RESUMEN
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.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Pirroles/química , Tiazinas/química , Animales , Sitios de Unión , Células CACO-2 , Perros , Diseño de Fármacos , Semivida , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Simulación de Dinámica Molecular , Mutación , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Permeabilidad/efectos de los fármacos , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Pirroles/farmacocinética , Pirroles/farmacología , Ratas , Relación Estructura-Actividad , Tiazinas/farmacocinética , Tiazinas/farmacologíaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Antígenos de Neoplasias/inmunología , Linfocitos B/efectos de los fármacos , Inmunotoxinas/uso terapéutico , Maitansina/análogos & derivados , Terapia Molecular Dirigida , Tetraspaninas/inmunología , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Anticuerpos Monoclonales de Origen Murino/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Linfocitos B/patología , Clorhidrato de Bendamustina , Línea Celular Tumoral/efectos de los fármacos , Ciclofosfamida/administración & dosificación , Citotoxicidad Inmunológica/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Inmunotoxinas/inmunología , Inmunotoxinas/farmacología , Maitansina/administración & dosificación , Maitansina/farmacología , Maitansina/uso terapéutico , Ratones , Ratones SCID , Compuestos de Mostaza Nitrogenada/uso terapéutico , Prednisona/administración & dosificación , Rituximab , Vincristina/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Anticuerpos/metabolismo , Disulfuros/química , Inmunoconjugados/metabolismo , Inmunoconjugados/uso terapéutico , Maitansina/metabolismo , Neoplasias/metabolismo , Sulfuros/química , Animales , Anticuerpos/química , Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Inmunoconjugados/química , Inmunoconjugados/inmunología , Maitansina/química , Maitansina/inmunología , Maitansina/uso terapéutico , Ratones , Ratones SCID , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Terapia Molecular Dirigida , Factor 88 de Diferenciación Mieloide/genética , Quinazolinas/química , Quinazolinas/farmacología , Administración Oral , Animales , Línea Celular Tumoral , Diseño de Fármacos , Femenino , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/química , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Ratones , Modelos Moleculares , Mutación , Conformación Proteica , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/administración & dosificación , Quinazolinas/farmacocinética , Ratas , Ratas Wistar , Relación Estructura-Actividad , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Cristalografía por Rayos X , Perros , Femenino , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/química , Linfoma de Células B Grandes Difuso/genética , Espectroscopía de Resonancia Magnética , Masculino , Ratones SCID , Mutación , Factor 88 de Diferenciación Mieloide/genética , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirimidinas/química , Pirroles/química , Ratas Wistar , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Inmunoconjugados/farmacología , Animales , Antineoplásicos Alquilantes/química , Efecto Espectador , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN/química , ADN/metabolismo , Aductos de ADN , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Inmunoconjugados/química , Ratones , Estructura Molecular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Ejercicio Físico/fisiología , Leptina/sangre , Tejido Adiposo/metabolismo , Adulto , Metabolismo Energético , Humanos , Leptina/fisiología , Masculino , Consumo de Oxígeno , Tiempo de Reacción , Factores de Tiempo , Levantamiento de PesoRESUMEN
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.
Asunto(s)
Dexametasona/análogos & derivados , Dexametasona/sangre , Iontoforesis , Adulto , Análisis de Varianza , Cromatografía Líquida de Alta Presión , Dexametasona/administración & dosificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Distribución Tisular , MuñecaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígeno CD56/inmunología , Inmunoterapia/métodos , Neoplasias Pulmonares/terapia , Maitansina/análogos & derivados , Maitansina/metabolismo , Carcinoma Pulmonar de Células Pequeñas/terapia , Moduladores de Tubulina/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Humanos , Neoplasias Pulmonares/inmunología , Maitansina/química , Maitansina/inmunología , Ratones , Ratones SCID , Carcinoma Pulmonar de Células Pequeñas/inmunología , Moduladores de Tubulina/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.