RESUMEN
A range of 4-substituted derivatives of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di-(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) were prepared in a search for more soluble analogs. 4-Aminoalkoxy substituents provided the most potent derivatives, with the 4-O(CH2)3NMe2 analog (compound 14) being identified as displaying the best overall activity in combination with good aqueous solubility (25 mg/mL for the hydrochloride salt). This compound was tested in a U87MG xenograft model, but displayed less potency than ZSTK474 as a result of an unfavorable pharmacokinetic profile.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Triazinas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Células HCT116 , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Estructura Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Transducción de Señal/efectos de los fármacos , Solubilidad , Relación Estructura-Actividad , Triazinas/síntesis química , Triazinas/químicaRESUMEN
PURPOSE: N-[2-(dimethylamino)ethyl]-2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b]-1,6-naphthyridine-4-carboxamide (SN 28049), a DNA-binding benzonaphthyridine, has shown curative activity against colon-38 adenocarcinoma after a single dose in mice. A homologous series of 5 compounds, where the 2-methyl group was replaced by a hydrogen, ethyl, propyl, or butyl, was used to evaluate the role of lipophilicity and tumour pharmacokinetics on their antitumour activity. METHODS: All analogues were administered (25 µmol/kg) to healthy and tumour-bearing C57 Bl/6 mice and concentrations were measured in plasma, brain, heart, kidney, liver, lung, and tumour tissues. Microsomal stability studies were performed with mouse livers and plasma protein binding studies by equilibrium dialysis. RESULTS: Plasma pharmacokinetics conformed to a model where increasing lipophilicity was associated with a decreasing area under the concentration-time curve (AUC), an increasing clearance and volume of distribution. In contrast, tumour pharmacokinetic parameters showed a very different relationship, where the AUC of the methyl derivative (2,334 µM h) was 89-fold higher than that of the hydrogen derivative (26.3 µM h), with other homologues having intermediate values. The tumour AUC correlated (r = -0.98, P = 2 × 10(-7)) with the in vivo antitumour activity of this series. The methyl derivative had a 22 min microsomal half-life, while other analogues ranged from 1.6 to 12.2 min. The plasma-free fraction decreased (17-5 %) significantly with lipophilicity (r = 0.96, P = 2 × 10(-7)). CONCLUSION: The plasma pharmacokinetics of this series is related to changes in drug lipophilicity. However, the tumour pharmacokinetics reveals a strong dependence on the nitrogen substituent on the benzonaphthyridine chromophore, with the methyl group providing by far the best tumour tissue retention.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Naftiridinas/farmacología , Adenocarcinoma/sangre , Adenocarcinoma/metabolismo , Animales , Área Bajo la Curva , Disponibilidad Biológica , Encéfalo/metabolismo , Neoplasias del Colon/sangre , Neoplasias del Colon/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Estructura Molecular , Miocardio/metabolismo , Naftiridinas/química , Naftiridinas/farmacocinética , Relación Estructura-Actividad , Distribución Tisular , Resultado del TratamientoRESUMEN
Drug lipophilicity is a vital physicochemical parameter that influences drug absorption, distribution, metabolism, excretion and toxicology. A comparative study of a homologous series based on a pharmaceutically active drug represents a powerful approach to the study of the effects of drug lipophilicity. We have developed a rapid and sensitive LC-MS/MS method suitable for such a homologous series and applied it to a series of DNA binding benzonaphthyridine-based antitumour drugs of differing lipophilicity. The method used a gradient elution with a run time of 7 min for simultaneous quantitation of five analogues in a pooled sample. Method validation was carried out in plasma (human and mouse) and mouse tissues (brain, heart, kidney, liver and lung). It had a limit of quantitation of 0.001 µmol/L and was linear (0.001-0.3 µmol/L) in all matrices with acceptable intra- and inter-assay precision and accuracy. This method allowed the pharmacokinetic parameters of these compounds in mice to be related to their lipophilicity as determined by their partition coefficient (LogD). Both the plasma CL (r=0.95; P=2×10â»7) and V(ss) (r=0.95; P=2×10â»7) exhibited a significant positive correlation with LogD values after intravenous bolus administration to mice. Consequently the plasma mean residence time for each of these five analogues decreased with increasing lipophilicity. There was also a significant positive correlation (r=0.91; P=2×10â»7) between LogD values and the brain to plasma AUC ratio indicating the importance of lipophilicity in the distribution of these compounds into the brain tissue.
Asunto(s)
Cromatografía Liquida , Descubrimiento de Drogas , Naftiridinas/farmacocinética , Espectrometría de Masas en Tándem , Inhibidores de Topoisomerasa II/farmacocinética , Animales , Área Bajo la Curva , Cromatografía Liquida/normas , Descubrimiento de Drogas/normas , Femenino , Humanos , Inyecciones Intravenosas , Límite de Detección , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Naftiridinas/administración & dosificación , Naftiridinas/sangre , Naftiridinas/química , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/normas , Distribución Tisular , Inhibidores de Topoisomerasa II/administración & dosificación , Inhibidores de Topoisomerasa II/sangre , Inhibidores de Topoisomerasa II/químicaRESUMEN
PURPOSE: The human tumour suppressor protein p53 is mutated in nearly half of human tumours and most mutant proteins have single amino acid changes. Several drugs including the quinazoline derivative 1 (CP-31398) have been reported to restore p53 activity in mutant cells. The side chain of 1 contains a styryl linkage that compromises its stability and we wished to explore the activity of analogues containing more stable side chains. METHODS: Reactivation of p53 function was measured by flow cytometry as the ability to potentiate radiation-induced G(1)-phase cell cycle arrest and by western blotting to determine expression of p21(WAF1). DNA binding was measured by competition with ethidium and preliminary pharmacological and xenograft studies were carried out. RESULTS: Screening of analogues for potentiation of radiation-induced G(1)-phase cell cycle arrest using NZOV11, an ovarian tumour cell line containing a p53(R248Q) mutation, demonstrated that the (2-benzofuranyl)-quinazoline derivative 5 was among the most active of the analogues. Compound 5 showed similar effects in several other p53 mutant human tumour cell lines but not in a p53 null cell line. 5 also potentiated p21(WAF1) expression induced by radiation. DNA binding affinity was measured and found to correlate with p53 reactivation activity. Plasma concentrations of 5 in mice were sufficient to suggest in vivo activity and a small induced tumour growth delay (7 days) of NZM4 melanoma xenografts was observed. CONCLUSION: Compound 5 restores p53-like function to a human tumour cells lines expressing a variety of mutant p53 proteins, thus providing a basis for the design of further new drugs.
Asunto(s)
Mutación/efectos de los fármacos , Quinazolinas/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Ratones , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismoRESUMEN
A structure-activity relationship (SAR) study of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) identified substitution at the 4 and 6 positions of the benzimidazole ring as having significant effects on the potency of substituted derivatives. The 6-amino-4-methoxy analogue displayed a greater than 1000-fold potency enhancement over the corresponding 6-aza-4-methoxy analogue against all three class Ia PI 3-kinase enzymes (p110α, p110ß, and p110δ) and also displayed significant potency against two mutant forms of the p110α isoform (H1047R and E545K). This compound was also evaluated in vivo against a U87MG human glioblastoma tumor xenograft model in Rag1(-/-) mice, and at a dose of 50 mg/kg given by ip injection at a qd × 10 dosing schedule it dramatically reduced cancer growth by 81% compared to untreated controls.
Asunto(s)
Antineoplásicos/síntesis química , Bencimidazoles/síntesis química , Inhibidores de las Quinasa Fosfoinosítidos-3 , Triazinas/síntesis química , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Bencimidazoles/farmacocinética , Bencimidazoles/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Noqueados , Modelos Moleculares , Mutación , Trasplante de Neoplasias , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Transducción de Señal/efectos de los fármacos , Solubilidad , Relación Estructura-Actividad , Trasplante Heterólogo , Triazinas/farmacocinética , Triazinas/farmacologíaRESUMEN
Wheat bran protects against mutations and cancer, but contains different plant cell types that are likely to have different protective effects. We previously described the production and chemical characterisation of an aleurone-rich fraction (ARF) and a pericarp-rich fraction (PRF) from wheat grain. We compared these with whole bran (WB), fed to rats as 10% of a high fat AIN-76 diet. All bran-supplemented diets increased faecal bulk, in the order PRF>WB>ARF. PRF increased the activity of NAD(P)H:quinone acceptor oxidoreductase only in the forestomach, whereas ARF and WB enhanced levels of glutathione S-transferase in the duodenum. ARF but not PRF was digested and fermented, and also encouraged bacterial growth. Rats were gavaged with the radioactive mutagen (14)C-labelled IQ (2-amino-3-methylimidazo[4,5-f]quinoline), and effects of the brans on plasma radioactivity measured. Compared with the control diet, all bran-supplemented diets reduced the concentration of radioactivity in plasma, in the order ARF>PRF>WB. All brans increased faecal elimination of radioactivity, but only ARF and PRF enhanced urinary radioactivity. These data suggest that wheat bran may reduce mutation and cancers through direct adsorption and enhanced elimination of a dietary mutagen and/or its metabolites, and that wheat bran enriched in pericarp or aleurone cell walls may exert protective effects through different mechanisms.
Asunto(s)
Antimutagênicos/farmacología , Fibras de la Dieta/farmacología , Quinolinas/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Carcinógenos/metabolismo , Heces/química , Femenino , Absorción Intestinal/efectos de los fármacos , Quinolinas/sangre , Quinolinas/toxicidad , Quinolinas/orina , Ratas , Ratas WistarRESUMEN
PURPOSE: N-[2-(Dimethylamino)ethyl]-2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b]-1,6-naphthyridine-4-carboxamide (SN 28049) is a potent DNA binding topoisomerase II poison that shows excellent antitumour activity in a colon-38 murine tumour model in comparison to standard topoisomerase II poisons. We report here the preclinical pharmacokinetics of SN 28049. METHODS: C57 Bl/6 mice (n = 3 per time point) were treated with a single i.v., i.p. or p.o. administration (8.9 mg/kg). Plasma and tissue samples were analysed using a validated LC/MS method utilizing a homologue as an internal standard. RESULTS: The assay range was 0.062-2.5 microM with a quantitation limit of 0.062 microM and a detection limit of 0.025 microM. Acceptable intra- and inter-assay accuracy (95-105%) and precision (<6.5% RSD) were achieved. Following i.v. administration, SN 28049 demonstrated 2-compartment model kinetics with a volume of distribution of 42.3 +/- 4.1 l/kg, a plasma clearance of 12.1 +/- 0.5 l/h per kg and distribution and elimination half-lives of 0.15 +/- 0.02 and 2.8 +/- 0.2 h (mean +/- SE), respectively. For all administration routes, SN 28049 concentrations in normal tissues (brain, heart, liver, lung, and kidney) were 12- to 120-fold higher than those in plasma, but half-lives and mean residence times were similar. The i.p. and p.o. bioavailabilities were 83.1 +/- 1.5 and 54.5 +/- 1.1%, respectively. In the tumour tissue, elimination half-life (9.1 +/- 0.7 h) and the mean residence time (18.2 +/- 0.7 h) were significantly (P < 0.001) longer than those of plasma and normal tissues. The tumour area under the concentration-time curve (AUC) (1,316 +/- 66 microM h) was also 693-fold greater than the plasma AUC, and considerably higher (approximately 5-fold) than any other tissue examined, indicating selective uptake and retention of SN 28049 in the tumour. CONCLUSION: We conclude that SN 28049's high tumour exposure and long tumour retention time is likely to contribute to its high antitumour activity in vivo.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/farmacocinética , Neoplasias del Colon/tratamiento farmacológico , Naftiridinas/farmacocinética , Adenocarcinoma/metabolismo , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Área Bajo la Curva , Disponibilidad Biológica , Cromatografía Liquida , Neoplasias del Colon/metabolismo , Femenino , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Masculino , Espectrometría de Masas , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Naftiridinas/administración & dosificación , Naftiridinas/química , Distribución Tisular , Inhibidores de Topoisomerasa IIRESUMEN
AIM: 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) (ASA404), a low molecular weight antivascular drug currently in clinical trial, acts both directly on the tumour vascular endothelium and indirectly through the induction of inflammatory cytokines and other vasoactive molecules from macrophages and other host cells. We wished to determine whether co-administration of non-steroidal anti-inflammatory drugs (NSAIDs) could modulate the antivascular effects of DMXAA in mice. METHODS: The effects of diclofenac, salicylate, ibuprofen, celecoxib and rofecoxib on the antitumour response to DMXAA were compared using growth delay assays of Colon 38 adenocarcinomas in C57Bl mice. Concentrations of DMXAA in mice were measured by high performance liquid chromatography. RESULTS: Administration of DMXAA alone (25 mg/kg i.p.) or of NSAIDs alone induced small tumour growth delays from 2 to 7 days. Co-administration of each of the NSAIDs augmented DMXAA effects with tumour growth delays from 4.5 to >20 days. The possibility of a pharmacokinetic interaction was investigated using diclofenac and it was found that diclofenac did not affect DMXAA pharmacokinetics. CONCLUSIONS: NSAIDs increase the antitumour activity of DMXAA in a murine tumour model. The effects are consistent with hypothesis that NSAIDs antagonises some of the protective effects of prostaglandins released in response to vascular injury. Co-administration of NSAIDs with DMXAA might be considered as a possible strategy for use in combination cancer therapy.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Antiinflamatorios no Esteroideos/uso terapéutico , Xantonas/uso terapéutico , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Neoplasias del Colon/tratamiento farmacológico , Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores de la Ciclooxigenasa/uso terapéutico , Diclofenaco/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Quimioterapia Combinada , Ratones , Ratones Endogámicos C57BL , Xantonas/administración & dosificación , Xantonas/sangreRESUMEN
N-[2-(Dimethylamino)ethyl]-2,6-dimethyl-1-oxo-1,2-dihydrobenzo[b]-1,6-naphthyridine-4-carboxamide (SN 28049) is a potent topoisomerase II poison being developed to treat solid tumours. A reliable and sensitive LC-MS method has been developed and validated for the determination of SN 28049 in plasma using a structurally similar internal standard. This method had acceptable intra- and inter-assay accuracy (95-105%) and precision (R.S.D.<6.5%) over the range 0.062-2.5 microM (using a 100 microl sample), and had a lower limit of quantitation of 0.062 microM. Both aqueous and plasma solutions of SN 28049 were stable during short-term (24h at room temperature or 4 degrees C) and long-term storage (8 months at -80 degrees C), and following freezing and thawing (three cycles). The method was applied to study the pharmacokinetics of SN 28049 in mice after iv administration (8.9 mg/kg; n=3 mice per time point). The maximum plasma concentration achieved was 1.22+/-0.05 microM, and concentrations were measurable up to 12h post-administration. A bi-exponential concentration-time curve was observed with an elimination half-life of 2.3+/-0.2h (mean+/-S.E.), a volume of distribution of 34.5+/-2.2l/kg, and a plasma clearance of 12+/-0.5l/h/kg.
Asunto(s)
Antineoplásicos/sangre , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Naftiridinas/sangre , Animales , Antineoplásicos/farmacocinética , Estabilidad de Medicamentos , Femenino , Semivida , Humanos , Infusiones Intravenosas , Ratones , Naftiridinas/análisis , Naftiridinas/metabolismo , Naftiridinas/farmacocinética , Estándares de Referencia , Análisis de Regresión , Sensibilidad y EspecificidadRESUMEN
The dose-dependent effects of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on rat GH3 prolactinomas were investigated in vivo. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to assess tumor blood flow/permeability pretreatment and 24 hours posttreatment with 0, 100, 200, or 350 mg/kg DMXAA. DCE-MRI data were analyzed using K(trans) and the integrated area under the gadolinium time curve (IAUGC) as response biomarkers. High-performance liquid chromatography (HPLC) was used to determine the plasma concentration of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) following treatment to provide an index of increased vessel permeability and vascular damage. Finally, tumor necrosis was assessed by grading hematoxylin and eosin-stained sections cut from the same tumors investigated by MRI. Both tumor K(trans) and IAUGC were significantly reduced 24 hours posttreatment with 350 mg/kg DMXAA only, with no evidence of dose response. HPLC demonstrated a significant increase in plasma 5-HIAA 24 hours posttreatment with 200 and 350 mg/kg DMXAA. Histologic analysis revealed some evidence of tumor necrosis following treatment with 100 or 200 mg/kg DMXAA, reaching significance with 350 mg/kg DMXAA. The absence of any reduction in K(trans) or IAUGC following treatment with 200 mg/kg, despite a significant increase in 5-HIAA, raises concerns about the utility of established DCE-MRI biomarkers to assess tumor response to DMXAA.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Indoles/sangre , Imagen por Resonancia Magnética , Neovascularización Patológica/tratamiento farmacológico , Neoplasias Hipofisarias/patología , Prolactinoma/irrigación sanguínea , Xantonas/uso terapéutico , Inhibidores de la Angiogénesis/farmacocinética , Inhibidores de la Angiogénesis/farmacología , Animales , Área Bajo la Curva , Biomarcadores , Permeabilidad Capilar , Línea Celular Tumoral/trasplante , Medios de Contraste , Relación Dosis-Respuesta a Droga , Femenino , Gadolinio DTPA , Necrosis , Trasplante de Neoplasias , Prolactinoma/tratamiento farmacológico , Prolactinoma/patología , Ratas , Ratas Endogámicas WF , Tejido Subcutáneo , Xantonas/farmacocinética , Xantonas/farmacologíaRESUMEN
1. The aim of the present study was to investigate the structural requirements for the inhibition of 6-methyl-hydroxylation of the antitumour agent 5,6-dimethyl-xanthenone-4-acetic acid (DMXAA) by acridine analogues and use a CYP1A2 homology model to provide some insight into this interaction. 2. Concentrations causing 50% inhibition (IC50) of the 6-methylhydroxylation of DMXAA were determined in human liver microsomes in the presence of various acridines. Some of the acridines were also tested for their ability to inhibit the CYP1A2-mediated 7-ethoxyresorufin O-de-ethylation. The molecular modelling studies of human CYP1A2 used the crystal structure of rabbit CYP2C5 as a template based on protein sequence homology and an interactive docking procedure using a dynamic hydrogen bond feature. 3. The in vitro IC50 studies for the inhibition of 6-methylhydroxylation of DMXAA indicated: (i) the importance of the position of the carboxamide side-chain on the acridine nucleus (and, to a lesser extent, its composition); (ii) the addition of hydroxyl groups to the 5-, 6- and 7-position of the acridine nucleus diminished the inhibitory potency; and (iii) amsacrine (acridine nucleus with methansulphonanilide side-chain at the 9-position) had no significant inhibitory effect. Similar structural trends were observed for the inhibition of O-de-ethylation of 7-ethoxyresorufin by acridines, supporting the involvement of CYP1A2 in DMXAA 6-methyl hydroxylation. 4. The molecular modelling studies indicated: (i) both DMXAA and N-[2-(dimethylamino)-ethyl]acridine-4-carboxamide (DACA) form two hydrogen bonds plus putative pi-pi stacking interactions with the CYP1A2-binding domain, typical of CYP1A2 substrates and inhibitors; (ii) the DMXAA 6-methyl group is 4.0 A from the central iron atom of the heme moiety and ideal for oxidation; (iii) the known oxidation sites for DACA are orientated away from the heme iron, supporting the non-involvement of CYP1A2; and (iv) amsacrine did not fit the putative CYP1A2 site owing to the steric hindrance of the bulky methanesulphonanilide side-chain. 5. These results suggest that docking studies with this homology model may be useful in the design of further acridine anticancer agents, in particular to identify agents that do not interact either as substrates or inhibitors with the CYP1A2-binding domain.
Asunto(s)
Acridinas/química , Acridinas/farmacología , Inhibidores del Citocromo P-450 CYP1A2 , Citocromo P-450 CYP1A2/metabolismo , Xantonas/metabolismo , Sitios de Unión , Citocromo P-450 CYP1A2/química , Humanos , Modelos Moleculares , Estructura Molecular , Oxidación-Reducción , Relación Estructura-ActividadRESUMEN
The novel vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) has completed phase 1 clinical trial and has shown tumor antivascular activity in both mice and humans. We have investigated its ability to change tumor vascular permeability, relating it to tumor vascular perfusion and other responses. The murine colon 38 adenocarcinoma was grown in C57Bl wild-type mice and mice lacking expression of either tumor necrosis factor receptor-1 (TNFR1(-/-)) or TNF (TNF-/-). Tumor vascular permeability, as measured by extravasation of albumin-Evans Blue complexes 4 hr after DMXAA treatment, was significantly increased in tumor tissue in C57Bl, TNFR1-/- and TNF-/- mice but not in normal (skin) tissue. Significant linear relationships were found between increased tumor vascular permeability, decreased functioning tumor blood vessels (measured by Hoechst 33342 staining at 4 hr), increased plasma 5-hydroxyindole-3-acetic acid concentrations (as a measure of serotonin release by platelets) and the degree of induced tumor hemorrhagic necrosis. The results support the hypothesis that DMXAA increases tumor vascular permeability both directly and through the induction of other vasoactive mediators, including TNF. DMXAA might be useful clinically to potentiate the vascular permeability of other anticancer modalities such as cytotoxic drugs, antibodies, drug conjugates and gene therapy.
Asunto(s)
Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/patología , Antineoplásicos/farmacología , Permeabilidad Capilar/efectos de los fármacos , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/patología , Neovascularización Patológica , Xantonas/farmacología , Adenocarcinoma/veterinaria , Animales , Neoplasias del Colon/veterinaria , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales , Receptores Tipo I de Factores de Necrosis Tumoral/biosíntesis , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a potent cytokine inducer, exhibited marked antitumor activity when given as multiple oral doses in mice. The aim of this study was to examine the transport of DMXAA and its acyl glucuronide (DMXAA-G) using the human Caco-2 cells. DMXAA was minimally metabolized by Caco-2 cells and both DMXAA and DMXAA-G were taken up to a minor extent by the cells. The permeability coefficient (Papp) values of DMXAA over 10-500 microM were 4x10(-5) cm/s to 4.3x10(-5) cm/s for both apical (AP) to basolateral (BL) and BL-AP transport, while the Papp values for the BL to AP flux of DMXAA-G were significantly greater than those for the AP to BL flux, with Rnet values of 4.5-17.6 over 50-200 microM. The BL to AP active efflux of DMXAA-G followed Michaelis-Menten kinetics, with a Km of 83.5+/-5.5 microM, and Vmax of 0.022+/-0.001 nmol/min. The flux of DMXAA-G was energy and Na+-dependent and MK-571 significantly (P<0.05) inhibited its BL to AP flux, with an estimated Ki of 130 microM. These data indicate that the transport of DMXAA across Caco-2 monolayers was through a passive process, whereas the transport of DMXAA-G was mediated by MRP1/2.
Asunto(s)
Antineoplásicos/farmacocinética , Glucurónidos/farmacocinética , Xantonas/farmacocinética , Adenosina Trifosfato/antagonistas & inhibidores , Transporte Biológico , Células CACO-2 , Estabilidad de Medicamentos , Humanos , Concentración de Iones de Hidrógeno , Temperatura , Xantonas/farmacologíaRESUMEN
A specific and efficient liquid chromatography-mass spectrometry (LC-MS) method was established for monitoring patient plasma cyclophosphamide levels in a phase I trial of an oral cyclophosphamide-based combination chemotherapy regimen. An Agilent 1100 Series LC-MSD system (Agilent Technologies, Avondale, PA, USA), with a single quadrupole mass detector using a positive atmospheric pressure chemical ionization (APCI) interface and single ion monitoring at m/z 261, was used. Chromatography was performed using a LUNA C8 5 microm 30 x 4.6 mm stainless steel column (Phenomenex, Torrance, CA, USA) and a mobile phase of aqueous acetonitrile pumped at a flow rate of 0.7 mL/min. High-throughput solid-phase sample extraction was performed using a Gilson ASPEC XL4 system (Gilson Medical, Middleton, WI, USA) controlled by prestored programs. The standard curve for cyclophosphamide was linear over the concentration range 0.026-1.08 microg/mL (r(2) > 0.994). Intra- and interassay accuracy and precision were 97-107 and 3-10%, respectively. The limit of detection was determined to be 0.01 microg/mL. Single ion monitoring at m/z 261 provided a high degree of specificity without interference from the matrix or other chemotherapy drugs. Automated sample processing allowed the analysis of a large number of plasma samples from a clinical trial of repeated daily oral dosing of cyclophosphamide. One hour after dosing, cyclophosphamide was detected in 98 of 106 plasma specimens at concentrations ranging between 0.03 and 4.88 microg/mL. Twenty-four hours after dosing, cyclophosphamide was detected in 72 of 77 plasma specimens at concentrations ranging between 0.06 and 3.13 microg/mL. There were no time-dependent changes in cyclophosphamide concentration during the 43 day period of repeated daily oral dosing. There was no correlation between cyclophosphamide dose and plasma concentration, despite the wide range of doses given in the clinical trial (50-125 mg/m(2)). We conclude that a solid-phase extraction LC-MS technique was validated for determining cyclophosphamide in human plasma. Interoccasion variability in the rate of oral absorption and in the clearance of systemically available drug may have contributed to the wide range of cyclophosphamide concentrations found at 1 and 24 h after tablet ingestion.
Asunto(s)
Antineoplásicos/sangre , Ciclofosfamida/sangre , Neoplasias/sangre , Antineoplásicos/uso terapéutico , Autoanálisis , Calibración , Cromatografía Liquida , Ensayos Clínicos Fase I como Asunto , Ciclofosfamida/uso terapéutico , Humanos , Espectrometría de Masas , Monitoreo Fisiológico , Neoplasias/tratamiento farmacológico , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
PURPOSE: Thalidomide has a variety of biological effects that vary considerably according to the species tested. We sought to establish whether differences in pharmacokinetics could form a basis for the species-specific effects of thalidomide. EXPERIMENTAL DESIGN: Mice and rabbits were administered thalidomide (2 mg/kg) p.o. or i.v., and plasma concentrations of thalidomide were measured after drug administration using high performance liquid chromotography. Plasma samples from five multiple myeloma patients over 24 hours after their first dose of thalidomide (200 mg) were similarly analyzed and all data were fitted to a one-compartment model. Metabolites of thalidomide in plasma were identified simultaneously using liquid chromatography-mass spectrometry. RESULTS: Plasma concentration-time profiles for the individual patients were very similar to each other, but widely different pharmacokinetic properties were found between patients compared with those in mice or rabbits. Area under the concentration curve values for mice, rabbits, and multiple myeloma patients were 4, 8, and 81 micromol/L. hour, respectively, and corresponding elimination half-lives were 0.5, 2.2, and 7.3 hours, respectively. Large differences were also observed between the metabolite profiles from the three species. Hydrolysis products were detected for all species, and the proportion of hydroxylated metabolites was higher in mice than in rabbits and undetectable in patients. CONCLUSIONS: Our results show major interspecies differences in the pharmacokinetics of thalidomide that are related to the altered degree of metabolism. We suggest that the interspecies differences in biological effects of thalidomide may be attributable, at least in part, to the differences in its metabolism and hence pharmacokinetics.
Asunto(s)
Inhibidores de la Angiogénesis/metabolismo , Inhibidores de la Angiogénesis/farmacocinética , Mieloma Múltiple/sangre , Talidomida/metabolismo , Talidomida/farmacocinética , Adulto , Anciano , Anciano de 80 o más Años , Inhibidores de la Angiogénesis/administración & dosificación , Animales , Área Bajo la Curva , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Femenino , Humanos , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Conejos , Especificidad de la Especie , Talidomida/administración & dosificaciónRESUMEN
5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a potent cytokine inducer, with a bioavailability of >70% in the mouse. The aim of this study was to develop and validate HPLC methods for the determination of DMXAA and DMXAA acyl glucuronide (DMXAA-G) in the human intestinal cell line Caco-2 monolayers. The developed HPLC methods were sensitive and reliable, with acceptable accuracy (85-115% of true values) and precision (intra- and inter-assay CV < 15%). The total running time was within 6.8 min, with acceptable separation of the compounds of interest. The limit of quantitation (LOQ) values for DMXAA and DMXAA-G were 14.2 and 24 ng/ml, respectively. The validated HPLC methods were applied to examine the epithelial transport of DMXAA and DMXAA-G by Caco-2 monolayers. The permeability coefficient (Papp) values (overall mean +/- S.D., n = 3-9) of DMXAA over 10-500 microM were independent of concentration for both apical (AP) to basolateral (BL) (4.0 +/- 0.4 x 10(-5)cm/s) and BL-AP (4.3 +/- 0.5 x 10(-5)cm/s) transport, and of similar magnitude in either direction, with net efflux ratio (Rnet) values of 1-1.3. However, the Papp values for the BL to AP transport of DMXAA-G were significantly greater than those for the AP to BL transport, with Rnet values of 17.6, 6.7 and 4.5 at 50, 100 and 200 microM, respectively. Further studies showed that the transport of DMXAA-G was Na+- and energy-dependent, and inhibited by MK-571 [a multidrug resistance associated protein (MRP) 1/2 inhibitor], but not by verapamil and probenecid. These data indicate that the HPLC methods for the determination of DMXAA and DMXAA-G in the transport buffer were simple and reliable, and the methods have been applied to the transport study of both compounds by Caco-2 monolayers. DMXAA across Caco-2 monolayers was through a passive transcellular process, whereas the transport of DMXAA-G was mediated by MRP1/2.
Asunto(s)
Antineoplásicos/análisis , Cromatografía Líquida de Alta Presión/métodos , Glucurónidos/análisis , Espectrometría de Fluorescencia/métodos , Xantonas/análisis , Células CACO-2 , Humanos , Sensibilidad y EspecificidadRESUMEN
The induction of haemorrhagic necrosis by 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in transplantable murine tumours depends on the in situ synthesis of cytokines, particularly tumour necrosis factor (TNF). Since the in vivo action of DMXAA would be greatly clarified by the development of an in vitro model, we investigated whether DMXAA could induce cytokines in cultured murine splenocytes. DMXAA alone induced low amounts of TNF with an optimal concentration of 10 microg/mL and an optimal time of 4 hr. When combined with low concentrations of lipopolysaccharide, deactivated-lipopolysaccharide (dLPS) or phorbol-12-myristate-13-acetate that did not elicit TNF production alone, synergistic TNF production was obtained. DMXAA also induced interferon-gamma at an optimal dose of 300 microg/mL, but the addition of dLPS had no further effect. Decreasing culture pH, although not changing the optimal concentrations for stimulation, increased both TNF and interferon-gamma production in response to DMXAA. The major DMXAA metabolites, DMXAA-glucuronide and 6-hydroxy-5-methylxanthenone-4-acetic acid, did not induce either cytokine alone, in combination with dLPS or at low pH. The results indicate that DMXAA rather than a metabolite is responsible for cytokine induction and suggest that the microenvironment of the tumour may be responsible for the observed selective induction of cytokines in tumour tissue.
Asunto(s)
Interferón gamma/metabolismo , Bazo/citología , Factor de Necrosis Tumoral alfa/metabolismo , Xantonas/farmacología , Animales , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Células Cultivadas , Citocinas/biosíntesis , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos C57BL , Xantonas/metabolismoRESUMEN
Thalidomide is increasingly important in clinical treatment, not only of various inflammatory conditions but also in multiple myeloma and other malignancies. Moreover, the metabolism of thalidomide varies considerably among different species, indicating a need to understand its mechanistic basis. Our previous in vivo studies showed the plasma half-life of thalidomide to be much shorter in mice than in humans, with rabbits showing intermediate values. We were unable to detect hydroxylated thalidomide metabolites in humans and suggested that interspecies differences in thalidomide hydroxylation might account for the differences in plasma half-life. We sought here to establish whether these species differences in the formation of hydroxylated thalidomide metabolites could be discerned from in vitro studies. Liver microsomes of mice, rabbit, and human donors were incubated with thalidomide and analyzed using liquid chromatography-mass spectrometry. Hydrolysis products were detected for all three species, and the rates of formation were similar to those for spontaneous hydrolysis, except in rabbits where phthaloylisoglutamine formation increased linearly with microsomal enzyme concentration. Multiple hydroxylation products were detected, including three dihydroxylated metabolites not observed in vivo. Thalidomide-5-O-glucuronide, detected in vivo, was absent in vitro. The amount of 5-hydroxythalidomide formed was high in mice, lower in rabbits, and barely detectable in humans. We conclude that major interspecies differences in hepatic metabolism of thalidomide relate closely to the rate of in vivo metabolite formation. The very low rate of in vitro and in vivo hydroxylation in humans strongly suggests that thalidomide hydroxylation is not a requirement for clinical anticancer activity.
Asunto(s)
Microsomas Hepáticos/metabolismo , Talidomida/metabolismo , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/química , Conejos , Especificidad de la Especie , Talidomida/análisis , Talidomida/químicaRESUMEN
PURPOSE: There is considerable current interest in the use of thalidomide as a single agent or in combination with drugs such as cyclophosphamide in the treatment of multiple myeloma and other cancers. Our previous work has shown that thalidomide potentiates the antitumour activity of both cyclophosphamide and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) against murine Colon 38 tumours. In both of these cases, thalidomide extends the half-life (t(1/2)) of the other drug. We wished to determine whether cyclophosphamide and DMXAA altered the t(1/2) of thalidomide. Since both thalidomide and DMXAA modulate tumour necrosis factor (TNF), we also wished to determine the role of TNF in this interaction. METHODS: Mice with Colon 38 tumours were treated with cyclophosphamide (220 mg/kg) and/or thalidomide (20 mg/kg) or DMXAA (25 mg/kg) and thalidomide (100 mg/kg), combinations that have previously demonstrated synergistic activity. Plasma and tumour tissue drug concentrations were analysed by high-performance liquid chromatography. To determine the role of TNF, similar experiments were performed using mice defective in the TNF gene (TNF(-/-)) or the TNF receptor-1 gene (TNFR1(-/-)). RESULTS: Coadministration of cyclophosphamide increased the thalidomide t(1/2) by 3.9- and 3.6-fold, respectively, in plasma and tumour tissue, with a corresponding increase in the concentration-time curve (AUC). The corresponding values following coadministration of DMXAA were 3.0- and 4.6-fold, respectively. Coadministration of cyclophosphamide had similar effects on thalidomide t(1/2) in C57Bl/6, TNF(-/-) and TNFR1(-/-) mice, while coadministration of DMXAA did not alter the t(1/2) or AUC in TNF(-/-) and TNFR1(-/-) mice. CONCLUSIONS: Both cyclophosphamide and DMXAA have a pharmacokinetic interaction with thalidomide, increasing t(1/2) and AUC. TNF mediates the effect of DMXAA on thalidomide pharmacokinetics but not that of cyclophosphamide.
Asunto(s)
Ciclofosfamida/farmacología , Talidomida/farmacocinética , Xantonas/farmacología , Animales , Área Bajo la Curva , Interacciones Farmacológicas , Ratones , Ratones Endogámicos C57BL , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
PURPOSE: 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a new anticancer drug that has recently completed Phase I clinical trial, is effective against transplantable murine tumors with established vasculature. We wished to determine the relationship between administration schedule and antitumor activity. EXPERIMENTAL DESIGN: C57Bl/6 mice with s.c. implanted Colon 38 tumors were used for determination of maximal tolerated doses and tumor growth delay. Plasma and tissue DMXAA concentrations were measured by high-performance liquid chromatography. RESULTS: Continuous infusion (30 mg/kg/day for 3 days) and daily i.p. administration schedules (7.5 mg/kg) were ineffective. A pharmacokinetically guided schedule was developed to increase tumor tissue drug concentrations without increasing the maximal plasma concentration. A schedule comprising a loading dose (25 mg/kg, i.p.) followed by supplementary doses (5 mg/kg after 4 and 8 h) provided a 1.6-fold increase in tumor tissue area under the concentration-time curve, no increased toxicity, and superior antitumor activity (100% cure rate, as compared with 55% for a single i.p. dose of 25 mg/kg). A similar strategy was developed for oral administration with a loading dose (30 mg/kg) and supplementary doses (15 mg/kg after 4 and 8 h). It provided a 90% cure rate, in contrast to a single oral dose (0% cure rate). CONCLUSIONS: The antitumor action of DMXAA is schedule dependent, and the achievement of an adequate tumor tissue DMXAA concentration above a threshold value appears to be critical for activity. The use of a pharmacokinetically guided schedule provides excellent oral activity against Colon 38 tumors and provides a basis for developing more effective administration schedules in clinical trials.