RESUMO
Immunoliposomes composed of hydrogenated soy phosphatidylcholine, cholesterol, methoxypoly(ethylene glycol)-distearoyl phosphatidylethanolamine (mPEG-DSPE), and hydrazide-PEG-DSPE (mole ratio, 57:38:3.3:1.7) linked to periodate-oxidized chimerized mouse IgG (C225, anti-human epidermal growth factor receptor) were prepared by an optimized aggregation-free procedure. The antigen-binding activity of the immunoliposomes was well preserved. When injected intravenously into naive rats, the immunoliposomes (approximately 18 IgG per 100 nm liposome) exhibited long circulation times (MRT = 8.5 h, Cl = 0.2 ml/h). Subsequent injections of the immunoliposomes into the same animals resulted in rapid clearance (MRT < or = 0.7 h, Cl > or = 7 ml/h), which was accompanied by a significant increase in anti-C225 specific titers. Upon repeated injection or coinjection with the parent liposomes free C225 consistently exhibited prolonged circulation without any increase in C225-specific antisera, but was cleared quickly when administered into animals that had been pretreated with the immunoliposomes. Screening of the immunoliposome induced antisera against human polyclonal IgG and C225-derived Fab' fragment revealed that the immune response was specifically triggered by the constant human region of C225. These results demonstrate that the preparations of PEG-grafted immunoliposomes are more immunogenic than the free IgG component, which is of profound importance to the antibody-mediated liposomal drug delivery effort.
Assuntos
Sistemas de Liberação de Medicamentos , Lipossomos/imunologia , Polietilenoglicóis , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/imunologia , Citometria de Fluxo , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Lipossomos/farmacocinética , Masculino , Camundongos , Fosfatidiletanolaminas , Ratos , Ratos Sprague-Dawley , Células Tumorais CultivadasRESUMO
The topoisomerase I inhibitor GL147211C [7-[(4-methylpiperazino)methyl]-10,11-(ethylenedioxy)-(20S)-campto thecin trifluoroacetate], a camptothecin analogue, has significant activity in tumor cell cytotoxicity assays in vitro and antitumor activity in both animal tumor models and human patients. Its toxicity is significant, however, effectively limiting the amount of drug that can be administered and its clinical utility. To determine whether the therapeutic index of GL147211C could be improved, the drug was encapsulated in long-circulating, pegylated (STEALTH) liposomes (SPI-355). The pharmacokinetics and antitumor activity of SPI-355 were compared to those of nonliposomal GL147211C. The plasma pharmacokinetics of SPI-355 in rats were typical of those of other pegylated liposomal formulations, with significantly increased blood circulation time; the dose-corrected area under the curve and Cmax of SPI-355 (10 mg/kg) were 1250- and 35-fold higher, respectively, than those of nonliposomal GL14711C (8.72 mg/kg). The comparative antitumor activity of SPI-355 and nonliposomal GL1472211C was evaluated in nude mice implanted with HT29 colon carcinoma xenografts. SPI-355 was 20-fold more effective than GL147211C in inhibiting tumor growth (1 mg/kg SPI-355 and 20 mg/kg GL147211C) and produced durable complete remissions of tumors at well-tolerated dose levels that were >5-fold lower than the maximally tolerated dose of GL147211C, which induced no durable complete responses. Signs of toxicity were similar between the two drugs, but liposome encapsulation increased the toxicity of drug approximately 4-fold, with increased weight loss and several deaths with SPI-355 (5 mg/kg SPI-355 versus 20 mg/kg GL147211C). Despite the increased toxicity seen with SPI-355, the therapeutic index of the liposomal formulation was increased approximately 5-fold over that of nonliposomal GL147211C, suggesting that such a pegylated liposomal formulation could demonstrate increased therapeutic index in human patients.
Assuntos
Antineoplásicos/uso terapêutico , Camptotecina/análogos & derivados , Neoplasias do Colo/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores da Topoisomerase I , Animais , Antineoplásicos/farmacocinética , Camptotecina/farmacocinética , Camptotecina/uso terapêutico , Neoplasias do Colo/metabolismo , Portadores de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HT29 , Humanos , Lipossomos , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Ratos , Ratos Sprague-Dawley , Transplante Heterólogo , Resultado do TratamentoRESUMO
A 20-fold increase in the relative expression of the hepatic GH-binding protein (GHBP)-encoding message between nonpregnant and 17-day pregnant mice was found. The hepatic GH receptor (GHR)-encoding message increased 8-fold between nonpregnant and pregnant mice. The increase in both messages began on day 9 of pregnancy. The steady state level of the GHBP-encoding message continued to increase steadily until day 17 of pregnancy; however, by day 13 of pregnancy, the steady state level of the GHR-encoding message reached a plateau that continued to day 17. The ratio between the GHBP- and GHR-encoding messages gradually increased during the second half of pregnancy, reaching a maximum on day 17. There was a 10- to 16-fold increase in GH-binding capacity in liver microsomes and a 30- to 50-fold increase in serum GH-binding capacity between nonpregnant and late pregnant mice. The increase in hepatic GH-binding capacity began on day 9 of pregnancy and reached a plateau on day 11, which was maintained until the end of gestation. The increase in serum GH-binding capacity began on day 9 of pregnancy and continued to increase until day 17. No significant change in mouse (m) GHR (mGHR) or mGHBP affinity constants were observed between nonpregnant and pregnant mice; however, the mGHR had a 20-fold greater affinity for mGH than did the mGHBP. The serum GH concentration increased in the second half of pregnancy. The GHBP-bound and the free fractions of GH during pregnancy were predicted. While the bound fraction of GH is predicted to parallel the total GH concentration measured by RIA, the concentration of free mouse GH remains unchanged during pregnancy.
Assuntos
Expressão Gênica , Prenhez/metabolismo , Receptores da Somatotropina/genética , Animais , Northern Blotting , Proteínas de Transporte/metabolismo , Feminino , Hormônio do Crescimento/sangue , Hormônio do Crescimento/metabolismo , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Receptores da Somatotropina/metabolismo , Fatores de TempoRESUMO
A RIA for mouse growth hormone-binding protein (mGHBP) was developed. A 28-amino acid synthetic peptide corresponding to the carboxyl-terminal 27 amino acids of mGHBP plus an additional cysteine residue at the amino-terminus was coupled to keyhole limpet hemocyanin and used as the antigen for antiserum production. The ability of the antiserum to recognize mGHBP was demonstrated by incubating mouse serum with 125I-iodomouse GH (mGH) in the presence of increasing concentrations of antiserum and subsequent immunoprecipitation. The antiserum precipitated mGHBP in a dose-dependent manner. The uncoupled synthetic peptide was used as the standard and radioligand in the RIA. Serial dilutions of sera from non-pregnant or 17-days-pregnant mice yielded displacement curves parallel to the synthetic peptide, with serum from 17-days-pregnant mice being 32 times more effective than serum from non-pregnant mice for a given dilution. The relative concentration of mGHBP in maternal serum on days 5, 11, and 15 of pregnancy was 1, 17, and 39, respectively.
Assuntos
Proteínas de Transporte/sangue , Prenhez/sangue , Animais , Ligação Competitiva , Feminino , Hormônio do Crescimento/metabolismo , Cinética , Camundongos , Gravidez , Coelhos , Radioimunoensaio/métodosRESUMO
PURPOSE: The pharmacokinetics (PK), biodistribution and therapeutic efficacy of cisplatin encapsulated in long-circulating pegylated (Stealth) liposomes (SPI-077) were compared with those of nonliposomal cisplatin in two murine (C26 colon carcinoma and Lewis lung) tumor models. METHODS: In therapeutic effectiveness studies, mice bearing murine C26 or Lewis lung tumors received multiple intravenous doses of SPI-077 or cisplatin in a variety of treatment schedules and cumulative doses. In the PK and biodistribution study, mice received a single intravenous bolus injection of 3 mg/kg of either SPI-077 or cisplatin 14 days after inoculation with 10(6) C26 tumor cells. Plasma and tissues were analyzed for total platinum (Pt) content by graphite furnace (flameless) atomic absorption spectrophotometery (GF-AAS). RESULTS: Efficacy studies showed that SPI-077 had superior antitumor activity compared to the same cumulative dose of cisplatin. When lower doses of SPI-077 were compared to cisplatin at its maximally tolerated dose in Lewis lung tumors, equivalent SPI-077 antitumor activity was seen at only half the cisplatin dose. Higher cumulative doses of SPI-077 were well tolerated and had increased antitumor effect. SPI-077 PK were characterized by a one-compartment model with nonlinear (saturable) elimination, whereas cisplatin PK were described by a two-compartment model with linear elimination. SPI-077 had a 55-fold lower [corrected] volume of distribution, 3-fold higher peak plasma levels, and a 60-fold larger plasma AUC compared with cisplatin. In addition, SPI-077-treated animals displayed a 4-fold reduction in Pt delivered to the kidneys (primary target organ of toxicity) relative to cisplatin, but a 28-fold higher tumor AUC than cisplatin. CONCLUSIONS: Based on the results of our studies, encapsulation of cisplatin in long-circulating pegylated liposomes has overcome limitations experienced with other liposomal cisplatin formulations. SPI-077 has a prolonged circulation time and increased tumor Pt disposition, and its antitumor effect is significantly improved compared to cisplatin in murine colon and lung cancer models.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Cisplatino/farmacologia , Cisplatino/farmacocinética , Algoritmos , Animais , Antineoplásicos/administração & dosagem , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/metabolismo , Cisplatino/administração & dosagem , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Portadores de Fármacos , Injeções Intravenosas , Lipossomos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Ratos , Espectrofotometria Atômica , Distribuição TecidualRESUMO
Poly(ethylene glycol) (PEG)-derivatized liposome vehicles improve antitumor effectiveness of entrapped anthracyclines and vinca alkaloids. However, the plasma clearance of entrapped vincristine is substantially faster than the lipid phase or other entrapped aqueous markers, suggesting leakage out of the liposome during transit in the blood compartment. We tested the effect of altering the drug's in vivo leakage rate on pharmacokinetics, toxicity, and antitumor activity of entrapped drug in rodent models. Suramin, heparin, and dextran sulfate were tested for their ability to produce a precipitable complex in vitro. PEG-derivatized liposomes were prepared with the complexing agent inside, and vincristine was driven inside using an ammonium gradient. The resulting preparations were found to have plasma distribution half-lives significantly longer than the formulation without a complex-forming agent. There was no increase in acute lethality, and in the case of the suramin-vincristine complex, the acute lethality was significantly reduced at the highest does level. Anti-tumor activity against the mouse mammary carcinoma MC2 was tested in a multiple-dose study. Free vincristine did not affect the tumor growth rate significantly, but at the same dose level all PEG-coated liposome formulations inhibited tumor growth markedly. The suramin containing formulation was as effective as the formulation lacking polyanion, but the heparin and dextran sulfate containing formulations were less effective. Thus, compounds which form insoluble complexes with vincristine alter in vivo plasma distribution phase pharmacokinetics without increasing acute lethality, but without a corresponding increase in anti-tumor activity.
Assuntos
Antineoplásicos Fitogênicos/farmacocinética , Antineoplásicos Fitogênicos/toxicidade , Neoplasias Mamárias Experimentais/metabolismo , Vincristina/farmacocinética , Vincristina/toxicidade , Animais , Ânions , Anticoagulantes , Antineoplásicos , Antineoplásicos Fitogênicos/sangue , Sulfato de Dextrana , Portadores de Fármacos , Feminino , Heparina , Injeções Intravenosas , Lipossomos , Masculino , Neoplasias Mamárias Experimentais/tratamento farmacológico , Camundongos , Camundongos Endogâmicos ICR , Polietilenoglicóis , Ratos , Ratos Sprague-Dawley , Suramina , Vincristina/sangueRESUMO
Lipid-conjugates of two amphipatic polymers, poly(2-methyl-2-oxazoline) (PMOZ) and poly(2-ethyl-2-oxazoline) (PEOZ) (degree of polymerization approximately 50) were synthesized by linking glutarate esters of the polymers to distearoylphosphatidylethanolamine (DSPE) or alternatively by termination of the polymerization process with DSPE. Surface-modified liposomes (90 +/- 5 nm) prepared from either conjugate (5 mol % of total lipid) were injected into rats and followed by blood level and tissue distribution measurements. Both polymers PEOZ and PMOZ were found to convey long circulation and low hepatosplenic uptake to liposomes to the same extent as polyethylene glycol (PEG), the best known material for this purpose. This is the first demonstration of protection from rapid recognition and clearance conveyed by alternative polymers, which is equal to the effect of PEG.
Assuntos
Lipídeos/síntese química , Lipossomos/química , Sistema Fagocitário Mononuclear/fisiologia , Polímeros/síntese química , Animais , Lipossomos/metabolismo , Fígado/metabolismo , Oxazóis/química , Fosfatidiletanolaminas/química , Polietilenoglicóis/metabolismo , Ratos , Baço/metabolismo , Distribuição TecidualRESUMO
The levels and characteristics of growth hormone (GH)-binding protein (GHBP) and the distribution of GH in peripheral circulation between the free and the bound fractions were studied in three lines of transgenic mice with various degrees of overexpression of bovine (b) GH gene. Two serum fractions bound GH specifically: one with low affinity and high capacity (GHBPI) and one with high affinity and low capacity (GHBPII). The GHBP binding capacity in normal mice (both sexes), transgenic male mice that express the metallothionein-I-hybrid bGH genes, transgenic female mice that express phosphoenolpyruvate carboxykinase (PEPCK)-bGH hybrid genes (PEPCK-bGH-1), and transgenic PEPCK-bGH-5 animals was 1.1 +/- 0.2, 2.0 +/- 0.1, 3.0 +/- 0.1, and 3.9 +/- 0.6 pmol/ml serum, respectively. The amount of GH bound to GHBP in transgenic animals vs. normal siblings was increased 1.8-, 2.5-, and 3.9-fold in these three lines. Consequently, the levels of GH-GHBP complexes in the circulation of PEPCK-bGH-1 transgenic mice were increased approximately 10-fold. Specific GHBP radioimmunoassay confirmed a threefold increase in GHBP in PEPCK-bGH-1 transgenic animals. The levels of GHBP were not significantly correlated to serum GH within or between lines, perhaps due to elevation of serum GH in PEPCK-bGH mice above the level producing maximal response. From these and previous studies, we conclude that life-long exposure to supranormal GH levels leads to major shifts in GH binding in the circulation and in the GH target organs.
Assuntos
Proteínas de Transporte/metabolismo , Expressão Gênica , Hormônio do Crescimento/genética , Animais , Bovinos , Feminino , Hormônio do Crescimento/metabolismo , Hibridização Genética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Valores de ReferênciaRESUMO
Poly(ethylene glycol) (PEG)-grafted liposomes offer new opportunities as long-circulating platforms presenting biologically relevant ligands. In pursuit of this goal, liposomal conjugates of YIGSR were prepared by mild periodate oxidation of TYIGSR-NH2 and incubation of the product with hydrazide-PEG-(distearoylphosphatidyl)ethanolamine-containing liposomes. The peptide-carrying liposomes, with up to 500 YIGSR residues per vesicle, despite exhibiting faster blood clearance rates than the parent liposomes in rats, remained in circulation for extended periods of time. Mean residence times for the parent liposomal formulation and conjugated preparations containing 200 and 500 YIGSR residues per vesicle were 28, 25, and 23 h, respectively. The results have important implications for systemic delivery of peptides and for their use as targeting moieties for PEG-grafted liposomes.