RESUMO
Arginase is an enzyme responsible for converting arginine, a semi-essential amino acid, to ornithine and urea. Arginine depletion suppresses immunity via multiple mechanisms including inhibition of T-cell and NK cell proliferation and activity. Arginase inhibition is therefore an attractive mechanism to potentially reverse immune suppression and thus has been explored as a therapy for oncology and respiratory indications. Small molecules targeting arginase present significant bioanalytical challenges for in vitro and in vivo characterization as inhibitors of arginase are typically hydrophilic in nature. The resulting low or negative LogD characteristics are incompatible with common analytical methods such as RP-ESI-MS/MS. Accordingly, a sensitive, high-throughput bioanalytical method was developed by incorporating benzoyl chloride derivatization to increase the hydrophobic characteristics of these polar analytes. Samples were separated by reversed phase chromatography on a Waters XBridge BEH C18 3.5 µm, 30 × 3â¯mm column using gradient elution. The mass spec was operated in positive mode using electrospray ionization. The m/z 434.1â176.1, 439.4â181.2, 334.9â150.0 and 339.9â150.0 for AZD0011, AZD0011 IS, AZD0011-PL and AZD0011-PL IS respectively were used for quantitation. The linear calibration range of the assay was 1.00-10,000â¯ng/mL with QC values of 5, 50 and 500â¯ng/mL. The qualified method presented herein exhibits a novel, robust analytical performance and was successfully applied to evaluate the in vivo ADME properties of boronic acid-based arginase inhibitor prodrug AZD0011 and its active payload AZD0011-PL.
Assuntos
Arginase , Espectrometria de Massas em Tandem , Arginase/antagonistas & inibidores , Espectrometria de Massas em Tandem/métodos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia de Fase Reversa/métodos , Animais , Cromatografia Líquida/métodos , Humanos , Cromatografia Líquida de Alta Pressão/métodos , Interações Hidrofóbicas e Hidrofílicas , Reprodutibilidade dos Testes , Espectrometria de Massa com Cromatografia LíquidaRESUMO
Antitumor immunity can be hampered by immunosuppressive mechanisms in the tumor microenvironment, including recruitment of arginase (ARG) expressing myeloid cells that deplete l-arginine essential for optimal T-cell and natural killer cell function. Hence, ARG inhibition can reverse immunosuppression enhancing antitumor immunity. We describe AZD0011, a novel peptidic boronic acid prodrug to deliver an orally available, highly potent, ARG inhibitor payload (AZD0011-PL). We demonstrate that AZD0011-PL is unable to permeate cells, suggesting that this compound will only inhibit extracellular ARG. In vivo, AZD0011 monotherapy leads to arginine increases, immune cell activation, and tumor growth inhibition in various syngeneic models. Antitumor responses increase when AZD0011 is combined with anti-PD-L1 treatment, correlating with increases in multiple tumor immune cell populations. We demonstrate a novel triple combination of AZD0011, anti-PD-L1, and anti-NKG2A, and combination benefits with type I IFN inducers, including polyI:C and radiotherapy. Our preclinical data demonstrate AZD0011's ability to reverse tumor immunosuppression and enhance immune stimulation and antitumor responses with diverse combination partners providing potential strategies to increase immuno-oncology therapies clinically.
Assuntos
Arginase , Linfócitos T , Humanos , Linhagem Celular Tumoral , Terapia de Imunossupressão , Tolerância Imunológica , Microambiente TumoralRESUMO
A physiologically based pharmacokinetic model was developed to describe the tissue distribution kinetics of a dendritic nanoparticle and its conjugated active pharmaceutical ingredient (API) in plasma, liver, spleen, and tumors. Tumor growth data from MV-4-11 tumor-bearing mice were incorporated to investigate the exposure/efficacy relationship. The nanoparticle demonstrated improved antitumor activity compared to the conventional API formulation, owing to the extended released API concentrations at the site of action. Model simulations further enabled the identification of critical parameters that influence API exposure in tumors and downstream efficacy outcomes upon nanoparticle administration. The model was utilized to explore a range of dosing schedules and their effect on tumor growth kinetics, demonstrating the improved antitumor activity of nanoparticles with less frequent dosing compared to the same dose of naked APIs in conventional formulations.
Assuntos
Antineoplásicos/administração & dosagem , Dendrímeros/farmacocinética , Nanopartículas/metabolismo , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos SCID , Transplante de Neoplasias , Distribuição Tecidual , Resultado do TratamentoRESUMO
Dual Bcl-2/Bcl-xL inhibitors are expected to deliver therapeutic benefit in many haematological and solid malignancies, however, their use is limited by tolerability issues. AZD4320, a potent dual Bcl-2/Bcl-xL inhibitor, has shown good efficacy however had dose limiting cardiovascular toxicity in preclinical species, coupled with challenging physicochemical properties, which prevented its clinical development. Here, we describe the design and development of AZD0466, a drug-dendrimer conjugate, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer. Mathematical modelling was employed to determine the optimal release rate of the drug from the dendrimer for maximal therapeutic index in terms of preclinical anti-tumour efficacy and cardiovascular tolerability. The optimised candidate is shown to be efficacious and better tolerated in preclinical models compared with AZD4320 alone. The AZD4320-dendrimer conjugate (AZD0466) identified, through mathematical modelling, has resulted in an improved therapeutic index and thus enabled progression of this promising dual Bcl-2/Bcl-xL inhibitor into clinical development.
Assuntos
Antineoplásicos , Dendrímeros , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Dendrímeros/síntese química , Dendrímeros/química , Dendrímeros/farmacocinética , Dendrímeros/uso terapêutico , Cães , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Ratos , Ratos Wistar , Índice Terapêutico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/antagonistas & inibidoresRESUMO
The aim of the present study was to evaluate and interpret the pharmacokinetic profiles after subcutaneous (s.c.) administration of crystalline AZ'72 nano- and microsuspensions to rodents. Both formulations were injected at 1.5 and 150 mg/kg to rats. For the lower dose, the profiles were similar after s.c. injection but extended as compared to oral administration. The overall exposure was higher for nanoparticles compared with microparticles during the investigated period. For the higher dose, injection of both suspensions resulted in maintained plateaus caused by the drug depots but, unexpectedly, at similar exposure levels. After addition of a further stabilizer, pluronic F127, nanosuspensions showed improved exposure with dose and higher exposure compared to larger particles in mice. Obviously, a stabilizer mixture that suits one delivery route is not necessarily optimal for another one. The differences in peak concentration (Cmax) between nano- and microparticles were mainly ascribed to differences in dissolution rate. Plasma profiles in mice showed curves with secondary absorption peaks after intravenous and oral administration, suggesting hepatic recirculation following both administration routes. This process, together with the depot formulation, complicates the analysis of absorption from s.c. administration, i.e. multiple processes were driving the plasma profile of AZ'72.
Assuntos
Excipientes/química , Refluxo Gastroesofágico/tratamento farmacológico , Fármacos Gastrointestinais/farmacocinética , Fígado/metabolismo , 2-Hidroxipropil-beta-Ciclodextrina/química , Administração Oral , Animais , Disponibilidade Biológica , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/farmacocinética , Relação Dose-Resposta a Droga , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Feminino , Fármacos Gastrointestinais/administração & dosagem , Humanos , Injeções Subcutâneas , Fígado/irrigação sanguínea , Masculino , Camundongos , Modelos Animais , Nanopartículas/química , Tamanho da Partícula , Poloxâmero/química , Ratos , Solubilidade , SuspensõesRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
The inaccessibility of living bone marrow (BM) hampers the study of its pathophysiology under myelotoxic stress induced by drugs, radiation or genetic mutations. Here, we show that a vascularized human BM-on-a-chip (BM chip) supports the differentiation and maturation of multiple blood cell lineages over 4 weeks while improving CD34+ cell maintenance, and that it recapitulates aspects of BM injury, including myeloerythroid toxicity after clinically relevant exposures to chemotherapeutic drugs and ionizing radiation, as well as BM recovery after drug-induced myelosuppression. The chip comprises a fluidic channel filled with a fibrin gel in which CD34+ cells and BM-derived stromal cells are co-cultured, a parallel channel lined by human vascular endothelium and perfused with culture medium, and a porous membrane separating the two channels. We also show that BM chips containing cells from patients with the rare genetic disorder Shwachman-Diamond syndrome reproduced key haematopoietic defects and led to the discovery of a neutrophil maturation abnormality. As an in vitro model of haematopoietic dysfunction, the BM chip may serve as a human-specific alternative to animal testing for the study of BM pathophysiology.
Assuntos
Células da Medula Óssea/citologia , Medula Óssea/patologia , Hematopoese , Microfluídica/métodos , Animais , Antígenos CD34 , Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos da radiação , Transplante de Medula Óssea , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Dispositivos Lab-On-A-Chip , Células-Tronco Mesenquimais , Microfluídica/instrumentaçãoRESUMO
PURPOSE: Cyclin-dependent kinase 9 (CDK9) is a transcriptional regulator and potential therapeutic target for many cancers. Multiple nonselective CDK9 inhibitors have progressed clinically but were limited by a narrow therapeutic window. This work describes a novel, potent, and highly selective CDK9 inhibitor, AZD4573. EXPERIMENTAL DESIGN: The antitumor activity of AZD4573 was determined across broad cancer cell line panels in vitro as well as cell line- and patient-derived xenograft models in vivo. Multiple approaches, including integrated transcriptomic and proteomic analyses, loss-of-function pathway interrogation, and pharmacologic comparisons, were employed to further understand the major mechanism driving AZD4573 activity and to establish an exposure/effect relationship. RESULTS: AZD4573 is a highly selective and potent CDK9 inhibitor. It demonstrated rapid induction of apoptosis and subsequent cell death broadly across hematologic cancer models in vitro, and MCL-1 depletion in a dose- and time-dependent manner was identified as a major mechanism through which AZD4573 induces cell death in tumor cells. This pharmacodynamic (PD) response was also observed in vivo, which led to regressions in both subcutaneous tumor xenografts and disseminated models at tolerated doses both as monotherapy or in combination with venetoclax. This understanding of the mechanism, exposure, and antitumor activity of AZD4573 facilitated development of a robust pharmacokinetic/PD/efficacy model used to inform the clinical trial design. CONCLUSIONS: Selective targeting of CDK9 enables the indirect inhibition of MCL-1, providing a therapeutic option for MCL-1-dependent diseases. Accordingly, AZD4573 is currently being evaluated in a phase I clinical trial for patients with hematologic malignancies (clinicaltrials.gov identifier: NCT03263637).See related commentary by Alcon et al., p. 761.
Assuntos
Antineoplásicos , Neoplasias Hematológicas , Apoptose/efeitos dos fármacos , Quinase 9 Dependente de Ciclina , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , ProteômicaRESUMO
Adenosine is known to be an important signaling molecule in many physiological processes and has recently been shown to be an important molecule in oncology. A fit for purpose method has been developed for the quantification of adenosine in murine tumor samples using pre-column derivatization and liquid chromatography-mass spectrometry (LC-MS/MS). To overcome adenosine quantification challenges, derivatization with dansyl chloride was employed. This derivatization technique, following protein precipitation and liquid-liquid extraction, improved the sensitivity and selectivity of adenosine in tumor samples through the reduction of endogenous interference and matrix effects. This method utilizes a mouse plasma calibration curve, qualified over a range of 0.019⯵M-37⯵M. The 15â¯min derivatization incubation time and 1â¯min chromatographic run time allow for higher throughput. The following established method overcomes challenges associated with the quantification of low molecular weight, polar, endogenous molecules, such as adenosine, using derivatization and LC-MS/MS. With the additional analysis of murine tumors, this method will contribute to the understanding of the impact adenosine plays in the tumor microenvironment and the bearing it has on targeted cancer therapies.
Assuntos
Adenosina/sangue , Neoplasias Encefálicas/sangue , Neoplasias Hepáticas/sangue , Adenosina/análogos & derivados , Adenosina/química , Animais , Neoplasias Encefálicas/diagnóstico , Calibragem , Cromatografia Líquida , Neoplasias Hepáticas/diagnóstico , Camundongos , Espectrometria de Massas em TandemRESUMO
Efficacies of ceftazidime-avibactam (4:1 w/w) and ceftazidime were tested against ceftazidime-susceptible (blaKPC-2-negative), and meropenem- and ceftazidime-resistant (blaKPC-2-positive), Klebsiella pneumoniae in a 52-h, multiple dose, abdominal abscess model in the rat. Efficacies corresponded to minimum inhibitory concentrations (MICs) measured in vitro and were consistent with drug exposures modelled from pharmacokinetics in infected animals. The ceftazidime, ceftazidime-avibactam and meropenem control treatments were effective in the rat abscess model against the susceptible strain, whereas only ceftazidime-avibactam was effective against K. pneumoniae harbouring blaKPC-2.
Assuntos
Abscesso Abdominal/tratamento farmacológico , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/efeitos dos fármacos , Resistência beta-Lactâmica , Abscesso Abdominal/metabolismo , Abscesso Abdominal/microbiologia , Animais , Compostos Azabicíclicos/farmacologia , Ceftazidima/farmacologia , Combinação de Medicamentos , Infecções por Klebsiella/metabolismo , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/metabolismo , Meropeném , Testes de Sensibilidade Microbiana , Ratos , Tienamicinas/farmacologia , beta-LactamasesRESUMO
There are many sources of analytical variability in ligand binding assays (LBA). One strategy to reduce variability has been duplicate analyses. With recent advances in LBA technologies, it is conceivable that singlet analysis is possible. We retrospectively evaluated singlet analysis using Gyrolab data. Relative precision of duplicates compared to singlets was evaluated using 60 datasets from toxicokinetic (TK) or pharmacokinetic (PK) studies which contained over 23,000 replicate pairs composed of standards, quality control (QC), and animal samples measured with 23 different bioanalytical assays. The comparison was first done with standard curve and QCs followed by PK parameters (i.e., Cmax and AUC). Statistical analyses were performed on combined duplicate versus singlets using a concordance correlation coefficient (CCC), a measurement used to assess agreement. Variance component analyses were conducted on PK estimates to assess the relative analytical and biological variability. Overall, 97.5% of replicate pairs had a %CV of <11% and 50% of the results had a %CV of ≤1.38%. There was no observable bias in concentration comparing the first replicate with the second (CCC of 0.99746 and accuracy value of 1). The comparison of AUC and Cmax showed no observable difference between singlet and duplicate (CCC for AUC and Cmax >0.99999). Analysis of variance indicated an AUC inter-subject variability 35.3-fold greater than replicate variability and 8.5-fold greater for Cmax. Running replicates from the same sample will not significantly reduce variation or change PK parameters. These analyses indicated the majority of variance was inter-subject and supported the use of a singlet strategy.
Assuntos
Bases de Dados Factuais , Estudos de Viabilidade , Ligantes , Preparações Farmacêuticas/metabolismo , Estatística como Assunto/métodos , Animais , Haplorrinos , Camundongos , Preparações Farmacêuticas/análise , Ligação Proteica/fisiologia , Ratos , Estudos RetrospectivosRESUMO
Recombinant human serum albumin (HSA) conjugates of a 15-amino-acid truncated peptide YY (PYY) analogue were prepared using three heterobifunctional linkers [succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (MHS), and N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (GMBS)] in 2 synthetic steps involving (1) reaction of succinimidyl ester on linker with ε-amine of Lys2 on the peptide and (2) reaction of maleimide on peptide linker with free thiol of Cysteine 34 (Cys34) on albumin. In-process controls using ESI LC-MS were used to follow reactions and identify reaction products. Proteolytic digests of the conjugate revealed that peptide conjugation occurs at Cys34 on HSA. Conjugates were assayed in cell-based assays to determine potency at the human Y2-receptor, and selectivity at the human Y1-, Y4-, and Y5-receptors using a calcium flux assay. All three conjugates assayed were selective agonists of the Y2-receptor, and displayed nanomolar potencies. MCC and MH conjugates were selected for acute PK/PD studies in DIO mice. Significant reduction in food intake was observed with the MH conjugate, which lasted for 24 h at the 10 mg (or 4 µmol)/kg dose. While the MCC conjugate exhibited greater potency in vitro, it was slightly less effective than the MH conjugate in vivo with respect to reduction in food intake. Both conjugates were significantly less active than the peptide coupled to a 30 kDa PEG. The observed T1/2 (8-9 h) for both conjugates was significantly lower than that observed for the PEGylated peptide (â¼25 h). These results suggest that, as compared with the unmodified and PEGylated peptide, the extended circulation half-life of albumin conjugates is mediated through uptake and recirculation by FcRn, and allometric scaling methods are necessary to account for interspecies variation in pharmacokinetic properties.