileal mucosa of AA rats, and the extent of the lesions, as well as iNOS and NO levels in AA rats are higher than in normal rats. On the other hand, the co-administration of 200 mg/kg Mg2+ attenuates intestinal ulceration and the elevation in the iNOS and NO levels in AA rats. Further, hemorrhagic lesioning and enhanced iNOS and NO levels in AA rats also result from the repetitive oral administration of 3 mg/kg IMC (therapeutic dose) for 42 d (once a day), and these changes are also prevented by the co-administration of 200 mg/kg Mg2+. In conclusion, the co-administration of Mg2+ suppresses the ulcerogenic response to IMC in the jejunal and ileal mucosa of AA rats, probably by preventing the elevation of iNOS and NO levels in the region.
Assuntos
Anti-Inflamatórios não Esteroides/efeitos adversos , Artrite Experimental/tratamento farmacológico , Indometacina/efeitos adversos , Magnésio/uso terapêutico , Úlcera Péptica/prevenção & controle , Substâncias Protetoras/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Quimioterapia Combinada , Íleo/efeitos dos fármacos , Íleo/metabolismo , Indometacina/uso terapêutico , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Úlcera Péptica/induzido quimicamente , Úlcera Péptica/metabolismo , Úlcera Péptica/patologia , RatosRESUMO
In the therapy of rheumatoid arthritis, ibuprofen (IBU) is widely used; however, it has been limited the clinical use by its systemic side effect, such as gastrointestinal lesions. Therefore, we prepared topical gel ointment used IBU solid nanoparticles (IBUnano-gel formulation). In addition, we demonstrated their anti-inflammatory effect by using arthritis model rat (adjuvant-induced arthritis rat, AA rat). The gel formulations were prepared using additives (Carbopol 934, 2-hydroxypropyl-ß-cyclodextrin and methylcellulose) and bead mill-method. The IBU particle size in the IBUnano-gel formulation was 208 nm. The increase in inflammation of the hind feet of AA rats was attenuated by the treatment with the IBUnano-gel formulation, and preventive effect was higher than that of a gel formulation containing IBUmicroparticles (IBUmicro-gel formulation, mean particle size 85.4 µm); the accumulation and permeability through the skin of IBU from the IBUnano-gel formulation were significantly larger in comparison with the IBUmicro-gel formulation. Further, no gastrointestinal lesions were observed in AA rats following the repetitive administration of the 5% IBUnano-gel formulation (0.30 g) for 42 days (once a day). These results suggest that the dermal application of IBU-nanoparticles provide effective and efficient therapy that spares patients from unwanted side effects.
Assuntos
Anti-Inflamatórios não Esteroides/farmacocinética , Ibuprofeno/farmacocinética , Nanopartículas/química , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Modelos Animais de Doenças , Géis/síntese química , Géis/química , Géis/farmacocinética , Ibuprofeno/química , Ibuprofeno/uso terapêutico , Inflamação/tratamento farmacológico , Masculino , Tamanho da Partícula , Ratos , Ratos Endogâmicos , Propriedades de SuperfícieRESUMO
Type 1 allergic diseases are characterized by elevated production of specific immunoglobulin E (IgE) for each antigen and have become a significant health problem worldwide. This study investigated the effect of IgE-mediated allergy on drug pharmacokinetics. To further understand differential suppression of hepatic cytochrome P450 (P450) activity, we examined the inhibitory effect of nitric oxide (NO), a marker of allergic conditions. Seven days after primary sensitization (PS7) or secondary sensitization (SS7), hepatic CYP1A2, CYP2C, CYP2E1, and CYP3A activities were decreased to 45%-75% of the corresponding control; however, CYP2D activity was not downregulated. PS7 and SS7 did not change the expression levels of five P450 proteins. Disappearance of CYP1A2 and CYP2D substrates from the plasma was not significantly different between allergic mice and control mice. In contrast, the area under the curve of a CYP1A2-mediated metabolite in PS7 and SS7 mice was reduced by 50% of control values. Total clearances of a CYP2E1 substrate in PS7 and SS7 mice were significantly decreased to 70% and 50% respectively, of the control without altering plasma protein binding. Hepatic amounts of CYP1A2 and CYP2E1 substrates were enhanced by allergic induction, being responsible for each downregulated activity. NO scavenger treatment completely improved the downregulated P450 activities. Therefore, our data suggest that the onset of IgE-mediated allergy alters the pharmacokinetics of major P450-metabolic capacity-limited drugs except for CYP2D drugs. NO is highly expected to participate in regulatory mechanisms of the four P450 isoforms.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Hipersensibilidade/metabolismo , Animais , Regulação para Baixo/fisiologia , Feminino , Imunoglobulina E/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Óxido Nítrico/metabolismoRESUMO
PURPOSE: We determined nitric oxide (NO) production via inducible NO synthase (iNOS) by hyperglycemia using the retina of Otsuka Long-Evans Tokushima Fatty rats (OLETF rats), and investigated the relationship between ATP contents and NO production in the retinas of OLETF rats. METHODS: Long-Evans Tokushima Otsuka rats (LETO rats, normal rats) and OLETF rats (model rat for diabetes mellitus) aged 60 weeks of age were used. Plasma glucose (Glu) levels were determined using an Accutrend GCT System, and NO levels were measured by the microdialysis method as nitrite ([Formula: see text]). Cytochrome c oxidase (CCO) activity was measured using a Mitochondrial Isolation Kit and Cytochrome c Oxidase Assay Kit, and ATP levels were determined using a Sigma ATP Bioluminescent Assay Kit and a luminometer AB-2200. RESULTS: [Formula: see text] levels in the retinas of OLETF rats were significantly higher than in LETO rats, and the [Formula: see text] levels in the retinas of 60-week-old OLETF rats increased with increasing Glu. CCO activity in the retinas of OLETF rats showed no significant difference from that in LETO rats; however, ATP levels in the retinas of OLETF rats were significantly lower than those in LETO rats. The oral administration of aminoguanidine or disulfiram, an iNOS inhibitor, attenuated the decrease in ATP levels in the retinas of 60-week-old OELTF rats. CONCLUSION: The present study demonstrates that NO production via iNOS in the retinas of 60-week-old OLETF rats is caused by hyperglycemia, and that NO causes a decrease in ATP contents in the retinas of 60-week-old OELTF rats. It is possible that the low ATP contents caused by NO may affect the normal functioning of the retina in OLETF rats.
Assuntos
Trifosfato de Adenosina/metabolismo , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2/metabolismo , Óxido Nítrico/biossíntese , Retina/metabolismo , Animais , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/genética , Regulação da Expressão Gênica , Masculino , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/genética , RNA/genética , Ratos , Ratos Endogâmicos OLETF , Retina/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
It is well known that rheumatoid arthritis patients taking nonsteroidal anti-inflammatory drugs (NSAIDs) are more susceptible to NSAIDs-induced gastroenteropathy in comparison with other patients. In this study we demonstrate that expression levels of interleukin (IL)-18 are related to aggravation of intestinal ulcerogenic lesions in adjuvant-induced arthritis (AA) rats following oral administration of indomethacin. AA rats were administered oral indomethacin (40 mg/kg) and killed under deep isoflurane anesthesia after 24 h. The small intestinal mucosa was then examined. Oral administration of indomethacin caused hemorrhagic lesions in the small intestinal mucosa of AA rats, and the lesion score of AA rats 24 h after indomethacin treatment was approximately 5.6-fold higher than for normal rats administered indomethacin. IL-18 expression in the small intestinal mucosa of AA rats administered indomethacin was also higher in comparison with normal rats receiving indomethacin. In addition, interferon-γ and nitric oxide levels in the small intestinal mucosa of AA rats were increased following oral administration of indomethacin. It is possible that IL-18 expression in AA rats renders the small intestinal mucosa more sensitive to indomethacin, and that IL-18 may play a role in aggravating intestinal ulcerogenic lesions in AA rats treated with this drug.
Assuntos
Anti-Inflamatórios não Esteroides/efeitos adversos , Indometacina/efeitos adversos , Interleucina-18/metabolismo , Úlcera/induzido quimicamente , Úlcera/metabolismo , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Células CACO-2 , Pé/patologia , Humanos , Íleo/efeitos dos fármacos , Íleo/metabolismo , Íleo/patologia , Interferon gama/metabolismo , Interleucina-18/genética , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Jejuno/efeitos dos fármacos , Jejuno/metabolismo , Jejuno/patologia , Lipopolissacarídeos , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Ratos , Úlcera/patologiaRESUMO
OBJECTIVES: The objective of this study was to address the beneficial effects of Cistanche tubulosa extract on improving the low intestinal permeability of echinacoside (ECH) and acteoside (ACT). METHODS: Absorption of ECH and ACT in C. tubulosa extract was characterized using human intestinal Caco-2 cell monolayers with intact compounds. Glucose transporter-dependent absorption of ECH and ACT was confirmed by an in-situ intestinal perfusion technique. KEY FINDINGS: The apparent permeability (Papp ) was not significantly different between intact ECH and intact ACT. In the presence of phloridzin, the Pap p of the ECH and ACT at a high dose was reduced to 20% of the respective non-treatment, but was not altered by phloretin and verapamil. C. tubulosa extract at low and high doses enhanced the Papp of ECH and ACT (both by threefold), resulting in their large participation in sodium-dependent glucose transporter-independent absorption. At a low concentration, concomitant ECH and ACT levels in portal blood were significantly suppressed by phloridzin. CONCLUSION: The dietary and medicinal C. tubulosa extract enhancing the intestinal absorption of ECH and ACT may serve to better manage human health, although the involvement of phloridzin-sensitive transport should be reduced.
Assuntos
Cistanche/química , Glucosídeos/farmacocinética , Glicosídeos/farmacocinética , Fenóis/farmacocinética , Extratos Vegetais/farmacologia , Animais , Células CACO-2 , Relação Dose-Resposta a Droga , Glucosídeos/administração & dosagem , Glicosídeos/administração & dosagem , Humanos , Absorção Intestinal/efeitos dos fármacos , Masculino , Fenóis/administração & dosagem , Floretina/farmacologia , Florizina/administração & dosagem , Florizina/farmacologia , Extratos Vegetais/administração & dosagem , Ratos , Ratos Wistar , Proteínas de Transporte de Sódio-Glucose/metabolismo , Verapamil/farmacologiaRESUMO
We investigated the protective effects of mannitol on corneal damage caused by benzalkonium chloride (BAC), which is used as a preservative in commercially available timolol maleate eye drops, using rat debrided corneal epithelium and a human cornea epithelial cell line (HCE-T). Corneal wounds were monitored using a fundus camera TRC-50X equipped with a digital camera; eye drops were instilled into rat eyes five times a day after corneal epithelial abrasion. The viability of HCE-T cells was calculated by TetraColor One; and Escherichia coli (ATCC 8739) were used to measure antimicrobial activity. The reducing effects on transcorneal penetration and intraocular pressure (IOP) of the eye drops were determined using rabbits. The corneal wound healing rate and rate constant (kH), as well as cell viability, were higher following treatment with 0.005% BAC solution containing 0.5% mannitol than in the case BAC solution alone; the antimicrobial activity was approximately the same for BAC solutions with and without mannitol. In addition, the kH for rat eyes instilled with commercially available timolol maleate eye drops containing 0.5% mannitol was significantly higher than that for eyes instilled with timolol maleate eye drops without mannitol, and the addition of mannitol did not affect the corneal penetration or IOP reducing effect of the timolol maleate eye drops. A preservative system comprising BAC and mannitol may provide effective therapy for glaucoma patients requiring long-term treatment with anti-glaucoma agents.
Assuntos
Compostos de Benzalcônio/toxicidade , Lesões da Córnea/induzido quimicamente , Lesões da Córnea/prevenção & controle , Manitol/administração & dosagem , Soluções Oftálmicas/química , Conservantes Farmacêuticos/toxicidade , Timolol/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Epitélio Corneano/citologia , Epitélio Corneano/efeitos dos fármacos , Humanos , Masculino , Manitol/farmacologia , Coelhos , Ratos Wistar , Cicatrização/efeitos dos fármacosRESUMO
OBJECTIVES: 2-Arylpropionic acid (profen) drugs are associated with severe hepatotoxicity; however, risk factors are still poorly understood. Acyl-coenzyme A (acyl-CoA) thioesters of profen drugs play a more important role in the covalent binding to rat hepatocyte proteins than the respective acyl-glucuronides. Therefore, we examined whether acyl-glucuronides, acyl-CoA thioesters and oxidative metabolites of profen drugs stereoselectively participated in liver damage. METHODS: Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) leakage from three-dimensional cultured rat hepatocytes. KEY FINDINGS: LDH leakage was not induced by R-2-phenylpropionic acid and R-ibuprofen greatly forming acyl-CoA thioesters. S-Naproxen metabolized mainly by Uridine 5'-diphosphate (UDP)-glucuronosyl-transferase did not enhance LDH leakage. However, flurbiprofen (FLP) induced LDH leakage. A selective cytochrome P450 (CYP) 2C11 inhibitor suppressed 40-50% of the R-FLP and S-FLP-induced cytotoxicity. Borneol non-stereoselectively accelerated the FLP-induced cytotoxicity. The R-FLP-induced cytotoxicity decreased intracellular adenosine triphosphate (ATP) levels to 50% of untreated hepatocytes. An inhibitor of mitochondrial permeability transition pore, cyclosporin A (Cys A), rescued ATP levels and LDH leakage back to control levels. CONCLUSION: The reactive acyl-CoA thioesters and acyl-glucuronides were not associated with liver damage, denying one of the leading hypotheses. CYP metabolism of FLP non-stereoselectively participated in Cys A-sensitive cytotoxicity, suggesting mitochondrial injury.
Assuntos
Ciclosporina/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Flurbiprofeno/toxicidade , Hepatócitos/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Hepatócitos/metabolismo , Ibuprofeno/toxicidade , L-Lactato Desidrogenase/metabolismo , Masculino , Mitocôndrias/patologia , Naproxeno/toxicidade , Fenilpropionatos/toxicidade , Ratos , Ratos Wistar , EstereoisomerismoRESUMO
The accumulation of amyloid ß(1-42) peptide (Aß(1-42)) in retina is implicated in the development of retinal ganglion cell apoptosis and diabetic retinopathy. In this study we demonstrate that spontaneous diabetes mellitus Otsuka Long-Evans Tokushima Fatty (OLETF) rats can be used as an animal model in studies to identify the expression of Aß in diabetic retinas. In addition, we investigated the relation between glucose level and Aß production in the retinas of OLETF rats. In the retinas of Long-Evans Tokushima Otsuka (LETO) rats used as normal controls and OLETF rats, no expression of neprilysin (NEP), which degrades Aß, was detected, and the expression levels of genes associated with Aß production (amyloid precursor protein, ß site APP cleaving enzyme, and presenilin) and Aß(1-42) levels in the retinas of 60-week-old OLETF rats with diabetes mellitus were significantly higher than in 60-week-old LETO rat retinas. Furthermore, the increase in the expression levels of genes associated with Aß production was enhanced by administration of glucose (3.0 g/kg; OGT test), and close relations between the retinal Aß(1-42) level and plasma blood glucose and HbA1c were observed. In conclusion, we have found that Aß accumulates easily in the retinas of LETO and OLETF rats due to the absence of NEP. In addition, we determined that the accumulation of Aß(1-42) in the retinas of OLETF rats is promoted by high plasma glucose levels. Therefore OLETF rats may be a suitable model for studies to identify the expression of Aß in diabetic retinas.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Diabetes Mellitus/metabolismo , Retinopatia Diabética/metabolismo , Glucose/farmacologia , Fragmentos de Peptídeos/metabolismo , Retina/efeitos dos fármacos , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Glicemia/análise , Colesterol/sangue , Diabetes Mellitus/sangue , Retinopatia Diabética/sangue , Hemoglobinas Glicadas/análise , Insulina/sangue , Masculino , Presenilina-1/genética , Presenilina-2/genética , RNA Mensageiro/metabolismo , Ratos Endogâmicos Lew , Ratos Endogâmicos OLETF , Ratos Sprague-Dawley , Ratos Wistar , Retina/metabolismo , Triglicerídeos/sangueRESUMO
Nevirapine (NVP) treatment is associated with a significant incidence of skin rash in humans, and it also causes a similar immune-mediated skin rash in Brown Norway (BN) rats. We have shown that the sulfate of a major oxidative metabolite, 12-OH-NVP, covalently binds in the skin. The fact that the sulfate metabolite is responsible for covalent binding in the skin does not prove that it is responsible for the rash. We used various inhibitors of sulfation to test whether this reactive sulfate is responsible for the skin rash. Salicylamide (SA), which depletes 3'-phosphoadenosine-5'-phosphosulfate (PAPS) in the liver, significantly decreased 12-OH-NVP sulfate in the blood, but it did not prevent covalent binding in the skin or the rash. Topical application of 1-phenyl-1-hexanol, a sulfotransferase inhibitor, prevented covalent binding in the skin as well as the rash, but only where it was applied. In vitro incubations of 12-OH-NVP with PAPS and cytosolic fractions from the skin of rats or from human skin also led to covalent binding that was inhibited by 1-phenyl-1-hexanol. Incubation of 12-OH-NVP with PAPS and sulfotransferase 1A1*1, a human isoform that is present in the skin, also led to covalent binding, and this binding was also inhibited by 1-phenyl-1-hexanol. We conclude that salicylamide did not deplete PAPS in the skin and was unable to prevent covalent binding or the rash, while topical 1-phenyl-1-hexanol inhibited sulfation of 12-OH-NVP in the skin and did prevent covalent binding and the rash. These results provide definitive evidence that 12-OH-NVP sulfate formed in skin is responsible for NVP-induced skin rashes. Sulfotransferase is one of the few metabolic enzymes with significant activity in the skin, and it may be responsible for the bioactivation of other drugs that cause skin rashes.
Assuntos
Exantema/induzido quimicamente , Exantema/metabolismo , Nevirapina/análogos & derivados , Nevirapina/efeitos adversos , Animais , Exantema/patologia , Feminino , Humanos , Estrutura Molecular , Nevirapina/química , Nevirapina/metabolismo , Ratos , Ratos Endogâmicos BN , Fatores de TempoRESUMO
If we could predict and prevent idiosyncratic drug reactions (IDRs) it would have a profound effect on drug development and therapy. Given our present lack of mechanistic understanding, this goal remains elusive. Hypothesis testing requires valid animal models with characteristics similar to the idiosyncratic reactions that occur in patients. Although it has not been conclusively demonstrated, it appears that almost all IDRs are immune-mediated, and a dominant characteristic is a delay between starting the drug and the onset of the adverse reaction. In contrast, most animal models are acute and therefore involve a different mechanism than idiosyncratic reactions. There are, however, a few animal models such as the nevirapine-induced skin rash in rats that have characteristics very similar to the idiosyncratic reaction that occurs in humans and presumably have a very similar mechanism. These models have allowed testing hypotheses that would be impossible to test in any other way. In addition there are models in which there is a delayed onset of mild hepatic injury that resolves despite continued treatment similar to the "adaptation" reactions that are more common than severe idiosyncratic hepatotoxicity in humans. This probably represents the development of immune tolerance. However, most attempts to develop animal models by stimulating the immune system have been failures. A specific combination of MHC and T cell receptor may be required, but it is likely more complex. Animal studies that determine the requirements for an immune response would provide vital clues about risk factors for IDRs in patients.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Modelos Animais , Animais , Autoimunidade , Doença Hepática Induzida por Substâncias e Drogas , Modelos Animais de Doenças , Hipersensibilidade a Drogas/etiologia , Exantema/induzido quimicamente , Doenças Hematológicas/induzido quimicamente , HumanosRESUMO
We previously demonstrated that HF10, which is a natural, non-engineered HSV-1, has potent oncolytic activity in the treatment of solid malignant tumors in vitro and in vivo [H. Takakuwa, F. Goshima, N. Nozawa, T. Yoshikawa, H. Kimata, A. Nakao, et al., Oncolytic viral therapy using a spontaneously generated herpes simplex virus type 1 variant for disseminated peritoneal tumor in immunocompetent mice, Arch. Virol. 148 (2003) 813-825; S. Kohno, C. Lou, F. Goshima, Y. Nishiyama, T. Sata, Y. Ono, Herpes simplex virus type 1 mutant HF10 oncolytic viral therapy for bladder cancer, Urology 66 (2005) 1116-1121; D. Watanabe, F. Goshima, I. Mori, Y. Tamada, Y. Matsumoto, Y. Nishiyama, Oncolytic virotherapy for malignant melanoma with herpes simplex virus type 1 mutant HF10, J. Dermatol. Sci. 50 (2008) 185-196; A. Nawa, C. Luo, L. Zhang, Y. Ushijima, D. Ishida, M. Kamakura, et al., Non-engineered, naturally oncolytic herpes simplex virus HSV1 HF10: applications for cancer gene therapy, Curr. Gene. Ther. 8 (2008) 208-221]. Previous reports have also shown that a combination of HF10 and paclitaxel (TAX) was more efficacious than either regimen alone for some types of malignant tumors [S. Shimoyama, F. Goshima, O. Teshigahara, H. Kasuya, Y. Kodera, A. Nakao, et al., Enhanced efficacy of herpes simplex virus mutant HF10 combined with paclitaxel in peritoneal cancer dissemination models, Hepatogastroenterology 54 (2007) 1038-1042]. In this study, we investigated the efficacy of gene-directed enzyme prodrug therapy (GDEPT) using a novel system that combines the paclitaxel-2'-ethylcarbonate prodrug (TAX-2'-Et) and an HSV amplicon expressing rabbit-carboxylesterase (CES) with HF10 as a helper virus. This GDEPT system aims to produce high level of CES at the tumor site, resulting in efficient local conversion of the TAX-2'-Et prodrug into the active drug TAX [A. Nawa, T. Tanino, C. Lou, M. Iwaki, H. Kajiyama, K. Shibata, et al., Gene directed enzyme prodrug therapy for ovarian cancer: could GDEPT become a promising treatment against ovarian cancer?, Anti-Cancer Agents Med Chem 8 (2008) 232-239]. We demonstrated that the green fluorescent protein (GFP) gene, as a trace maker, was more efficiently introduced by the HSV amplicon compared to the expression vector, pHGCX, and that the HSV amplicon system expressed an active CES enzyme that could convert TAX-2'-Et to TAX in Cos7 cells. Furthermore, although the cytotoxicity of this amplicon system was not enhanced in virus-sensitive tumor cells, it was significantly enhanced in low virus-sensitive tumor cells in the presence of the prodrug in a concentration-dependent manner, compared to the control virus alone (p<0.05). These results indicate that the addition of a prodrug converting enzyme may be a feasible approach to further enhance the efficacy of HF10 as a cancer therapeutics in low HF10-sensitive malignancies.