RESUMEN
HSP90 (Heat shock protein 90) is a molecular chaperone protein ubiquitously expressed throughout all tissues in the body. HSP90 has been proposed as a target to increase turnover of pathological proteins leading to neurodegeneration in Huntington's disease, Parkinson's disease and Alzheimer's disease. The mechanism of how HSP90 inhibition leads to clearance of misfolded proteins is not fully understood. It may involve direct effects of inhibiting ATPase function, indirect effects by inducing the heat-shock-response resulting in upregulation of other chaperone proteins like HSP70 or a combination of both. In the current work we established a methodology to investigate the relationship between HSP90 target occupancy and HSP70 induction in vivo. We also characterized the acute effect of two different HSP90 inhibitors in the rTg4510 transgenic mouse model of Alzheimer's disease which displays a tau-mediated synaptic dysfunction. We show that reversal of synaptic impairments in this model can be obtained with a compound which has a high HSP70 induction capacity. The current developed assay methodologies may thus be of significant use in the further elucidation of the mechanism involved in the in vivo effect of HSP90 inhibition in models of neurodegeneration. Further on, the ability of HSP90 inhibitors to normalize synaptic dysfunction in an in vivo disease model of Alzheimer's disease could have therapeutic relevance and further strengthens the usefulness of this animal model to establish pharmacodynamic effect of HSP90 inhibition.
Asunto(s)
Encéfalo/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Animales , Línea Celular , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Ratones Transgénicos , Proteínas tau/genéticaRESUMEN
APO866 inhibits nicotinamide phosphoribosyltransferase (NMPRTase), a key enzyme involved in nicotinamide adenine dinucleotide (NAD) biosynthesis from the natural precursor nicotinamide. Intracellular NAD is essential for cell survival, and NAD depletion resulting from APO866 treatment elicits tumor cell death. Here, we determine the in vitro and in vivo sensitivities of hematologic cancer cells to APO866 using a panel of cell lines (n = 45) and primary cells (n = 32). Most cancer cells (acute myeloid leukemia [AML], acute lymphoblastic leukemia [ALL], mantle cell lymphoma [MCL], chronic lymphocytic leukemia [CLL], and T-cell lymphoma), but not normal hematopoietic progenitor cells, were sensitive to low concentrations of APO866 as measured in cytotoxicity and clonogenic assays. Treatment with APO866 decreased intracellular NAD and adenosine triphosphate (ATP) at 24 hours and 48 to72 hours, respectively. The NAD depletion led to cell death. At 96 hours, APO866-mediated cell death occurred in a caspase-independent mode, and was associated with mitochondrial dysfunction and autophagy. Further, in vivo administration of APO866 as a single agent prevented and abrogated tumor growth in animal models of human AML, lymphoblastic lymphoma, and leukemia without significant toxicity to the animals. The results support the potential of APO866 for treating hematologic malignancies.
Asunto(s)
Acrilamidas/uso terapéutico , Antineoplásicos/uso terapéutico , Citocinas/antagonistas & inhibidores , Neoplasias Hematológicas/tratamiento farmacológico , NAD/biosíntesis , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Piperidinas/uso terapéutico , Acrilamidas/farmacología , Animales , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HL-60 , Neoplasias Hematológicas/metabolismo , Humanos , Células Jurkat , Células K562 , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , NAD/efectos de los fármacos , Piperidinas/farmacología , Células Tumorales Cultivadas , Células U937 , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Alpha-synuclein is a 15 kDa protein associated with neurodegenerative diseases such as Parkinson disease and multiple-system atrophy where pathological forms of alpha-synuclein aggregate and become neurotoxic. Here we describe the nonclinical program to support a first-in-human (FIH) single ascending dose (SAD) study for Lu AF82422, a human recombinant, anti-alpha-synuclein monoclonal antibody (mAb) in development for treatment of synucleinopathies. Alpha-synuclein is primarily expressed in brain, peripheral nerves and in blood cells. A tissue cross-reactivity assessment showed that Lu AF82422 binding was generally restricted to nervous tissues. Flow cytometry analysis did not show extracellular surface binding of Lu AF82422 to human platelets, erythrocytes, granulocytes, or lymphocytes, but to a low fraction of monocytes, without any functional consequences on activation or phagocytic capacity. A single dose pharmacokinetic (PK) study in cynomolgus monkeys with dose levels of 1-30 mg/kg confirmed PK properties in the expected range for a mAb with a soluble target, and target engagement was shown as a decrease in free alpha-synuclein in plasma. Four-week repeat-dose toxicity studies were conducted in rats and cynomolgus monkeys at doses up to 600 mg/kg administered intravenously every 10 days. Results showed no treatment-related adverse findings and the no-observed-adverse-effect-level was the highest dose tested. Target engagement was shown in plasma and cerebrospinal fluid. Taken together, the nonclinical data indicated no safety signal of concern and provided adequate safety margins between observed safe doses in animals and the planned dose levels in the FIH SAD study.
Asunto(s)
Enfermedad de Parkinson , Sinucleinopatías , Animales , Anticuerpos Monoclonales/farmacocinética , Inmunoglobulina G , Ratas , alfa-SinucleínaRESUMEN
There remains an insufficient number of P2X7 receptor antagonists with adequate rodent potency, CNS permeability, and pharmacokinetic properties from which to evaluate CNS disease hypotheses preclinically. Herein, we describe the molecular pharmacology, safety, pharmacokinetics, and functional CNS target engagement of Lu AF27139, a novel rodent-active and CNS-penetrant P2X7 receptor antagonist. Lu AF27139 is highly selective and potent against rat, mouse, and human forms of the receptors. The rat pharmacokinetic profile is favorable with high oral bioavailability, modest clearance (0.79 L/(h kg)), and good CNS permeability. In vivo mouse CNS microdialysis studies of lipopolysaccharide (LPS)-primed and 2'(3')-O-(benzoylbenzoyl)adenosine-5'-triphosphate (BzATP)-induced IL-1ß release demonstrate functional CNS target engagement. Importantly, Lu AF27139 was without effect in standard in vitro and in vivo toxicity studies. Based on these properties, we believe Lu AF27139 will be a valuable tool for probing the role of the P2X7 receptor in rodent models of CNS diseases.
Asunto(s)
Sistema Nervioso Central/metabolismo , Antagonistas del Receptor Purinérgico P2X/síntesis química , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Línea Celular , Sistema Nervioso Central/efectos de los fármacos , Perros , Femenino , Semivida , Humanos , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Microglía/efectos de los fármacos , Microglía/metabolismo , Microsomas Hepáticos/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Antagonistas del Receptor Purinérgico P2X/metabolismo , Antagonistas del Receptor Purinérgico P2X/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2X7/químicaRESUMEN
BACKGROUND: Inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) are promising cancer drugs currently in clinical trials in oncology, including APO866, CHS-828 and the CHS-828 prodrug EB1627/GMX1777, but cancer cell resistance to these drugs has not been studied in detail. METHODS: Here, we introduce an analogue of CHS-828 called TP201565 with increased potency in cellular assays. Further, we describe and characterize a panel of cell lines with acquired stable resistance towards several NAMPT inhibitors of 18 to 20,000 fold compared to their parental cell lines. RESULTS: We find that 4 out of 5 of the resistant sublines display mutations of NAMPT located in the vicinity of the active site or in the dimer interface of NAMPT. Furthermore, we show that these mutations are responsible for the resistance observed. All the resistant cell lines formed xenograft tumours in vivo. Also, we confirm CHS-828 and TP201565 as competitive inhibitors of NAMPT through docking studies and by NAMPT precipitation from cellular lysate by an analogue of TP201565 linked to sepharose. The NAMPT precipitation could be inhibited by addition of APO866. CONCLUSION: We found that CHS-828 and TP201565 are competitive inhibitors of NAMPT and that acquired resistance towards NAMPT inhibitors can be expected primarily to be caused by mutations in NAMPT.
Asunto(s)
Acrilamidas/farmacología , Antineoplásicos/farmacología , Cianuros/farmacología , Citocinas/antagonistas & inhibidores , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Mutación , Neoplasias/tratamiento farmacológico , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Piperidinas/farmacología , Acrilamidas/metabolismo , Animales , Antineoplásicos/metabolismo , Sitios de Unión , Unión Competitiva , Dominio Catalítico , Línea Celular Tumoral , Cianuros/metabolismo , Citocinas/química , Citocinas/genética , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/metabolismo , Femenino , Guanidinas/metabolismo , Células HCT116 , Humanos , Concentración 50 Inhibidora , Ratones , Ratones Desnudos , Modelos Moleculares , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Nicotinamida Fosforribosiltransferasa/química , Nicotinamida Fosforribosiltransferasa/genética , Nicotinamida Fosforribosiltransferasa/metabolismo , Piperidinas/metabolismo , Conformación Proteica , Factores de Tiempo , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Anthracycline-induced cardiomyopathy is a major problem in anti-cancer therapy. The only approved agent for alleviating this serious dose limiting side effect is ICRF-187 (dexrazoxane). The current thinking is that the ring-opened hydrolysis product of this agent, ADR-925, which is formed inside cardiomyocytes, removes iron from its complexes with anthracyclines, hereby reducing the concentration of highly toxic iron-anthracycline complexes that damage cardiomyocytes by semiquinone redox recycling and the production of free radicals. However, the 2 carbon linker ICRF-187 is also is a catalytic inhibitor of topoisomerase II, resulting in the risk of additional myelosuppression in patients receiving ICRF-187 as a cardioprotectant in combination with doxorubicin. The development of a topoisomerase II-inactive iron chelating compound thus appeared attractive. In the present paper we evaluate the topoisomerase II-inactive 3 carbon linker bisdioxopiperazine analog ICRF-161 as a cardioprotectant. We demonstrate that this compound does chelate iron and protects against doxorubicin-induced LDH release from primary rat cardiomyocytes in vitro, similarly to ICRF-187. The compound does not target topoisomerase II in vitro or in cells, it is well tolerated and shows similar exposure to ICRF-187 in rodents, and it does not induce myelosuppression when given at high doses to mice as opposed to ICRF-187. However, when tested in a model of chronic anthracycline-induced cardiomyopathy in spontaneously hypertensive rats, ICRF-161 was not capable of protecting against the cardiotoxic effects of doxorubicin. Modulation of the activity of the beta isoform of the topoisomerase II enzyme by ICRF-187 has recently been proposed as the mechanism behind its cardioprotection. This concept is thus supported by the present study in that iron chelation alone does not appear to be sufficient for protection against anthracycline-induced cardiomyopathy.
Asunto(s)
Antibióticos Antineoplásicos/antagonistas & inhibidores , Antibióticos Antineoplásicos/toxicidad , Antineoplásicos/farmacología , Cardiomiopatías/inducido químicamente , Cardiomiopatías/prevención & control , ADN-Topoisomerasas de Tipo II/metabolismo , Doxorrubicina/antagonistas & inhibidores , Doxorrubicina/toxicidad , Razoxano/farmacología , Animales , Animales Recién Nacidos , Antineoplásicos/farmacocinética , Cardiomiopatías/patología , Ensayo de Unidades Formadoras de Colonias , Crithidia fasciculata/metabolismo , ADN/efectos de los fármacos , Compuestos Férricos/farmacología , Estimación de Kaplan-Meier , L-Lactato Deshidrogenasa/metabolismo , Ratones , Mitocondrias Cardíacas/efectos de los fármacos , Miocardio/patología , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Ratas , Ratas Endogámicas SHR , Razoxano/farmacocinética , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/patología , Troponina I/metabolismoRESUMEN
Parkinson's disease (PD) is a progressive neurodegenerative disorder for which there is no existing therapeutic approach to delay or stop progression. Genetic, biochemical and pre-clinical studies have provided evidence that leucine-rich-repeat-kinase-2 (LRRK2) kinase is involved in the pathogenesis of PD, and small molecule LRRK2 inhibitors represent a novel potential therapeutic approach. However, potentially adverse target-related effects have been discovered in the lung and kidneys of LRRK2 knock-out (ko) mice and rats. It is unclear if the LRRK2 ko effect in the kidneys and lung is also induced by pharmacological inhibition of the LRRK2 kinase. Here, we show that treatment with the LRRK2 inhibitor PFE-360 in rats induces a morphological kidney phenotype resembling that of the LRRK2 ko rats, whereas no effects were observed in the lung. The PFE-360 treatment induced morphological changes characterised by darkened kidneys and progressive accumulation of hyaline droplets in the renal proximal tubular epithelium. However, no histopathological evidence of renal tubular injury or changes in the blood and urine parameters that would be indicative of kidney toxicity or impaired kidney function were observed after up to 12â¯weeks of treatment. Morphological changes were detected in the kidney after 2 weeks of treatment and were partially reversible within a 30â¯day treatment-free period. Our findings suggest that pharmacological LRRK2 inhibition may not have adverse consequences for kidney function.
Asunto(s)
Inhibidores Enzimáticos/toxicidad , Riñón/efectos de los fármacos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Morfolinas/toxicidad , Pirimidinas/toxicidad , Pirroles/toxicidad , Animales , Peso Corporal/efectos de los fármacos , Femenino , Riñón/anatomía & histología , Riñón/metabolismo , Pruebas de Función Renal , Túbulos Renales Proximales/anatomía & histología , Túbulos Renales Proximales/efectos de los fármacos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/biosíntesis , Pulmón/anatomía & histología , Pulmón/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
PURPOSE: Histone deacetylase inhibitors (HDACi) inhibit the growth of cancer cells, and combinations of HDACi with established chemotherapeutics can lead to synergistic effects. We have investigated effects of PXD101 (HDACi in phase II clinical trials) in combination with 5-fluorouracil, on tumour cell proliferation and apoptosis both in vitro and in vivo. EXPERIMENTAL DESIGN: HCT116 cells were studied using proliferation and clonogenic assays. Synergistic inhibition of proliferation and clonogenicity was determined by incubation with PXD101 and 5-fluorouracil, and analysis using CalcuSyn software. The effect of combining PXD101 and 5-fluorouracil on apoptosis was examined in vitro using PARP-cleavage and TUNEL. Finally, the effectiveness of combining PXD101 and 5-fluorouracil in vivo was tested using both HT-29 and HCT116 xenograft models. RESULTS: Synergistic inhibition of proliferation and clonogenicity was obtained when HCT116 cells were incubated with PXD101 and 5-fluorouracil. 5-fluorouracil combined with PXD101 also increased DNA fragmentation and PARP cleavage in HCT116 cells. Incubation with PXD101 down regulated thymidylate synthase expression in HCT116 cells. In vivo studies, using mouse HT29 and HCT116 xenograft models, showed improved reductions in tumour volume compared to single compound, when PXD101 and 5-fluorouracil were combined. CONCLUSIONS: PXD101 and 5-fluorouracil synergistically combine in their anti-tumour effects against colon cancer cells in vitro and show enhanced activity when combined in vivo. Based on the results presented herein, a rationale for the use of PXD101 and 5-fluorouracil in combination in the clinic has been demonstrated.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Fluorouracilo/farmacología , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/patología , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Sinergismo Farmacológico , Femenino , Humanos , Ratones , Neoplasias Experimentales/patología , Sulfonamidas , Ensayo de Tumor de Célula Madre/métodos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
By screening 1,990 compounds from the National Cancer Institute diversity set library against human topoisomerase IIalpha, we identified a novel catalytic topoisomerase II inhibitor NSC35866, a S6-substituted analogue of thioguanine. In addition to inhibiting the DNA strand passage reaction of human topoisomerase IIalpha, NSC35866 also inhibited its ATPase reaction. NSC35866 primarily inhibited DNA-stimulated ATPase activity, whereas DNA-independent ATPase activity was less sensitive to inhibition. We compared the mode of topoisomerase II ATPase inhibition induced by NSC35866 with that of 12 other substituted purine analogues of different chemical classes. The ability of thiopurines with free SH functionalities to inhibit topoisomerase II ATPase activity was completely abolished by DTT, suggesting that these thiopurines inhibit topoisomerase II ATPase activity by covalently modifying free cysteine residues. In contrast, NSC35866 as well as two O6-substituted guanine analogues, O6-benzylguanine and NU2058, could inhibit topoisomerase II ATPase activity in the presence of DTT, indicating that they have a different mechanism of inhibition. NSC35866 did not increase the level of topoisomerase II covalent cleavable complexes with DNA, indicating that it is a catalytic inhibitor and not a poison. NSC35866 was also capable of inducing a salt-stable complex of topoisomerase II on closed circular DNA. In accordance with these biochemical data, NSC35866 could antagonize etoposide-induced cytotoxicity and DNA breaks in human and murine cancer cells, confirming that NSC35866 also functions as a catalytic topoisomerase II inhibitor in cells.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Purinas/farmacología , Tioguanina/análogos & derivados , Inhibidores de Topoisomerasa II , Antígenos de Neoplasias/metabolismo , Carcinoma de Células Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/enzimología , Catálisis , Línea Celular Tumoral , ADN-Topoisomerasas de Tipo II/metabolismo , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/metabolismo , Ensayos de Selección de Medicamentos Antitumorales/métodos , Histonas/antagonistas & inhibidores , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Nocodazol/farmacología , Fosforilación/efectos de los fármacos , Tioguanina/farmacologíaRESUMEN
Histone deacetylase inhibitors represent a promising new class of anticancer agents. In the current investigation, we examined the activity of PXD101, a potent histone deacetylase inhibitor, used alone or in combination with clinically relevant chemotherapeutics (docetaxel, paclitaxel, and carboplatin), in preclinical in vitro and in vivo models of ovarian cancer. In vitro activity was examined in ovarian cancer and multidrug-resistant cell lines grown in monolayer culture, and in primary clinical ovarian cancer specimens grown in three-dimensional organoid culture. PXD101 was found to inhibit in vitro cancer cell growth at sub- to low micromolar IC(50) potency, exhibited synergistic activity when used in combination with relevant chemotherapeutics, and effectively inhibited the growth of multidrug-resistant cells. In vivo, PXD101 displayed single-agent antitumor activity on human A2780 ovarian cancer s.c. xenografts which was enhanced via combination therapy with carboplatin. In support of these findings, PXD101 was shown to increase the acetylation of alpha-tubulin induced by docetaxel and the phosphorylation of H2AX induced by carboplatin. Taken together, these results support the clinical evaluation of PXD101 used alone or in combination therapy for the treatment of ovarian cancer.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Acetilación , Animales , Protocolos de Quimioterapia Combinada Antineoplásica , Carboplatino/administración & dosificación , Línea Celular Tumoral , Docetaxel , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Histonas/efectos de los fármacos , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/administración & dosificación , Ratones , Ratones Desnudos , Neoplasias Ováricas/patología , Fosforilación , Sulfonamidas , Taxoides/administración & dosificación , Taxoides/farmacología , Tubulina (Proteína)/efectos de los fármacos , Tubulina (Proteína)/metabolismoRESUMEN
Dexrazoxane has been reported to be protective against anthracycline induced subcutaneous ulceration in mice. It is currently under clinical investigation as an acute antidote in accidental anthracycline extravasation, for which indication topical dimethylsulfoxide (DMSO) and intralesional hydrocortisone are used empirically. We studied the effect in 72 mice of monotherapy with and combined therapy of intraperitoneal dexrazoxane, topical DMSO, and intralesional hydrocortisone as acute antidotes against ulceration after subcutaneous daunorubicin. Dexrazoxane completely prevented wounds from occurring, while neither DMSO nor hydrocortisone had any preventive effect. The addition of topical DMSO actually reduced the efficacy of dexrazoxane. In conclusion, the present study does not support the concomitant use of topical DMSO + systemic dexrazoxane or intralesional hydrocortisone + systemic dexrazoxane. Monotherapy with systemic dexrazoxane seems preferable and is highly efficacious in preventing ulceration.
Asunto(s)
Antibióticos Antineoplásicos/efectos adversos , Daunorrubicina/efectos adversos , Dimetilsulfóxido/uso terapéutico , Extravasación de Materiales Terapéuticos y Diagnósticos/tratamiento farmacológico , Hidrocortisona/uso terapéutico , Razoxano/uso terapéutico , Administración Tópica , Animales , Dimetilsulfóxido/administración & dosificación , Quimioterapia Combinada , Extravasación de Materiales Terapéuticos y Diagnósticos/etiología , Femenino , Hidrocortisona/administración & dosificación , Inyecciones Intralesiones , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos , Razoxano/administración & dosificaciónRESUMEN
PURPOSE: The anthracyclines daunorubicin and doxorubicin and the epipodophyllotoxin etoposide are potent DNA cleavage-enhancing drugs that are widely used in clinical oncology; however, myelosuppression and cardiac toxicity limit their use. Dexrazoxane (ICRF-187) is recommended for protection against anthracycline-induced cardiotoxicity. EXPERIMENTAL DESIGN: Because of their widespread use, the hematologic toxicity following coadministration of dexrazoxane and these three structurally different DNA cleavage enhancers was investigated: Sensitivity of human and murine blood progenitor cells to etoposide, daunorubicin, and doxorubicin +/- dexrazoxane was determined in granulocyte-macrophage colony forming assays. Likewise, in vivo, B6D2F1 mice were treated with etoposide, daunorubicin, and doxorubicin, with or without dexrazoxane over a wide range of doses: posttreatment, a full hematologic evaluation was done. RESULTS: Nontoxic doses of dexrazoxane reduced myelosuppression and weight loss from daunorubicin and etoposide in mice and antagonized their antiproliferative effects in the colony assay; however, dexrazoxane neither reduced myelosuppression, weight loss, nor the in vitro cytotoxicity from doxorubicin. CONCLUSION: Although our findings support the observation that dexrazoxane reduces neither hematologic activity nor antitumor activity from doxorubicin clinically, the potent antagonism of daunorubicin activity raises concern; a possible interference with anticancer efficacy certainly would call for renewed attention. Our data also suggest that significant etoposide dose escalation is perhaps possible by the use of dexrazoxane. Clinical trials in patients with brain metastases combining dexrazoxane and high doses of etoposide is ongoing with the aim of improving efficacy without aggravating hematologic toxicity. If successful, this represents an exciting mechanism for pharmacologic regulation of side effects from cytotoxic chemotherapy.
Asunto(s)
Anemia/prevención & control , Antibióticos Antineoplásicos/efectos adversos , Antineoplásicos/efectos adversos , Fármacos Cardiovasculares/farmacología , Daunorrubicina/efectos adversos , Doxorrubicina/efectos adversos , Etopósido/efectos adversos , Leucopenia/prevención & control , Neutropenia/prevención & control , Razoxano/farmacología , Trombocitopenia/prevención & control , Anemia/inducido químicamente , Animales , Técnicas de Cultivo de Célula , Ensayo de Unidades Formadoras de Colonias , Femenino , Leucopenia/inducido químicamente , Ratones , Neutropenia/inducido químicamente , Trombocitopenia/inducido químicamenteRESUMEN
PURPOSE: The treatment of patients with brain metastases is presently ineffective, but cerebral chemoradiotherapy using radiosensitizing agents seems promising. Etoposide targets topoisomerase II, resulting in lethal DNA breaks; such lesions may increase the effect of irradiation, which also depends on DNA damage. Coadministration of the topoisomerase II catalytic inhibitor dexrazoxane in mice allows for more than 3-fold higher dosing of etoposide. We hypothesized that dexrazoxane combined with escalated etoposide doses might improve the efficacy of cerebral radiotherapy. EXPERIMENTAL DESIGN: Mice with cerebrally inoculated Ehrlich ascites tumor (EHR2) cells were treated with combinations of etoposide + dexrazoxane + cerebral radiotherapy. Similar chemotherapy and radiation combinations were investigated by clonogenic assays using EHR2 cells, and by DNA double-strand break assay through quantification of phosphorylated histone H2AX (gammaH2AX). RESULTS: Escalated etoposide dosing (90 mg/kg) combined with dexrazoxane (125 mg/kg) and cerebral radiotherapy (10 Gy x 1) increased the median survival by 60% (P = 0.001) without increased toxicity, suggesting that escalated etoposide levels may indeed represent a new strategy for improving radiotherapy. Interestingly, 125 mg/kg dexrazoxane combined with normal etoposide doses (34 mg/kg) also increased survival from radiotherapy, but only by 27% (P = 0.002). This indicates a direct dexrazoxane modulation of the combined effects of etoposide and radiation in brain tumors. Further, in vitro, concurrent dexrazoxane, etoposide, and irradiation significantly increased DNA double-strand breaks. CONCLUSION: Combining etoposide (high or normal doses) and dexrazoxane synergizes with cerebral radiotherapy and significantly improves survival in mice with central nervous system tumors. This regimen may thus improve radiation therapy of central nervous system tumors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Neoplasias del Sistema Nervioso Central/radioterapia , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/patología , Barrera Hematoencefálica/efectos de la radiación , Neoplasias del Sistema Nervioso Central/patología , Terapia Combinada , Daño del ADN , ADN de Neoplasias/efectos de los fármacos , ADN de Neoplasias/genética , ADN de Neoplasias/efectos de la radiación , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Etopósido/administración & dosificación , Femenino , Ratones , Ratones Endogámicos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Neoplasias Experimentales/radioterapia , Razoxano/administración & dosificación , Análisis de Supervivencia , Factores de Tiempo , Resultado del Tratamiento , Células Tumorales CultivadasRESUMEN
BACKGROUND: Tetranectin (TN) is a 67 kDa glycoprotein thought to play a prominent role in the regulation of proteolytic processes via its binding to plasminogen and indirect activation of plasminogen. The TN concentration in serum is approximately 10 mg/l and is reduced in patients with several cancers. The TN concentration in the normal CSF has not been examined. METHODS: The TN concentration in the serum and CSF of 47 normal subjects without neurological disorders was established using a polyclonal sandwich ELISA. RESULTS: The median TN concentration (quartile range) was 10.8 mg/l (9.0-12.1) in serum and 0.43 mg/l (0.3-0.53) in CSF. The TN index median (quartile range), defined as (TN CSF concentration/TN serum concentration)/(Albumin CSF concentration/Albumin serum concentration), was found to be 5.5 (4.7-7.6), suggesting intrathecal synthesis or selective uptake of TN in CNS. Immunohistochemistry showed TN immunoreactivity in neurons and dendrites, but no staining in glial cells in the cerebrum and cerebellum. In plexus choroideus, the ependymal cells exhibited strong immunoreactivity. TN in serum and CSF were immunochemically identical and of similar size. CONCLUSION: TN is present in normal brain and CSF, and the TN index is very high, but further studies are necessary to decide whether TN is synthesised in the CNS or selectively transported over the blood-brain barrier.
Asunto(s)
Lectinas Tipo C/análisis , Líquido Cefalorraquídeo , Cromatografía en Gel , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Lectinas Tipo C/biosíntesisRESUMEN
Genotoxicity is an unacceptable property for new drug candidates and we employ three screening assays during the drug discovery process to identify genotoxicity early and optimize chemical series. One of these methods is the flow cytometric in vitro micronucleus assay for which protocol optimizations have been described recently. Here, we report further validation of the assay in TK6 cells including assessment of metabolic activation. We first optimized assay conditions to allow for testing with and without metabolic activation in parallel in a 96-well plate format. Then, we tested a set of 48 compounds carefully selected to contain known in vivo genotoxins, nongenotoxins and drugs. Avoidance of irrelevant positives, a known issue with mammalian cell-based genotoxicity assays, is important to prevent early deselection of potentially promising compounds. Therefore, we enriched the validation set with compounds that were previously reported to produce irrelevant positive results in mammalian cell-based genotoxicity assays. The resulting dataset was used to set the relevant cut-off values for scoring a compound positive or negative, such that we obtained an optimal balance of high sensitivity (88%) and high specificity (87%). Finally, we tested an additional set of 16 drugs to further probe assay performance and 14 of them were classified correctly. To our knowledge, the present study is the most comprehensive validation of the in vitro flow cytometric micronucleus assay and the first to report parallel assessment with metabolic activation in reasonable throughput. The assay allows for rapidly screening novel compounds for genotoxicity and is therefore well-suited for use in early drug discovery projects. Environ.
Asunto(s)
Citometría de Flujo/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Pruebas de Micronúcleos/métodos , Animales , Células Cultivadas , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , RatasRESUMEN
Toxicity and clinical safety have major impact on drug development success. Moving toxicological studies into earlier phases of the R&D chain prevents drug candidates with a safety risk from entering clinical development. However, to identify candidates without such risk, safety has to be designed actively. Therefore, we argue that toxicology should be fully integrated into the discovery process. We describe our strategy, including safety assessment of novel targets, selection of chemical series without inherent liabilities, designing out risk factors and profiling of candidates, and we discuss considerations regarding what to screen for. We aim to provide timely go/no-go decisions (fail early) and direction to the discovery teams, by steering away from safety risk (showing what will not fail).
Asunto(s)
Descubrimiento de Drogas/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Animales , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Humanos , SeguridadRESUMEN
In an effort to reduce toxicity-related attrition, different strategies have been implemented throughout the pharmaceutical industry. Previously (in Part I), we have outlined our 'integrated toxicology' strategy, which aims to provide timely go/no-go decisions (fail early) but also to show a direction to the drug discovery teams (showing what will not fail). In this review (Part II of the series) we describe our compound testing strategies with respect to cardiovascular safety, hepatotoxicity, genotoxicity, immunotoxicity and exploratory in vivo toxicity. We discuss the in vitro, ex vivo and in vivo assays and models we employ to assess safety risks and optimize compound series during the drug discovery process, including their predictivity and the decisions they generate.
Asunto(s)
Descubrimiento de Drogas/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Animales , Evaluación Preclínica de Medicamentos/métodos , Industria Farmacéutica/métodos , Humanos , SeguridadRESUMEN
High-content imaging/analysis has emerged as a powerful tool for predictive toxicology as it can be used for identifying and mitigating potential safety risks during drug discovery. By careful selection of end-points, some cellular assays can show better predictivity than routine animal toxicity testing for certain adverse events. Here, we present the perhaps most utilized high-content screening assays for predictive toxicology in the pharmaceutical industry. Multi-parametric imaging of cell health in simple and cost-effective model systems can be used to predict human hepatotoxicity and elucidate mechanisms of toxicity, and imaging of bile salt transport inhibition in sandwich-cultured hepatocytes can be used to predict cholestasis-inducing compounds. Imaging of micronuclei formation in simple cell models can be used to detect genotoxic potential and elucidate anuegenic or clastogenic mode of actions. The hope is that application of these relatively predictive assays during drug discovery will reduce toxicity and safety-related attrition of drug development programmes at later stages.
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Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Daño del ADN/efectos de los fármacos , Hígado/efectos de los fármacos , Pruebas de Toxicidad/métodos , Animales , Ácidos y Sales Biliares/antagonistas & inhibidores , Ácidos y Sales Biliares/metabolismo , Colestasis/diagnóstico , Análisis Costo-Beneficio , Hepatocitos/efectos de los fármacos , Humanos , Hígado/metabolismoRESUMEN
Existing pharmacological inhibitors for nicotinamide phosphoribosyltransferase (NAMPT) are promising therapeutics for treating cancer. By using medicinal and computational chemistry methods, the structure-activity relationship for novel classes of NAMPT inhibitors is described, and the compounds are optimized. Compounds are designed inspired by the NAMPT inhibitor APO866 and cyanoguanidine inhibitor scaffolds. In comparison with recently published derivatives, the new analogues exhibit an equally potent antiproliferative activity in vitro and comparable activity in vivo. The best performing compounds from these series showed subnanomolar antiproliferative activity toward a series of cancer cell lines (compound 15: IC50 0.025 and 0.33 nM, in A2780 (ovarian carcinoma) and MCF-7 (breast), respectively) and potent antitumor in vivo activity in well-tolerated doses in a xenograft model. In an A2780 xenograft mouse model with large tumors (500 mm(3)), compound 15 reduced the tumor volume to one-fifth of the starting volume at a dose of 3 mg/kg administered ip, bid, days 1-9. Thus, compounds found in this study compared favorably with compounds already in the clinic and warrant further investigation as promising lead molecules for the inhibition of NAMPT.
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Antineoplásicos/química , Antineoplásicos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Guanidinas/química , Humanos , Enlace de Hidrógeno , Ácidos Hidroxámicos/química , Concentración 50 Inhibidora , Ratones , Simulación del Acoplamiento Molecular , Nicotinamida Fosforribosiltransferasa/química , Nicotinamida Fosforribosiltransferasa/metabolismo , Conformación Proteica , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Dexrazoxane is an established treatment option in extravasation of the classic anthracyclines such as doxorubicin, epirubicin, and daunorubicin. However, it is not known whether the protection against the devastating tissue injuries extends into extravasation with new types of anthracyclines, the anthracenediones, or the liposomal pegylated anthracycline formulations. We therefore tested the antidotal efficacy of dexrazoxane against extravasation of amrubicin, mitoxantrone, and liposomal pegylated doxorubicin in mice. METHODS: A total of 80 female B6D2F1 mice were tested in an established mouse extravasation model. The mice had experimental extravasations of amrubicin, mitoxtanrone, and Caelyx and were immediately hereafter treated with systemic dexrazoxane or saline. RESULTS AND CONCLUSION: Systemic treatment with dexrazoxane resulted in significant protection against extravasation injuries from all three drugs. Moreover, the vesicant potential of the three test drugs was weaker than seen in previous experiments with the classic anthracyclines.