RESUMEN
The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, [(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-[(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability. To probe the basis of its in vivo efficacy, the metabolism of PPI-2458 was studied in detail. Reaction phenotyping identified CYP3A4/5 as the major source of metabolism in humans. Six metabolites were isolated from liver microsomes and characterized by mass spectrometry and nuclear resonance spectroscopy, and their structures were confirmed by chemical synthesis. The synthetic metabolites showed correlated inhibition of MetAP2 enzymatic activity and vascular endothelial cell growth. In an ex vivo experiment, MetAP2 inhibition in white blood cells, thymus, and lymph nodes in rats after single dosing with PPI-2458 and the isolated metabolites was found to correlate with the in vitro activity of the individual species. In a phase 1 clinical study, PPI-2458 was administered to patients with non-Hodgkin lymphoma. At 15 mg administered orally every other day, MetAP2 in whole blood was 80% inactivated for up to 48 hours, although the exposure of the parent compound was only â¼10% that of the summed cytochrome P450 metabolites. Taken together, the data confirm the participation of active metabolites in the in vivo efficacy of PPI-2458. The structures define a metabolic pathway for PPI-2458 that is distinct from that of TNP-470 ([(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-(2-chloroacetyl)carbamate). The high level of MetAP2 inhibition achieved in vivo supports the value of fumagillin-derived therapeutics for angiogenic diseases.
Asunto(s)
Aminopeptidasas/antagonistas & inhibidores , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/farmacocinética , Compuestos Epoxi/farmacocinética , Metaloendopeptidasas/antagonistas & inhibidores , Valina/análogos & derivados , Aminopeptidasas/sangre , Animales , Esquema de Medicación , Compuestos Epoxi/administración & dosificación , Compuestos Epoxi/análisis , Compuestos Epoxi/farmacología , Humanos , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/metabolismo , Linfoma no Hodgkin/sangre , Linfoma no Hodgkin/tratamiento farmacológico , Metaloendopeptidasas/sangre , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Ratas , Relación Estructura-Actividad , Timo/efectos de los fármacos , Timo/metabolismo , Valina/administración & dosificación , Valina/análisis , Valina/farmacocinética , Valina/farmacologíaRESUMEN
In pursuit of potent and selective sphingosine-1-phosphate receptor agonists, we have utilized previously reported phenylamide and phenylimidazole scaffolds to explore extensive side-chain modifications to generate new molecular entities. A number of designed molecules demonstrate good selectivity and excellent in vitro and in vivo potency in both mouse and rat models. Oral administration of the lead molecule 11c (PPI-4667) demonstrated potent and dose-responsive lymphopenia.
Asunto(s)
Amidas/síntesis química , Imidazoles/síntesis química , Receptores de Lisoesfingolípidos/agonistas , Amidas/farmacología , Animales , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Clorhidrato de Fingolimod , Imidazoles/farmacología , Ratones , Glicoles de Propileno/química , Glicoles de Propileno/farmacología , Subunidades de Proteína/agonistas , Subunidades de Proteína/fisiología , Receptores de Lisoesfingolípidos/fisiología , Esfingosina/análogos & derivados , Esfingosina/química , Esfingosina/farmacologíaRESUMEN
PURPOSE: Fumagillin and related compounds have potent antiproliferative activity through inhibition of methionine aminopeptidase-2 (MetAP-2). It has recently been reported that MetAP-2 is highly expressed in germinal center B cells and germinal center-derived non-Hodgkin's lymphomas (NHL), suggesting an important role for MetAP-2 in proliferating B cells. Therefore, we determined the importance of MetAP-2 in normal and transformed germinal center B cells by evaluating the effects of MetAP-2 inhibition on the form and function of germinal centers and germinal center-derived NHL cells. EXPERIMENTAL DESIGN: To examine the activity of PPI-2458 on germinal center morphology, spleen sections from cynomolgus monkeys treated with oral PPI-2458 were analyzed. Antiproliferative activity of PPI-2458 was assessed on germinal center-derived NHL lines in culture. A MetAP-2 pharmacodynamic assay was used to determine cellular MetAP-2 inhibition following PPI-2458 treatment. Finally, inhibition of MetAP-2 and proliferation by PPI-2458 was examined in the human SR NHL line in culture and in implanted xenografts. RESULTS: Oral PPI-2458 caused a reduction in germinal center size and number in lymphoid tissues from treated animals. PPI-2458 potently inhibited growth (GI(50) = 0.2-1.9 nmol/L) of several NHL lines in a manner that correlated with MetAP-2 inhibition. Moreover, orally administered PPI-2458 significantly inhibited SR tumor growth, which correlated with inhibition of tumor MetAP-2 (>85% at 100 mg/kg) in mice. CONCLUSIONS: These results show the potent antiproliferative activity of PPI-2458 on NHL lines in vitro and oral antitumor activity in vivo and suggest the therapeutic potential of PPI-2458 as a novel agent for treatment of NHL should be evaluated in the clinical setting.
Asunto(s)
Aminopeptidasas/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Compuestos Epoxi/farmacología , Linfoma no Hodgkin/tratamiento farmacológico , Metaloendopeptidasas/antagonistas & inhibidores , Valina/análogos & derivados , Aminopeptidasas/metabolismo , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/patología , Western Blotting , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Compuestos Epoxi/uso terapéutico , Femenino , Centro Germinal/efectos de los fármacos , Centro Germinal/patología , Humanos , Recuento de Linfocitos , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Linfoma no Hodgkin/metabolismo , Linfoma no Hodgkin/patología , Macaca fascicularis , Metaloendopeptidasas/metabolismo , Ratones , Ratones SCID , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Tiempo , Valina/farmacología , Valina/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The dependence of cell growth on methionine aminopeptidase (MetAP) function in bacteria and yeast is firmly established. Here we report experimental evidence that the control of cell proliferation in mammalian cells is directly linked and strictly dependent on the activity of both MetAP-1 and MetAP-2. The targeted downregulation of either methionine aminopeptidase MetAP-1 or MetAP-2 protein expression by small interfering RNA (siRNA) significantly inhibited the proliferation of human umbilical vein endothelial cells (HUVEC) (70%-80%), while A549 human lung carcinoma cell proliferation was less inhibited (20%-30%). The cellular levels of MetAP-2 enzyme were measured after MetAP-2 siRNA treatment and found to decrease over time from 4 to 96 h, while rapid and complete depletion of MetAP-2 enzyme activity was observed after 4 h treatment with two pharmacological inhibitors of MetAP-2, PPI-2458 and fumagillin. When HUVEC and A549 cells were treated simultaneously with MetAP-2 siRNA and PPI-2458, or fumagillin, which irreversibly inhibit MetAP-2 enzyme activity, no additive effect on maximum growth inhibition was observed. This strongly suggests that MetAP-2 is the single critical cellular enzyme affected by either MetAP-2 targeting approach. Most strikingly, despite their significantly different sensitivity to growth inhibition after targeting of either MetAP-1 or MetAP-2, HUVEC, and A549 cells, which were made functionally deficient in both MetAP-1 and MetAP-2 were completely or almost completely inhibited in their growth, respectively. This closely resembled the observed growth inhibition in genetically double-deficient map1map2 yeast strains. These results suggest that MetAP-1 and MetAP-2 have essential functions in the control of mammalian cell proliferation and that MetAP-dependent growth control is evolutionarily highly conserved.