RESUMO
PURPOSE: To evaluate the effectiveness of a neutral DOPC nanoliposome system for the delivery of siRNA to tumor cells in an obese murine cervical cancer model. METHODS: In vitro silencing of E6-E7 mRNA and E7 protein using siRNAE6 or siRNAE7 was analyzed in TC-1 cells by RT-PCR and Western blot. Silencing and antitumor capacities of siRNAE7-DOPC-nanoparticles (NP) were tested in vivo in both normal and obese mice using qPCR. These NPs were administered twice a week for 15 days and tumor volume and weight were recorded. RESULTS: Levels of in vitro E6-E7 silencing were 90% for mRNA and 60% for protein when siRNAE7 was used. On the other hand when siRNAE6 was used, the levels of silencing were 50% for E6-E7 mRNA and only 20% for protein. In vivo E7 mRNA silencing by siRNAE7-DOPC-NP was similar (60%) in both non-obese and obese mouse models. The therapeutic study showed a 65% decrease in tumor volume and a 57% reduction in tumor weight as compared to the control groups. CONCLUSION: There was no negative impact of obesity on the antitumor activity of siRNA-DOPC-NP in obese mice.
Assuntos
Obesidade/complicações , Proteínas E7 de Papillomavirus/genética , Fosfatidilcolinas/administração & dosagem , RNA Interferente Pequeno/genética , Neoplasias do Colo do Útero/terapia , Animais , Feminino , Inativação Gênica , Lipossomos , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas , Neoplasias do Colo do Útero/virologiaRESUMO
Small interfering RNA (siRNA) technology holds great promise as a therapeutic intervention for targeted gene silencing in cancer and other diseases. However, in vivo systemic delivery of siRNA-based therapeutics to tumour tissues/cells remains a challenge. The major limitations against the use of siRNA as a therapeutic tool are its degradation by serum nucleases, poor cellular uptake and rapid renal clearance following systemic administration. Several siRNA-based loco-regional therapeutics are already in clinical trials. Further development of siRNAs for anti-cancer therapy depends on the development of safe and effective nanocarriers for systemic administration. To overcome these hurdles, nuclease-resistant chemically modified siRNAs and variety of synthetic and natural biodegradable lipids and polymers have been developed to systemically deliver siRNA with different efficacy and safety profiles. Cationic liposomes have emerged as one of the most attractive carriers because of their ability to form complexes with negatively charged siRNA and high in vitro transfection efficiency. However, their effectiveness as potential therapeutic carriers is limited by potential for pulmonary toxicity. Recently, our laboratories described the use of neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine based nanoliposomes in murine tumour models. We found this approach to be safe and 10- and 30-fold more effective than cationic liposomes and naked siRNA, respectively, for systemic delivery of siRNA into tumour tissues. Here, we review potential approaches for systemic delivery of siRNA for cancer therapy.
Assuntos
Lipídeos/administração & dosagem , Nanocápsulas/administração & dosagem , Neoplasias/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , Animais , Regulação para Baixo , Inativação Gênica , Humanos , Lipídeos/química , Lipídeos/farmacocinética , Lipossomos , Camundongos , Camundongos Nus , Nanocápsulas/química , Nanomedicina/métodos , Neoplasias/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Activation of muscarinic acetylcholine receptors (mAChRs) inhibits spinal nociceptive transmission by potentiation of GABAergic tone through M(2), M(3), and M(4) subtypes. To study the signaling mechanisms involved in this unique mAChR action, GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of lamina II neurons were recorded using whole-cell patch clamp techniques in rat spinal cord slices. The mAChR agonist oxotremorine-M caused a profound increase in the frequency of GABAergic sIPSCs, which was abolished in the Ca(2+)-free solution. Inhibition of voltage-gated Ca(2+) channels with Cd(2+) and Ni(2+) largely reduced the effect of oxotremorine-M on sIPSCs. Blocking nonselective cation channels (NSCCs) with SKF96365 or 2-APB also largely attenuated the effect of oxotremorine-M. However, the KCNQ channel blocker XE991 and the adenylyl cyclase inhibitor MDL12330A had no significant effect on oxotremorine-M-induced increases in sIPSCs. Furthermore, the phosphoinositide-3-kinase (PI3K) inhibitor wortmannin or LY294002 significantly reduced the potentiating effect of oxotremorine-M on sIPSCs. In the spinal cord in which the M(3) subtype was specifically knocked down by intrathecal small interfering RNA (siRNA) treatment, SKF96365 and wortmannin still significantly attenuated the effect of oxotremorine-M. In contrast, SKF96365 and wortmannin both failed to alter the effect of oxotremorine-M on sIPSCs when the M(2)/M(4) mAChRs were blocked. Therefore, our study provides new evidence that activation of mAChRs increases synaptic GABA release through Ca(2+) influx and voltage-gated Ca(2+) channels. The PI3K-NSCC signaling cascade is primarily involved in the excitation of GABAergic interneurons by the M(2)/M(4) mAChRs in the spinal dorsal horn.
Assuntos
Potenciais Pós-Sinápticos Inibidores/fisiologia , Neurônios/fisiologia , Receptores Muscarínicos/fisiologia , Transdução de Sinais/fisiologia , Medula Espinal/citologia , Ácido gama-Aminobutírico/metabolismo , Animais , Antracenos/farmacologia , Biofísica , Cádmio/farmacologia , Cálcio/metabolismo , Interações Medicamentosas , Estimulação Elétrica/métodos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Masculino , Agonistas Muscarínicos/farmacologia , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Neurônios/efeitos dos fármacos , Níquel/farmacologia , Oxotremorina/análogos & derivados , Oxotremorina/farmacologia , Técnicas de Patch-Clamp/métodos , Bloqueadores dos Canais de Potássio/farmacologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Muscarínicos/classificação , Receptores Muscarínicos/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Anti-vascular endothelial growth factor (VEGF) therapy has demonstrated efficacy in treating human metastatic cancers, but therapeutic resistance is a practical limitation and most tumors eventually become unresponsive. To identify microenvironmental factors underlying the resistance of cancer to antiangiogenesis therapy, we conducted genomic analyses of intraperitoneal ovarian tumors in which adaptive resistance to anti-VEGF therapy (B20 antibody) developed. We found that expression of the microseminoprotein, prostate-associated (MSMP) gene was substantially upregulated in resistant compared with control tumors. MSMP secretion from cancer cells was induced by hypoxia, triggering MAPK signaling in endothelial cells to promote tube formation in vitro. Recruitment of the transcriptional repressor CCCTC-binding factor (CTCF) to the MSMP enhancer region was decreased by histone acetylation under hypoxic conditions in cancer cells. MSMP siRNA, delivered in vivo using the DOPC nanoliposomes, restored tumor sensitivity to anti-VEGF therapy. In ovarian cancer patients treated with bevacizumab, serum MSMP concentration increased significantly only in non-responders. These findings imply that MSMP inhibition combined with the use of antiangiogenesis drugs may be a new strategy to overcome resistance to antiangiogenesis therapy.
Assuntos
Bevacizumab/farmacologia , Carcinoma Epitelial do Ovário/patologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias das Tubas Uterinas/patologia , Proteínas de Neoplasias/metabolismo , Neoplasias Peritoneais/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Animais , Apoptose , Biomarcadores Tumorais , Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/metabolismo , Hipóxia Celular , Proliferação de Células , Neoplasias das Tubas Uterinas/tratamento farmacológico , Neoplasias das Tubas Uterinas/metabolismo , Feminino , Seguimentos , Humanos , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Neovascularização Patológica , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/metabolismo , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
KRas is mutated in a significant number of human cancers and so there is an urgent therapeutic need to target KRas signalling. To target KRas in lung cancers we used a systems approach of integrating a genome-wide miRNA screen with patient-derived phospho-proteomic signatures of the KRas downstream pathway, and identified miR-193a-3p, which directly targets KRas. Unique aspects of miR-193a-3p biology include two functionally independent target sites in the KRas 3'UTR and clinically significant correlation between miR-193a-3p and KRas expression in patients. Rescue experiments with mutated KRas 3'UTR showed very significantly that the anti-tumour effect of miR-193a-3p is via specific direct targeting of KRas and not due to other targets. Ex vivo and in vivo studies utilizing nanoliposome packaged miR-193a-3p demonstrated significant inhibition of tumour growth, circulating tumour cell viability and decreased metastasis. These studies show the broader applicability of using miR-193a-3p as a therapeutic agent to target KRas-mutant cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias/genética , Neoplasias/patologia , Células Neoplásicas Circulantes/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA , Regiões 3' não Traduzidas , Ciclo Celular , Linhagem Celular Tumoral , Bases de Dados Genéticas , Genes Reporter , Humanos , Metástase Neoplásica , Células Neoplásicas Circulantes/patologia , RNA Mensageiro/genéticaRESUMO
Fatty acid binding protein 4 (FABP4) is a fatty acid chaperone, which is induced during adipocyte differentiation. Previously we have shown that FABP4 in endothelial cells is induced by the NOTCH1 signalling pathway, the latter of which is involved in mechanisms of resistance to antiangiogenic tumour therapy. Here, we investigated the role of FABP4 in endothelial fatty acid metabolism and tumour angiogenesis. We analysed the effect of transient FABP4 knockdown in human umbilical vein endothelial cells on fatty acid metabolism, viability and angiogenesis. Through therapeutic delivery of siRNA targeting mouse FABP4, we investigated the effect of endothelial FABP4 knockdown on tumour growth and blood vessel formation. In vitro, siRNA-mediated FABP4 knockdown in endothelial cells led to a marked increase of endothelial fatty acid oxidation, an increase of reactive oxygen species and decreased angiogenesis. In vivo, we found that increased NOTCH1 signalling in tumour xenografts led to increased expression of endothelial FABP4 that decreased when NOTCH1 and VEGFA inhibitors were used in combination. Angiogenesis, growth and metastasis in ovarian tumour xenografts were markedly inhibited by therapeutic siRNA delivery targeting mouse endothelial FABP4. Therapeutic targeting of endothelial FABP4 by siRNA in vivo has antiangiogenic and antitumour effects with minimal toxicity and should be investigated further.
Assuntos
Inibidores da Angiogênese/metabolismo , Cistadenocarcinoma Seroso/prevenção & controle , Proteínas de Ligação a Ácido Graxo/antagonistas & inibidores , Neovascularização Patológica/prevenção & controle , Neoplasias Ovarianas/prevenção & controle , Animais , Apoptose , Biomarcadores Tumorais/metabolismo , Movimento Celular , Proliferação de Células , Cistadenocarcinoma Seroso/irrigação sanguínea , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Seguimentos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Camundongos , Camundongos Nus , Gradação de Tumores , Invasividade Neoplásica , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Prognóstico , Estudos Prospectivos , Receptor Notch1/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To ensure the success of systemic gene therapy, it is critical to enhance the tumor specificity and activity of the promoter. In the current study, we determined that topoisomerase IIalpha promoter is selectively activated in breast cancer cells. An element containing an inverted CCAAT box (ICB) was shown to be responsible for the breast cancer specificity. When the ICB-harboring topoisomerase IIalpha minimal promoter was linked with an enhancer sequence from the cytomegalovirus immediate early gene promoter (CMV promoter), this composite promoter, CT90, exhibited activity comparable to or higher than the CMV promoter in breast cancer cells in vitro and in vivo, yet expresses much lower activity in normal cell lines and normal organs than the CMV promoter. A CT90-driven construct expressing BikDD, a potent proapoptotic gene, was shown to selectively kill breast cancer cells in vitro, and to suppress mammary tumor development in an animal model of intravenously administrated, liposome-delivered gene therapy. Expression of BikDD was readily detectable in the tumors but not in the normal organs (such as heart) of CT90-BikDD-treated animals. The results indicate that liposomal CT90-BikDD is an effective systemic breast cancer-targeting gene therapy.
Assuntos
Antígenos de Neoplasias/genética , Proteínas Reguladoras de Apoptose/genética , Neoplasias da Mama/terapia , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Terapia Genética , Proteínas de Membrana/genética , Mutação , Elementos de Resposta/genética , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Citomegalovirus/genética , Elementos Facilitadores Genéticos/genética , Feminino , Expressão Gênica , Vetores Genéticos , Humanos , Lipossomos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Proteínas Mitocondriais , Neoplasias ExperimentaisRESUMO
Human monocytes obtained from healthy volunteers and isolated by centrifugal elutriation were not cytotoxic to allogeneic tumorigenic cells. These freshly isolated monocytes were rendered tumoricidal following interaction in vitro for 24 hours with greater than 0.01 micrograms lipopolysaccharide (LPS)/ml or over 1 microgram nor-muramyl dipeptide/ml. Monocytes activated by this procedure produced a soluble factor that lysed tumor cells. Full expression of tumor cell lysis required a minimum of 18 hours' exposure of tumor cells to the factor. The degree of tumor cytotoxic factor (TCF) production was closely related to the intensity of monocyte activation to become tumoricidal. Significant production of TCF by monocytes was detected in the supernatants after treatment for 3 hours with LPS. TCF was also released by activated monocytes when cocultivated with tumorigenic cells. Similarly, the level of TCF production correlated with the monocyte density. TCF destroyed human allogeneic tumor cell lines (melanoma, glioblastoma, colon carcinoma, prostatic carcinoma, and breast carcinoma), but it did not affect nontumorigenic cell lines (lung and skin fibroblasts). TCF activity was not blocked by superoxide dismutase, catalase, or protease inhibitors; it was destroyed by being heated at 100 degrees C for 2 minutes. The ability of activated monocytes to release TCF could enhance host defense against cancer.
Assuntos
Citotoxicidade Imunológica , Citotoxinas/biossíntese , Monócitos/imunologia , Comunicação Celular , Células Cultivadas , Citotoxinas/farmacologia , Temperatura Alta , Humanos , Cinética , Lipopolissacarídeos/farmacologia , Monócitos/metabolismoRESUMO
Multilamellar vesicles (MLVs) composed of dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol at a molar ratio of 7:3 were used as carriers of cis-bis-cyclopentenecarboxylato-trans-R,R-1,2-diaminocyclohexane-p latinum (II) (CPDP). The encapsulation efficiency of liposomal CPDP (L-CPDP) was 87.6%, and its stability in normal saline at 14 days was 94.4%. The in vitro and in vivo effects on the function of the monocyte-macrophage system and the antitumor activity against L1210 leukemia were investigated in CD-1 and (C57BL/6J X DBA/2J)F1 mice. L-CPDP and cisplatin (CDDP) caused a comparable inhibition of murine-resident peritoneal macrophage (PM) protein and RNA synthesis and superoxide anion release. PM-mediated tumor cell cytotoxicity was completely inhibited at a concentration of 10 micrograms CDDP and L-CPDP/ml but not at concentrations of 1 and 5 micrograms/ml. The differences in plasma clearance of 99mTc-labeled MLV and phagocytic capacity of the liver among animals pretreated with the maximum tolerated doses of L-CPDP (25 mg/kg), empty liposomes, or CDDP (10 mg/kg) were not statistically significant (plasma clearance % of control: 105, 110, and 100, respectively: P greater than .05; liver uptake % of control: 87, 96, and 104, respectively: P greater than .05). At the maximum tolerated doses, the antitumor activity of L-CPDP against L1210 leukemia was similar to that of CDDP when a single dose was administered [median survival of treated mice/median survival of control mice X 100 (%T/C): 181 vs. 175] and slightly higher with the use of a triple-dose schedule (%T/C: 275 vs. 225). L-CPDP is easy to prepare, has a high-encapsulation efficiency and stability, is not more toxic than CDDP to the monocyte-macrophage system, and is at least as effective as CDDP against L1210 leukemia.
Assuntos
Leucemia L1210/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Compostos Organoplatínicos/administração & dosagem , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Lipossomos/administração & dosagem , Fígado/metabolismo , Taxa de Depuração Metabólica , Camundongos , Fagocitose , Biossíntese de Proteínas , RNA/biossíntese , Baço/metabolismo , Superóxidos/metabolismoRESUMO
Retinoic acid (RA) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced differentiation of a human monocytic leukemia cell line, THP-1. RA- or TPA-treated cells stopped proliferating, became adherent to plastic surfaces, and acquired the ability to phagocytose yeast cells, plain sheep RBCs, and IgG-coated sheep RBCs. The morphological and functional changes, induced by RA or TPA, were associated with a 20-50-fold increase in cellular transglutaminase activity. This increase in enzyme activity was found to be due to the induction of a specific intracellular transglutaminase, tissue transglutaminase. The induction of tissue transglutaminase was a specific response of THP-1 cells to differentiation and was not observed with agents that did not induce their morphological or functional differentiation. Dibutyryl cyclic AMP potentiated the RA-induced expression of tissue transglutaminase. A 15-min exposure to TPA was sufficient to induce differentiation and expression of tissue transglutaminase in THP-1 cells. In contrast, RA required a continuous exposure (48 h) to induce similar changes in morphology or enzyme activity. These results support the view that differentiation of cells of the monocytic lineage is associated with an induction and accumulation of the protein cross-linking enzyme tissue transglutaminase.
Assuntos
Leucemia Mieloide/enzimologia , Macrófagos/enzimologia , Transglutaminases/metabolismo , Bucladesina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Humanos , Técnicas de Imunoadsorção , Interferon gama/farmacologia , Leucemia Mieloide/patologia , Macrófagos/citologia , Fagocitose/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tretinoína/farmacologia , Vitamina A/farmacologiaRESUMO
Human peripheral blood monocytes, isolated in high purity by centrifugal counterflow elutriation from normal donors, were stimulated in vitro to release cell toxins, herein termed human monocyte toxin(s) (HMT). Bacterial lipopolysaccharide, the lipophilic 6-O-stearoyl derivative of muramyl dipeptide, and 4 beta-phorbol-12 beta-myristate-13 alpha-acetate served as effective induction signals. Induction involved a sequence of transcription, translation, and secretion, all necessary for HMT synthesis and release into the supernatant as determined by blocking of these functions with the drugs actinomycin D, cycloheximide, and monensin, respectively; HMT levels reached a peak within 4-6 h and thereafter declined. The levels of HMT produced varied considerably from donor to donor; one parameter causing this variability appeared to be the plateletapheresis history of the donor. Monocytes from donors subjected to pheresis for the first time were responsive to induction signals immediately after adherence and could not be brought to a higher state of priming for HMT production by further in vitro culture for up to 9 days, with or without recombinant human gamma-interferon. In contrast, monocytes from donors who had recently undergone pheresis (up to 1 wk earlier) were poorly responsive initially to triggering with lipopolysaccharide; however, these cells could be brought to a highly primed state for HMT production by a combination of culture in vitro for several days and a subsequent 24-h exposure to recombinant gamma-interferon (0.1-1.0 units/ml). These primed cells could then be effectively triggered by lipopolysaccharide to release HMT. HMT was found to be cytotoxic (cytostatic/cytolytic) for human and murine tumor cells in vitro.
Assuntos
Citotoxicidade Imunológica , Citotoxinas/biossíntese , Monócitos/imunologia , Animais , Adesão Celular , Separação Celular , Citotoxinas/genética , Humanos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Camundongos , Biossíntese de Proteínas , Transcrição GênicaRESUMO
cis-Bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum++ +-(II) (NDDP) was encapsulated in multilamellar vesicles composed of dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol at a 7:3 molar ratio. Compared with cisplatin, i.v. administration of an equimolar dose of liposome-encapsulated NDDP (L-NDDP) resulted in 15-fold higher peak platinum levels in the spleen (204.7 versus 13.3 micrograms/g dry tissue), 5-fold higher in the lungs (116.4 versus 21.0 micrograms/g dry tissue), 3-fold higher in the liver (71.6 versus 23.9 micrograms/g dry tissue), and 4-fold higher in the blood (14.8 versus 3.9 micrograms/ml). At the optimal dose and schedule, L-NDDP administered i.p. in mice bearing peritoneal L1210 leukemia resulted in the percentage of median survival time of treated mice divided by median survival time of control mice (%T/C) of 312 versus 225 for cisplatin and free NDDP. When administered i.v., L-NDDP was also more active than cisplatin against L1210 leukemia inoculated i.v. (%T/C 186 versus 142). L-NDDP was markedly active against L1210 leukemia resistant to cisplatin (%T/C, 200 versus 112 for cisplatin). In mice bearing liver metastases of M5076 reticulosarcoma, L-NDDP was significatnly more effective than cisplatin at equimolar doses (mean survival time, 57 +/- 9 (SD) days for L-NDDP versus 42 +/- 3 days for cisplatin, P less than 0.05). L-NDDP was also effective in preventing liver metastases of M5076 when administered up to 24 h prior to tumor inoculation (mean survival, 28 +/- 2 days for L-NDDP versus 22 +/- 2 days for cisplatin, P less than 0.05). L-NDDP is significantly non-cross-resistant with cisplatin and more effective against phagocytic and nonphagocytic murine tumors.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Hepáticas/secundário , Linfoma não Hodgkin/secundário , Compostos Organoplatínicos/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Linhagem Celular , Leucemia L1210/tratamento farmacológico , Neoplasias Hepáticas/prevenção & controle , Neoplasias Hepáticas/terapia , Linfoma não Hodgkin/prevenção & controle , Linfoma não Hodgkin/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Transplante de Neoplasias , Compostos Organoplatínicos/farmacocinética , Distribuição Tecidual , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The potential of multilamellar vesicles (MLVs) as carriers of cis-bis-cyclopentenecarboxylato-1,2-diaminocyclohexane platinum(II) (CPDP), a lipophilic cisplatin derivative, was assessed. MLVs composed of dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), and cholesterol at different molar ratios were tested. The MLV-CPDP preparation with the highest antitumor activity against L1210 leukemia in vivo was DMPC:DMPG 7:3-CPDP. The encapsulation efficiency of this preparation was 66 +/- 4% (SD); the stability in 0.9% NaCl solution at 4 degrees C was 89% at 14 days and 93% 18 h after incubation in human AB serum at 37 degrees C. The toxicities of DMPC:DMPG 7:3-CPDP and free CPDP (suspended in hydroxypropyl cellulose) administered i.p. were similar (50% lethal dose = 75 versus 91 mg/kg; blood urea nitrogen values 96 h after the administration of the 50% lethal dose = 32.0 versus 34.4 mg/dl). The mean %T/C [(median survival time of treated mice divided by median survival time of control mice) X 100] obtained after a single i.p. injection of the optimal dose of each preparation tested was 215 (range 200 to 232) for DMPC:DMPG 7:3-CPDP, 175 (range 158 to 209) for DMPG-CPDP, 162 (range 136 to 179) for free CPDP, and 178 (range 169 to 189) for cisplatin. Using a multiple i.p. injection schedule (injections on Days 1, 5, and 9), DMPC:DMPG 7:3-CPDP was more active than free CPDP and cisplatin (%T/C: 403, 284, and 253% respectively). DMPC:DMPG 7:3-CPDP is less toxic and more active against L1210 leukemia in vivo than is cisplatin. The encapsulation of CPDP in MLVs composed of DMPC:DMPG 7:3 provides an adequate vehicle for the administration of this lipophilic compound and enhances its antitumor activity against L1210 leukemia.
Assuntos
Antineoplásicos/administração & dosagem , Lipossomos/administração & dosagem , Compostos Organoplatínicos/administração & dosagem , Animais , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Dimiristoilfosfatidilcolina/análise , Estabilidade de Medicamentos , Rim/efeitos dos fármacos , Leucemia L1210/tratamento farmacológico , Leucemia L1210/patologia , Lipossomos/análise , Camundongos , Camundongos Endogâmicos , Compostos Organoplatínicos/farmacologia , Veículos Farmacêuticos , Fosfatidilgliceróis/análiseRESUMO
All-trans-retinoic acid (ATRA) induces complete remissions in the majority of patients with acute promyelocytic leukemia. Despite continuous p.o. ATRA administration, many patients relapse after a short remission duration. In these patients, ATRA plasma concentrations were found to be very low, which was related to induction of retinoic acid-binding protein and increased drug catabolism by cytochrome P-450-mediated reactions. An i.v. ATRA formulation, which can be achieved by encapsulating ATRA into liposomes, may have the potential to overcome these unwanted effects. We investigated the ability of liposomal ATRA (L-ATRA) to induce differentiation of human myeloid leukemia cell lines (HL-60, KG-1, and THP-1). Cellular differentiation, as assessed by morphological criteria and by the expression of a mature myeloid cell surface antigen (CD11b on HL-60 and KG-1 cells), was induced by culture in the presence of L-ATRA. The ability of L-ATRA-treated HL-60 cells to reduce nitroblue tetrazolium demonstrated that they were functionally differentiated cells. Cell cycle analysis revealed significant growth inhibition of the cells after L-ATRA treatment. Following culture with L-ATRA, cells from five patients with acute promyelocytic leukemia were found to be morphologically and immunophenotypically mature myeloid cells. L-ATRA was as effective as free ATRA in inducing differentiation of the cell lines and of the cells from patients with acute promyelocytic leukemia. We conclude that L-ATRA effectively induces differentiation and may be a useful parenteral ATRA formulation for overcoming the pharmacological mechanisms that lead to "retinoid resistance."
Assuntos
Leucemia Mieloide/patologia , Tretinoína/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Granulócitos/citologia , Humanos , Técnicas In Vitro , Lipossomos , Estereoisomerismo , Tretinoína/química , Células Tumorais CultivadasRESUMO
Highly purified human blood monocytes, isolated by centrifugal elutriation under endotoxin-free conditions, were activated in vitro by combining subthreshold amounts of human recombinant gamma-interferon (r-IFN-gamma) and muramyl dipeptide (MDP) to become tumor cytotoxic against allogeneic A375 melanoma cells. Only intact r-IFN-gamma and MDP produced synergism for human monocyte activation. Neither pH 2-treated r-IFN-gamma and intact MDP nor heat-treated IFN-gamma and intact MDP, nor intact IFN-gamma and the biologically inactive stereoisomer of MDP, N-acetylmuramyl-D-alanyl-D-isoglutamine, produced activation of blood monocytes. The encapsulation of intact r-IFN-gamma and MDP within the same preparation of multilamellar liposomes was synergistic for monocyte activation. These data show that synergism for monocyte activation can be produced by human r-IFN-gamma and MDP produced synthetically can be simultaneously delivered to monocytes. Because both r-IFN-gamma and MDP can now be produced in large standardized quantities their synergism for activation of tumoricidal properties in human monocytes could be of clinical significance.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/administração & dosagem , Interferon gama/administração & dosagem , Monócitos/imunologia , Neoplasias Experimentais/imunologia , Proteínas Recombinantes/administração & dosagem , Células Cultivadas , Citotoxicidade Imunológica/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Lipossomos , Ativação de Macrófagos/efeitos dos fármacosRESUMO
Human peripheral blood monocytes, isolated in high purity by centrifugal counterflow elutriation from normal donors, release cell toxins, herein termed human monocyte toxins (HMTs) upon further stimulation in vitro. The principal form of HMTs produced by these human peripheral blood monocytes has been subjected to biochemical, functional, and serological characterization. By molecular sieving on Sephacryl S-200, HMTs can be resolved into two molecular weight classes. The larger, termed alpha, has a molecular weight of about 120,000, and the smaller, termed beta, has a molecular weight of about 65,000. The beta class is by far the most predominant species and has been further characterized. Chromatofocusing of beta-HMT indicates a slightly acidic nature, since this species is eluted at pH 5.8. Functional characterization of beta-HMT suggests that it is not a trypsin-like protease, since neither alpha,N-tosyl-L-lysylchloromethylketone nor alpha,N-tosyl-L-arginyl methyl ester are capable of causing significant inhibition of the cell-lytic activity of the molecule. Furthermore, cell lysis induced by beta-HMT appears to be independent of oxygen-dependent mechanisms, since catalase is incapable of blocking lysis, and since hydrogen peroxide and superoxide anion are not produced in detectable amounts during lysis. Finally, beta-HMT does not appear to be an arginase, since it is active in arginine-containing medium and further addition of arginine to the assay medium does not inhibit lysis significantly. beta-HMT is serologically related to recombinant human tumor necrosis factor (rHuTNF), since its cell lytic activity can be blocked by a rabbit antiserum against rHuTNF. However, much higher levels of this antiserum are required to achieve neutralization than are required to neutralize a comparable number of cell lytic units of rHuTNF. Furthermore, the results of preliminary immunoprecipitation experiments using the rabbit anti-rHuTNF antiserum suggest that a peptide in the Mr 60,000-70,000 range is recognized by this serum, whereas no signal at Mr 17,000 corresponding to rHuTNF is detectable. Thus, human peripheral blood monocytes can be triggered to release cell toxins, the principal form of which, beta-HMT, appears to be functionally distinct from the cytotoxic proteases reported in the murine system and appears to be molecularly distinct from, but serologically related to rHuTNF.
Assuntos
Citotoxinas/isolamento & purificação , Monócitos/análise , Neoplasias/imunologia , Cromatografia em Gel , Citotoxicidade Imunológica , Citotoxinas/análise , Citotoxinas/imunologia , Glicoproteínas/imunologia , Humanos , Soros Imunes/imunologia , Técnicas In Vitro , Peso Molecular , Monócitos/imunologia , Fator de Necrose Tumoral alfaRESUMO
The in vitro and in vivo interaction of liposomal cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum++ + (II) (L-NDDP) with mouse resident peritoneal macrophages (RPM), Kupffer cells (KC), and hepatocytes was studied. The peak in vitro uptake of L-NDDP by RPM was 12.5 ng elemental platinum/100 micrograms cell protein and constituted 0.2% of the platinum available for phagocytosis. The subsequent release of platinum by RPM was rapid initially, with a 20-fold increase over the first 4 h, followed by a plateau; ultrafilterable (free) platinum constituted 50% of the total platinum released at 24 h. The retained intracellular platinum in RPM at 24 h was close to 50% of that initially present. The peak in vitro uptake of L-NDDP by KC was 11.3 ng platinum/100 micrograms cell protein and amounted to 0.2% of the platinum available for phagocytosis. The release of platinum by KC was detectable only after 4 h of incubation and increased 3-fold over the next 14 h. The ultrafilterable platinum released by KC at 18 h was 40% of the total platinum released. The retained intracellular platinum in KC at 18 h was 33% of that initially present. The peak in vitro uptake of L-NDDP by hepatocytes was almost 50 ng platinum/100 micrograms cell protein and constituted 0.8% of the platinum available for intake. Following the i.v. injection of L-NDDP, hepatocytes contained up to 6-fold higher platinum concentrations than KC. This observation was supported by transmission electron microscopy showing a higher concentration of multilamellar vesicles within hepatocytes than in KC, 5 min after i.v. injection of L-NDDP. These findings suggest that L-NDDP becomes available to the liver following i.v. injection, that both macrophages and hepatocytes play a role in the metabolism of L-NDDP, and that Kupffer cells could mediate a sustained release of platinum in the liver following the interaction with L-NDDP, indicating the potential of L-NDDP for the treatment of tumors in the liver.
Assuntos
Antineoplásicos/farmacocinética , Células de Kupffer/metabolismo , Lipossomos/administração & dosagem , Fígado/metabolismo , Macrófagos/metabolismo , Compostos Organoplatínicos/farmacocinética , Animais , Antineoplásicos/administração & dosagem , Técnicas In Vitro , Fígado/ultraestrutura , Masculino , Camundongos , Platina/farmacocinéticaRESUMO
cis-Bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum++ +(II) (NDDP) is a liposome dependent cisplatin analogue since the liposome carrier is required for its i.v. administration and for its biological activity. A Phase I study of liposome entrapped NDDP (L-NDDP) was performed using a single i.v. injection every 4 weeks. L-NDDP was prepared and characterized at M. D. Anderson Cancer Center. The maximum tolerated dose of L-NDDP was 312.5 mg/m2. The dose-limiting toxicity was myelosuppression, affecting all three blood cell lineages. The granulocyte nadir occurred on days 14-18, and the platelet nadir consistently earlier (days 11-12). The median day of recovery of blood cell counts was day 21 (range, 18-32). Other toxicities included grade 2 nausea and vomiting, fever consisting of a single temperature spike in most patients, grade 1 diarrhea after 60% of courses, and grade 1-2 malaise lasting for 5-10 days after the infusion in 73% of courses. Transient alanine aminotransferase elevations without clinical relevance were common. No signs of renal dysfunction or ototoxicity were observed. One patient with a preexisting peripheral neuropathy showed some progression of the neuropathy after a cumulative dose of 1605 mg/m2. Except for fever and transient liver dysfunction, no liposome related side effects were observed in spite of the high doses of lipid administered. The blood clearance of L-NDDP fits a two-compartment model at lower doses and a single-compartment model at the maximum tolerated dose, suggesting that saturation of the reticuloendothelial organs occurs at the maximum tolerated dose. Two minimal responses were observed. L-NDDP has a toxicity profile similar to that of carboplatin. Phase II studies to address the issue of how the therapeutic index of platinum compounds is affected by liposome entrapment are being planned.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias/tratamento farmacológico , Compostos Organoplatínicos/administração & dosagem , Portadores de Fármacos/administração & dosagem , Avaliação de Medicamentos , Feminino , Hemoglobinas/metabolismo , Humanos , Contagem de Leucócitos/efeitos dos fármacos , Lipossomos , Masculino , Compostos Organoplatínicos/efeitos adversos , Compostos Organoplatínicos/farmacocinética , Contagem de Plaquetas/efeitos dos fármacosRESUMO
The pharmacokinetics, organ distribution, and 24-hr urinary excretion of negatively charged 99mTc-labeled multilamellar liposomes, composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol in a 7:3 molar ratio, were studied in seven patients with cancer. The radiolabeled liposomes were administered i.v. in three doses: 150 mg/sq m of body surface area; 300 mg/sq m; and 450 mg/sq m of lipid. The dose of 99mTc was 4.8 to 7.6 mCi per patient. The plasma disappearance curve was biphasic (half-life alpha = 5.53 min, half-life beta = 289 min), suggesting a two-compartmental model of distribution. The calculated volume of distribution indicated considerable tissue retention of liposomes. This was confirmed by body imaging. Twenty-four hr after injection, liposomes were localized in organs rich in reticuloendothelial cells, i.e., liver [44.5 +/- 9.1% (S.E.)], spleen [25.5 +/- 7.7%], lung [14.5 +/- 4.9%], and bone marrow. Although the hepatic uptake accounted for more than 40% of the total uptake, the spleen retained liposomes at a higher density. Cumulative urinary excretion of radioactivity was 13.4 +/- 1.5% over 24 hr. Liposome administration was safe and devoid of any adverse side effects. The results provide a basis for the use of liposomes as potential target-specific and safe drug carriers in the treatment of pathological conditions that involve organs rich in reticuloendothelial cells.