RESUMEN
Approximately 3.3 billion people live with the threat of Plasmodium vivax malaria. Infection can result in liver-localized hypnozoites, which when reactivated cause relapsing malaria. This work demonstrates that an enzyme-cleavable polymeric prodrug of tafenoquine addresses key requirements for a mass administration, eradication campaign: excellent subcutaneous bioavailability, complete parasite control after a single dose, improved therapeutic window compared to the parent oral drug, and low cost of goods sold (COGS) at less than $1.50 per dose. Liver targeting and subcutaneous dosing resulted in improved liver:plasma exposure profiles, with increased efficacy and reduced glucose 6-phosphate dehydrogenase-dependent hemotoxicity in validated preclinical models. A COGS and manufacturability analysis demonstrated global scalability, affordability, and the ability to redesign this fully synthetic polymeric prodrug specifically to increase global equity and access. Together, this polymer prodrug platform is a candidate for evaluation in human patients and shows potential for P. vivax eradication campaigns.
Asunto(s)
Antimaláricos , Malaria Vivax , Malaria , Humanos , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Aminoquinolinas/efectos adversos , Malaria/tratamiento farmacológico , Malaria Vivax/tratamiento farmacológico , Malaria Vivax/inducido químicamente , HígadoRESUMEN
Primaquine and tafenoquine are the two 8-aminoquinoline (8-AQ) antimalarial drugs approved for malarial radical cure - the elimination of liver stage hypnozoites after infection with Plasmodium vivax. A single oral dose of tafenoquine leads to high efficacy against intra-hepatocyte hypnozoites after efficient first pass liver uptake and metabolism. Unfortunately, both drugs cause hemolytic anemia in G6PD-deficient humans. This toxicity prevents their mass administration without G6PD testing given the approximately 400 million G6PD deficient people across malarial endemic regions of the world. We hypothesized that liver-targeted delivery of 8-AQ prodrugs could maximize liver exposure and minimize erythrocyte exposure to increase their therapeutic window. Primaquine and tafenoquine were first synthesized as prodrug vinyl monomers with self-immolative hydrolytic linkers or cathepsin-cleavable valine-citrulline peptide linkers. RAFT polymerization was exploited to copolymerize these prodrug monomers with hepatocyte-targeting GalNAc monomers. Pharmacokinetic studies of released drugs after intravenous administration showed that the liver-to-plasma AUC ratios could be significantly improved, compared to parent drug administered orally. Single doses of the liver-targeted, enzyme-cleavable tafenoquine polymer were found to be as efficacious as an equivalent dose of the oral parent drug in the P. berghei causal prophylaxis model. They also elicited significantly milder hemotoxicity in the humanized NOD/SCID mouse model engrafted with red blood cells from G6PD deficient donors. The clinical application is envisioned as a single subcutaneous administration, and the lead tafenoquine polymer also showed excellent bioavailability and liver-to-blood ratios exceeding the IV administered polymer. The liver-targeted tafenoquine polymers warrant further development as a single-dose therapeutic via the subcutaneous route with the potential for broader patient administration without a requirement for G6PD diagnosis.
Asunto(s)
Antimaláricos , Malaria Vivax , Malaria , Profármacos , Aminoquinolinas , Animales , Hígado , Malaria/tratamiento farmacológico , Malaria Vivax/tratamiento farmacológico , Ratones , Ratones Endogámicos NOD , Ratones SCID , Polímeros/uso terapéutico , Primaquina , Profármacos/uso terapéuticoRESUMEN
PURPOSE: Transcription factor C/EBP-α (CCAAT/enhancer-binding protein alpha) acts as a master regulator of hepatic and myeloid functions and multiple oncogenic processes. MTL-CEBPA is a first-in-class small activating RNA oligonucleotide drug that upregulates C/EBP-α. PATIENTS AND METHODS: We conducted a phase I, open-label, dose-escalation trial of MTL-CEBPA in adults with advanced hepatocellular carcinoma (HCC) with cirrhosis, or resulting from nonalcoholic steatohepatitis or with liver metastases. Patients received intravenous MTL-CEBPA once a week for 3 weeks followed by a rest period of 1 week per treatment cycle in the dose-escalation phase (3+3 design). RESULTS: Thirty-eight participants have been treated across six dose levels (28-160 mg/m2) and three dosing schedules. Thirty-four patients were evaluable for safety endpoints at 28 days. MTL-CEBPA treatment-related adverse events were not associated with dose, and no maximum dose was reached across the three schedules evaluated. Grade 3 treatment-related adverse events occurred in nine (24%) patients. In 24 patients with HCC evaluable for efficacy, an objective tumor response was achieved in one patient [4%; partial response (PR) for over 2 years] and stable disease (SD) in 12 (50%). After discontinuation of MTL-CEBPA, seven patients were treated with tyrosine kinase inhibitors (TKIs); three patients had a complete response with one further PR and two with SD. CONCLUSIONS: MTL-CEBPA is the first saRNA in clinical trials and demonstrates an acceptable safety profile and potential synergistic efficacy with TKIs in HCC. These encouraging phase I data validate targeting of C/EBP-α and have prompted MTL-CEBPA + sorafenib combination studies in HCC.
Asunto(s)
Antineoplásicos/administración & dosificación , Proteínas Potenciadoras de Unión a CCAAT/agonistas , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Oligorribonucleótidos/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Proteínas Potenciadoras de Unión a CCAAT/genética , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Relación Dosis-Respuesta a Droga , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Infusiones Intravenosas , Liposomas , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Nanopartículas/administración & dosificación , Estadificación de Neoplasias , Oligorribonucleótidos/efectos adversos , Oligorribonucleótidos/farmacocinética , Resultado del Tratamiento , Microambiente Tumoral/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
A series of [1,2,4]triazolo[3,4-f][1,6]naphthyridine allosteric dual inhibitors of Akt1 and 2 have been developed. These compounds have been shown to have potent dual Akt1 and 2 cell potency. The representative compound 13 provided potent inhibitory activity against Akt1 and 2 in vivo in a mouse model.
Asunto(s)
Química Farmacéutica/métodos , Naftiridinas/química , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Triazoles/química , Sitio Alostérico , Animales , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/metabolismo , Concentración 50 Inhibidora , Pulmón/efectos de los fármacos , Ratones , Modelos Químicos , Proteínas Proto-Oncogénicas c-akt/química , Relación Estructura-ActividadRESUMEN
This letter describes the development of potent, allosteric dual Akt1 and Akt2 inhibitors with improved aqueous solubility (approximately 18 mg/mL) that translates into enhanced cell activity and caspase-3 induction.
Asunto(s)
Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Quinoxalinas/química , Quinoxalinas/farmacología , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Caspasa 3/metabolismo , Línea Celular Tumoral , Activación Enzimática , Células HT29 , Humanos , Cinética , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinoxalinas/síntesis química , Inhibidores de Serina Proteinasa/síntesis química , Solubilidad , Agua/químicaRESUMEN
This letter shows inhibitor SAR on a pyridine series of allosteric Akt inhibitors to optimize enzymatic and cellular potency. We have optimized 2,3,5-trisubstituted pyridines to give potent Akt1 and Akt2 inhibitors in both enzyme and cell based assays. In addition, we will also highlight the pharmacokinetic profile of an optimized inhibitor that has low clearance and long half-life in dogs.
Asunto(s)
Sitio Alostérico , Neoplasias de la Próstata/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Piridinas/química , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Perros , Humanos , Masculino , Tasa de Depuración Metabólica , Estructura Molecular , Neoplasias de la Próstata/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Relación Estructura-Actividad , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Células Tumorales CultivadasRESUMEN
This communication reports a new synthetic route of pyridopyrimidines to facilitate their structural optimization in a library fashion and describes the development of pyridopyrimidines that have excellent enzymatic and cell potency against Akt1 and Akt2. This series also shows a high level of selectivity over other closely related kinases and significantly improved caspase-3 activity with the more optimized compounds.
Asunto(s)
Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Pirimidinas/síntesis química , Pirimidinas/farmacología , Aminas/síntesis química , Aminas/química , Aminas/farmacología , Caspasa 3/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Piperidinas/síntesis química , Piperidinas/química , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/química , Pirimidinas/química , Relación Estructura-Actividad , Ligando Inductor de Apoptosis Relacionado con TNF/farmacologíaRESUMEN
A series of naphthyridine and naphthyridinone allosteric dual inhibitors of Akt1 and 2 have been developed. These compounds have been optimized to have potent dual activity against the activated kinase as well as the activation of Akt in cells. One molecule in particular, compound 17, has potent inhibitory activity against Akt1 and 2 in vivo in a mouse lung and efficacy in a tumor xenograft model.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Naftiridinas/síntesis química , Naftiridinas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Animales , Antineoplásicos/química , Técnicas Químicas Combinatorias , Modelos Animales de Enfermedad , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Naftiridinas/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Relación Estructura-ActividadRESUMEN
This paper describes the rapid assembly of four different classes of potent Akt inhibitors from a common intermediate. Among them, a pyridopyrimidine series displayed the best intrinsic and cell potency against Akt1 and Akt2. This series also showed a promising pharmacokinetic profile and excellent selectivity over other closely related kinases.
Asunto(s)
Sitio Alostérico , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias de la Próstata/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Piridinas/química , Pirimidinas/química , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Perros , Femenino , Humanos , Masculino , Tasa de Depuración Metabólica , Estructura Molecular , Neoplasias Ováricas/metabolismo , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Relación Estructura-Actividad , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Células Tumorales CultivadasRESUMEN
We developed a high-throughput HTRF (homogeneous time-resolved fluorescence) assay for Akt kinase activity and screened approx. 270000 compounds for their ability to inhibit the three isoforms of Akt. Two Akt inhibitors were identified that exhibited isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 (IC50 4.6 microM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC50 values of 2.7 and 21 microM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 microM. These compounds were reversible inhibitors, and exhibited a linear mixed-type inhibition against ATP and peptide substrate. In addition to inhibiting kinase activity of individual Akt isoforms, both inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1 (phosphoinositide-dependent kinase 1). A model is proposed in which these inhibitors bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region blocked the inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be cell-active and to block phosphorylation of Akt at Thr308 and Ser473, reduce the levels of active Akt in cells, block the phosphorylation of known Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in LNCap prostate cancer cells.
Asunto(s)
Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Péptidos/química , Péptidos/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Homología de Secuencia de Aminoácido , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Adenosina Trifosfato/metabolismo , Proteínas Reguladoras de la Apoptosis , Bencilaminas/farmacología , Unión Competitiva , Proteínas Sanguíneas/inmunología , Carcinoma/química , Carcinoma/metabolismo , Carcinoma/patología , Caspasas/metabolismo , Línea Celular Tumoral , Clonación Molecular , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Compuestos Heterocíclicos con 2 Anillos/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Masculino , Glicoproteínas de Membrana/farmacología , Estructura Molecular , Péptidos/inmunología , Péptidos/metabolismo , Fosfoproteínas/inmunología , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/química , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Quinoxalinas/farmacología , Transducción de Señal/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/farmacología , Neoplasias del Cuello Uterino/química , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
Recent studies indicate that dysregulation of the Akt/PKB family of serine/threonine kinases is a prominent feature of many human cancers. The Akt/PKB family is composed of three members termed Akt1/PKBalpha, Akt2/PKBbeta, and Akt3/PKBgamma. It is currently not known to what extent there is functional overlap between these family members. We have recently identified small molecule inhibitors of Akt. These compounds have pleckstrin homology domain-dependent, isozyme-specific activity. In this report, we present data showing the relative contribution that inhibition of the different isozymes has on the apoptotic response of tumor cells to a variety of chemotherapies. In multiple cell backgrounds, maximal induction of caspase-3 activity is achieved when both Akt1 and Akt2 are inhibited. This induction is not reversed by overexpression of functionally active Akt3. The level of caspase-3 activation achieved under these conditions is equivalent to that observed with the phosphatidylinositol-3-kinase inhibitor LY294002. We also show that in different tumor cell backgrounds inhibition of mammalian target of rapamycin, a downstream substrate of Akt, is less effective in inducing caspase-3 activity than inhibition of Akt1 and Akt2. This shows that the survival phenotype conferred by Akt can be mediated by signaling pathways independent of mammalian target of rapamycin in some tumor cell backgrounds. Finally, we show that inhibition of both Akt1 and Akt2 selectively sensitizes tumor cells, but not normal cells, to apoptotic stimuli.
Asunto(s)
Apoptosis/fisiología , Neoplasias/enzimología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Antibióticos Antineoplásicos/farmacología , Caspasa 3 , Caspasas/metabolismo , Cromonas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Activación Enzimática , Humanos , Morfolinas/farmacología , Isoformas de Proteínas/química , Isoformas de Proteínas/farmacología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Sirolimus/farmacología , Células Tumorales CultivadasRESUMEN
We report here the first inhibitor-bound structure of a mitotic motor protein. The 1.9 A resolution structure of the motor domain of KSP, bound with the small molecule monastrol and Mg2+ x ADP, reveals that monastrol confers inhibition by "induced-fitting" onto the protein some 12 A away from the catalytic center of the enzyme, resulting in the creation of a previously non-existing binding pocket. The structure provides new insights into the biochemical and mechanical mechanisms of the mitotic motor domain. Inhibition of KSP provides a novel mechanism to arrest mitotic spindle formation, a target of several approved and investigative anti-cancer agents. The structural information gleaned from this novel pocket offers a new angle for the design of anti-mitotic agents.
Asunto(s)
Cinesinas/antagonistas & inhibidores , Cinesinas/química , Pirimidinas/farmacología , Tionas/farmacología , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Humanos , Magnesio/metabolismo , Microtúbulos/química , Mitosis , Modelos Moleculares , Proteínas Motoras Moleculares , Unión Proteica/genética , Conformación Proteica , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
A series of novel diaryl ether lactams have been identified as very potent dual inhibitors of protein farnesyltransferase (FTase) and protein geranylgeranyltransferase I (GGTase-I), enzymes involved in the prenylation of Ras. The structure of the complex formed between one of these compounds and FTase has been determined by X-ray crystallography. These compounds are the first reported to inhibit the prenylation of the important oncogene Ki-Ras4B in vivo. Unfortunately, doses sufficient to achieve this endpoint were rapidly lethal.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Antineoplásicos/síntesis química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Proteínas del Choque Térmico HSP40 , Proteínas de Choque Térmico/metabolismo , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Trasplante de Neoplasias , Neoplasias Experimentales/tratamiento farmacológico , Prenilación de Proteína , Relación Estructura-Actividad , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteínas de Unión al GTP rap1/metabolismo , Proteínas ras/metabolismoRESUMEN
A series of macrocyclic 3-aminopyrrolidinone farnesyltransferase inhibitors (FTIs) has been synthesized. Compared with previously described linear 3-aminopyrrolidinone FTIs such as compound 1, macrocycles such as 49 combined improved pharmacokinetic properties with a reduced potential for side effects. In dogs, oral bioavailability was good to excellent, and increases in plasma half-life were due to attenuated clearance. It was observed that in vivo clearance correlated with the flexibility of the molecules and this concept proved useful in the design of FTIs that exhibited low clearance, such as FTI 78. X-ray crystal structures of compounds 49 and 66 complexed with farnesyltransferase (FTase)-farnesyl diphosphate (FPP) were determined, and they provide details of the key interactions in such ternary complexes. Optimization of this 3-aminopyrrolidinone series of compounds led to significant increases in potency, providing 83 and 85, the most potent inhibitors of FTase in cells described to date.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Hidrocarburo de Aril Hidroxilasas , Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Inhibidores Enzimáticos/síntesis química , Naftalenos/síntesis química , Canales de Potasio con Entrada de Voltaje , Pirrolidinas/síntesis química , Transactivadores , Animales , Línea Celular , Cromatografía Liquida , Cristalografía por Rayos X , Citocromo P-450 CYP3A , Inhibidores Enzimáticos del Citocromo P-450 , Perros , Canal de Potasio ERG1 , Electrocardiografía , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Canales de Potasio Éter-A-Go-Go , Farnesiltransferasa , Humanos , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Modelos Moleculares , Estructura Molecular , Naftalenos/química , Naftalenos/farmacocinética , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Canales de Potasio/metabolismo , Unión Proteica , Pirrolidinas/química , Pirrolidinas/farmacocinética , Estereoisomerismo , Relación Estructura-Actividad , Regulador Transcripcional ERGRESUMEN
Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in a glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia-produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependant cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.
Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales/inmunología , Pichia/genética , Receptor ErbB-2/inmunología , Proteínas Recombinantes/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Área Bajo la Curva , Unión Competitiva/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Evaluación Preclínica de Medicamentos , Fucosa/metabolismo , Ingeniería Genética , Humanos , Macaca fascicularis , Ratones , Ratones Endogámicos C57BL , Pichia/metabolismo , Polisacáridos/metabolismo , Unión Proteica/inmunología , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Trastuzumab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The PI3K-Akt pathway is dysregulated in the majority of solid tumors. Pharmacological inhibition of Akt is a promising strategy for treating tumors resistant to growth factor receptor antagonists due to mutations in PI3K or PTEN. We have developed allosteric, isozyme-specific inhibitors of Akt activity and activation, as well as ex vivo kinase assays to measure inhibition of individual Akt isozymes in tissues. Here we describe the relationship between PK, Akt inhibition, hyperglycemia and tumor efficacy for a selective inhibitor of Akt1 and Akt2 (AKTi). In nude mice, AKTi treatment caused transient insulin resistance and reversible, dose-dependent hyperglycemia and hyperinsulinemia. Akt1 and Akt2 phosphorylation was inhibited in mouse lung with EC50 values of 1.6 and 7 µM, respectively, and with similar potency in other tissues and xenograft tumors. Weekly subcutaneous dosing of AKTi resulted in dose-dependent inhibition of LNCaP prostate cancer xenografts, an AR-dependent tumor with PTEN deletion and constitutively activated Akt. Complete tumor growth inhibition was achieved at 200 mpk, a dose that maintained inhibition of Akt1 and Akt2 of greater than 80% and 50%, respectively, for at least 12 hours in xenograft tumor and mouse lung. Hyperglycemia could be controlled by reducing C(max), while maintaining efficacy in the LNCaP model, but not by insulin administration. AKTi treatment was well tolerated, without weight loss or gross toxicities. These studies supported the rationale for clinical development of allosteric Akt inhibitors and provide the basis for further refining of pharmacokinetic properties and dosing regimens of this class of inhibitors.
Asunto(s)
Glucosa/metabolismo , Indazoles/farmacología , Indoles/farmacología , Insulina/metabolismo , Naftiridinas/farmacología , Neoplasias de la Próstata/prevención & control , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Regulación Alostérica , Animales , Humanos , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/metabolismo , Indazoles/farmacocinética , Indoles/farmacocinética , Isoenzimas , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Naftiridinas/farmacocinética , Fosfohidrolasa PTEN/metabolismo , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target. PRINCIPAL FINDINGS: Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors. CONCLUSIONS/SIGNIFICANCE: Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Mutación , Receptor Notch1/inmunología , Transducción de Señal/efectos de los fármacos , Células 3T3 , Animales , Especificidad de Anticuerpos/inmunología , Sitios de Unión/genética , Sitios de Unión/inmunología , Unión Competitiva , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Jagged-2 , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptor Notch1/genética , Receptor Notch1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Dysregulated PI3K/Akt signaling occurs commonly in breast cancers and is due to HER2 amplification, PI3K mutation or PTEN inactivation. The objective of this study was to determine the role of Akt activation in breast cancer as a function of mechanism of activation and whether inhibition of Akt signaling is a feasible approach to therapy. METHODOLOGY/PRINCIPAL FINDINGS: A selective allosteric inhibitor of Akt kinase was used to interrogate a panel of breast cancer cell lines characterized for genetic lesions that activate PI3K/Akt signaling: HER2 amplification or PI3K or PTEN mutations in order to determine the biochemical and biologic consequences of inhibition of this pathway. A variety of molecular techniques and tissue culture and in vivo xenograft models revealed that tumors with mutational activation of Akt signaling were selectively dependent on the pathway. In sensitive cells, pathway inhibition resulted in D-cyclin loss, G1 arrest and induction of apoptosis, whereas cells without pathway activation were unaffected. Most importantly, the drug effectively inhibited Akt kinase and its downstream effectors in vivo and caused complete suppression of the growth of breast cancer xenografts with PI3K mutation or HER2 amplification, including models of the latter selected for resistance to Herceptin. Furthermore, chronic administration of the drug was well-tolerated, causing only transient hyperglycemia without gross toxicity to the host despite the pleiotropic normal functions of Akt. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that breast cancers with PI3K mutation or HER2 amplification are selectively dependent on Akt signaling, and that effective inhibition of Akt in tumors is feasible and effective in vivo. These findings suggest that direct inhibition of Akt may represent a therapeutic strategy for breast and other cancers that are addicted to the pathway including tumors with resistant to Herceptin.
Asunto(s)
Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Amplificación de Genes , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/fisiología , Receptor ErbB-2/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Ciclo Celular/efectos de los fármacos , Dimetilsulfóxido/farmacología , Femenino , Fase G1 , Humanos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Inhibition of kinesin spindle protein (KSP) is a novel mechanism for treatment of cancer with the potential to overcome limitations associated with currently employed cytotoxic agents. Herein, we describe a C2-hydroxymethyl dihydropyrrole KSP inhibitor ( 11) that circumvents hERG channel binding and poor in vivo potency, issues that limited earlier compounds from our program. However, introduction of the C2-hydroxymethyl group caused 11 to be a substrate for cellular efflux by P-glycoprotein (Pgp). Utilizing knowledge garnered from previous KSP inhibitors, we found that beta-fluorination modulated the p K a of the piperidine nitrogen and reduced Pgp efflux, but the resulting compound ( 14) generated a toxic metabolite in vivo. Incorporation of fluorine in a strategic, metabolically benign position by synthesis of an N-methyl-3-fluoro-4-(aminomethyl)piperidine urea led to compound 30 that has an optimal in vitro and metabolic profile. Compound 30 (MK-0731) was recently studied in a phase I clinical trial in patients with taxane-refractory solid tumors.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Cinesinas/antagonistas & inhibidores , Neoplasias/enzimología , Piperidinas/farmacología , Pirroles/farmacología , Taxoides/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Humanos , Neoplasias/tratamiento farmacológico , Taxoides/uso terapéuticoRESUMEN
Installation of a C2-aminopropyl side chain to the 2,4-diaryl-2,5-dihydropyrrole series of kinesin spindle protein (KSP) inhibitors results in potent, water soluble compounds, but the aminopropyl group induces susceptibility to cellular efflux by P-glycoprotein (Pgp). We show that by carefully modulating the basicity of the amino group by beta-fluorination, this series of inhibitors maintains potency against KSP and has greatly improved efficacy in a Pgp-overexpressing cell line. The discovery that cellular efflux by Pgp can be overcome by carefully modulating the basicity of an amine may be of general use to medicinal chemists attempting to transform leading compounds into cancer cell- or CNS-penetrant drugs.