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1.
Br J Cancer ; 128(2): 245-254, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36352028

RESUMO

BACKGROUND: Epidermal growth factor receptor (EGFR) is a therapeutic target to which HER2/HER3 activation may contribute resistance. This Phase I/II study examined the toxicity and efficacy of high-dose pulsed AZD8931, an EGFR/HER2/HER3 inhibitor, combined with chemotherapy, in metastatic colorectal cancer (CRC). METHODS: Treatment-naive patients received 4-day pulses of AZD8931 with irinotecan/5-FU (FOLFIRI) in a Phase I/II single-arm trial. Primary endpoint for Phase I was dose limiting toxicity (DLT); for Phase II best overall response. Samples were analysed for pharmacokinetics, EGFR dimers in circulating exosomes and Comet assay quantitating DNA damage. RESULTS: Eighteen patients received FOLFIRI and AZD8931. At 160 mg bd, 1 patient experienced G3 DLT; 160 mg bd was used for cohort expansion. No grade 5 adverse events (AE) reported. Seven (39%) and 1 (6%) patients experienced grade 3 and grade 4 AEs, respectively. Of 12 patients receiving 160 mg bd, best overall response rate was 25%, median PFS and OS were 8.7 and 21.2 months, respectively. A reduction in circulating HER2/3 dimer in the two responding patients after 12 weeks treatment was observed. CONCLUSIONS: The combination of pulsed high-dose AZD8931 with FOLFIRI has acceptable toxicity. Further studies of TKI sequencing may establish a role for pulsed use of such agents rather than continuous exposure. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov number: NCT01862003.


Assuntos
Neoplasias Colorretais , Receptor ErbB-3 , Humanos , Receptor ErbB-3/metabolismo , Transdução de Sinais , Quinazolinas/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/induzido quimicamente , Fluoruracila , Leucovorina/efeitos adversos , Camptotecina , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo
2.
BMC Vet Res ; 11: 215, 2015 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-26282406

RESUMO

BACKGROUND: Cancer is the leading cause of death in older dogs and its prevalence is increasing. There is clearly a need to develop more effective anti-cancer drugs in dogs. SG2000 (SJG-136) is a sequence selective DNA minor groove cross-linking agent. Based on its in vitro potency, the spectrum of in vivo and clinical activity against human tumours, and its tolerability in human patients, SG2000 has potential as a novel therapeutic against spontaneously occurring canine malignancies. RESULTS: In vitro cytotoxicity was assessed using SRB and MTT assays, and in vivo activity was assessed using canine tumour xenografts. DNA interstrand cross-linking (ICL) was determined using a modification of the single cell gel electrophoresis (comet) assay. Effects on cell cycle distribution were assessed by flow cytometry and measurement of γ-H2AX by immunofluorescence and immunohistochemistry. SG2000 had a multi-log differential cytotoxic profile against a panel of 12 canine tumour cell lines representing a range of common tumour types in dogs. In the CMeC-1 melanoma cell line, DNA ICLs increased linearly with dose following a 1 h treatment. Peak ICL was achieved within 1 h and no removal was observed over 48 h. A relationship between DNA ICL formation and cytotoxicity was observed across cell lines. The formation of γ-H2AX foci was slow, becoming evident after 4 h and reaching a peak at 24 h. SG2000 exhibited significant anti-tumour activity against two canine melanoma tumour models in vivo. Anti-tumour activity was observed at 0.15 and 0.3 mg/kg given i.v. either once, or weekly x 3. Dose-dependent DNA ICL was observed in tumours (and to a lower level in peripheral blood mononuclear cells) at 2 h and persisted at 24 h. ICL increased following the second and third doses in a repeated dose schedule. At 24 h, dose dependent γ-H2AX foci were more numerous than at 2 h, and greater in tumours than in peripheral blood mononuclear cells. SG2000-induced H2AX phosphorylation measured by immunohistochemistry showed good correspondence, but less sensitivity, than measurement of foci. CONCLUSIONS: SG2000 displayed potent activity in vitro against canine cancer cell lines as a result of the formation and persistence of DNA ICLs. SG2000 also had significant in vivo antitumour activity against canine melanoma xenografts, and the comet and γ-H2AX foci methods were relevant pharmacodynamic assays. The clinical testing of SG2000 against spontaneous canine cancer is warranted.


Assuntos
Antineoplásicos/farmacologia , Benzodiazepinonas/farmacologia , Doenças do Cão/tratamento farmacológico , Pirróis/farmacologia , Animais , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/farmacologia , DNA , Cães , Relação Dose-Resposta a Droga , Esquema de Medicação , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Citometria de Fluxo , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Oncotarget ; 6(12): 10548-62, 2015 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-25868852

RESUMO

Treatment options for ovarian cancer patients remain limited and overall survival is less than 50% despite recent clinical advances. The lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B) has been described as a tumor suppressor in the PI3K/Akt pathway with loss of expression found most pronounced in breast, ovarian cancer and melanoma. Using microarray technology we identified a DNA repair defect in INPP4B-deficient cells, which we further characterized by comet assays and quantification of γH2AX, RAD51 and 53BP1 foci formation. INPP4B loss resulted in significantly increased sensitivity towards PARP inhibition, comparable to loss of BRCA1 in two- and three-dimensional in vitro models, as well as in in vivo xenograft models. Mechanistically, we discovered that INPP4B forms a protein complex with the key players of DNA repair, ATR and BRCA1, in GST pulldown and 293T overexpression assays, and INPP4B loss affects BRCA1, ATM and ATR protein stability resulting in the observed DNA repair defect. Given that INPP4B loss has been found in 40% of ovarian cancer patients, this study provides the rationale for establishing INPP4B as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients. Loss of the tumor suppressor inositol polyphosphate 4-phosphatase type II (INPP4B) results in a DNA repair defect due to concomitant loss of BRCA1, ATR and ATM and can be therapeutically targeted with PARP inhibitors.


Assuntos
Neoplasias da Mama/genética , Reparo do DNA , Neoplasias Ovarianas/genética , Monoéster Fosfórico Hidrolases/deficiência , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Genes Supressores de Tumor , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Transcriptoma , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Cell Rep ; 10(12): 2006-18, 2015 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-25818292

RESUMO

Deficiencies in DNA double-strand break (DSB) repair lead to genetic instability, a recognized cause of cancer initiation and evolution. We report that the retinoblastoma tumor suppressor protein (RB1) is required for DNA DSB repair by canonical non-homologous end-joining (cNHEJ). Support of cNHEJ involves a mechanism independent of RB1's cell-cycle function and depends on its amino terminal domain with which it binds to NHEJ components XRCC5 and XRCC6. Cells with engineered loss of RB family function as well as cancer-derived cells with mutational RB1 loss show substantially reduced levels of cNHEJ. RB1 variants disabled for the interaction with XRCC5 and XRCC6, including a cancer-associated variant, are unable to support cNHEJ despite being able to confer cell-cycle control. Our data identify RB1 loss as a candidate driver of structural genomic instability and a causative factor for cancer somatic heterogeneity and evolution.


Assuntos
Antígenos Nucleares/metabolismo , Reparo do DNA por Junção de Extremidades/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica/genética , Proteína do Retinoblastoma/metabolismo , Ciclo Celular/fisiologia , Quebras de DNA de Cadeia Dupla , Humanos , Autoantígeno Ku , Recombinação Genética/genética , Proteínas Supressoras de Tumor/metabolismo
5.
Clin Cancer Res ; 19(3): 721-30, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23251007

RESUMO

PURPOSE: To evaluate γ-H2AX foci as a pharmacodynamic marker for DNA damage induced by DNA interstrand cross-linking drugs. EXPERIMENTAL DESIGN: γ-H2AX foci formation was validated preclinically in comparison with the Comet assay, and evaluated pharmacodynamically in two phase I studies of different dosing schedules of the novel cross-linking agent SJG-136 (SG2000). RESULTS: The measurement of γ-H2AX foci in human fibroblasts and lymphocytes in vitro was more than 10-fold more sensitive than Comet assay measurement of cross-linking, with peak γ-H2AX response 24 hours after the peak of cross-linking. In lymphocytes from a phase I study (every three week schedule), γ-H2AX foci were detectable 1 hour following the end of administration, and in all patients, maximum response was observed at 24 hours. Significant levels of foci were still evident at days 8 and 15 consistent with the known persistence of the DNA damage produced by this agent. In two tumor biopsy samples, foci were detected 4 hours postinfusion with levels higher than in lymphocytes. Extensive foci formation was also observed before the third dose in cycle 1 in lymphocytes from a second phase I study (daily × 3 schedule). These foci also persisted with a significant level evident before the second cycle (day 21). An increased γ-H2AX response was observed during the second cycle consistent with a cumulative pharmacodynamic effect. No clear relationship between foci formation and administered drug dose was observed. CONCLUSION: This is the first use of γ-H2AX as a pharmacodynamic response to a DNA cross-linking agent in a clinical trial setting.


Assuntos
Benzodiazepinonas/farmacologia , Dano ao DNA/efeitos dos fármacos , Histonas/genética , Pirróis/farmacologia , Ensaio Cometa , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Histonas/metabolismo , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Farmacogenética
6.
BMC Cancer ; 12: 436, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-23020514

RESUMO

BACKGROUND: DNA interstrand cross-links (ICLs) are critical lesions produced by several cancer chemotherapy agents including platinum drugs and nitrogen mustards. We have previously shown in haematological (multiple myeloma) and solid tumours (ovarian cancer) that clinical sensitivity to such agents can result from a defect in DNA ICL processing leading to their persistence. Conversely, enhanced repair can result in clinical acquired resistance following chemotherapy. The repair of ICLs is complex but it is assumed that the 'unhooking' step is common to all ICLs. METHODS: Using a modification of the single cell gel electrophoresis (Comet) assay we measured the formation and unhooking of melphalan and cisplatin-induced ICLs in cell lines and clinical samples. DNA damage response in the form of γ-H2AX foci formation and the formation of RAD51 foci as a marker of homologous recombination were also determined. Real-time PCR of 84 genes involved in DNA damage signalling pathways was also examined pre- and post-treatment. RESULTS: Plasma cells from multiple myeloma patients known to be clinically resistant to melphalan showed significant unhooking of melphalan-induced ICLs at 48 hours, but did not unhook cisplatin-induced ICLs. In ovarian cancer cells obtained from patients following platinum-based chemotherapy, unhooking of cisplatin-induced ICLs was observed at 48 hours, but no unhooking of melphalan-induced ICLs. In vitro, A549 cells were proficient at unhooking both melphalan and cisplatin-induced ICLs. γ-H2AX foci formation closely followed the formation of ICLs for both drugs, and rapidly declined following the peak of formation. RPMI8226 cells unhooked melphalan, but not cisplatin-induced ICLs. In these cells, although cross-links form with cisplatin, the γ-H2AX response is weak. In A549 cells, addition of 3nM gemcitabine resulted in complete inhibition of cisplatin-induced ICL unhooking but no effect on repair of melphalan ICLs. The RAD51 foci response was both drug and cell line specific. Real time PCR studies highlighted differences in the damage response to melphalan and cisplatin following equi-ICL forming doses. CONCLUSIONS: These data suggest that the mechanisms by which melphalan and cisplatin-induced ICLs are 'unhooked' in vitro are distinct, and the mechanisms of clinical acquired resistance involving repair of ICLs, are drug specific.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Dano ao DNA , Reparo do DNA , DNA/efeitos dos fármacos , Melfalan/farmacologia , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Histonas/genética , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/genética , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Rad51 Recombinase/genética , Transdução de Sinais , Gencitabina
7.
Methods Mol Biol ; 731: 309-20, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21516417

RESUMO

The Single Cell Gel Electrophoresis (Comet) assay is a simple, versatile and sensitive method for measuring DNA damage in individual cells, allowing the determination of heterogeneity of response within a cell population. The basic alkaline technique described is for the determination of DNA strand break damage and its repair at a single cell level. Specific modifications to the method use a lower pH ('neutral' assay), or allow the measurement of DNA interstrand cross-links. It can be further adapted to, for example, study specific DNA repair mechanisms, be combined with fluorescent in situ hybridisation, or incorporate lesion specific enzymes.


Assuntos
Ensaio Cometa/métodos , Dano ao DNA , Análise de Célula Única/métodos , Adesão Celular , Linhagem Celular Tumoral , DNA Glicosilases/metabolismo , Reparo do DNA , Desoxirribonuclease I/metabolismo , Humanos , Hibridização in Situ Fluorescente , Imagem Molecular , Coloração e Rotulagem
8.
Clin Cancer Res ; 17(11): 3794-802, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21346148

RESUMO

PURPOSE: This phase I study assessed the maximum tolerated dose (MTD), safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of SJG-136, a sequence-specific DNA cross-linking agent, in patients with advanced cancer. EXPERIMENTAL DESIGN: In schedule A, seven patients received escalating doses of SJG-136 (6, 12, 24, and 48 µg/m(2)) daily for 5 of 21 days. Blood samples were collected for PK analysis on days 1 and 5 of cycle 1. In schedule B, SJG-136 was given daily for 3 of 21 days (N = 17; doses 20, 25, 30, and 35 µg/m(2)). Blood samples were collected on days 1 and 3 of cycles 1 and 2 for PK and PD analysis. Patients in schedule B received dexamethasone and early diuretic care. RESULTS: Schedule A-dose-limiting toxicities included grade 3 edema, dyspnea, fatigue, and delayed liver toxicity (grade 3-4). PK analysis revealed dose-dependent increases in AUC and C(max). Substantial changes in volume of distribution at steady-state occurred after repeated dosing in some patients prior to the onset of edema. Schedule B-the same toxicities were manageable with steroid premedication and diuretic support. No significant myelosuppression occurred on either schedule. DNA interstrand cross-links correlated with systemic exposure of SJG-136 following the second dose in cycle 1 and were still detectable immediately before cycle 2. CONCLUSIONS: The MTD of SJG-136 in this study was 30 µg/m(2) administered on a daily 3× basis with no myelosuppression effects. Coupled with supportive management, SJG-136 is now advancing to a phase II trial in ovarian cancer.


Assuntos
Benzodiazepinonas/farmacologia , Dose Máxima Tolerável , Neoplasias/tratamento farmacológico , Pirróis/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Benzodiazepinonas/efeitos adversos , Benzodiazepinonas/metabolismo , Benzodiazepinonas/farmacocinética , Dexametasona/administração & dosagem , Dispneia/induzido quimicamente , Edema/induzido quimicamente , Fadiga/induzido quimicamente , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pirróis/efeitos adversos , Pirróis/metabolismo , Pirróis/farmacocinética
9.
Cancer Chemother Pharmacol ; 68(3): 777-86, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21188379

RESUMO

PURPOSE: The dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501, SG2000) has potent in vitro antiproliferative activity and in vivo antitumor activity associated with binding in the minor groove of DNA and formation of covalent interstrand DNA cross-links. The pharmacokinetics and in vitro metabolism of SJG-136 and as well as the feasibility of using the Comet assay to measure in vivo interstrand DNA cross-links, was assessed in the rat. METHODS: SJG-136 pharmacokinetics and pharmacodynamics were characterized in rats following single-dose administration of 15 and 50 µg/kg or multiple-dose administration of 25 µg/kg/day for 5 days. DNA damage was measured in peripheral blood mononuclear cells using the Comet assay. SJG-136 oxidative metabolism was characterized in rat liver microsomes. RESULTS: SJG-136 half-life, clearance and volume of distribution values were 9 min, 190 ml/min/m(2), and 1780 ml/m(2), respectively. SJG-136 did not accumulate in plasma during treatment with 25 µg/kg/day for 5 days. Treatment with SJG-136 produced the anticipated DNA interstrand cross-links, as well as DNA strand breaks, in rat PBMCs. Oxidative metabolism of SJG-136 in rat liver microsomes was catalyzed by CYP3A isoforms and produced a previously unreported monomeric metabolite. CONCLUSIONS: Plasma concentrations of SJG-136 associated with pharmacological activity and in vitro antiproliferative activity were achieved with doses that were tolerated by rats. CYP3A isoforms are the predominant P450s catalyzing SJG-136 metabolism. The comet assay detects DNA damage in PBMCs from rats treated with SJG-136 and is being used in clinical trials to monitor in vivo lesions produced by SJG-136.


Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Benzodiazepinonas/farmacologia , Benzodiazepinonas/farmacocinética , Pirróis/farmacologia , Pirróis/farmacocinética , Animais , Antineoplásicos/metabolismo , Área Sob a Curva , Benzodiazepinonas/metabolismo , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , Ensaio Cometa , DNA/efeitos dos fármacos , Dano ao DNA , Meia-Vida , Técnicas In Vitro , Indicadores e Reagentes , Masculino , Espectrometria de Massas , Microssomos Hepáticos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/ultraestrutura , Soluções Farmacêuticas , Pirróis/metabolismo , Ratos , Ratos Endogâmicos F344
10.
Clin Cancer Res ; 16(19): 4899-905, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20719935

RESUMO

BACKGROUND: The potential of gemcitabine to interact with carboplatin was explored in a phase II trial in platinum-resistant ovarian cancer. Peripheral blood lymphocytes were sampled after drug administration to measure DNA interstrand cross-link formation and repair. PATIENTS AND METHODS: Forty patients received carboplatin target area under concentration-time curve (AUC 4) followed by gemcitabine 1,000 mg/m(2) with a second dose of gemcitabine on day 8. Peripheral blood lymphocytes were obtained in 12 patients before and at intervals during the first cycle of chemotherapy. DNA cross-link formation and repair (unhooking) were measured by the single-cell gel electrophoresis (comet) assay following ex vivo incubation. RESULTS: The global response rate was 47% (Response Evaluation Criteria in Solid Tumors rate, 29%; CA125 rate, 63%). Delays in treatment were seen in 24% of cycles largely due to myelosuppression; 15% of day 8 administration was omitted. Peak carboplatin-induced DNA cross-linking was seen by 24 hours. Significant reduction was seen in the repair of in vivo carboplatin-induced DNA cross-links following administration of gemcitabine. CONCLUSION: An enhanced activity of carboplatin in platinum-resistant ovarian cancer may be due to synergy with gemcitabine through inhibition of repair of DNA cross-links. Future studies should explore coadministration of these drugs, as this may be a more effective schedule.


Assuntos
Antineoplásicos/uso terapêutico , Carboplatina/antagonistas & inibidores , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Compostos de Platina/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Carboplatina/administração & dosagem , Carboplatina/farmacologia , Carboplatina/uso terapêutico , DNA de Neoplasias/metabolismo , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Relação Dose-Resposta a Droga , Feminino , Humanos , Pessoa de Meia-Idade , Compostos de Platina/farmacologia , Fatores de Tempo , Resultado do Tratamento , Gencitabina
11.
Sci Transl Med ; 2(40): 40ra50, 2010 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-20630857

RESUMO

DNA-damaging agents are widely used in cancer treatment despite their lack of tumor specificity. Human NQO2 (quinone oxidoreductase-2) is an atypical oxidoreductase because no endogenous electron donor has been identified to date. The enzyme converts CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], in the presence of the synthetic nicotinamide cofactor analog EP0152R, to a cytotoxic bifunctional alkylating agent. NQO2 activity in hepatocellular tumor tissue is higher than that in other cancer types by a factor of 6 and higher than that in bone marrow by a factor of 20. Structural data from x-ray crystallography and nuclear magnetic resonance spectroscopy allowed us to construct a model of CB1954 and EP0152R binding to NQO2, which suggested an optimal infusion schedule for a phase I trial combining the two agents. Thirty-two patients were treated, and diarrhea and serum transaminase concentrations defined a maximum tolerated dose for the drug combination. There was a clear pharmacokinetic interaction, with EP0152R inducing a marked increase in clearance of CB1954, in keeping with model predictions. We detected DNA interstrand cross-links caused by nitroreduced CB1954 in tumor biopsies from treated patients, demonstrating that the activated prodrug exerts its cytotoxic properties through DNA base alkylation.


Assuntos
Antineoplásicos/uso terapêutico , Aziridinas/uso terapêutico , Neoplasias/tratamento farmacológico , Pró-Fármacos/uso terapêutico , Quinona Redutases/metabolismo , Adulto , Idoso , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Antineoplásicos/farmacocinética , Aziridinas/efeitos adversos , Aziridinas/química , Aziridinas/farmacocinética , Morte Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Cristalografia por Raios X , DNA de Neoplasias/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Quinona Redutases/química
12.
Methods Mol Biol ; 613: 267-82, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19997890

RESUMO

The Single Cell Gel Electrophoresis (Comet) assay, originally developed to allow visualisation of DNA strand break damage in individual cells, has been adapted to measure DNA interstrand cross-links. DNA interstrand cross-links are formed in cells by a number of commonly used cancer chemotherapy agents and are considered to be the critical lesion formed by such agents. This technique allows the analysis of DNA interstrand cross-link formation and repair at a single cell level, requires few cells, allows the determination of heterogeneity of response within a cell population and is sensitive enough to measure DNA interstrand cross-links at pharmacologically relevant doses. The method can be applied to any in vitro or in vivo application where a single cell suspension can be obtained. The method has also become invaluable in studies using human tissue and can be used as a method for pharmacodynamic analysis in early clinical trials.


Assuntos
Ensaio Cometa/métodos , Reagentes de Ligações Cruzadas/farmacologia , DNA/química , DNA/metabolismo , Ascite/metabolismo , Linhagem Celular Tumoral , DNA/genética , Dano ao DNA , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Melfalan/farmacologia , Coloração e Rotulagem
13.
Clin Cancer Res ; 15(6): 2140-7, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19276288

RESUMO

PURPOSE: This phase I dose-escalation study was undertaken to establish the maximum tolerated dose of the sequence-selective minor groove DNA binding agent SJG-136 in patients with advanced solid tumors. The study also investigated antitumor activity and provided pharmacokinetic and pharmacodynamic data. EXPERIMENTAL DESIGN: Sixteen patients were assigned sequentially to escalating doses of SJG-136 (15-240 microg/m(2)) given as a 10-minute i.v. infusion every 21 days. The dose was subsequently reduced in incremental steps to 45 microg/m(2) due to unexpected toxicity. RESULTS: The maximum tolerated dose of SJG-136 was 45 microg/m(2). The main drug-related adverse event was vascular leak syndrome (VLS) characterized by hypoalbuminemia, pleural effusions, ascites, and peripheral edema. Other unexpected adverse events included elevated liver function tests and fatigue. The VLS and liver toxicity had delayed onset and increased in severity with subsequent cycles. Disease stabilization was achieved for >6 weeks in 10 patients; in 2 patients this was maintained for >12 weeks. There was no evidence of DNA interstrand cross-linking in human blood lymphocytes with the use of the comet assay. Evidence of DNA interaction in lymphocytes and tumor cells was shown through a sensitive gamma-H2AX assay. SJG-136 had linear pharmacokinetics across the dose range tested. CONCLUSIONS: SJG-136 was associated with dose-limiting VLS and hepatotoxicity when administered by short injection every 21 days. DNA damage was noted, at all dose levels studied, in circulating lymphocytes. The etiology of the observed toxicities is unclear and is the subject of further preclinical research. Alternative clinical dosing strategies are being evaluated.


Assuntos
Antineoplásicos/uso terapêutico , Benzodiazepinonas/uso terapêutico , Neoplasias/tratamento farmacológico , Pirróis/uso terapêutico , Adulto , Idoso , Benzodiazepinonas/efeitos adversos , Benzodiazepinonas/farmacocinética , DNA/metabolismo , Histonas/análise , Humanos , Dose Máxima Tolerável , Pessoa de Meia-Idade , Pirróis/efeitos adversos , Pirróis/farmacocinética
14.
Mol Cancer Ther ; 5(6): 1602-9, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16818520

RESUMO

SJG-136 is a new pyrrolobenzodiazepine dimer inducing time-dependent cytotoxicity. HCT 116 cells were exposed to 50 nmol/L of SJG-136 for 1 hour or 1 nmol/L of SJG-136 for 24 hours to achieve similar levels of interstrand cross-links (ICL). The short exposure led to a rapid formation of ICLs (1 hour), early H2AX foci formation (4 hours), prominent S phase arrest, and greater phosphorylation of Nbs1 (on serine 343) and Chk1 (on serine 317) than a 24-hour exposure. The prolonged exposure at low concentrations of SJG-136 induced a gradual formation of ICLs (up to 24 hours) which was associated with a limited S phase arrest and delayed Nbs1 phosphorylation. Prolonged exposure was also associated with a reduced phosphorylation of p53 on serines 15 and 20, a limited and delayed phosphorylation on serine 392, and a less prominent increase in p21 levels. These data suggest that the 24-hour exposure to a low concentration of SJG-136 led to delayed and reduced DNA damage signaling compared with a higher concentration of SJG-136 for 1 hour, resulting in greater cytotoxicity and contributing to the time-dependent cytotoxic effect of SJG-136.


Assuntos
Benzodiazepinonas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA/efeitos dos fármacos , Pirróis/farmacologia , Transdução de Sinais , Quinase 1 do Ponto de Checagem , DNA/metabolismo , Células HCT116/efeitos dos fármacos , Células HCT116/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Fase S/efeitos dos fármacos , Fatores de Tempo
15.
Bioorg Med Chem Lett ; 16(2): 252-6, 2006 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-16290933

RESUMO

The design, synthesis and evaluation of four novel pyrrolo[2,1-c][1,4]benzodiazepine (PBD) prodrugs (1a,b and 2a,b; ) for potential use in carboxypeptidase G2 (CPG2)-based antibody-directed enzyme prodrug therapy (ADEPT) is reported. Although all four prodrugs were shown to be less cytotoxic than the released parent PBDs 3 and 4, the urea prodrugs 1b and 2b were found to be too unstable for use in ADEPT, whereas carbamates 1a and 2a are both stable in an aqueous environment and are good substrates for CPG2.


Assuntos
Anticorpos/uso terapêutico , Antineoplásicos , Benzodiazepinas , Neoplasias do Colo/tratamento farmacológico , Pró-Fármacos , Pirróis , gama-Glutamil Hidrolase/uso terapêutico , Anticorpos/química , Antígenos de Neoplasias/efeitos dos fármacos , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzodiazepinas/síntese química , Benzodiazepinas/farmacologia , Benzodiazepinas/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Compostos de Mostarda Nitrogenada/química , Compostos de Mostarda Nitrogenada/farmacologia , Compostos de Mostarda Nitrogenada/uso terapêutico , Pró-Fármacos/síntese química , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Pirróis/síntese química , Pirróis/farmacologia , Pirróis/uso terapêutico , Relação Estrutura-Atividade , Fatores de Tempo , gama-Glutamil Hidrolase/química
16.
Cancer Res ; 64(18): 6693-9, 2004 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-15374986

RESUMO

SJG-136 (NSC 694501) is a rationally designed pyrrolobenzodiazepine dimer that binds in the minor groove of DNA. It spans 6 bp with a preference for binding to purine-GATC-pyrimidine sequences. The agent has potent activity in the National Cancer Institute (NCI) anticancer drug screen with 50% net growth inhibition conferred by 0.14 to 320 nmol/L (7.4 nmol/L mean). Sensitive cell lines exhibit total growth inhibition and 50% lethality after treatment with as little as 0.83 and 7.1 nmol/L SJG-136, respectively. COMPARE and molecular target analysis of SJG-136 data versus that of >60,000 compounds tested in the NCI 60 cell line screen shows that, although the agent has similarity to other DNA binding agents, the pattern of activity for SJG-136 does not fit within the clusters of any known agents, suggesting that SJG-136 possesses a distinct mechanism of action. Testing in the NCI standard hollow fiber assay produced prominent growth inhibition in 20 of 24 i.p. and 7 of 24 s.c. test combinations with 5 of 12 cell lines exhibiting cell kill. In addition, SJG-136 produced antitumor activity in mice bearing CH1 and CH1cisR xenografts, a cisplatin-resistant human ovarian tumor model, and also in mice bearing LS174T xenografts, a human colon tumor model. SJG-136 produces DNA interstrand cross-links between two N-2 guanine positions on opposite strands and separated by 2 bp. In human tumor cell lines, the cross-links form rapidly and persist compared with those produced by conventional cross-linking agents such as nitrogen mustards. In mice bearing the LS174T human colon xenograft, DNA interstrand cross-links can be detected in tumor cells using a modification of the single cell gel electrophoresis (comet) assay after administration of a therapeutic dose. Cross-links in the tumor increase with dose and are clearly detectable at 1 hour after i.v. administration. The level of cross-linking persists over a 24-hour period in this tumor in contrast to cross-links produced by conventional cross-linking agents observed over the same time period.


Assuntos
Antineoplásicos/farmacologia , Benzodiazepinonas/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , DNA/efeitos dos fármacos , Pirróis/farmacologia , Animais , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral , Ensaio Cometa , DNA/metabolismo , Cães , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Blood ; 100(1): 224-9, 2002 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-12070031

RESUMO

Melphalan is widely used as a preparative agent in patients with multiple myeloma (MM) undergoing autologous stem cell transplantation (SCT). Although disease relapse is the major cause of death after a melphalan-conditioned autograft, the mechanism remains unclear. Melphalan produces a number of DNA adducts with the DNA interstrand crosslink (ICL) considered to be the critical cytotoxic lesion. By using a modification of the single-cell gel electrophoresis (Comet) assay, we have measured formation and repair of DNA ICL in plasma cells from melphalan- naive and melphalan-treated patients (ie, those who have relapsed after a melphalan-conditioned autologous SCT or oral melphalan therapy). Similar levels of dose-dependent DNA interstand crosslinking were observed in cells from both melphalan-naive and -treated patients. However, marked differences in ICL repair were observed: cells from naive patients showed no repair, whereas those from treated patients exhibited between 42% and 100% repair at 40 hours. In vitro sensitivity to melphalan in plasma cells was found to correlate with ICL repair. These findings suggest that ICL repair may be an important mechanism by which melphalan resistance emerges after autologous SCT or oral therapy. This mechanism may have implications for MM patients undergoing melphalan therapy.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Reparo do DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos , Melfalan/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Adulto , Idoso , Antineoplásicos Alquilantes/administração & dosagem , Ensaio Cometa , Adutos de DNA/farmacologia , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Cinética , Masculino , Melfalan/administração & dosagem , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/patologia
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