RESUMEN
Arsenic is a proven human carcinogen and is associated with a myriad of other adverse health effects. This metalloid is methylated in human liver to monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), dimethylarsinic acid (DMA(V)), and dimethylarsinous acid (DMA(III)) and eliminated predominantly in urine. Hepatic basolateral transport of arsenic species is ultimately critical for urinary elimination; however, these pathways are not fully elucidated in humans. A potentially important human hepatic basolateral transporter is the ATP-binding cassette (ABC) transporter multidrug resistance protein 4 (MRP4/ABCC4) that in vitro is a high-affinity transporter of DMA(V) and the diglutathione conjugate of MMA(III) [MMA(GS)(2)]. In rats, the related canalicular transporter Mrp2/Abcc2 is required for biliary excretion of arsenic as As(GS)(3) and MMA(GS)(2). The current study used sandwich cultured human hepatocytes (SCHH) as a physiological model of human arsenic hepatobiliary transport. Arsenic efflux was detected only across the basolateral membrane for 9 out of 14 SCHH preparations, 5 had both basolateral and canalicular efflux. Basolateral transport of arsenic was temperature- and GSH-dependent and inhibited by the MRP inhibitor MK-571. Canalicular efflux was completely lost after GSH depletion suggesting MRP2-dependence. Treatment of SCHH with As(III) (0.1-1 µM) dose-dependently increased MRP2 and MRP4 levels, but not MRP1, MRP6, or aquaglyceroporin 9. Treatment of SCHH with oltipraz (Nrf2 activator) increased MRP4 levels and basolateral efflux of arsenic. In contrast, oltipraz increased MRP2 levels without increasing biliary excretion. These results suggest arsenic basolateral transport prevails over biliary excretion and is mediated at least in part by MRPs, most likely including MRP4.
Asunto(s)
Arsénico/metabolismo , Conductos Biliares/metabolismo , Hepatocitos/metabolismo , Conductos Biliares/efectos de los fármacos , Transporte Biológico , Técnicas de Cultivo de Célula , Relación Dosis-Respuesta a Droga , Glutatión/metabolismo , Células HEK293 , Hepatocitos/efectos de los fármacos , Humanos , Cinética , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Factor 2 Relacionado con NF-E2/agonistas , Factor 2 Relacionado con NF-E2/metabolismo , Propionatos/farmacología , Pirazinas/farmacología , Quinolinas/farmacología , Temperatura , Tionas , Tiofenos , TransfecciónRESUMEN
PURPOSE: Pharmacokinetic studies on liposomal drugs have previously measured total drug levels in tumors, which include non-bioavailable drug. However, drugs must be released from liposomes to have activity. We have developed a method for measuring levels of bioavailable (released) doxorubicin in vivo in tumors that will allow therapeutic activity to be correlated with bioavailable drug levels. EXPERIMENTAL DESIGN: Mice orthotopically implanted with mammary carcinoma (4T1) were injected i.v. 10 days after implantation with free doxorubicin or formulations of liposomal doxorubicin with different drug release rates. Tumors were excised at various times after injection, and total tumor doxorubicin levels were determined by acidified isopropanol extraction of whole tumor homogenates. Bioavailable doxorubicin levels were determined by extraction of doxorubicin from isolated tumor nuclei. RESULTS: Free doxorubicin had high levels of bioavailability in tumor tissue; 95% of the total doxorubicin in tumors was bound to nuclear DNA by 24 hours after injection. Administration of Doxil, a slow release liposomal formulation of doxorubicin, gave an area under the time-versus-concentration curve (AUC) for total doxorubicin 7 days after injection that was 87-fold higher than that obtained for free doxorubicin, and 49% of the liposomal doxorubicin was bioavailable. For liposomes with a more rapid doxorubicin release rate, by 7 days after injection, the AUC(0-7 days) for total doxorubicin was only 14-fold higher than that for free doxorubicin and only 27% of liposomal doxorubicin was bioavailable. CONCLUSIONS: This technique allows correlations to be made between drug bioavailability and therapeutic activity and will help in the rational design of drug carriers.
Asunto(s)
Antibióticos Antineoplásicos/farmacocinética , Núcleo Celular/metabolismo , Doxorrubicina/farmacocinética , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/metabolismo , Animales , Antibióticos Antineoplásicos/química , Área Bajo la Curva , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Doxorrubicina/química , Femenino , Liposomas/química , Ratones , Ratones Endogámicos BALB C , Factores de TiempoRESUMEN
Some formulations of liposomal doxorubicin with intermediate rates of drug release have shown increased levels of toxicity in mice. Because antibody-mediated targeting of liposomal drugs influences the pharmacokinetics, mechanism of uptake, and selectivity of the associated drugs, we hypothesized that anti-CD19-mediated targeting of liposomal doxorubicin might moderate the toxicity of the problem formulations. Phosphatidylcholine/cholesterol liposomal formulations of doxorubicin having faster, intermediate, and slower drug release rates were prepared by altering the fatty acyl chain length or degree of saturation of the phosphatidylcholine component. Pharmacokinetic and biodistribution studies and in vivo drug release rates were determined in mice using liposomes dual labeled with [3H]cholesteryl hexadecylether and [14C]doxorubicin. Therapeutic studies were done in xenograft models of human B lymphoma (Namalwa cells). The rate of clearance of the liposomal lipid was similar for all formulations (average t1/2, 18 hours), but the rate of clearance of doxorubicin was dependent on the release rate of the formulation (t1/2, 2-315 hours). Liposomes with the slowest drug release rates showed no toxicity and exhibited therapeutic activity that was superior to the other formulations when targeted with anti-CD19; liposomes with the most rapid drug release rates also showed no toxicity but showed little therapeutic effect even when targeted. Liposomes with intermediate drug release rates exhibited varying degrees of toxicity. The toxicities could be reduced and even overcome by targeting with anti-CD19 antibodies. For these formulations, therapeutic effects were intermediate between those found for liposomes with the fastest and slowest drug release rates.
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD19/inmunología , Doxorrubicina/uso terapéutico , Linfoma de Células B/tratamiento farmacológico , Animales , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/farmacocinética , Anticuerpos Monoclonales/química , Área Bajo la Curva , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacocinética , Sistemas de Liberación de Medicamentos/métodos , Femenino , Humanos , Liposomas/química , Linfoma de Células B/patología , Ratones , Ratones SCID , Fosfolípidos/química , Análisis de Supervivencia , Distribución Tisular , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The pharmacokinetics (PK), biodistribution (BD), and therapeutic activity of pegylated liposomal doxorubicin formulations with different drug release rates were studied in an orthotopic 4T1 murine mammary carcinoma model. The focus of these experiments was to study the effects of different release rates on the accumulation of liposomal lipid and doxorubicin (DXR) into the tumor and cutaneous tissues of mice (skin and paws). These tissues were chosen because the clinical formulation of pegylated liposomal doxorubicin (Caelyx)/Doxi) causes mucocutaneous reactions such as palmar-plantar erythrodysesthesia (PPE). Liposomes with different doxorubicin (DXR) leakage rates were prepared by altering liposome fluidity through changing the fatty acyl chain length and/or degree of saturation of the phosphatidylcholine component of the liposome. Liposomes with fast, intermediate, and slow rates of drug release were studied. The plasma PK of the liposomal lipid was similar for all formulations, while the plasma PK of the DXR component was dependent on the liposome formulation. Liposomal lipid accumulated to similar levels in tumor and cutaneous tissues for all three formulations tested, while the liposomes with the slowest rates of DXR release produced the highest DXR concentrations in both cutaneous tissues and in tumor. Liposomes with the fastest drug release rates resulted in low DXR concentrations in cutaneous tissues and tumor. The formulation with intermediate release rates produced unexpected toxicity that was not related to the lipid content of the formulation. The liposomes with the slowest rate of drug leakage had the best therapeutic activity of the formulations tested.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/farmacocinética , Doxorrubicina/farmacología , Doxorrubicina/farmacocinética , Liposomas/química , Polietilenglicoles/química , Animales , Antibióticos Antineoplásicos/sangre , Antibióticos Antineoplásicos/química , Área Bajo la Curva , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Doxorrubicina/sangre , Doxorrubicina/química , Femenino , Semivida , Inyecciones Intravenosas , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Fosfatidilcolinas/química , Distribución Tisular , Temperatura de TransiciónRESUMEN
Effects of multiple injections of liposomal doxorubicin on pharmacokinetics, therapeutic outcome, and toxicity were studied in mice using different dosing schedules and dose intensities. Biodistribution of doxorubicin to the cutaneous tissues of mice (skin and paws) and to orthotopically implanted mammary tumors (4T1) was examined. Weekly intravenous administration of pegylated (STEALTH) liposomal doxorubicin (SL-DXR) at a dose of 9 mg/kg (every week x 4 doses) resulted in accumulation of doxorubicin in cutaneous tissues of mice and development of lesions resembling palmar-plantar erythrodysesthesia (PPE). Lengthening the dose interval to every 2 weeks x 4 doses reduced the accumulation of doxorubicin and lowered the incidence of PPE-like lesions. A dose interval of every 4 weeks x 4 resulted in complete clearance of doxorubicin from tissues between subsequent doses and a negligible incidence of PPE-like lesions. Doses of 9 mg/kg SL-DXR given at every week x 2 or every 2 weeks x 2 had similar therapeutic activities, whereas prolonging the dose interval to every 4 weeks x 2 reduced therapeutic activity. Pharmacokinetics, biodistribution, and therapeutic activity were studied in tumor-bearing mice for three dose schedules having the same dose intensity (4.5 mg/kg every 3 days x 4, 9 mg/kg every week x 2, or 18 mg/kg every 2 weeks x 1). For these schedules, larger doses administered less often tended to be superior therapeutically to smaller doses given more often. These data provide the first pharmacokinetic measurements of doxorubicin concentrations in cutaneous tissues and tumors with repeat administration of liposomal formulations, and they provide a useful model for the study of factors leading to PPE in humans.
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Doxorrubicina/uso terapéutico , Neoplasias Experimentales/tratamiento farmacológico , Animales , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/farmacocinética , Modelos Animales de Enfermedad , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacocinética , Femenino , Liposomas , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Neoplasias Experimentales/metabolismo , Distribución Tisular , Resultado del TratamientoRESUMEN
The influence of diameter on the pharmacokinetic and biodistribution of STEALTH liposomes into the tumor (4T1 murine mammary carcinoma) and cutaneous tissues (skin and paws) of mice was studied to ascertain the time course of liposome accumulation and to determine if a preferential accumulation of liposomes into tumor over skin or paws could be achieved by altering liposome size. These tissues were chosen as the dose-limiting toxicity for Caelyx/Doxil in humans is palmar-plantar erythrodysesthesia, a cutaneous toxicity. We examined liposomes of four diameters: 82, 101, 154, or 241 nm. Liposomes with the three smallest diameters showed similar accumulation profiles that were significantly higher than the largest liposomes in all three tissues of interest. We were unable to achieve a preferential accumulation of liposomes into tumor over skin or paws based on size alone, as evidenced by the tumor to skin and tumor to paw ratios. However, there were differences in the time courses of liposome accumulation in these three tissues. Liposome levels plateaued in tumors and paws within 24 h, whereas skin levels plateaued between 24 and 48 h. The therapeutic activity of liposomal doxorubicin of three diameters (100, 157, and 255 nm) was tested in the same model. All formulations delayed tumor growth, with liposomes of 100 or 157 nm being equally efficacious and superior to liposomes of 255 nm.