RESUMO
Objective: The investigate the effects and mechanism of exosomes derived from human umbilical vein endothelial cells (HUVECs) on wound healing in diabetes rabbits. Methods: The experimental research methods were used. The primary vascular endothelial cells (VECs) and human skin fibroblasts (HSFs) were extracted from skin tissue around ulcer by surgical excision of two patients with diabetic ulcer (the male aged 49 years and the female aged 58 years) admitted to Xiangya Third Hospital of Central South University in June 2019. The cells were successfully identified through morphological observation and flow cytometry. The HUVEC exosomes were extracted by ultracentrifugation and identified successfully by morphological observation, particle size detection, and Western blotting detection. Twenty female 3-month-old New Zealand rabbits were taken to create one type 2 diabetic full-thickness skin defect wound respectively on both sides of the back. The wounds were divided into exosomes group and phosphate buffer solution (PBS) group and treated accordingly, with 20 wounds in each group, the time of complete tissue coverage of wound was recorded. On PID 14, hematoxylin-eosin staining or Masson staining was performed to observe angiogenesis or collagen fiber hyperplasia (n=20). The VECs and HSFs were co-cultured with HUVEC exosomes for 24 h to observe the uptake of HUVEC exosomes by the two kinds of cells. The VECs and HSFs were divided to exosome group treated with HUVEC exosomes and PBS group treated with PBS to detect the cell proliferation on 4 d of culture with cell count kit 8, to detect and calculate the cell migration rate at 24 and 48 h after scratch by scratch test, to detect the cell migration number at 24 h of culture with Transwell test, and to detect the mRNA expressions of nuclear factor-erythroid 2-related factor 2 (NRF2) and transcription activating factor 3 (ATF3) by real time fluorescence quantitative reverse transcription polymerase chain reaction. Besides, the number of vascular branches and vascular length were observed in the tube forming experiment after 12 h of culture of VECs (n=3). The VECs and HSFs were taken and divided into PBS group and exosome group treated as before, and NRF2 interference group, ATF3 interference group, and no-load interference group with corresponding gene interference. The proliferation and migration of the two kinds of cells, and angiogenesis of VECs were detected as before (n=3). Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, and least significant difference test. Results: The time of complete tissue coverage of wound in exosome group was (17.9±1.9) d, which was significantly shorter than (25.2±2.3) d in PBS group (t=4.54, P<0.05). On PID14, the vascular density of wound in PBS group was significantly lower than that in exosome group (t=10.12, P<0.01), and the collagen fiber hyperplasia was less than that in exosome group. After 24 h of culture, HUVEC exosomes were successfully absorbed by VECs and HSFs. The proliferative activity of HSFs and VECs in exosome group was significantly higher than that in PBS group after 4 d of culture (with t values of 54.73 and 7.05, respectively, P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs (with t values of 3.42 and 11.87, respectively, P<0.05 or P<0.01) and VECs (with t values of 21.42 and 5.49, respectively, P<0.05 or P<0.01) in exosome group were significantly higher than those in PBS group. After 24 h of culture, the migration numbers of VECs and HSFs in exosome group were significantly higher than those in PBS group (with t values of 12.31 and 16.78, respectively, P<0.01). After 12 h of culture, the mRNA expressions of NRF2 in HSFs and VECs in exosome group were significantly higher than those in PBS group (with t values of 7.52 and 5.78, respectively, P<0.05 or P<0.01), and the mRNA expressions of ATF3 were significantly lower than those in PBS group (with t values of 13.44 and 8.99, respectively, P<0.01). After 12 h of culture, the number of vascular branches of VECs in exosome group was significantly more than that in PBS group (t=17.60, P<0.01), and the vascular length was significantly longer than that in PBS group (t=77.30, P<0.01). After 4 d of culture, the proliferation activity of HSFs and VECs in NRF2 interference group was significantly lower than that in PBS group and exosome group (P<0.05 or P<0.01); the proliferation activity of HSFs and VECs in ATF3 interference group was significantly higher than that in PBS group (P<0.05 or P<0.01) and significantly lower than that in exosome group (P<0.05 or P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs and VECs in ATF3 interference group were significantly higher than those in PBS group (P<0.05 or P<0.01) and significantly lower than those in exosome group (P<0.05 or P<0.01). At 24 and 48 h after scratch, the migration rates of HSFs and VECs in NRF2 interference group were significantly lower than those in PBS group and exosome group (P<0.05 or P<0.01). After 24 h of culture, the migration numbers of VECs and HSFs in ATF3 interference group were significantly more than those in PBS group (P<0.05) and significantly less than those in exosome group (P<0.05 or P<0.01); the migration numbers of VECs and HSFs in NRF2 interference group were significantly less than those in PBS group and exosome group (P<0.01). After 12 h of culture, the vascular length and number of branches of VECs in NRF2 interference group were significantly decreased compared with those in PBS group and exosome group (P<0.01); the vascular length and number of branches of VECs in ATF3 interference group were significantly increased compared with those in PBS group (P<0.01) and were significantly decreased compared with those in exosome group (P<0.01). Conclusions: HUVEC exosomes can promote the wound healing of diabetic rabbits by promoting the proliferation and migration of VECs and HSFs, and NRF2 and ATF3 are obviously affected by exosomes in this process, which are the possible targets of exosome action.
Assuntos
Diabetes Mellitus , Exossomos , Animais , Feminino , Humanos , Masculino , Coelhos , Colágeno/metabolismo , Exossomos/metabolismo , Células Endoteliais da Veia Umbilical Humana , Hiperplasia/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , RNA Mensageiro/metabolismo , Úlcera , Cicatrização , Pessoa de Meia-IdadeRESUMO
Purpose. Our study analyses clinical trials and evaluates the efficacy of adding cetuximab in systematic chemotherapy for unresectable colorectal cancer liver-confined metastases patients. Materials and Methods. Search EMBASE, PubMed, and the Cochrane Central Register of Controlled Trials for RCTs comparing chemotherapy plus cetuximab with chemotherapy alone for KRAS wild type patients with colorectal cancer liver metastases (CRLMs). We calculated the relative risks (RRs) with 95% confidence interval and performed meta-analysis of hazard ratios (HRs) for the R0 resection rate, the overall response rate (ORR), the progression-free survival (PFS) and overall survival (OS). Results. 1173 articles were retrieved and 4 RCTs were available for our study. The four studies involved 504 KRAS wild type patients with CRLMs. The addition of cetuximab significantly improved all the 4 outcomes: the R0 resection rate (RR 2.03, p = 0.004), the ORR (RR 1.76, p < 0.00001), PFS (HR 0.63, p < 0.0001), and also OS (HR 0.74, p = 0.04); the last outcome is quite different from the conclusion published before. Conclusions. Although the number of patients analysed was limited, we found that the addition of cetuximab significantly improves the outcomes in KRAS wild type patients with unresectable colorectal cancer liver-confined metastases. Cetuximab combined with systematic chemotherapy perhaps suggests a promising choice for KRAS wild type patients with unresectable liver metastases.
RESUMO
Extracellular activation of hydrophilic glucuronide prodrugs by ß-glucuronidase (ßG) was examined to increase the therapeutic efficacy of bacteria-directed enzyme prodrug therapy (BDEPT). ßG was expressed on the surface of Escherichia coli by fusion to either the bacterial autotransporter protein Adhesin (membrane ßG (mßG)/AIDA) or the lipoprotein (lpp) outermembrane protein A (mßG/lpp). Both mßG/AIDA and mßG/lpp were expressed on the bacterial surface, but only mßG/AIDA displayed enzymatic activity. The rate of substrate hydrolysis by mßG/AIDA-BL21cells was 2.6-fold greater than by pßG-BL21 cells, which express periplasmic ßG. Human colon cancer HCT116 cells that were incubated with mßG/AIDA-BL21 bacteria were sensitive to a glucuronide prodrug (p-hydroxy aniline mustard ß-D-glucuronide, HAMG) with an half maximal inhibitory concentration (IC50) value of 226.53±45.4 µM, similar to the IC50 value of the active drug (p-hydroxy aniline mustard, pHAM; 70.6±6.75 µM), indicating that mßG/AIDA on BL21 bacteria could rapidly and efficiently convert HAMG to an active anticancer agent. These results suggest that surface display of functional ßG on bacteria can enhance the hydrolysis of glucuronide prodrugs and may increase the effectiveness of BDEPT.
Assuntos
Escherichia coli/enzimologia , Glucuronatos/farmacocinética , Glucuronidase/metabolismo , Glucuronídeos/farmacocinética , Nitrofenóis/farmacocinética , Pró-Fármacos/farmacocinética , Proteínas de Transporte/farmacocinética , Escherichia coli/genética , Glucuronidase/biossíntese , Glucuronidase/genética , Células HCT116 , Humanos , Proteínas Recombinantes , Células Tumorais CultivadasRESUMO
Non-invasive gene monitoring is important for most gene therapy applications to ensure selective gene transfer to specific cells or tissues. We developed a non-invasive imaging system to assess the location and persistence of gene expression by anchoring an anti-dansyl (DNS) single-chain antibody (DNS receptor) on the cell surface to trap DNS-derivatized imaging probes. DNS hapten was covalently attached to cross-linked iron oxide (CLIO) to form a 39+/-0.5 nm DNS-CLIO nanoparticle imaging probe. DNS-CLIO specifically bound to DNS receptors but not to a control single-chain antibody receptor. DNS-CLIO (100 microM Fe) was non-toxic to both B16/DNS (DNS receptor positive) and B16/phOx (control receptor positive) cells. Magnetic resonance (MR) imaging could detect as few as 10% B16/DNS cells in a mixture in vitro. Importantly, DNS-CLIO specifically bound to a B16/DNS tumor, which markedly reduced signal intensity. Similar results were also shown with DNS quantum dots, which specifically targeted CT26/DNS cells but not control CT26/phOx cells both in vitro and in vivo. These results demonstrate that DNS nanoparticles can systemically monitor the expression of DNS receptor in vivo by feasible imaging systems. This targeting strategy may provide a valuable tool to estimate the efficacy and specificity of different gene delivery systems and optimize gene therapy protocols in the clinic.
Assuntos
Meios de Contraste/farmacologia , Compostos de Dansil/farmacologia , Compostos Férricos/farmacologia , Corantes Fluorescentes/farmacologia , Haptenos/farmacologia , Imageamento por Ressonância Magnética/métodos , Nanopartículas , Neoplasias Experimentais/patologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Meios de Contraste/química , Compostos de Dansil/química , Compostos Férricos/química , Corantes Fluorescentes/química , Terapia Genética , Haptenos/química , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência/métodos , Nanopartículas/química , Neoplasias Experimentais/terapia , Sensibilidade e EspecificidadeRESUMO
Increasing the specificity of chemotherapy may improve the efficacy of cancer treatment. Toward this aim, we developed a strain of bacteria to express enzymes for selective prodrug activation and non-invasive imaging in tumors. beta-glucuronidase and the luxCDABE gene cluster were expressed in the DH5alpha strain of Escherichia coli to generate DH5alpha-lux/betaG. These bacteria emitted light for imaging and hydrolyzed the glucuronide prodrug 9ACG to the topoisomerase I inhibitor 9-aminocamptothecin (9AC). By optical imaging, colony-forming units (CFUs) and staining for betaG activity, we found that DH5alpha-lux/betaG preferentially localized and replicated within CL1-5 human lung tumors in mice. The intensity of luminescence, CFU and betaG activity increased with time, indicating bacterial replication occurred in tumors. In comparison with DH5alpha-lux/betaG, 9AC or 9ACG treatment, combined systemic administration of DH5alpha-lux/betaG followed by 9ACG prodrug treatment significantly (P<0.005) delayed the growth of CL1-5 tumors. Our results demonstrate that prodrug-activating bacteria may be useful for selective cancer chemotherapy.
Assuntos
Bactérias/metabolismo , Neoplasias/terapia , Pró-Fármacos/uso terapêutico , Animais , Bactérias/genética , Glucuronidase/genética , Glucuronidase/metabolismo , Glucuronídeos/metabolismo , Humanos , Modelos Biológicos , Neoplasias/microbiologia , Neoplasias/patologia , Pró-Fármacos/metabolismoRESUMO
Development of nonimmunogenic and specific reporter genes to monitor gene expression in vivo is important for the optimization of gene therapy protocols. We developed a membrane-anchored form of mouse beta-glucuronidase (mbetaG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-beta-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional beta-glucuronidase (betaG) was stably expressed on the surface of murine CT26 colon adenocarcinoma cells where it selectively hydrolyzed the cell-impermeable FDGlcU probe. FDGlcU was also preferentially converted to fluorescent probe by (betaG) on CT26 tumors. The fluorescent intensity in betaG-expressing CT26 tumors was 240 times greater than the intensity in control tumors. Selective imaging of gene expression was also observed after intratumoral injection of adenoviral betaG vector into carcinoma xenografts. Importantly, mbetaG did not induce an antibody response after hydrodynamic plasmid immunization of Balb/c mice, indicating that the reporter gene product displayed low immunogenicity. A membrane-anchored form of human betaG also allowed in vivo imaging, demonstrating that human betaG can be employed for imaging. This imaging system therefore, displays good selectivity with low immunogenicity and may help assess the location, magnitude and duration of gene expression in living animals and humans.
Assuntos
Membrana Celular/enzimologia , Corantes Fluorescentes/metabolismo , Genes Reporter , Terapia Genética , Glucuronidase/metabolismo , Animais , Catálise , Linhagem Celular Tumoral , Citometria de Fluxo , Fluoresceínas/metabolismo , Expressão Gênica , Vetores Genéticos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência , Neoplasias ExperimentaisRESUMO
Gene-mediated enzyme prodrug therapy (GDEPT) seeks to increase the therapeutic index of anti-neoplastic agents by promoting selective activation of relatively nontoxic drug derivatives at sites of specific enzyme expression. Glucuronide prodrugs are attractive for GDEPT due to their low toxicity, bystander effect in the interstitial tumor space and the large range of possible glucuronide drug targets. In this study, we expressed human, murine and Esherichia coli beta-glucuronidase on tumor cells and examined their in vitro and in vivo efficacy for the activation of glucuronide prodrugs of 9-aminocamptothecin and p-hydroxy aniline mustard. We show that (1) fusion of beta-glucuronidase to the Ig-like C(2)-type and Ig-hinge-like domains of the B7-1 antigen followed by the B7-1 transmembrane domain anchored high levels of active murine and human beta-glucuronidase on cells, (2) strong bystander killing of tumor cells was achieved in vitro by murine beta-glucuronidase activation of prodrug, (3) potent in vivo anti-tumor activity was achieved by prodrug treatment of tumors that expressed murine beta-glucuronidase and (4) the p-hydroxy aniline prodrug was more effective in vivo than the 9-aminocamptothecin prodrug. Our results demonstrate that surface expression of murine beta-glucuronidase for activation of a glucuronide prodrug of p-hydroxy aniline mustard may be useful for more selective therapy of cancer.
Assuntos
Glucuronidase/metabolismo , Glucuronídeos/metabolismo , Pró-Fármacos/farmacocinética , Células 3T3 , Animais , Western Blotting , Linhagem Celular Tumoral , DNA Complementar , Citometria de Fluxo , Humanos , Camundongos , Proteínas Recombinantes/metabolismoRESUMO
Monitoring gene expression is important to optimize gene therapy protocols and ensure that the proper tissue distribution is achieved in clinical practice. We developed a noninvasive imaging system based on the expression of artificial antibody receptors to trap hapten-labeled imaging probes. Functional membrane-bound anti-dansyl antibodies (DNS receptor) were stably expressed on melanoma cells in vitro and in vivo. A bivalent (DNS)2-diethylenetriaminepentaacetic 111Indium probe specifically bound to cells that expressed DNS receptors but not control scFv receptors. Importantly, the 111In probe preferentially localized to DNS receptors but not control receptors on tumors in mice as assessed by gamma camera imaging. By 48 h after intravenous injection, the uptake of the probe in tumors expressing DNS receptors was 72 times greater than the amount of probe in the blood. This targeting strategy may allow noninvasive assessment of the location, extent and persistence of gene expression in living animals and in the clinic.
Assuntos
Terapia Genética/métodos , Fosfatidilcolinas/imunologia , Receptores de Superfície Celular/metabolismo , Animais , Especificidade de Anticorpos , Expressão Gênica , Engenharia Genética , Vetores Genéticos/administração & dosagem , Haptenos , Células HeLa , Humanos , Radioisótopos de Índio , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Contraste de Fase , Ácido Pentético , Ligação Proteica , Receptores Fc/metabolismo , Retroviridae/genéticaRESUMO
Cancer chemotherapy is limited by the modest therapeutic index of most antineoplastic drugs. Some glucuronide prodrugs may display selective anti-tumour activity against tumours that accumulate beta-glucuronidase. We examined the toxicity and anti-tumour activity of 9-aminocamptothecin glucuronide, a new glucuronide prodrug of 9-aminocamptothecin, to evaluate its potential clinical utility. 9-aminocamptothecin glucuronide was 25-60 times less toxic than 9-aminocamptothecin to five human cancer cell lines. Beta-glucuronidase activated 9-aminocamptothecin glucuronide to produce similar cell killing as 9-aminocamptothecin or topotecan. The in vivo toxicity of 9-aminocamptothecin glucuronide in BALB/c mice was dose-, route-, sex- and age-dependent. 9-aminocamptothecin glucuronide was significantly less toxic to female than to male mice but the difference decreased with age. 9-aminocamptothecin glucuronide and 9-aminocamptothecin produced similar inhibition (approximately 80%) of LS174T human colorectal carcinoma tumours. 9-aminocamptothecin glucuronide cured a high percentage of CL1-5 human lung cancer xenografts with efficacy that was similar to or greater than 9-aminocamptothecin, irinotecan and topotecan. The potent anti-tumour activity of 9-aminocamptothecin glucuronide suggests that this prodrug should be further evaluated for cancer treatment.
Assuntos
Antineoplásicos/uso terapêutico , Camptotecina/uso terapêutico , Glucuronídeos/uso terapêutico , Pró-Fármacos/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Camptotecina/toxicidade , Estabilidade de Medicamentos , Feminino , Glucuronídeos/farmacocinética , Glucuronídeos/farmacologia , Glucuronídeos/toxicidade , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Pró-Fármacos/farmacologia , Pró-Fármacos/toxicidade , Fatores Sexuais , Solubilidade , Topotecan/farmacologia , Topotecan/toxicidade , Células Tumorais Cultivadas/efeitos dos fármacos , Redução de Peso , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antibody-directed enzyme prodrug therapy (ADEPT) has displayed antitumor activity in animal models and clinical trials. We examined whether antitumor immunity is generated during ADEPT by employing an immunoenzyme composed of the monoclonal antibody (MAb) RH1 conjugated to beta-glucuronidase to target rat AS-30D hepatocellular carcinoma tumors. A glucuronide prodrug of p-hydroxyaniline mustard was used to treat malignant ascites after immunoenzyme localization at the cancer cells. ADEPT cured more than 96% of Sprague-Dawley rats bearing advanced malignant ascites, and all cured rats were protected from a lethal challenge of AS-30D cells. Immunization with radiation-killed AS-30D cells or AS-30D cells coated with immunoenzyme did not provide tumor protection. Likewise, ex vivo treatment of tumor cells by ADEPT before injection into rats did not protect against a tumor challenge. AS-30D and N1-S1 hepatocellular carcinoma cells but not unrelated syngeneic tumor cells were lysed by peritoneal exudate cells isolated from ADEPT-cured rats. Depletion of CD8(+) but not CD4(+) T cells or natural killer (NK) cells reduced the cytolytic activity of peritoneal lymphocytes. ADEPT did not cure tumor-bearing rats depleted of CD4(+) and CD8(+) T cells even though it was curative when given 7 days after tumor transplantation in rats with an intact immune system, indicating that ADEPT can synergize with host immunity to increase therapeutic efficacy. These results have important implications for the clinical application of ADEPT.
Assuntos
Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Glucuronidase/uso terapêutico , Neoplasias Hepáticas Experimentais/terapia , Pró-Fármacos/uso terapêutico , Animais , Citosina Desaminase , Neoplasias Hepáticas Experimentais/imunologia , Nucleosídeo Desaminases/fisiologia , Ratos , Ratos Sprague-Dawley , Linfócitos T Citotóxicos/imunologiaRESUMO
OBJECTIVE: The aim of this study was to determine whether the activity of topoisomerase I (topo I), the target of the anti-neoplastic drug camptothecin (CPT), is elevated in cervical cancer and whether CPT can radiosensitize cervical tumors. METHODS: The topo I activity of 11 normal cervix and 30 cervical carcinoma tumors was assayed by measuring the relaxation of supercoiled DNA. Subconfluent or postconfluent CaSki human cervical carcinoma cells were exposed to CPT (1-5000 ng/ml) and immediately X-irradiated (0-800 cGy). Cell survival was determined by clonogenic assay. RESULTS: Mean topo I activity in cervical cancer (3.0 +/- 0.06 h(-1)) was significantly greater than in normal cervix tissue (0.29 +/- 0.06 h(-1)). Stage 3 and 4 cervical carcinoma specimens displayed a trend of greater topo I activity (5.88 +/- 3.7 h(-1)) than stage 1 and 2 tumors (2.57 +/- 0.47 h(-1)). No correlation between topo I protein levels and catalytic activity was found. Combined treatment of subconfluent CaSki cells with CPT and ionizing radiation resulted in additive killing of cells. Combined treatment of postconfluent CaSki cells with low doses of radiation (200 and 400 cGy) and 1 or 10 ng/ml CPT for 2 or 48 h produced significant cytotoxicity compared to CPT or radiation alone, which were ineffective at these doses. CONCLUSIONS: Topo I activity is elevated in cervical cancer compared to normal cervix. The radiosensitivity of noncycling cells within cervical tumors may be increased by simultaneous treatment with low doses of CPT or other topo I inhibitors.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Inibidores Enzimáticos/farmacologia , Radiossensibilizantes/farmacologia , Neoplasias do Colo do Útero/enzimologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Terapia Combinada , Relação Dose-Resposta à Radiação , Feminino , Humanos , Concentração Inibidora 50 , Inibidores da Topoisomerase I , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos da radiação , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/radioterapiaRESUMO
The F(ab')(2) fragment of the anti-TAG-72 antibody, B72.3, was covalently linked to Escherichia coli-derived beta-glucuronidase that was modified with methoxypoly(ethylene glycol). The conjugate (B72.3-betaG-PEG) localized to a peak concentration in LS174T xenografts within 48 h after injection, but enzyme activity persisted in plasma such that prodrug administration had to be delayed for at least 4 days to avoid systemic prodrug activation and associated toxicity. Conjugate levels in tumors decreased to 36% of peak levels at this time. Intravenous administration of AGP3, an IgM mAb against methoxypoly(ethylene glycol), accelerated clearance of conjugate from serum and increased the tumor/blood ratio of B72. 3-betaG-PEG from 3.9 to 29.6 without significantly decreasing the accumulation of conjugate in tumors. Treatment of nude mice bearing established human colon adenocarcinoma xenografts with B72. 3-betaG-PEG followed 48 h later with AGP3 and a glucuronide prodrug of p-hydroxyaniline mustard significantly (p< or =0.0005) delayed tumor growth with minimal toxicity compared to therapy with a control conjugate or conventional chemotherapy.
Assuntos
Anticorpos Monoclonais/farmacocinética , Antineoplásicos/uso terapêutico , Glucuronidase/farmacocinética , Polietilenoglicóis/farmacocinética , Pró-Fármacos/farmacocinética , Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/química , Mostarda de Anilina/uso terapêutico , Mostarda de Anilina/toxicidade , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/química , Antineoplásicos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Escherichia coli/enzimologia , Glucuronidase/química , Glucuronidase/imunologia , Humanos , Técnicas Imunoenzimáticas/métodos , Fragmentos Fab das Imunoglobulinas/química , Radioisótopos do Iodo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Polietilenoglicóis/química , Pró-Fármacos/química , Pró-Fármacos/uso terapêutico , Distribuição Tecidual , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The specificity of tumor therapy may be improved by preferentially activating antineoplastic prodrugs at tumor cells pretargeted with antibody-enzyme conjugates. In this study, the conditions required for the efficient activation of p-hydroxyaniline mustard glucuronide (BHAMG) to p-hydroxyaniline mustard (pHAM) were investigated. pHAM induced cross-links in linearized double-stranded DNA at about 180-fold lower concentrations than BHAMG, indicating that the nucleophilicity of pHAM was decreased by the presence of a glucuronide group. The partition coefficient of BHAMG was about 1890 times lower than pHAM in an octanol-water two-phase system, suggesting that the reduced toxicity of BHAMG was due to both hindered diffusion across the lipid bilayer of cells and decreased reaction with nuclear DNA. BHAMG was significantly less toxic to BHK cells that expressed cytosolic Escherichia coli-derived beta-glucuronidase (betaG) compared with cells that were engineered to secrete betaG, demonstrating that extracellular localization of betaG was required for optimal activation of BHAMG. The extended retention of mAb RH1 on the surface of AS-30D cells was also consistent with extracellular activation of BHAMG. Taken together, our results indicate that the low toxicity of BHAMG was due to hindered cellular uptake and low alkylating activity. BHAMG must be enzymatically activated outside of tumor cells for maximum cytotoxicity, and non-internalizing antibodies are preferred for human tumor therapy by targeted antibody-enzyme activation of BHAMG.
Assuntos
Mostarda de Anilina/análogos & derivados , Antineoplásicos Alquilantes/metabolismo , Glucuronatos/metabolismo , Glucuronidase/metabolismo , Pró-Fármacos/metabolismo , Mostarda de Anilina/metabolismo , Mostarda de Anilina/farmacologia , Animais , Anticorpos/imunologia , Antineoplásicos Alquilantes/farmacologia , Glucuronidase/genética , Humanos , Ratos , Transfecção , Células Tumorais CultivadasRESUMO
RHI-betaG-PEG, formed by linking poly(ethylene glycol)-modified beta-glucuronidase to Mab RH1, was employed to examine bystander killing of antigen-negative N1S1 rat hepatoma cells by activation of a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at antigen-positive AS-30D rat hepatoma cells. Sequential treatment of cells with 10 microg ml(-1) RH1-betaG-PEG and 20 microM BHAMG was not toxic to N1S1 cells but killed 99% of AS-30D cells. Over 98% of N1S1 cells, however, were killed in mixed populations containing as few as 2% AS-30D cells after identical treatment, demonstrating an in vitro bystander effect. Subcutaneous injection of AS-30D and N1S1 cells in BALB/c nu/nu mice produced solid tumours containing both cells. Uptake of radiolabelled RH1-betaG-PEG in solid AS-30D and mixed AS-30D/N1S1 tumours was 11.6 and 9.3 times greater than a control antibody conjugate 120 h after i.v. injection. Intravenous treatment with RH1-betaG-PEG and BHAMG cured seven of seven nude mice bearing solid s.c. AS-30D tumours and significantly delayed, compared with control conjugate and prodrug treatment, the growth of mixed N1S1/AS-30D tumours with one cure, showing that targeted activation of BHAMG kills bystander tumour cells in vivo.
Assuntos
Mostarda de Anilina/análogos & derivados , Antineoplásicos Alquilantes/uso terapêutico , Glucuronidase/uso terapêutico , Imunotoxinas/uso terapêutico , Polietilenoglicóis/uso terapêutico , Pró-Fármacos/uso terapêutico , Mostarda de Anilina/metabolismo , Mostarda de Anilina/uso terapêutico , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Alquilantes/metabolismo , Difusão , Ensaios de Seleção de Medicamentos Antitumorais , Glucuronidase/metabolismo , Imuno-Histoquímica , Imunotoxinas/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Polietilenoglicóis/metabolismo , Pró-Fármacos/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Células Tumorais CultivadasRESUMO
We examined the in vivo efficacy of targeting beta-glucuronidase (betaG) to activate a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at hepatoma ascites in Sprague-Dawley rats. Injection i.p. of 500 microg RH1-betaG, a conjugate formed between recombinant betaG and monoclonal antibody RH1 with specificity for an antigen expressed on AS-30D rat hepatoma cells, into rats bearing AS-30D ascites resulted in the accumulation of 54 microg conjugate per 10(9) tumor cells after 2 hr. Ascites fluid and serum contained 0.53 and 0 microg/ml, respectively, RH1-betaG 2 hr after injection of the conjugate. Conjugate binding to AS-30D cells was heterogeneous and non-saturated, as determined by flow cytometry. BHAMG was less toxic than pHAM to SD rats based on measures of animal mortality, weight loss and hematological toxicity. Treatment of rats bearing established hepatoma ascites with 500 microg RH1-betaG followed 2 hr later with a single i.p. injection of 30 mg/kg BHAMG or 3 i.p. injections of 10 mg/kg BHAMG 2, 3 and 4 hr later resulted in the cure of 6/8 and 8/8 animals, respectively. Treatment with BHAMG or pHAM alone did not produce cures, whereas treatment with a control antibody-betaG conjugate and BHAMG produced significantly greater hematological toxicity compared to treatment with RH1-betaG and BHAMG. All cured rats were completely protected from rechallenge with 2 x 10(7) AS-30D cells, indicating that successful treatment of animals induced protective immunity.
Assuntos
Mostarda de Anilina/análogos & derivados , Antineoplásicos/uso terapêutico , Ascite/terapia , Carcinoma Hepatocelular/terapia , Glucuronidase/metabolismo , Imunotoxinas/uso terapêutico , Neoplasias Hepáticas/terapia , Pró-Fármacos/uso terapêutico , Mostarda de Anilina/metabolismo , Mostarda de Anilina/uso terapêutico , Mostarda de Anilina/toxicidade , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Ascite/metabolismo , Carcinoma Hepatocelular/metabolismo , Imunotoxinas/metabolismo , Leucócitos/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Pró-Fármacos/metabolismo , Pró-Fármacos/toxicidade , Ratos , Ratos Sprague-Dawley , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Methoxypoly(ethylene glycol) (PEG) modification of Escherichia coli beta-glucuronidase (betaG) was examined as a method to improve the stability and pharmacokinetics of antibody-betaG conjugates for the targeted activation of glucuronide prodrugs at tumor cells. Introduction of 3 PEG molecules did not affect betaG activity whereas higher degrees of PEG modification produced progressively greater loss of enzymatic activity. The enzyme was found to be stable in serum regardless of PEG modification. PEG-modified betaG was coupled via a thioether bond to mAb RH1, an IgG2a antibody that binds to the surface of AS-30D hepatoma cells, to produce conjugates with 3 (RH1-betaG-3PEG), 5.2 (RH1-betaG-5PEG) or 9.8 (RH1-betaG-10PEG) PEG molecules per betaG with retention of 75%, 45% and 40% of the combined antigen-binding and enzymatic activity of the unmodified conjugate RH1-betaG. In contrast to the rapid serum clearance of RH1-betaG observed in mice, the PEG-modified conjugates displayed extended serum half-lives. RH1-betaG-3PEG and RH1-betaG-5PEG also exhibited reduced spleen uptake and greater tumor accumulation than RH1-betaG. BHAMG, the glucuronide prodrug of p-hydroxyaniline mustard (pHAM), was relatively nontoxic in vivo. Injection of 6 mg/kg or 12 mg/kg pHAM i.v. depressed white blood cell numbers by 46% and 71% whereas 80 mg/kg BHAMG reduced these levels by 22%. Although the tumor/blood ratio of RH1-betaG-5PEG was adversely affected by slow clearance from serum, combined therapy of small solid hepatoma tumors with this conjugate, followed 4 and 5 days later with i.v. injections of BHAMG, cured all of seven mice with severe combined immunodeficiency. Combined treatment with a control antibody-betaG conjugate and BHAMG delayed tumor growth and cured two of six mice while treatment with pHAM or BHAMG alone was ineffective.
Assuntos
Antineoplásicos/farmacocinética , Glucuronidase/farmacocinética , Imunoconjugados/farmacocinética , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/metabolismo , Polietilenoglicóis/farmacocinética , Pró-Fármacos/farmacocinética , Mostarda de Anilina/análogos & derivados , Mostarda de Anilina/toxicidade , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Antineoplásicos/toxicidade , Estabilidade Enzimática , Glucuronatos/farmacocinética , Glucuronatos/farmacologia , Glucuronidase/sangue , Glucuronidase/farmacologia , Imunoconjugados/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Polietilenoglicóis/farmacologia , Ratos , Transplante HeterólogoAssuntos
Medicamentos de Ervas Chinesas/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Vírus da Hepatite B/genética , Hepatoblastoma/patologia , Humanos , Neoplasias Hepáticas/patologia , Quinolinas/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
Pseudomonas exotoxin A (PE) linked to the F(ab')2 fragment of 1H10, a murine monoclonal antibody recognizing a carbohydrate epitope of a glycoconjugate expressed on the surface of human cervical carcinoma tumor cells, was evaluated for in vitro and in vivo activity. PE can kill cells by ADP-ribosylating elongation factor 2 thus inhibiting protein synthesis. Disulfide- as well as thioether-linked immunotoxins (1H10-PE) killed cervical carcinoma cells in vitro and were 20-160 times more inhibitory to target than to control cells. Cell killing was antibody mediated as demonstrated by the reduction of 1H10-PE growth inhibition to target CaSki cells by free 1H10 F(ab')2. In addition, a control antibody immunotoxin was nontoxic to CaSki cells. Thioether-linked 1H10-PE administered either i.v. or i.p. suppressed the growth of established solid s.c. cervical carcinoma tumors xenografted in nude mice for over 30 days. Treatment with antibody alone or a control immunotoxin had no significant effect on tumor growth. Administration of immunotoxin i.p. was associated with less toxicity than administration i.v., but i.v. injections were more effective at suppressing the growth of established solid tumors.