RESUMEN
The present study aimed to orally deliver methylthioadenosine (MTA) to the brain employing solid lipid nanoparticles (SLNs) for the management of neurological conditions like multiple sclerosis. The stearic acid-based SLNs were below 100 nm with almost neutral zeta potential and offered higher drug entrapment and drug loading. Cuprizone-induced demyelination model in mice was employed to mimic the multiple sclerosis-like conditions. It was observed that the MTA-loaded SLNs were able to maintain the normal metabolism, locomotor activity, motor coordination, balancing, and grip strength of the rodents in substantially superior ways vis-à-vis plain MTA. Histopathological studies of the corpus callosum and its subsequent staining with myelin staining dye luxol fast blue proved the potential of MTA-loaded SLNs in the remyelination of neurons. The pharmacokinetic studies provided the evidences for improved bioavailability and enhanced bioresidence supporting the pharmacodynamic findings. The studies proved that SLN-encapsulated MTA can be substantially delivered to the brain and can effectively remyelinate the neurons. It can reverse the multiple sclerosis-like symptoms in a safer and effective manner, that too by oral route.
Asunto(s)
Encéfalo/efectos de los fármacos , Desoxiadenosinas/administración & dosificación , Sistemas de Liberación de Medicamentos , Actividad Motora/efectos de los fármacos , Esclerosis Múltiple/tratamiento farmacológico , Nanopartículas/administración & dosificación , Ácidos Esteáricos/administración & dosificación , Tionucleósidos/administración & dosificación , Administración Oral , Animales , Disponibilidad Biológica , Encéfalo/patología , Desoxiadenosinas/farmacocinética , Ratones , Ratas , Ratas Wistar , Tionucleósidos/farmacocinéticaRESUMEN
Therapeutic drug monitoring (TDM), which involves measurement of drug or active metabolite levels and anti-drug antibodies, is a promising strategy that can be used to optimize inflammatory bowel disease therapeutics. It is based on the premise that there is a relationship between drug exposure and outcomes, and that considerable inter-individual variability exists in how patients metabolize the drug (pharmacokinetics) and the magnitude and duration of response to therapy (pharmacodynamics). Therefore, the American Gastroenterological Association has prioritized clinical guidelines on the role of TDM in the management of inflammatory bowel disease. To inform these clinical guidelines, this technical review was developed in accordance with the GRADE (Grading of Recommendations Assessment, Development, and Evaluation) framework for interventional and prognostic studies, and focused on the application of TDM for biologic therapy, specifically anti-tumor necrosis factor-α agents, and for thiopurines. Focused questions address the benefits and risks of a strategy of reactive TDM (in patients with active inflammatory bowel disease) to guide treatment changes compared with empiric treatment changes, and the benefits and risks of a strategy of routine proactive TDM (during routine clinical care in patients with quiescent disease) compared with no routine TDM. Additionally, the review addresses the benefits and risks of routine measurement of thiopurine methyltransferase enzyme activity or genotype before starting thiopurine therapy compared with empiric weight-based dosing and explores the performance of different trough drug concentrations for anti-tumor necrosis factor agents and thiopurines to inform clinical decision making when applying TDM in a reactive setting. Due to a paucity of data, this review does not address the role of TDM for more recently approved biologic agents, such as vedolizumab or ustekinumab.
Asunto(s)
Anticuerpos Monoclonales/sangre , Monitoreo de Drogas , Inmunosupresores/sangre , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Tionucleósidos/sangre , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/uso terapéutico , Azatioprina/sangre , Azatioprina/farmacocinética , Peso Corporal , Monitoreo de Drogas/métodos , Humanos , Inmunosupresores/farmacocinética , Inmunosupresores/uso terapéutico , Metiltransferasas/sangre , Tionucleósidos/farmacocinética , Tionucleósidos/uso terapéuticoRESUMEN
LJ-2698, a highly potent human A3 adenosine receptor antagonist with nucleoside structure, was designed to have a minimal species dependence. For further pre-clinical studies, analytical method for the detection of LJ-2698 in rat plasma was developed by liquid chromatography-tandem mass. Plasma samples were processed by protein precipitation method with acetonitrile, using losartan as the internal standard (IS). Chromatographic separation was carried out using a Kinetex C18 column (100 × 4.6 mm; 100 Å; 2.6 µ) with acetonitrile/water with 0.2% (v/v) formic acid (65:35, v/v) in the isocratic mode at a flow rate of 0.4 mL/min. Mass spectrometric detection in multiple reaction monitoring mode was performed with positive electrospray ionization. The mass transitions of LJ-2698 and IS were m/z 412.3 â 294.1 and m/z 423.1 â 207.2, respectively. The calibration curves were linear in the range 5.00-5000 ng/mL (r 2 ≥ 0.998). The lower limit of quantification was established as 5.00 ng/mL. Within- and between-run precisions were <7.01%, as relative standard deviation; and accuracies were in the range 3.37-3.64%, as relative error. The validated method was successfully applied to its pharmacokinetic evaluation after intravenous and oral administration in rats, and the dose-dependent pharmacokinetic behavior of LJ-2698 was elucidated for the first time.
Asunto(s)
Antagonistas del Receptor de Adenosina A3/farmacocinética , Tionucleósidos/farmacocinética , Antagonistas del Receptor de Adenosina A3/administración & dosificación , Administración Intravenosa , Administración Oral , Animales , Calibración , Cromatografía Liquida , Relación Dosis-Respuesta a Droga , Límite de Detección , Masculino , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Tionucleósidos/administración & dosificaciónRESUMEN
6-Thiopurine (6-TP) prodrugs include 6-thioguanine and azathioprine. Both are widely used to treat autoimmune disorders and certain cancers. This study showed that a 6-thioguanosine triphosphate (6-TGTP), converted in T-cells from 6-TP, targets Rac1 to form a disulfide adduct between 6-TGTP and the redox-sensitive GXXXXGK(S/T)C motif of Rac1. This study also showed that, despite the conservation of the catalytic activity of RhoGAP (Rho-specific GAP) on the 6-TGTP-Rac1 adduct to produce the biologically inactive 6-thioguanosine diphosphate (6-TGDP)-Rac1 adduct, RhoGEF (Rho-specific GEF) cannot exchange the 6-TGDP adducted on Rac1 with free guanine nucleotide. The biologically inactive 6-TGDP-Rac1 adduct accumulates in cells because of the ongoing combined actions of RhoGEF and RhoGAP. Because other Rho GTPases, such as RhoA and Cdc42, also possess the GXXXXGK(S/T)C motif, the proposed mechanism for the inactivation of Rac1 also applies to RhoA and Cdc42. However, previous studies have shown that CD3/CD28-stimulated T-cells contain more activated Rac1 than other Rho GTPases such as RhoA and Cdc42. Accordingly, Rac1 is the main target of 6-TP in activated T-cells. This explains the T-cell-specific Rac1-targeting therapeutic action of 6-TP that suppresses the immune response. This proposed mechanism for the action of 6-TP on Rac1 performs a critical role in demonstrating the capability to design a Rac1-targeting chemotherapeutic agent(s) for autoimmune disorders. Nevertheless, the results also suggest that the targeting action of other Rho GTPases in other organ cells, such as RhoA in vascular cells, may be linked to cytotoxicities because RhoA plays a key role in vasculature functions.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Inmunosupresores/farmacocinética , Profármacos/farmacología , Tionucleósidos/farmacocinética , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/metabolismo , Secuencias de Aminoácidos , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Thiopurines have been used in inflammatory bowel disease (IBD) for >30 years, and measurements of both thiopurine methyltransferase (TPMT) and thiopurine (TP) metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP), have been readily available. The North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition (NASPGHAN) Committee on Inflammatory Bowel Disease thought it appropriate to review the present indications for use of TPMT and TP metabolite testing. Substantial evidence demonstrates that TP therapy is useful for both Crohn disease and ulcerative colitis. Review of the existing data yielded the following recommendations. TPMT testing is recommended before initiation of TPs to identify individuals who are homozygote recessive or have extremely low TPMT activity, with the latter having more reliability than the former. Individuals who are homozygous recessive or have extremely low TPMT activity should avoid the use of TPs because of concerns for significant leukopenia. TMPT testing does not predict all cases of leukopenia and has no value to predict hypersensitivity adverse effects such as pancreatitis. Any potential value to reduce the risk of malignancy has not been studied. All individuals taking TPs should have routine monitoring with complete blood cell count and white blood cell count differential to evaluate for leukopenia regardless of TPMT testing results. Metabolite testing can be used to determine adherence with TP therapy. Metabolite testing can be used to guide dose increases or modifications in patients with active disease. Consideration would include either increasing the dose, changing therapy or for those with elevated transaminases or an elevated 6-MMP, using adjunctive allopurinol to help raise 6-thioguanine metabolites and suppress formation of 6-MMP. Routine and repetitive metabolite testing has little or no role in patients who are doing well and taking an acceptable dose of a TP.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacocinética , Colitis Ulcerosa/tratamiento farmacológico , Enfermedad de Crohn/tratamiento farmacológico , Metiltransferasas/metabolismo , Purinas/farmacocinética , Compuestos de Sulfhidrilo/farmacocinética , Tionucleósidos/farmacocinética , Antiinflamatorios no Esteroideos/efectos adversos , Antiinflamatorios no Esteroideos/sangre , Antiinflamatorios no Esteroideos/uso terapéutico , Biotransformación , Colitis Ulcerosa/sangre , Colitis Ulcerosa/metabolismo , Consenso , Enfermedad de Crohn/sangre , Enfermedad de Crohn/metabolismo , Interacciones Farmacológicas , Monitoreo de Drogas , Medicina Basada en la Evidencia , Humanos , Enfermedades Inflamatorias del Intestino/sangre , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/metabolismo , Metiltransferasas/sangre , Guías de Práctica Clínica como Asunto , Purinas/efectos adversos , Purinas/sangre , Purinas/uso terapéutico , Sociedades Científicas , Compuestos de Sulfhidrilo/efectos adversos , Compuestos de Sulfhidrilo/sangre , Compuestos de Sulfhidrilo/uso terapéutico , Tionucleósidos/efectos adversos , Tionucleósidos/sangre , Tionucleósidos/uso terapéuticoRESUMEN
Methylthioadenosine (MTA) is a precursor of the methionine salvage pathway and has been shown to have anti-inflammatory properties in various models of acute and chronic inflammation. However, the anti-inflammatory properties of MTA in models of intestinal inflammation are not defined. We hypothesized that orally administered MTA would be bioavailable and reduce morbidity associated with experimental colitis. We examined clinical, histological, and molecular markers of disease in mice provided oral MTA before (preventative) or after (therapy) the induction of colitis with 3% dextran sulfate sodium (DSS). We found a reduction in disease activity, weight loss, myeloperoxidase activity, and histological damage in mice given preventative MTA compared with DSS alone. We also found that equivalent supplementation with methionine could not reproduce the anti-inflammatory effects of MTA, and that MTA had no detectable adverse effects in control or DSS mice. Expression microarray analysis of colonic tissue showed several dominant pathways related to inflammatory cytokines/chemokines and extracellular matrix remodeling were upregulation by DSS and suppressed in MTA-supplemented mice. MTA is rapidly absorbed in the gastrointestinal tract and disseminated throughout the body, based on a time course analysis of an oral bolus of MTA. This effect is transient, with MTA levels falling to near baseline within 90 min in most organs. Moreover, MTA did not lead to increased blood or tissue methionine levels, suggesting that its effects are specific. However, MTA provided limited therapeutic benefit when administered after the onset of colitis. Our results show that oral MTA supplementation is a safe and effective strategy to prevent inflammation and tissue injury associated with DSS colitis in mice. Additional studies in chronic inflammatory models are necessary to determine if MTA is a safe and beneficial option for the maintenance of remission in human inflammatory bowel disease.
Asunto(s)
Adenosina/análogos & derivados , Antiinflamatorios no Esteroideos , Colitis/prevención & control , Tionucleósidos/farmacología , Adenosina/efectos adversos , Adenosina/farmacocinética , Adenosina/farmacología , Animales , Disponibilidad Biológica , Colitis/inducido químicamente , Colitis/patología , Sulfato de Dextran , Dieta , Expresión Génica/fisiología , Inflamación/inducido químicamente , Inflamación/patología , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Peroxidasa/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/aislamiento & purificación , Tionucleósidos/efectos adversos , Tionucleósidos/farmacocinéticaRESUMEN
BACKGROUND: Methylthioadenosine (MTA) is a natural metabolite with immunomodulatory properties. MTA improves the clinical course and pathology of the animal model of multiple sclerosis, even when therapy is started after disease onset. OBJECTIVE: Our aim was to compare the efficacy of MTA in ameliorating experimental autoimmune encephalomyelitis (EAE) compared with first line approved therapies, to develop an oral formulation of MTA and to assess its pharmacokinetic profile. METHODS: EAE was induced in C57BL/6 mice by immunization with MOG(35-55) peptide in Freund's Adjuvant. Animals were treated with MTA, interferon-beta or glatiramer acetate starting the day of immunization and the clinical score was collected blind. Pharmacokinetic studies were performed in Sprague Dawley rats by administering MTA by intraperitoneal injection and orally, and collecting blood at different intervals. MTA levels were measured by high-performance liquid chromatography. RESULTS: We found that MTA ameliorated EAE in a dose-response manner. Moreover, the highest dose of MTA (60 mg/kg) was more efficacious than mouse interferon-beta or glatiramer acetate. We developed a salt of MTA for oral administration, with similar dose-response effect in the EAE model. Combination therapy assays between MTA and interferon-beta or glatiramer acetate were more effective than the individual therapies. Finally, oral MTA half-life was 20 min, with a C(max) of 80 mg/L and without signs of obvious toxicity (animal death, behavioural changes, liver enzymes). CONCLUSIONS: In the EAE model MTA is more efficacious than first line therapies for multiple sclerosis, with a dose- response effect and higher efficacy when combined with interferon-beta or glatiramer acetate. Oral MTA was also effective in the animal model of multiple sclerosis.
Asunto(s)
Adenosina/farmacología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Factores Inmunológicos/farmacología , Tionucleósidos/farmacología , Adenosina/administración & dosificación , Adenosina/análogos & derivados , Adenosina/farmacocinética , Adenosina/toxicidad , Administración Oral , Animales , Química Farmacéutica , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Quimioterapia Combinada , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Acetato de Glatiramer , Glicoproteínas , Semivida , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/farmacocinética , Factores Inmunológicos/toxicidad , Inyecciones Intraperitoneales , Interferón beta/farmacología , Ratones , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos , Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Tionucleósidos/administración & dosificación , Tionucleósidos/farmacocinética , Tionucleósidos/toxicidadRESUMEN
UNLABELLED: We evaluated 4'-[methyl-11C]thiothymidine ([methyl-11C]S-dThd) to obtain a thymidine analog that might prove simpler to use for imaging DNA synthesis and that might follow the same biochemistry as its surrogate. METHODS: [Methyl-14C]S-dThd was synthesized by rapid methylation of 5-trimethylstannyl-4'-thio-2'-deoxyuridine via a palladium-mediated Stille coupling reaction with 14C-methyl iodide. Degradation of [methyl-14C]S-dThd, when incubated in human blood, was analyzed by high-performance liquid chromatography (HPLC). The in vivo potential of [methyl-14C]S-dThd was evaluated by studying its distribution in EMT-6 mammary carcinoma-bearing mice. 2-Fluoro-2'-deoxycytidine, a potent inhibitor of DNA synthesis, was used to modulate cell proliferation. Tissue extraction was also performed to investigate the incorporation of [methyl-14C]S-dThd into DNA. RESULTS: [Methyl-14C]S-dThd was obtained in a 31%-41% radiochemical yield (calculated from 14C-methyl iodide) at 130 degrees C, 5-min reaction in N,N-dimethylformamide followed by semipreparative HPLC purification. The radiochemical purity of [methyl-14C]S-dThd was >99% and the specific activity was 2.04 GBq/mmol (according to the specific activity of 14C-methyl iodide). [2-14C]Thymidine, when incubated with human blood, demonstrated rapid degradation. In contrast, [methyl-14C]S-dThd was stable with <3% degradation at 60 min. An in vivo distribution study showed the accumulation of radioactivity in proliferating tissues (spleen, thymus, duodenum, and tumor). On the other hand, the radioactivity of nonproliferating tissues (lung, liver, kidney, and muscle) was rapidly cleared in parallel with the clearance of blood radioactivity. The tumor uptake of [methyl-14C]S-dThd was high (8.8 percentage injected dose per gram at 60 min) and selective (tumor-to-blood ratio, 12.2 at 60 min). 2-Fluoro-2'-deoxycytidine pretreatment significantly reduced the tumor uptake of [methyl-14C]S-dThd. Relative blood flow as measured by the uptake of 4-[N-methyl-14C]iodoantipyrine was similar between the treated and untreated groups. Tissue extraction studies showed that most of the total tissue radioactivity of rapidly proliferating tissues was recovered in the DNA fraction at 60 min after [methyl-14C]S-dThd injection. CONCLUSION: The labeling procedure is rapid and suitable for 11C labeling. Positron-labeled 4'-thiothymidine should be useful for imaging DNA synthesis by PET.
Asunto(s)
ADN/biosíntesis , Radiofármacos , Tionucleósidos , Timidina/análogos & derivados , Animales , Radioisótopos de Carbono , Línea Celular Tumoral , Proliferación Celular , ADN/antagonistas & inhibidores , Humanos , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Tomografía de Emisión de Positrones , Radiofármacos/farmacocinética , Tionucleósidos/farmacocinética , Timidina/farmacocinética , Distribución Tisular , Trasplante HeterólogoRESUMEN
5'-methylthioadenosine (MTA) is a nucleoside generated from S-adenosylmethionine (AdoMet) during polyamine synthesis. Recent evidence indicates that AdoMet modulates in vivo the production of inflammatory mediators. We have evaluated the anti-inflammatory properties of MTA in bacterial lipopolysaccharide (LPS) challenged mice, murine macrophage RAW 264.7 cells, and isolated rat hepatocytes treated with pro-inflammatory cytokines. MTA administration completely prevented LPS-induced lethality. The life-sparing effect of MTA was accompanied by the suppression of circulating tumor necrosis factor-alpha (TNF-alpha), inducible NO synthase (iNOS) expression, and by the stimulation of IL-10 synthesis. These responses to MTA were also observed in LPS-treated RAW 264.7 cells. MTA prevented the transcriptional activation of iNOS by pro-inflammatory cytokines in isolated hepatocytes, and the induction of cyclooxygenase 2 (COX2) in RAW 264.7 cells. MTA inhibited the activation of p38 mitogen-activated protein kinase (MAPK), c-jun phosphorylation, inhibitor kappa B alpha (IkappaBalpha) degradation, and nuclear factor kappaB (NFkappaB) activation, all of which are signaling pathways related to the generation of inflammatory mediators. These effects were independent of the metabolic conversion of MTA into AdoMet and the potential interaction of MTA with the cAMP signaling pathway, central to the anti-inflammatory actions of its structural analog adenosine. In conclusion, these observations demonstrate novel immunomodulatory properties for MTA that may be of value in the management of inflammatory diseases.
Asunto(s)
Antiinflamatorios/farmacología , Desoxiadenosinas/farmacocinética , Hepatocitos/efectos de los fármacos , Hepatocitos/inmunología , Lipopolisacáridos/farmacología , Choque Séptico/tratamiento farmacológico , Tionucleósidos/farmacocinética , Animales , Línea Celular Tumoral , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Hepatocitos/citología , Humanos , Macrófagos del Hígado/citología , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/inmunología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , S-Adenosilmetionina/metabolismo , Choque Séptico/inmunologíaRESUMEN
The availability of a noninvasive method to detect and quantify apoptosis in tumours will enable tumour response to several cancer therapies to be assessed. We have synthesised two radiotracers, annexin V and the N-succinimidyl-3-iodobenzoic acid (SIB) derivative of annexin V, labelled with radio-iodine ((124)I and (125)I) and provided proof of the concept by assessing specific binding and biodistribution of these probes to apoptotic cells and tumours. We have also assessed the tumour uptake of [(124)I]annexin V in a mouse model of apoptosis. RIF-1 cells induced to undergo apoptosis in vitro showed a drug concentration-dependent increased binding of [(125)I]annexin V and [(125)I]SIB-annexin V. In the same model system, there was an increase in terminal deoxynucleotidyl transferase-mediated nick end labelling (TUNEL)-positive cells and a decrease in clonogenic survival. Radiotracer binding was completely inhibited by preincubation with unlabelled annexin V. In RIF-1 tumour-bearing mice, rapid distribution of [(125)I]SIB-annexin V-derived radioactivity to kidneys was observed and the radiotracer accumulated in urine. The binding of [(125)I]SIB-annexin V to RIF-1 tumours increased by 2.3-fold at 48 h after a single intraperitoneal injection of 5-fluorouracil (165 mg kg(-1) body weight), compared to a 4.4-fold increase in TUNEL-positive cells measured by immunostaining. Positron emission tomography images with both radiotracers demonstrated intense localisation in the kidneys and bladder. Unlike [(124)I]SIB-annexin V, [(124)I]annexin V also showed localisation in the thyroid region presumably due to deiodination of the radiolabel. [(124)I]SIB-annexin V is an attractive candidate for in vivo imaging of apoptosis by PET.
Asunto(s)
Anexina A5/farmacocinética , Inhibidores Enzimáticos/farmacocinética , Fibrosarcoma/metabolismo , Sarcoma Experimental/metabolismo , Animales , Antimetabolitos Antineoplásicos/uso terapéutico , Apoptosis , Desoxiadenosinas/farmacocinética , Fibrosarcoma/diagnóstico por imagen , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/patología , Citometría de Flujo , Fluorouracilo/uso terapéutico , Humanos , Etiquetado Corte-Fin in Situ , Técnicas In Vitro , Radioisótopos de Yodo , Marcaje Isotópico/métodos , Ratones , Ratones Endogámicos C3H , Unión Proteica , Sarcoma Experimental/diagnóstico por imagen , Sarcoma Experimental/tratamiento farmacológico , Sarcoma Experimental/patología , Tionucleósidos/farmacocinética , Distribución Tisular , Tomografía Computarizada de Emisión , Células Tumorales CultivadasRESUMEN
The objective of this study was to evaluate the effect of food on the pharmacokinetics of racemic dOTC, a nucleoside analogue reverse transcriptase inhibitor, in adult male volunteers. Twelve healthy adult male subjects were enrolled in a randomized, open-label, single-dose crossover study. All were nonsmoking, within 15% of ideal body weight, and between 18 and 50 years of age. Subjects received single oral doses of 800mg racemic dOTC, in random order, either fed or fasted. The meal given to fed subjects was the standard Food and Drug Administration high-fat breakfast, and all subjects completed both study periods. Sixteen plasma samples for pharmacokinetic assessments were collected for 72 hours following dosing and assayed for (-) and (+) dOTC concentrations. Area under the plasma concentration-time curve (AUC), maximum observed plasma concentration (Cmax), and time to maximum concentration (tm) were determined for each enantiomer by standard noncompartmental techniques. Statistical hypothesis testing was by Wilcoxon signed rank, and the two one-sided tests procedure was used to determine bioequivalence between thefed and fasted study periods. The only effect of coadministration of racemic dOTC with food was a delay in time to peak concentration (t(max) of between 0.6 and 0.7 hours for both (-) and (+) dOTC stereoisomers (p < or =0.02). Neither AUC (p > or = 0.10) nor Cmax (p > or = 0.35) differed significantly between the fed and fasted study periods for either (-) or (+) dOTC. Both AUC and Cmax were equivalent between the fed and fasted study periods. It was concluded that there is no clinically significant effect of a high-fat meal on the pharmacokinetics of either (-) or (+) dOTC when administered orally as a racemic mixture.
Asunto(s)
Desoxicitidina/análogos & derivados , Desoxicitidina/farmacocinética , Inhibidores de la Transcriptasa Inversa/farmacocinética , Tionucleósidos/farmacocinética , Administración Oral , Adulto , Anciano , Área Bajo la Curva , Estudios Cruzados , Desoxicitidina/administración & dosificación , Alimentos , Humanos , Masculino , Persona de Mediana Edad , Estereoisomerismo , Tionucleósidos/administración & dosificaciónRESUMEN
African trypanosomes are parasitic flagellates that live in the connective tissues of the host. Trypanosomes must obtain from their host adenine/adenosine and other nucleosides that can be salvaged through enzymatic cleavage. Methylthioadenosine (MTA) is a byproduct of polyamine metabolism, formed from the donation of an aminopropyl moiety by decarboxylated S-adenosylmethionine (dcAdoMet) to form spermidine. MTA is then cleaved phosphorolytically by MTA phosphorylase to methylthioribose-1-phosphate (MTR-1-P) and adenine. The uptake of MTA was compared with that of adenosine in two strains: Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense. The K(m) values for MTA and adenosine (with 5 mM inosine) transport by T. b. brucei were 1.4 and 0.175 mM, and the V(max) values were 70 and 7.8 micromol/L/min, respectively. The K(m) values for T. b. rhodesiense MTA and adenosine (with 5 mM inosine) transport were 1.2 and 0.11 mM, and the V(max) values were 52.6 and 2.9 micromol/L/min, respectively. Since MTA was not competitive with either AdoMet (100 microM), inosine (100 microM), or the methionine precursor ketomethylthiobutyrate (100 microM), it appears that MTA enters through the P(2) (adenosine/adenine) transport site. From this study and our previous work, we determined that these organisms transport adenylated intermediates of methionine metabolism found in sera for purine salvage and as an ancillary source of methionine. The significant ability of African trypanosomes to transport MTA and related intermediates is an important consideration in the design and development of selective chemotherapeutic agents.
Asunto(s)
Adenosina/análogos & derivados , Adenosina/farmacocinética , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Tionucleósidos/farmacocinética , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei rhodesiense/metabolismo , Adenosina/metabolismo , Animales , Unión Competitiva , Transporte Biológico , Cinética , Metionina/metabolismo , Proteínas de Transporte de Nucleósidos , Poliaminas/metabolismo , S-Adenosilhomocisteína/metabolismo , Tionucleósidos/metabolismoRESUMEN
Racemic dOTC (BCH-10652) is a novel nucleoside reverse transcriptase inhibitor consisting of two enantiomers of 2'-deoxy-3'-oxa-4'-thiocytidine, (-)dOTC and (+)dOTC, that have both shown activity against human immunodeficiency virus type 1. The objectives of this study were to characterize the safety, tolerability, and stereospecific pharmacokinetics of single oral doses of racemic dOTC in healthy, nonsmoking adult male volunteers. Subjects received single oral doses of 100, 200, 400, 800, and 1,600 mg of racemic dOTC in a placebo-controlled, dose-rising, incomplete crossover study design, and the pharmacokinetics of both (+)dOTC and (-)dOTC were determined. At least six subjects were studied at each dose level, with each subject studied in three of five periods, receiving two different doses of racemic dOTC and one placebo dose. Plasma and urine drug concentrations were measured for 24 to 48 h after each dose. Pharmacokinetic models were fitted to the plasma concentrations of (+)dOTC and (-)dOTC using maximum likelihood and maximum a posteriori Bayesian procedures. Statistical hypothesis testing was by nonparametric analysis of variance (where possible) and, when tests with dose as a covariate were performed, by linear mixed-effects modeling. The mean terminal elimination half-lives for (+)dOTC and (-)dOTC were 15.3 h (coefficient of variation [CV], 28%) and 11.3 h (CV, 43%), respectively (P<0.05). The mean CV for total oral clearance (liter/h/65 kg) was 17.5 (25%) for (+)dOTC and 21.5 (24%) for (-)dOTC; for oral steady-state volume of distribution (liter/65 kg), values were 61.8 (24%) for (+)dOTC and 34.1 (33%) for (-)dOTC (P<0.05). The mean CV for renal clearance (liter/h/65 kg) of (+)dOTC was 10.4 (19%) and for (-)dOTC was 13.6 (20%) (P<0.05). There was no significant effect of dose size on the pharmacokinetics of racemic dOTC. All doses were well tolerated, and no serious adverse events or laboratory abnormalities were observed.
Asunto(s)
Desoxicitidina/análogos & derivados , Inhibidores de la Transcriptasa Inversa/efectos adversos , Inhibidores de la Transcriptasa Inversa/farmacocinética , Tionucleósidos/efectos adversos , Tionucleósidos/farmacocinética , Adolescente , Adulto , Área Bajo la Curva , Estudios Cruzados , Desoxicitidina/efectos adversos , Desoxicitidina/farmacocinética , Humanos , Masculino , Persona de Mediana Edad , Método Simple Ciego , Espectrofotometría Ultravioleta , EstereoisomerismoRESUMEN
The purpose of this study was to characterize the pharmacokinetics and determine the absolute bioavailability of 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC) (BCH-10652), a novel nucleoside analogue reverse transcriptase inhibitor, in humans. dOTC belongs to the 4'-thio heterosubstituted class of compounds and is a 1:1 mixture of its two enantiomers, (-) and (+) dOTC. Twelve healthy adult male volunteers each received oral (800-mg) and intravenous (100-mg) doses of dOTC in two study periods separated by at least 7 days. Sixteen plasma samples were obtained over 72 h and assayed for (-) and (+) dOTC, and the resultant data fit by candidate pharmacokinetic models. Data were weighted by the fitted inverse of the observation variance; model discrimination was by AIC. The pharmacokinetic model was a linear, three compartment model, with absorption occurring during one to three first-order input phases, each following a fitted lag time. The model goodness-of-fit was excellent; r(2) ranged from 0.995 to 1.0. The mean absolute bioavailabilities of (+) and (-) dOTC were 77.2% (coefficient of variation [given as a percentage] [CV%], 14) and 80.7% (CV%, 15), respectively. The median steady-state volume of distribution for (+) dOTC, 74.7 (CV%, 19.2) liters/65 kg, was greater than that for (-) dOTC, 51.7 (CV%, 16.7) liters/65 kg (P<0.05). The median total clearance of (+) dOTC was less than that of (-) dOTC, 11.7 (CV%, 17.3) versus 15.4 (CV%, 18.6) liters/h/65 kg, respectively (P< 0.05). The intersubject variability of these parameters was very low. The median terminal half-life of (+) dOTC was 18.0 (CV%, 31.5) h, significantly longer than the 6.8 (CV%, 69.9) h observed for (-) dOTC (P<0.01). No serious adverse events were reported during the study. These results suggest that dOTC is well absorbed, widely distributed, and well tolerated. The terminal half-lives indicate that dosing intervals of 12 to 24 h would be reasonable.
Asunto(s)
Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/farmacocinética , Desoxicitidina/análogos & derivados , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Inhibidores de la Transcriptasa Inversa/farmacocinética , Tionucleósidos/administración & dosificación , Tionucleósidos/farmacocinética , Administración Oral , Adulto , Fármacos Anti-VIH/efectos adversos , Desoxicitidina/administración & dosificación , Desoxicitidina/efectos adversos , Desoxicitidina/farmacocinética , Humanos , Inyecciones Intravenosas , Masculino , Inhibidores de la Transcriptasa Inversa/efectos adversos , Tionucleósidos/efectos adversosRESUMEN
The racemic nucleoside analogue 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC) is in clinical development for the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection. dOTC is structurally related to lamivudine (3TC), but the oxygen and sulfur in the furanosyl ring are transposed. Intracellular metabolism studies showed that dOTC is phosphorylated within cells via the deoxycytidine kinase pathway and that approximately 2 to 5% of dOTC is converted into the racemic triphosphate derivatives, which had measurable half-lives (2 to 3 hours) within cells. Both 5'-triphosphate (TP) derivatives of dOTC were more potent than 3TC-TP at inhibiting HIV-1 reverse transcriptase (RT) in vitro. The K(i) values for dOTC-TP obtained against human DNA polymerases alpha, beta, and gamma were 5,000-, 78-, and 571-fold greater, respectively, than those for HIV RT (28 nM), indicating a good selectivity for the viral enzyme. In culture experiments, dOTC is a potent inhibitor of primary isolates of HIV-1, which were obtained from antiretroviral drug-naive patients as well as from nucleoside therapy-experienced (3TC- and/or zidovudine [AZT]-treated) patients. The mean 50% inhibitory concentration of dOTC for drug-naive isolates was 1.76 microM, rising to only 2.53 and 2.5 microM for viruses resistant to 3TC and viruses resistant to 3TC and AZT, respectively. This minimal change in activity is in contrast to the more dramatic changes observed when 3TC or AZT was evaluated against these same viral isolates. In tissue culture studies, the 50% toxicity levels for dOTC, which were determined by using [(3)H]thymidine uptake as a measure of logarithmic-phase cell proliferation, was greater than 100 microM for all cell lines tested. In addition, after 14 days of continuous culture, at concentrations up to 10 microM, no measurable toxic effect on HepG2 cells or mitochondrial DNA replication within these cells was observed. When administered orally to rats, dOTC was well absorbed, with a bioavailability of approximately 77%, with a high proportion (approximately 16.5% of the levels in serum) found in the cerebrospinal fluid.
Asunto(s)
Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/farmacocinética , Desoxicitidina/análogos & derivados , VIH-1/efectos de los fármacos , Tionucleósidos/farmacocinética , Animales , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/toxicidad , Disponibilidad Biológica , Células de la Médula Ósea/efectos de los fármacos , Líquido Cefalorraquídeo/metabolismo , Técnicas de Cultivo , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxicitidina/farmacocinética , Desoxicitidina/farmacología , Desoxicitidina/toxicidad , Farmacorresistencia Microbiana , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/toxicidad , Femenino , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , VIH-1/aislamiento & purificación , Humanos , Líquido Intracelular/metabolismo , Cinética , Masculino , Ratones , Mitocondrias Hepáticas/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico , Ratas , Ratas Sprague-Dawley , Inhibidores de la Transcriptasa Inversa/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacocinética , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/toxicidad , Células Madre/efectos de los fármacos , Estereoisomerismo , Tionucleósidos/farmacología , Tionucleósidos/toxicidad , Células Tumorales CultivadasRESUMEN
The effects of 5'-deoxy-5'-(hydroxyethylthio)adenosine (HETA), a trypanocidal analog of 5'-deoxy-5'-(methylthio)adenosine (MTA), on polyamine synthesis and S-adenosylmethionine (AdoMet) metabolism were examined in bloodstream forms of Trypanosoma brucei brucei. HETA was cleaved by trypanosome MTA phosphorylase at the same rate as the natural substrate, MTA, in a phosphate-dependent reaction. Fluorine substitution at the 2-position of the purine ring increased activity by approximately 50%, whereas substitution with an amino group reduced activity to about one-third of the control. HETA was accumulated by trypanosomes with internal concentrations of 100-250 microM and >800 microM after a 15-min incubation with 1 and 10 microM, respectively. Trypanosomes preincubated with HETA metabolized it at a rate of 21.9 nmol/hr/mg protein. Preincubation of cells with HETA at 1 or 10 microM inhibited spermidine synthesis from [3H]ornithine by 22-37%, and increased the cytosolic levels of AdoMet by 2- to 5-fold and that of MTA by up to 8-fold. S-Adenosylhomocysteine (AdoHcy) levels also increased 1.5- to 7-fold in treated cells, whereas decarboxylated AdoMet decreased 65%. Preincubation of trypanosomes with HETA for 4 hr also reduced the incorporation of [35S]methionine in trichloroacetic acid-precipitable material by 50-60%, and reduced the methyl group incorporation into protein from [U-14C]methionine by 65-70%. Thus, HETA interferes with a series of biochemical events involving the participation of AdoMet and methionine in polyamine synthesis, protein synthesis, and transmethylation reactions.
Asunto(s)
Adenosina/análogos & derivados , Desoxiadenosinas/farmacocinética , Poliaminas/metabolismo , Purina-Nucleósido Fosforilasa/metabolismo , Tionucleósidos/farmacocinética , Tripanocidas/farmacología , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei rhodesiense/metabolismo , Adenosina/farmacocinética , Adenosina/farmacología , Animales , Transporte Biológico , Desoxiadenosinas/farmacología , Metionina/metabolismo , Modelos Químicos , Estructura Molecular , Putrescina/metabolismo , S-Adenosilmetionina/metabolismo , Espermidina/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Tionucleósidos/farmacología , Tripanocidas/farmacocinética , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei rhodesiense/efectos de los fármacosRESUMEN
The synthesis of new potential PFA-BCH-189 conjugate analogues is described and their molecular structure clearly identified through NMR and mass spectra techniques. The anti-HIV-1 activity was determined according to the inhibition of syncytium formation in MT-4 cells, while the anti-HBV activity was determined in infected duck hepatocytes. Both antiviral activities of the PFA-BCH-189 conjugates were much lower than those of the parent BCH-189 (2',3'-dideoxy-3'-thiacytidine) (1). Whereas a prodrug effect, following cleavage and release of the free BCH-189 and PFA, cannot be ruled out, poor cellular permeation of the drug seems to be the most likely reason for the reduced activities against HIV and DHBV. The presence of the PFA moiety appears to be detrimental for both the anti-HIV and anti-DHBV activity of PFA-BCH-189 cases.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Foscarnet/farmacología , VIH-1/efectos de los fármacos , Tionucleósidos/síntesis química , Tionucleósidos/farmacología , Animales , Antivirales/farmacocinética , Células Cultivadas , Efecto Citopatogénico Viral/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Patos , Foscarnet/farmacocinética , Células Gigantes/efectos de los fármacos , Virus de la Hepatitis B del Pato/efectos de los fármacos , Humanos , Lamivudine , Hígado/citología , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Estructura Molecular , Relación Estructura-Actividad , Tionucleósidos/farmacocinética , Zalcitabina/análogos & derivados , Zalcitabina/farmacologíaRESUMEN
Intracellular metabolism of [3H]guanosine was minimal (< 15%) during the first 22 s of incubation, and hence reasonable estimates of initial-rate influx kinetics could be derived by using metabolically active cells. Na(+)-dependent concentrative [3H]guanosine uptake was not observed. Data suggest that [3H]guanosine was accumulated primarily via the nitrobenzylthioguanosine (NBTGR)-sensitive subtype of facilitated nucleoside transporter. Incubation of cells with 100 nM-NBTGR significantly decreased the potency of guanosine as an inhibitor of [3H]uridine influx. The Vmax. for [3H]guanosine influx (9.2 pmol/s per microliters) was significantly lower than that for [3H]uridine influx (16 pmol/s per microliters). The Km for transporter-mediated [3H]guanosine influx determined in the presence of 100 nM-NBTGR was 16-fold higher (1780 microM) than that determined in its absence, whereas the Km for [3H]uridine influx was shifted by only 2-fold. In other respects, the cellular accumulations of [3H]guanosine and [3H]uridine were similar; both had Km values of approx. 140 microM for total mediated influx, and both were inhibited similarly by other nucleosides and transport inhibitors. These characteristics, and the fact that guanosine is an endogenous nucleoside, suggest that [3H]guanosine may prove useful as a poorly metabolized, relatively selective, substrate for study of the NBTGR-sensitive nucleoside transport systems of mammalian cells.
Asunto(s)
Carcinoma de Ehrlich/metabolismo , Proteínas Portadoras/metabolismo , Guanosina/farmacocinética , Proteínas de la Membrana/metabolismo , Adenosina Trifosfato/deficiencia , Adenosina Trifosfato/fisiología , Animales , Guanosina/análogos & derivados , Guanosina/farmacología , Cinética , Masculino , Ratones , Proteínas de Transporte de Nucleósidos , Nucleótidos/metabolismo , Sensibilidad y Especificidad , Sodio/farmacología , Tionucleósidos/farmacocinética , Tionucleósidos/farmacología , Tritio , Células Tumorales Cultivadas , Uridina/farmacocinéticaRESUMEN
Preclinical investigations in vivo revealed unexpected differences in the biological characteristics of 2-amino-9-beta-D-ribofuranosylpurine-6-sulfenamide (sulfenosine, 1) and 2-amino-9-beta-D-ribofuranosylpurine-6-sulfonamide (sulfonosine, 2), two novel but structurally related derivatives of 6-thioguanosine (6TGR). Strikingly, the addition of a fully oxidized sulfur atom at the 6 position of sulfenosine produced a purine derivative (sulfonosine) that was remarkably active against experimental leukemia resistant to treatment with either sulfenosine or 6TGR. This slight structural modification also appeared to influence solubility, scheduling capability, and oral activity as well as penetration of the central nervous system (CNS) and the onset of cellular resistance. These findings underscore the dramatic changes in biologic activity that can be produced by subtle modifications in molecular structure. We trust they may also contribute to the development of improved clinical therapy.