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1.
J Med Chem ; 52(22): 7170-85, 2009 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-19873981

RESUMO

We disclose the development of a novel series of 2-phenyl-2H-indazole-7-carboxamides as poly(ADP-ribose)polymerase (PARP) 1 and 2 inhibitors. This series was optimized to improve enzyme and cellular activity, and the resulting PARP inhibitors display antiproliferation activities against BRCA-1 and BRCA-2 deficient cancer cells, with high selectivity over BRCA proficient cells. Extrahepatic oxidation by CYP450 1A1 and 1A2 was identified as a metabolic concern, and strategies to improve pharmacokinetic properties are reported. These efforts culminated in the identification of 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide 56 (MK-4827), which displays good pharmacokinetic properties and is currently in phase I clinical trials. This compound displays excellent PARP 1 and 2 inhibition with IC(50) = 3.8 and 2.1 nM, respectively, and in a whole cell assay, it inhibited PARP activity with EC(50) = 4 nM and inhibited proliferation of cancer cells with mutant BRCA-1 and BRCA-2 with CC(50) in the 10-100 nM range. Compound 56 was well tolerated in vivo and demonstrated efficacy as a single agent in a xenograft model of BRCA-1 deficient cancer.


Assuntos
Amidas/farmacologia , Descoberta de Drogas , Genes BRCA1 , Genes BRCA2 , Indazóis/farmacologia , Mutação , Neoplasias/genética , Piperidinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Administração Oral , Amidas/administração & dosagem , Amidas/química , Amidas/farmacocinética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica , Estabilidade de Medicamentos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Indazóis/administração & dosagem , Indazóis/química , Indazóis/farmacocinética , Concentração Inibidora 50 , Neoplasias/enzimologia , Neoplasias/patologia , Piperidinas/administração & dosagem , Piperidinas/química , Piperidinas/farmacocinética , Ratos
2.
J Gene Med ; 9(10): 862-74, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17685494

RESUMO

BACKGROUND: Ocular neovascular disorders, such as diabetic retinopathy and age-related macular degeneration, are the principal causes of blindness in developed countries. Current treatments are of limited efficacy, whereas a therapy based on intraocular gene transfer of angiostatic factors represents a promising alternative. For the first time we have explored the potential of helper-dependent adenovirus (HD-Ad), the last generation of Ad vectors, in the therapy of retinal neovascularization. METHODS: We first analyzed efficiency and stability of intraretinal gene transfer following intravitreous injection in mice. A HD-Ad vector expressing green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter (HD-Ad/GFP) was compared with a first-generation (E1/E3-deleted) Ad vector carrying an identical GFP expression cassette (FG-Ad/GFP). We also constructed HD-Ad vectors expressing a soluble form of the VEGF receptor (sFlt-1) in a constitutive (HD-Ad/sFlt-1) or doxycycline (dox)-inducible (HD-Ad/S-M2/sFlt-1) manner and tested their therapeutic efficacy upon intravitreous delivery in a rat model of oxygen-induced retinopathy (OIR). RESULTS: HD-Ad/GFP promoted long-lasting (up to 1 year) transgene expression in retinal Müller cells, in marked contrast with the short-term expression observed with FG-Ad/GFP. Intravitreous injection of HD-Ad vectors expressing sFlt-1 resulted in detectable levels of sFlt-1 and inhibited retinal neovascularization by more than 60% in a rat model of OIR. Notably, the therapeutic efficacy of the inducible vector HD-Ad/S-M2/sFlt-1 was strictly dox-dependent. CONCLUSIONS: HD-Ad vectors enable stable gene transfer and regulated expression of angiostatic factors following intravitreous injection and thus are attractive vehicles for the gene therapy of neovascular diseases of the retina.


Assuntos
Adenoviridae/genética , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vírus Auxiliares/genética , Neovascularização Retiniana/terapia , Animais , Doxiciclina/farmacologia , Vetores Genéticos/administração & dosagem , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hipóxia/metabolismo , Camundongos , Modelos Animais , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Retina/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo
3.
Mol Vis ; 11: 366-73, 2005 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-15951738

RESUMO

PURPOSE: Inhibition of vascular endothelial growth factor (VEGF) signaling has shown great promise for the treatment of ocular neovascular disease. Current anti-VEGF therapies in late-stage development, while efficacious, require dosing by frequent intravitreal injections that are inconvenient to patients. VEGF signaling inhibitors that demonstrate more convenient dosing regimens could lead to the improved treatment of neovascular diseases such as wet age related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR). Here we describe the assessment of a KDR (VEGFR2) kinase inhibitor in two well-established models of ocular neovascularization following oral administration. METHODS: A novel KDR kinase inhibitor was dosed by oral gavage for 12 days at 0, 10, 30, or 100 mg/kg in an adult male Brown Norway rat laser induced choroidal neovascularization (CNV) model. The areas of CNV lesions were quantitated by fluorescence image analysis of FITC-dextran perfused animals. The kinase inhibitor was also assessed in a rat oxygen induced retinopathy (OIR) model in which neonatal rats were placed in an oxygen chamber that delivered alternating 24 h cycles of 50% and 10% oxygen for 14 days. After 14 days of oxygen treatment, the animals were returned to room air and dosed orally for 7 days with 0, 10, or 30 mg/kg kinase inhibitor. The extent of retinal neovascularization was assessed by counting pre-retinal neovascular nuclei on histological sections. RESULTS: At doses of 100 mg/kg, the KDR kinase inhibitor resulted in a 98% reduction in lesion size in the rat CNV model. 30 mg/kg doses of the inhibitor showed a 70% and 80% reduction in lesion size in the laser CNV and OIR models, respectively. CONCLUSIONS: Oral dosing of the described KDR kinase inhibitor effectively inhibits neovascularization in two well-established animal models of ocular neovascularization. These data suggest that compounds of this class may prove to be useful for the treatment of a variety of ocular neovascular diseases using a convenient oral dosing regimen.


Assuntos
Neovascularização de Coroide/prevenção & controle , Modelos Animais de Doenças , Inibidores Enzimáticos/administração & dosagem , Indóis/administração & dosagem , Quinolonas/administração & dosagem , Neovascularização Retiniana/prevenção & controle , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Administração Oral , Animais , Animais Recém-Nascidos , Neovascularização de Coroide/diagnóstico , Neovascularização de Coroide/enzimologia , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/síntese química , Angiofluoresceinografia , Indóis/síntese química , Terapia a Laser , Masculino , Microscopia de Fluorescência , Oxigênio/toxicidade , Quinolonas/síntese química , Ratos , Ratos Endogâmicos BN , Ratos Sprague-Dawley , Neovascularização Retiniana/diagnóstico , Neovascularização Retiniana/enzimologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/sangue
4.
J Gene Med ; 6(9): 992-1002, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15352072

RESUMO

BACKGROUND: In mouse models of retinopathy of prematurity (ROP) inhibitors of vascular endothelial growth factor (VEGF) functions administered systemically completely block retinal neovascularization. In contrast, selective ocular VEGF depletion has achieved an approx. 50% inhibition of retinal neovascular growth. It is unclear whether a more complete inhibition of new blood vessel development can be obtained with an anti-VEGF therapy localized to the eye. Therefore, the objective of the present study was to determine the effect of local anti-VEGF therapy in a different animal model which closely mimics human ROP. METHODS: Rats were exposed to alternating cycles of high and low levels of oxygen for 14 days immediately after birth; thereafter, they were intravitreally injected with an adenoviral vector expressing a secreted form of the VEGF receptor flt-1 (Ad.sflt), which acts by sequestering VEGF. Contralateral eyes were injected with the control vector carrying the reporter gene expressing beta-galactosidase (Ad.betaGal). RESULTS: At the peak of retinal neovascular growth, i.e. post-natal day 21 (P21), we observed up to 97.5% decrease in retinal neovascularization in animals injected with Ad.sflt. At the end of observation (P28), no significant difference in retinal vessel number was detected in both oxygen-injured and normoxic Ad.sflt-treated retinas compared with untreated or Ad.betaGal-treated retinas. CONCLUSION: Adenoviral-mediated sflt-1 gene transfer induces a near-complete inhibition of ischemia-induced retinal neovascularization in rats without affecting pre-existing retinal vessels.


Assuntos
Terapia Genética/métodos , Neovascularização Retiniana/prevenção & controle , Retinopatia da Prematuridade/terapia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Adenoviridae/genética , Animais , Modelos Animais de Doenças , Vetores Genéticos/uso terapêutico , Humanos , Recém-Nascido , Ratos , Retina/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Corpo Vítreo/metabolismo
5.
Mol Ther ; 7(2): 271-80, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12597916

RESUMO

The tetracycline (Tc)-dependent system in its "on" version (rtTA system) displays a baseline activity in the uninduced state, severely limiting its potential applicability in human gene therapy. So far, two different strategies to circumvent this limitation have been described. On one side, co-expression of the tetracycline regulated repressor tTS(kid) has proved capable of substantially reducing the baseline activity of rtTA. On the other, novel versions of the activator, namely rtTA2(s)-S2 and rtTA2(s)-M2, with a lower basal activity have been engineered. We have combined these two approaches by co-expressing TS(kid) with the novel transactivators. Bicistronic vectors were constructed that co-express TS(kid) with rtTA, rtTA2(s)-S2, or rtTA2(s) M2, through an internal ribosome entry site (plasmids IRES-A, IRES-S2, and IRES-M2, respectively). IRES-M2 proved to be the most effective construct EX VIVO: it displayed a negligible basal activity, > 1000 fold inducibility, and high responsiveness to doxycycline (Dox). Upon delivery as plasmid DNA in mouse muscles, IRES-M2 facilitated 1000-fold induction of serum alkaline phosphatase (SEAP) gene expression and long-term, stringent, and strictly Dox-dose-dependent regulation of erythropoietin (Epo) gene expression. Tight regulation of the gene encoding SEAP was demonstrated also in non-human primates. Notably, the system was induced in animals by Dox-dosing regimens comparable to those used in humans.


Assuntos
Doxiciclina/farmacologia , Vetores Genéticos , Transcrição Genética , Animais , Antibacterianos/farmacologia , Regulação da Expressão Gênica , Células HeLa , Humanos , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/metabolismo , Tetraciclina/farmacologia , Fatores de Tempo , Ativação Transcricional , Transfecção , Transgenes , Células Tumorais Cultivadas
6.
Mol Ther ; 6(5): 653-63, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12409264

RESUMO

Diseases requiring frequent and lifelong injections of recombinant proteins would be more efficaciously treated by intramuscular delivery of genes encoding secretable proteins. However, the success of this approach largely depends on our capability to temporally regulate transcription of delivered genes. Therefore, we sought to generate a humanized transcription factor to regulate transgene expression in muscle. A novel 4-hydroxytamoxifen (4-OHT)-dependent transcriptional regulator (called HEA-3) was constructed by fusing in-frame the DNA binding domain of the human hepatocyte nuclear factor-1alpha (HNF1alpha), which is not expressed in muscle cells, a G(521)R mutant of the ligand binding domain of human estrogen receptor-alpha (ERalpha), and the activation domain derived from human nuclear factor-kappaB p65 subunit (NF-kappaB p65). We demonstrate that an artificial promoter containing multimeric HNF1alpha binding sites is silent in muscles and in cell lines that lack endogenous HNF1alpha. HEA-3 stimulated transcription from this target promoter in a stringent 4-OHT-dependent manner. The dynamic range of transgene regulation was high, because of the low basal activity and high inducibility of the system. Ex vivo, HEA-3 increased expression of the transfected reporter gene by more than 1000-fold in a ligand-dependent manner. In vivo, HEA-3 stimulated by more than 100-fold, the expression of secreted alkaline phosphatase after delivery as plasmid DNA into mouse muscles. Moreover, long-term modulation of the expression of intramuscularly delivered mouse erythropoietin was achieved in immunocompetent mice.


Assuntos
Proteínas de Ligação a DNA , Técnicas de Transferência de Genes , Vetores Genéticos , Proteínas Nucleares , Tamoxifeno/análogos & derivados , Animais , Western Blotting , DNA/metabolismo , Relação Dose-Resposta a Droga , Terapia Genética/métodos , Células HeLa , Fator 1 Nuclear de Hepatócito , Fator 1-alfa Nuclear de Hepatócito , Fator 1-beta Nuclear de Hepatócito , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Músculo Esquelético/metabolismo , NF-kappa B/metabolismo , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tamoxifeno/farmacologia , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Transfecção , Transgenes
7.
Hum Gene Ther ; 13(2): 199-210, 2002 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-11812277

RESUMO

The tetracycline (Tet)-dependent regulatory system has been widely used for controlling gene expression. The Tet-on version of the system, in which the reverse Tet-responsive transcriptional activator (rtTA) is positively regulated by Tet or its analogs, such as doxycycline (Dox), is of potential utility for gene therapy applications in humans. However, rtTA may display a high basal activity, especially when delivered in vivo by using episomal vectors such as plasmids. Two novel Dox-inducible activators, called rtTA2(S)-S2 and rtTA2(S)-M2, which have a significantly lower basal activity than rtTA in stably transfected cell lines, have been described. In this study we tested the capability of these trans-activators to control expression of mouse erythropoietin (mEpo) and to modulate hematocrit (Hct) increase in vivo on delivery of plasmids into quadriceps muscles of adult mice by DNA electroinjection. Both rtTA2(S)-M2 and rtTA2(S)-S2 displayed a considerably lower background activity and higher window of induction than rtTA in vivo. Moreover, a stringent control of mEpo gene expression and Hct levels in the absence of any background activity was maintained over a 10-month period by injecting as little as 1 microg of a single plasmid containing the rtTA2(S)-S2 expression cassette and the Tet-responsive mEpo cDNA. This constitutes the first report of a stringent ligand-dependent control of gene expression in vivo obtained by delivering a single plasmid encoding both the trans-activator and the regulated gene. Notably, the rtTA2(S)-S2-based system was induced by oral doses of doxycycline comparable to those normally used in clinical practice in humans.


Assuntos
Doxiciclina/farmacologia , Eritropoetina/genética , Regulação da Expressão Gênica , Transativadores , Animais , Eritropoetina/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Terapia Genética , Células HeLa/fisiologia , Hematócrito , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Transgenes
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