RESUMO
Activated B-cell-like (ABC) and germinal center B-cell-like diffuse large B-cell lymphoma (DLBCL) represent the 2 major molecular DLBCL subtypes. They are characterized by differences in clinical course and by divergent addiction to oncogenic pathways. To determine activity of novel compounds in these 2 subtypes, we conducted an unbiased pharmacologic in vitro screen. The phosphatidylinositol-3-kinase (PI3K) α/δ (PI3Kα/δ) inhibitor AZD8835 showed marked potency in ABC DLBCL models, whereas the protein kinase B (AKT) inhibitor AZD5363 induced apoptosis in PTEN-deficient DLBCLs irrespective of their molecular subtype. These in vitro results were confirmed in various cell line xenograft and patient-derived xenograft mouse models in vivo. Treatment with AZD8835 induced inhibition of nuclear factor κB signaling, prompting us to combine AZD8835 with the Bruton's tyrosine kinase inhibitor ibrutinib. This combination was synergistic and effective both in vitro and in vivo. In contrast, the AKT inhibitor AZD5363 was effective in PTEN-deficient DLBCLs through downregulation of the oncogenic transcription factor MYC. Collectively, our data suggest that patients should be stratified according to their oncogenic dependencies when treated with PI3K and AKT inhibitors.
Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Oxidiazóis/farmacologia , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Animais , Apoptose/efeitos dos fármacos , Combinação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Linfoma Difuso de Grandes Células B/classificação , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Camundongos , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , Especificidade de Órgãos , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have developed a series of orally efficacious IRAK4 inhibitors, based on a scaffold hopping strategy and using rational structure based design. Efforts to tackle low permeability and high efflux in our previously reported pyrrolopyrimidine series (Scott et al., 2017) led to the identification of pyrrolotriazines which contained one less formal hydrogen bond donor and were intrinsically more lipophilic. Further optimisation of substituents on this pyrrolotriazine core culminated with the discovery of 30 as a promising in vivo probe to assess the potential of IRAK4 inhibition for the treatment of MyD88 mutant DLBCL in combination with a BTK inhibitor. When tested in an ABC-DLBCL model with a dual MyD88/CD79 mutation (OCI-LY10), 30 demonstrated tumour regressions in combination with ibrutinib.
Assuntos
Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Pirróis/química , Tiazinas/química , Animais , Sítios de Ligação , Células CACO-2 , Cães , Desenho de Fármacos , Meia-Vida , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Simulação de Dinâmica Molecular , Mutação , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Permeabilidade/efeitos dos fármacos , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Pirróis/farmacocinética , Pirróis/farmacologia , Ratos , Relação Estrutura-Atividade , Tiazinas/farmacocinética , Tiazinas/farmacologiaRESUMO
CD37 has gathered renewed interest as a therapeutic target in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL); however, CD37-directed antibody-drug conjugates (ADCs) have not been explored. Here, we identified a novel anti-CD37 antibody, K7153A, with potent in vitro activity against B-cell lines through multiple mechanisms including apoptosis induction, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity. The antibody was conjugated to the maytansinoid, DM1, a potent antimicrotubule agent, via the thioether linker, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and the resulting ADC, IMGN529, retained the intrinsic antibody activities and showed enhanced cytotoxic activity from targeted payload delivery. In lymphoma cell lines, IMGN529 induced G2/M cell cycle arrest after internalization and lysosomal processing to lysine-N(ε)-SMCC-DM1 as the sole intracellular maytansinoid metabolite. IMGN529 was highly active against subcutaneous B-cell tumor xenografts in severe combined immunodeficient mice with comparable or better activity than rituximab, a combination of cyclophosphamide, vincristine, and prednisone, or bendamustine. In human blood cells, CD37 is expressed in B cells at similar levels as CD20, and IMGN529 resulted in potent and specific depletion of normal and CLL B cells. These results support evaluation of the CD37-targeted ADC, IMGN529, in clinical trials in patients with B-cell malignancies including NHL and CLL.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Murinos/uso terapêutico , Antígenos de Neoplasias/imunologia , Linfócitos B/efeitos dos fármacos , Imunotoxinas/uso terapêutico , Maitansina/análogos & derivados , Terapia de Alvo Molecular , Tetraspaninas/imunologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Murinos/administração & dosagem , Anticorpos Monoclonais Murinos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Linfócitos B/patologia , Cloridrato de Bendamustina , Linhagem Celular Tumoral/efeitos dos fármacos , Ciclofosfamida/administração & dosagem , Citotoxicidade Imunológica/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Imunotoxinas/imunologia , Imunotoxinas/farmacologia , Maitansina/administração & dosagem , Maitansina/farmacologia , Maitansina/uso terapêutico , Camundongos , Camundongos SCID , Compostos de Mostarda Nitrogenada/uso terapêutico , Prednisona/administração & dosagem , Rituximab , Vincristina/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Developing therapies for the activated B-cell like (ABC) subtype of diffuse large B-cell lymphomas (DLBCL) remains an area of unmet medical need. A subset of ABC DLBCL tumors is driven by activating mutations in myeloid differentiation primary response protein 88 (MYD88), which lead to constitutive activation of interleukin-1 receptor associated kinase 4 (IRAK4) and cellular proliferation. IRAK4 signaling is driven by its catalytic and scaffolding functions, necessitating complete removal of this protein and its escape mechanisms for complete therapeutic suppression. Herein, we describe the identification and characterization of a dual-functioning molecule, KT-413 and show it efficiently degrades IRAK4 and the transcription factors Ikaros and Aiolos. KT-413 achieves concurrent degradation of these proteins by functioning as both a heterobifunctional degrader and a molecular glue. Based on the demonstrated activity and safety of KT-413 in preclinical studies, a phase 1 clinical trial in B-cell lymphomas, including MYD88 mutant ABC DLBCL, is currently underway.
Assuntos
Quinases Associadas a Receptores de Interleucina-1 , Linfoma Difuso de Grandes Células B , Mutação , Fator 88 de Diferenciação Mieloide , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Fator 88 de Diferenciação Mieloide/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Humanos , Animais , Linhagem Celular Tumoral , Descoberta de Drogas , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Camundongos , Imidazóis/química , Imidazóis/farmacologia , Imidazóis/metabolismo , Proteólise/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Interleukin-1 receptor associated kinase 4 (IRAK4) is an essential mediator of the IL-1R and TLR signaling pathways, both of which have been implicated in multiple autoimmune conditions. Hence, blocking the activity of IRAK4 represents an attractive approach for the treatment of autoimmune diseases. The activity of this serine/threonine kinase is dependent on its kinase and scaffolding activities; thus, degradation represents a potentially superior approach to inhibition. Herein, we detail the exploration of structure-activity relationships that ultimately led to the identification of KT-474, a potent, selective, and orally bioavailable heterobifunctional IRAK4 degrader. This represents the first heterobifunctional degrader evaluated in a nononcology indication and dosed to healthy human volunteers. This molecule successfully completed phase I studies in healthy adult volunteers and patients with atopic dermatitis or hidradenitis suppurativa. Phase II clinical trials in both of these indications have been initiated.
RESUMO
Antibody-maytansinoid conjugates (AMCs) are targeted chemotherapeutic agents consisting of a potent microtubule-depolymerizing maytansinoid (DM1 or DM4) attached to lysine residues of a monoclonal antibody (mAb) using an uncleavable thioether linker or a stable disulfide linker. Most of the administered dose of an antibody-based therapeutic is slowly catabolized by the liver and other tissues of the reticuloendothelial system. Maytansinoids released from an AMC during this catabolic process could potentially be a source of toxicity. To investigate this, we isolated and identified liver metabolites in mice for three different [(3)H]AMCs with structures similar to those currently undergoing evaluation in the clinic. We then synthesized each metabolite to confirm the identification and assessed their cytotoxic potencies when added extracellularly. We found that the uncleavable mAb-SMCC-[(3)H]DM1 conjugate was degraded to a single major maytansinoid metabolite, lysine-SMCC-[(3)H]DM1, that was nearly 50-fold less cytotoxic than maytansine. The two disulfide-linked conjugates, mAb-SPP-[(3)H]DM1 and mAb-SPDB-[(3)H]DM4, were also found to be catabolized to the analogous lysine-linked maytansinoid metabolites. However, subsequent reduction, S-methylation, and NADPH-dependent oxidation steps in the liver yielded the corresponding S-methyl sulfoxide and S-methyl sulfone derivatives. The cytotoxic potencies of the oxidized maytansinoids toward several human carcinoma cell lines were found to be 5- to 50-fold less potent than maytansine. Our results suggest that liver plays an important role in the detoxification of both cleavable and uncleavable AMCs.
Assuntos
Anticorpos Monoclonais/metabolismo , Desenho de Fármacos , Fígado/metabolismo , Maitansina/metabolismo , Animais , Anticorpos Monoclonais/química , Feminino , Fígado/química , Maitansina/análogos & derivados , Maitansina/química , Camundongos , Camundongos Endogâmicos , Estrutura MolecularRESUMO
Antibody-drug conjugates (ADCs) are designed to eradicate cancer cells that express the target antigen on their cell surface. A key component of an ADC is the linker that covalently connects the cytotoxic agent to the antibody. Several antibody-maytansinoid conjugates prepared with disulfide-based linkers such as those targeting the CanAg antigen have been shown to display more activity in preclinical mouse xenograft models than corresponding conjugates prepared with uncleavable thioether-based linkers. To investigate how the linker influences delivery and activation of antibody-maytansinoid conjugates, we isolated and characterized the [(3)H]maytansinoids from CanAg-positive tumor tissues following a single intravenous administration of 300 microg/kg (based on maytansinoid dose) of anti-CanAg antibody (huC242)-(3)H-maytansinoid conjugates prepared with cleavable disulfide linkers and an uncleavable thioether linker. We identified three target-dependent tumor metabolites of the disulfide-linked huC242-SPDB-DM4, namely, lysine-N(epsilon)-SPDB-DM4, DM4, and S-methyl-DM4. We found similar metabolites for the less hindered disulfide-linked huC242-SPP-DM1 conjugate with the exception that no S-methyl-DM1 was detected. The sole metabolite of the uncleavable thioether-linked huC242-SMCC-DM1 was lysine-N(epsilon)-SMCC-DM1. The AUC for the metabolites of huC242-SMCC-DM1 at the tumor over 7 d was about 2-fold greater than the corresponding AUC for the metabolites of the disulfide-linked conjugates. The lipophilic metabolites of the disulfide-linked conjugates were found to be nearly 1000 times more cytotoxic than the more hydrophilic lysine-N(epsilon)-linker-maytansinoids in cell-based viability assays when added extracellularly. The cell killing properties associated with the lipophilic metabolites of the disulfide-linked conjugates (DM4 and S-methyl-DM4, and DM1) provide an explanation for the superior in vivo efficacy that is often observed with antibody-maytansinoid conjugates prepared with disulfide-based linkers in xenograft mouse models.
Assuntos
Anticorpos/metabolismo , Dissulfetos/química , Imunoconjugados/metabolismo , Imunoconjugados/uso terapêutico , Maitansina/metabolismo , Neoplasias/metabolismo , Sulfetos/química , Animais , Anticorpos/química , Anticorpos/imunologia , Anticorpos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Maitansina/química , Maitansina/imunologia , Maitansina/uso terapêutico , Camundongos , Camundongos SCID , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this article, we report the discovery of a series of 5-azaquinazolines as selective IRAK4 inhibitors. From modestly potent quinazoline 4, we introduced a 5-aza substitution to mask the 4-NH hydrogen bond donor (HBD). This allowed us to substitute the core with a 2-aminopyrazole, which showed large gains in cellular potency despite the additional formal HBD. Further optimization led to 6-cyanomethyl-5-azaquinazoline 13, a selective IRAK4 inhibitor, which proved efficacious in combination with ibrutinib, while showing very little activity as a single agent up to 100 mg/kg. This contrasted to previously reported IRAK4 inhibitors that exhibited efficacy in the same model as single agents and was attributed to the enhanced specificity of 13 toward IRAK4.
Assuntos
Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Terapia de Alvo Molecular , Fator 88 de Diferenciação Mieloide/genética , Quinazolinas/química , Quinazolinas/farmacologia , Administração Oral , Animais , Linhagem Celular Tumoral , Desenho de Fármacos , Feminino , Humanos , Quinases Associadas a Receptores de Interleucina-1/química , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Modelos Moleculares , Mutação , Conformação Proteica , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/administração & dosagem , Quinazolinas/farmacocinética , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
: PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity. SIGNIFICANCE: These findings describe a new inhibitor of PARP6 and identify a novel function of PARP6 in regulating activation of Chk1 in breast cancer cells.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Neoplasias da Mama/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 1 do Ponto de Checagem/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases/química , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Herein we report the optimization of a series of pyrrolopyrimidine inhibitors of interleukin-1 receptor associated kinase 4 (IRAK4) using X-ray crystal structures and structure based design to identify and optimize our scaffold. Compound 28 demonstrated a favorable physicochemical and kinase selectivity profile and was identified as a promising in vivo tool with which to explore the role of IRAK4 inhibition in the treatment of mutant MYD88L265P diffuse large B-cell lymphoma (DLBCL). Compound 28 was shown to be capable of demonstrating inhibition of NF-κB activation and growth of the ABC subtype of DLBCL cell lines in vitro at high concentrations but showed greater effects in combination with a BTK inhibitor at lower concentrations. In vivo, the combination of compound 28 and ibrutinib led to tumor regression in an ABC-DLBCL mouse model.
Assuntos
Antineoplásicos/farmacologia , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Cristalografia por Raios X , Cães , Feminino , Humanos , Quinases Associadas a Receptores de Interleucina-1/química , Linfoma Difuso de Grandes Células B/genética , Espectroscopia de Ressonância Magnética , Masculino , Camundongos SCID , Mutação , Fator 88 de Diferenciação Mieloide/genética , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/química , Pirróis/química , Ratos Wistar , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The promise of tumor-selective delivery of cytotoxic agents in the form of antibody-drug conjugates (ADC) has now been realized, evidenced by the approval of two ADCs, both of which incorporate highly cytotoxic tubulin-interacting agents, for cancer therapy. An ongoing challenge remains in identifying potent agents with alternative mechanisms of cell killing that can provide ADCs with high therapeutic indices and favorable tolerability. Here, we describe the development of a new class of potent DNA alkylating agents that meets these objectives. Through chemical design, we changed the mechanism of action of our novel DNA cross-linking agent to a monofunctional DNA alkylator. This modification, coupled with linker optimization, generated ADCs that were well tolerated in mice and demonstrated robust antitumor activity in multiple tumor models at doses 1.5% to 3.5% of maximally tolerated levels. These properties underscore the considerable potential of these purpose-created, unique DNA-interacting conjugates for broadening the clinical application of ADC technology. Mol Cancer Ther; 15(8); 1870-8. ©2016 AACR.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Imunoconjugados/farmacologia , Animais , Antineoplásicos Alquilantes/química , Efeito Espectador , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA/química , DNA/metabolismo , Adutos de DNA , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Imunoconjugados/química , Camundongos , Estrutura Molecular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Leptin is an important metabolic hormone providing the brain with information concerning energy balance. Most studies have reported that circulating leptin concentrations are unaltered by acute, moderate exercise. We hypothesized that these studies have been limited by short sampling schemes (<4 h) postexercise and may have missed a time-delayed reduction in circulating leptin concentrations. METHODS: Ten men (age = 21 +/- 1 yr, height = 177 +/- 2 cm, body mass = 79 +/- 3 kg, body fat = 11 +/- 1%BF, .VO(2max) = 51 +/- 1 mL.kg-1.min-1) completed an acute heavy-resistance exercise protocol (AHREP) (50 total sets comprised of the squat, bench press, leg press, and lat pull-down) from 1500 to 1700 h. Blood was sampled hourly postexercise until 0600 h the next morning and also during a time-matched control period. Leptin concentrations were measured by an immunoradiometric assay. Resting energy expenditure (REE) was measured via indirect calorimetry using a ventilated hood beginning approximately 0600 h after both overnight conditions. RESULTS: The estimated caloric expenditure from the AHREP was 856 +/- 114 kcal. No significant differences (P > 0.05) between the control and exercise conditions were observed for serum leptin concentrations until 9 h postexercise. Significant interaction effects (P < 0.05) indicated lower serum leptin concentrations postexercise at hours 9 (2.9 vs 2.2 ng.mL-1), 10 (2.7 vs 2.0 ng.mL-1), 12 (2.5 vs 1.8 ng.mL-1), and 13 (2.6 vs 1.8 ng.mL-1). This delayed reduction was accompanied by a 12% elevation (P < 0.05) in morning-after REE (0.25 +/- 0.02 vs 0.28 +/- 0.02 L.min-1). CONCLUSION: Leptin concentrations experience a delayed ( approximately 9 h) reduction in the systemic circulation after acute resistance exercise. This decline is likely associated with the disruption in metabolic homeostasis created by the high-intensity, long-duration, energy expenditure and subsequent excess post oxygen consumption from the AHREP and is not due to losses in fat mass.
Assuntos
Exercício Físico/fisiologia , Leptina/sangue , Tecido Adiposo/metabolismo , Adulto , Metabolismo Energético , Humanos , Leptina/fisiologia , Masculino , Consumo de Oxigênio , Tempo de Reação , Fatores de Tempo , Levantamento de PesoRESUMO
STUDY DESIGN: A single-blind, 2-factor (4 treatments by 8 time points) repeated-measures study design. OBJECTIVE: To analytically determine dexamethasone and dexamethasone phosphate concentrations in plasma derived from proximal effluent venous blood, following cathodic iontophoresis. METHODS AND MEASURES: Six volunteers received the following dexamethasone phosphate (2.5 ml, 4 mg/ml) treatments to their wrists on separate occasions: cathodic iontophoresis (4 mA, 10 minutes or 4 mA, 20 minutes), passive application (10 or 20 minutes). Plasma samples from the ipsilateral antecubital vein were obtained 10 minutes prior to and half way through the treatment (5 or 10 minutes), at the end of the treatment (10 or 20 minutes), and posttreatment (15, 30, 60, 90, and 120 minutes). The present investigation examined: (1) the sensitivity and linearity of extraction and analysis of dexamethasone and dexamethasone phosphate; (2) the necessity for determining both; and (3) the plasma levels from proximal effluent venous blood following cathodic iontophoresis. RESULTS: The aggregate (n = 18) of the 6-point standard curves were linear for dexamethasone (r > 0.974) and dexamethasone phosphate (r > 0.829). In vitro dephosphorylation of dexamethasone phosphate to dexamethasone occurred in plasma at 37 degrees C and during freeze-thaw. Measurable dexamethasone or dexamethasone phosphate concentrations were absent at all time points and under all conditions in the human subjects. CONCLUSIONS: These results demonstrate the sensitivity of the current assay and the need for evaluating both forms of the drug, as in vitro dephosphorylation results in the presence of dexamethasone and dexamethasone phosphate in samples. Absence of measurable dexamethasone or dexamethasone phosphate in the proximal effluent venous blood may require re-evaluation of the extent of drug delivery during the clinical iontophoresis of dexamethasone phosphate.
Assuntos
Dexametasona/análogos & derivados , Dexametasona/sangue , Iontoforese , Adulto , Análise de Variância , Cromatografia Líquida de Alta Pressão , Dexametasona/administração & dosagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Distribuição Tecidual , PunhoRESUMO
Lorvotuzumab mertansine (LM) is an antibody-drug conjugate composed of a humanized anti-CD56 antibody, lorvotuzumab, linked via a cleavable disulfide linker to the tubulin-binding maytansinoid DM1. CD56 is expressed on most small cell lung cancers (SCLC), providing a promising therapeutic target for treatment of this aggressive cancer, which has a poor five-year survival rate of only 5-10%. We performed immunohistochemical staining on SCLC tumor microarrays, which confirmed that CD56 is expressed at high levels on most (~74%) SCLC tumors. Conjugation of lorvotuzumab with DM1 did not alter its specific binding to cells and LM demonstrated potent target-dependent cytotoxicity against CD56-positive SCLC cells in vitro. The anti-tumor activity of LM was evaluated against SCLC xenograft models in mice, both as monotherapy and in combination with platinum/etoposide and paclitaxel/carboplatin. Dose-dependent and antigen-specific anti-tumor activity of LM monotherapy was demonstrated at doses as low as 3 mg/kg. LM was highly active in combination with standard-of-care platinum/etoposide therapies, even in relatively resistant xenograft models. LM demonstrated outstanding anti-tumor activity in combination with carboplatin/etoposide, with superior activity over chemotherapy alone when LM was used in combinations at significantly reduced doses (6-fold below the minimally efficacious dose for LM monotherapy). The combination of LM with carboplatin/paclitaxel was also highly active. This study provides the rationale for clinical evaluation of LM as a promising novel targeted therapy for SCLC, both as monotherapy and in combination with chemotherapy.
Assuntos
Anticorpos Monoclonais/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno CD56/imunologia , Imunoterapia/métodos , Neoplasias Pulmonares/terapia , Maitansina/análogos & derivados , Maitansina/metabolismo , Carcinoma de Pequenas Células do Pulmão/terapia , Moduladores de Tubulina/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Maitansina/química , Maitansina/imunologia , Camundongos , Camundongos SCID , Carcinoma de Pequenas Células do Pulmão/imunologia , Moduladores de Tubulina/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Diffuse large B cell lymphoma is generally treated by chemotherapy and there is an unmet medical need for novel targeted therapies or combination therapies. Using in vitro screening, we have identified the combination of ibrutinib, an inhibitor of the tyrosine kinase BTK, and AZD2014, an mTOR catalytic inhibitor, as being highly synergistic in killing ABC-subtype DLBCL cell lines. Simultaneous inhibition of BTK and mTOR causes apoptosis both in vitro and in vivo and results in tumor regression in a xenograft model. We identify two parallel mechanisms that underlie apoptosis in this setting: cooperative inhibition of cap-dependent translation, and the inhibition of an NF-κB/IL10/STAT3 autocrine loop. Combined disruption of these pathways is required for apoptosis. These data represent a rational basis for the dual inhibition of BTK and mTOR as a potential treatment for ABC-subtype DLBCL.
Assuntos
Apoptose/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Complexos Multiproteicos/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Animais , Benzamidas , Western Blotting , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Células HEK293 , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos SCID , Morfolinas/farmacologia , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Piperidinas , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Conjugation of cytotoxic compounds to antibodies that bind to cancer-specific antigens makes these drugs selective in killing cancer cells. However, many of the compounds used in such antibody-drug conjugates (ADC) are substrates for the multidrug transporter MDR1. To evade the MDR1-mediated resistance, we conjugated the highly cytotoxic maytansinoid DM1 to antibodies via the maleimidyl-based hydrophilic linker PEG(4)Mal. Following uptake into target cells, conjugates made with the PEG(4)Mal linker were processed to a cytotoxic metabolite that was retained by MDR1-expressing cells better than a metabolite of similar conjugates prepared with the nonpolar linker N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). In accord, PEG(4)Mal-linked conjugates were more potent in killing MDR1-expressing cells in culture. In addition, PEG(4)Mal-linked conjugates were markedly more effective in eradicating MDR1-expressing human xenograft tumors than SMCC-linked conjugates while being tolerated similarly, thus showing an improved therapeutic index. This study points the way to the development of ADCs that bypass multidrug resistance.