RESUMO
Effective treatments for primary brain tumors and brain metastases represent a major unmet medical need. Targeting the CDK4/CDK6-cyclin D1-Rb-p16/ink4a pathway using a potent CDK4 and CDK6 kinase inhibitor has potential for treating primary central nervous system tumors such as glioblastoma and some peripheral tumors with high incidence of brain metastases. We compared central nervous system exposures of two orally bioavailable CDK4 and CDK6 inhibitors: abemaciclib, which is currently in advanced clinical development, and palbociclib (IBRANCE; Pfizer), which was recently approved by the U.S. Food and Drug Administration. Abemaciclib antitumor activity was assessed in subcutaneous and orthotopic glioma models alone and in combination with standard of care temozolomide (TMZ). Both inhibitors were substrates for xenobiotic efflux transporters P-glycoprotein and breast cancer resistant protein expressed at the blood-brain barrier. Brain Kp,uu values were less than 0.2 after an equimolar intravenous dose indicative of active efflux but were approximately 10-fold greater for abemaciclib than palbociclib. Kp,uu increased 2.8- and 21-fold, respectively, when similarly dosed in P-gp-deficient mice. Abemaciclib had brain area under the curve (0-24 hours) Kp,uu values of 0.03 in mice and 0.11 in rats after a 30 mg/kg p.o. dose. Orally dosed abemaciclib significantly increased survival in a rat orthotopic U87MG xenograft model compared with vehicle-treated animals, and efficacy coincided with a dose-dependent increase in unbound plasma and brain exposures in excess of the CDK4 and CDK6 Ki values. Abemaciclib increased survival time of intracranial U87MG tumor-bearing rats similar to TMZ, and the combination of abemaciclib and TMZ was additive or greater than additive. These data show that abemaciclib crosses the blood-brain barrier and confirm that both CDK4 and CDK6 inhibitors reach unbound brain levels in rodents that are expected to produce enzyme inhibition; however, abemaciclib brain levels are reached more efficiently at presumably lower doses than palbociclib and are potentially on target for a longer period of time.
Assuntos
Aminopiridinas/farmacologia , Benzimidazóis/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Aminopiridinas/administração & dosagem , Aminopiridinas/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzimidazóis/administração & dosagem , Benzimidazóis/uso terapêutico , Neoplasias Encefálicas/patologia , Dacarbazina/administração & dosagem , Dacarbazina/análogos & derivados , Cães , Feminino , Glioblastoma/patologia , Células Madin Darby de Rim Canino , Masculino , Camundongos , Piperazinas/administração & dosagem , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/administração & dosagem , Piridinas/uso terapêutico , Ratos , Temozolomida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The G1 restriction point is critical for regulating the cell cycle and is controlled by the Rb pathway (CDK4/6-cyclin D1-Rb-p16/ink4a). This pathway is important because of its inactivation in a majority of human tumors. Transition through the restriction point requires phosphorylation of retinoblastoma protein (Rb) by CDK4/6, which are highly validated cancer drug targets. We present the identification and characterization of a potent CDK4/6 inhibitor, LY2835219. LY2835219 inhibits CDK4 and CDK6 with low nanomolar potency, inhibits Rb phosphorylation resulting in a G1 arrest and inhibition of proliferation, and its activity is specific for Rb-proficient cells. In vivo target inhibition studies show LY2835219 is a potent inhibitor of Rb phosphorylation, induces a complete cell cycle arrest and suppresses expression of several Rb-E2F-regulated proteins 24 hours after a single dose. Oral administration of LY2835219 inhibits tumor growth in human tumor xenografts representing different histologies in tumor-bearing mice. LY2835219 is effective and well tolerated when administered up to 56 days in immunodeficient mice without significant loss of body weight or tumor outgrowth. In calu-6 xenografts, LY2835219 in combination with gemcitabine enhanced in vivo antitumor activity without a G1 cell cycle arrest, but was associated with a reduction of ribonucleotide reductase expression. These results suggest LY2835219 may be used alone or in combination with standard-of-care cytotoxic therapy. In summary, we have identified a potent, orally active small-molecule inhibitor of CDK4/6 that is active in xenograft tumors. LY2835219 is currently in clinical development.
Assuntos
Aminopiridinas/farmacologia , Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Aminopiridinas/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Benzimidazóis/uso terapêutico , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Quimioterapia Combinada , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
The HGF/MET signaling pathway regulates a wide variety of normal cellular functions that can be subverted to support neoplasia, including cell proliferation, survival, apoptosis, scattering and motility, invasion, and angiogenesis. MET over-expression (with or without gene amplification), aberrant autocrine or paracrine ligand production, and missense MET mutations are mechanisms that lead to activation of the MET pathway in tumors and are associated with poor prognostic outcome. We report here preclinical development of a potent, orally bioavailable, small-molecule inhibitor LY2801653 targeting MET kinase. LY2801653 is a type-II ATP competitive, slow-off inhibitor of MET tyrosine kinase with a dissociation constant (Ki) of 2 nM, a pharmacodynamic residence time (Koff) of 0.00132 min(-1) and t1/2 of 525 min. LY2801653 demonstrated in vitro effects on MET pathway-dependent cell scattering and cell proliferation; in vivo anti-tumor effects in MET amplified (MKN45), MET autocrine (U-87MG, and KP4) and MET over-expressed (H441) xenograft models; and in vivo vessel normalization effects. LY2801653 also maintained potency against 13 MET variants, each bearing a single-point mutation. In subsequent nonclinical characterization, LY2801653 was found to have potent activity against several other receptor tyrosine oncokinases including MST1R, FLT3, AXL, MERTK, TEK, ROS1, DDR1/2 and against the serine/threonine kinases MKNK1/2. The potential value of MET and other inhibited targets within a number of malignancies (such as colon, bile ducts, and lung) is discussed. LY2801653 is currently in phase 1 clinical testing in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037).
Assuntos
Indazóis/farmacologia , Niacinamida/análogos & derivados , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Tetrazóis/farmacologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacologia , Disponibilidade Biológica , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Indazóis/administração & dosagem , Indazóis/química , Camundongos , Mutação/genética , Niacinamida/administração & dosagem , Niacinamida/química , Niacinamida/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Tetrazóis/administração & dosagem , Tetrazóis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Studies have shown that nanoparticles (NPs) are cleared through the mononuclear phagocyte system (MPS). Pharmacokinetic studies of Doxil, DaunoXome, micellar doxorubicin (SP1049C) and small molecule (SM) doxorubicin were performed in SCID mice, Sprague-Dawley rats, and beagle dogs. An ex vivo MPS profiling platform was used to evaluate the interaction between the same agents, as well as colloid-forming and non-colloid forming SM drugs. In all species, the systemic clearance was highest for SP1049C and lowest for Doxil. With the exception of dog blood, the MPS screening results of mouse and rat blood showed that the greatest reduction in phagocytosis occurred after the ex vivo addition of SM-doxorubicin>SP1049C>DaunoXome>Doxil. The MPS profiling platform in rats, but not dogs, could differentiate between colloid forming and non-colloid forming drugs. The results of the MPS profiling platform were generally consistent with in vivo clearance rates of NP and SM anticancer drugs in mice and rats. This study suggests the MPS profiling platform is an effective method to screen and differentiate the important characteristics of NPs and colloid-forming drugs that affect their in vivo clearance. Implications of these findings on preclinical prediction of human clearance are discussed.
Assuntos
Coloides/farmacologia , Sistema Fagocitário Mononuclear/efeitos dos fármacos , Nanopartículas/química , Animais , Cães , Humanos , Camundongos , Camundongos SCID , Ratos , Ratos Sprague-DawleyRESUMO
The PI3K/AKT/mTOR pathway is among the most frequently altered pathways in cancer cell growth and survival. LY3023414 is a complex fused imidazoquinolinone with high solubility across a wide pH range designed to inhibit class I PI3K isoforms and mTOR kinase. Here, we describe the in vitro and in vivo activity of LY3023414. LY3023414 was highly soluble at pH 2-7. In biochemical testing against approximately 266 kinases, LY3023414 potently and selectively inhibited class I PI3K isoforms, mTORC1/2, and DNA-PK at low nanomolar concentrations. In vitro, inhibition of PI3K/AKT/mTOR signaling by LY3023414 caused G1 cell-cycle arrest and resulted in broad antiproliferative activity in cancer cell panel screens. In vivo, LY3023414 demonstrated high bioavailability and dose-dependent dephosphorylation of PI3K/AKT/mTOR pathway downstream substrates such as AKT, S6K, S6RP, and 4E-BP1 for 4 to 6 hours, reflecting the drug's half-life of 2 hours. Of note, equivalent total daily doses of LY3023414 given either once daily or twice daily inhibited tumor growth to similar extents in multiple xenograft models, indicating that intermittent target inhibition is sufficient for antitumor activity. In combination with standard-of-care drugs, LY3023414 demonstrated additive antitumor activity. The novel, orally bioavailable PI3K/mTOR inhibitor LY3023414 is highly soluble and exhibits potent in vivo efficacy via intermittent target inhibition. It is currently being evaluated in phase I and II trials for the treatment of human malignancies. Mol Cancer Ther; 15(10); 2344-56. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Disponibilidade Biológica , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Modelos Moleculares , Conformação Molecular , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Inibidores de Proteínas Quinases/química , Transdução de Sinais/efeitos dos fármacos , Solubilidade , Serina-Treonina Quinases TOR/química , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Selective inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) represents a promising therapeutic strategy. However, despite documented evidence of clinical activity, limited information is available on the optimal dosing strategy of CDK4/6 inhibitors. Here, we present an integrated semi-mechanistic pharmacokinetic/pharmacodynamic model to characterize the quantitative pharmacology of LY2835219, a CDK4/6 inhibitor, in xenograft tumors. EXPERIMENTAL DESIGN: LY2835219 plasma concentrations were connected to CDK4/6 inhibition and cell-cycle arrest in colo-205 human colorectal xenografts by incorporating the biomarkers, phospho-(ser780)-Rb, topoisomerase II α, and phosphohistone H3, into a precursor-dependent transit compartment model. This biomarker model was then connected to tumor growth inhibition (TGI) by: (i) relating the rate of tumor growth to mitotic cell density, and (ii) incorporating a concentration-dependent mixed cytostatic/cytotoxic effect driving quiescence and cell death at high doses. Model validation was evaluated by predicting LY2835219-mediated antitumor effect in A375 human melanoma xenografts. RESULTS: The model successfully described LY2835219-mediated CDK4/6 inhibition, cell-cycle arrest, and TGI in colo-205, and was validated in A375. The model also demonstrated that a chronic dosing strategy achieving minimum steady-state trough plasma concentrations of 200 ng/mL is required to maintain durable cell-cycle arrest. Quiescence and cell death can be induced by further increasing LY2835219 plasma concentrations. CONCLUSIONS: Our model provides mechanistic insight into the quantitative pharmacology of LY2835219 and supports the therapeutic dose and chronic dosing strategy currently adopted in clinical studies.
Assuntos
Aminopiridinas/farmacocinética , Antineoplásicos/farmacocinética , Benzimidazóis/farmacocinética , Administração Oral , Aminopiridinas/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Benzimidazóis/uso terapêutico , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Humanos , Concentração Inibidora 50 , Camundongos Nus , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
DNA-dependent RNA polymerase II (RNAP II) largest subunit RPB1 C-terminal domain (CTD) kinases, including CDK9, are serine/threonine kinases known to regulate transcriptional initiation and elongation by phosphorylating Ser 2, 5, and 7 residues on CTD. Given the reported dysregulation of these kinases in some cancers, we asked whether inhibiting CDK9 may induce stress response and preferentially kill tumor cells. Herein, we describe a potent CDK9 inhibitor, LY2857785, that significantly reduces RNAP II CTD phosphorylation and dramatically decreases MCL1 protein levels to result in apoptosis in a variety of leukemia and solid tumor cell lines. This molecule inhibits the growth of a broad panel of cancer cell lines, and is particularly efficacious in leukemia cells, including orthotopic leukemia preclinical models as well as in ex vivo acute myeloid leukemia and chronic lymphocytic leukemia patient tumor samples. Thus, inhibition of CDK9 may represent an interesting approach as a cancer therapeutic target, especially in hematologic malignancies.
Assuntos
Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Quinase 9 Dependente de Ciclina/genética , Cicloexilaminas/administração & dosagem , Indazóis/administração & dosagem , Leucemia/tratamento farmacológico , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Humanos , Leucemia/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Fosforilação/efeitos dos fármacos , Serina/metabolismoRESUMO
Definitive plasma protein binding (PB) studies in drug development are routinely conducted with radiolabeled material, where the radiochemical purity limits quantitative PB measurement. Recent and emerging regulatory guidances increasingly expect quantitative determination of the fraction unbound (Fu) for key decision making. In the present study, PB of 11 structurally- and therapeutically-diverse drugs spanning the full range of plasma binding was determined by equilibrium dialysis of non-radiolabeled compound and was validated against the respective definitive values obtained by accepted radiolabeled protocols. The extent of plasma binding was in agreement with the radiolabeled studies; however, the methodology reported herein enables reliable quantification of Fu values for highly-bound drugs and is not limited by the radiochemical purity. In order to meet the rigor of a development study, equilibrium dialysis of unlabeled drug must be supported by an appropriately validated bioanalytical method along with studies to determine compound solubility and stability in matrix and dialysis buffer, nonspecific binding to the dialysis device, and ability to achieve equilibrium in the absence of protein. The presented methodology establishes an experimental protocol for definitive PB measurement, which enables quantitative determination of low Fu values, necessary for navigation of new regulatory guidances in clinical drug development.
Assuntos
Proteínas Sanguíneas/metabolismo , Descoberta de Drogas , Preparações Farmacêuticas/sangue , Cromatografia Líquida , Diálise/instrumentação , Diálise/métodos , Descoberta de Drogas/instrumentação , Descoberta de Drogas/métodos , Interações Medicamentosas , Estabilidade de Medicamentos , Humanos , Técnicas In Vitro , Limite de Detecção , Preparações Farmacêuticas/química , Reprodutibilidade dos Testes , Solubilidade , Espectrometria de Massas em TandemRESUMO
Novel tetrahydroisoquinolines have been developed as potent PPAR ligands. Evaluation of these compounds in PPARgamma responsive models of type 2 diabetes is described.