RESUMO
PURPOSE: A same-day PET imaging agent capable of measuring PD-L1 status in tumors is an important tool for optimizing PD-1 and PD-L1 treatments. Herein we describe the discovery and evaluation of a novel, fluorine-18 labeled macrocyclic peptide-based PET ligand for imaging PD-L1. METHODS: [18F]BMS-986229 was synthesized via copper mediated click-chemistry to yield a PD-L1 PET ligand with picomolar affinity and was tested as an in-vivo tool for assessing PD-L1 expression. RESULTS: Autoradiography showed an 8:1 binding ratio in L2987 (PD-L1 (+)) vs. HT-29 (PD-L1 (-)) tumor tissues, with >90% specific binding. Specific radioligand binding (>90%) was observed in human non-small-cell lung cancer (NSCLC) and cynomolgus monkey spleen tissues. Images of PD-L1 (+) tissues in primates were characterized by high signal-to-noise, with low background signal in non-expressing tissues. PET imaging enabled clear visualization of PD-L1 expression in a murine model in vivo, with 5-fold higher uptake in L2987 (PD-L1 (+)) than in control HT-29 (PD-L1 (-)) tumors. Moreover, this imaging agent was used to measure target engagement of PD-L1 inhibitors (peptide or mAb), in PD-L1 (+) tumors as high as 97%. CONCLUSION: A novel 18F-labeled macrocyclic peptide radioligand was developed for PET imaging of PD-L1 expressing tissues that demonstrated several advantages within a nonhuman primate model when compared directly to adnectin- or mAb-based ligands. Clinical studies are currently evaluating [18F]BMS-986229 to measure PD-L1 expression in tumors.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Domínio de Fibronectina Tipo III , Radioisótopos de Flúor , Neoplasias Pulmonares , Proteínas Recombinantes , Humanos , Camundongos , Animais , Antígeno B7-H1/metabolismo , Ligantes , Macaca fascicularis/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Peptídeos/químicaRESUMO
PURPOSE: In cancer immunotherapy, the blockade of the interaction between programmed death-1 and its ligand (PD-1:PD-L1) has proven to be one of the most promising strategies. However, as mechanisms of resistance to PD-1/PD-L1 inhibition include variability in tumor cell PD-L1 expression in addition to standard tumor biopsy PD-L1 immunohistochemistry (IHC), a comprehensive and quantitative approach for measuring PD-L1 expression is required. Herein, we report the development and characterization of an 18F-PD-L1-binding macrocyclic peptide as a PET tracer for the comprehensive evaluation of tumor PD-L1 expression in cancer patients. PROCEDURES: 18F-BMS-986229 was characterized for PD-L1 expression assessment by autoradiography or PET imaging. 18F-BMS-986229 was utilized to evaluate tumor PD-L1 target engagement in competition with a macrocyclic peptide inhibitor of PD-L1 (BMS-986189) over a range of doses using PET imaging. A whole-body radiation dosimetry study of 18F-BMS-986229 in healthy non-human primates (NHPs) was performed. RESULTS: In vitro autoradiography showed an 8:1 binding ratio in L2987(PD-L1 +) vs. HT-29 (PD-L1-) tumors, more than 90% of which could be blocked with 1 nM of BMS-986189. Ex vivo autoradiography showed that 18F-BMS-986229 detection was penetrant over a series of sections spanning the entire L2987 tumor. In vivo PET imaging in mice demonstrated a 5:1 tracer uptake ratio (at 90-100 min after tracer administration) in L2987 vs. HT-29 tumors and demonstrated 83%-93% specific binding of BMS-986189 within those dose ranges. In a healthy NHP dosimetry study, the resultant whole-body effective dose was 0.025 mSv/MBq. CONCLUSION: 18F-BMS-986229 has been preclinically characterized and exhibits high target specificity, low background uptake, and a short blood half-life supportive of same day imaging in the clinic. As the PET tracer, 18F-BMS-986229 shows promise in the quantification of PD-L1 expression, and its use in monitoring longitudinal changes in patients may provide insights into PD-1:PD-L1 immuno-therapy treatment outcomes.
Assuntos
Antígeno B7-H1 , Neoplasias , Humanos , Animais , Camundongos , Antígeno B7-H1/metabolismo , Receptor de Morte Celular Programada 1 , Tomografia por Emissão de Pósitrons/métodos , Radiometria , PeptídeosRESUMO
Ibrutinib is a first-generation Bruton's tyrosine kinase (BTK) inhibitor that has shown efficacy in autoimmune diseases and has consequently been developed as a positron emission tomography (PET) radiotracer. Herein, we report the automated radiosynthesis of [11C]ibrutinib through 11C-carbonylation of the acrylamide functional group, by reaction of the secondary amine precursor with [11C]CO, iodoethylene, and palladium-NiXantphos. [11C]Ibrutinib was reliably formulated in radiochemical yields of 5.4% ± 2.5% (non-decay corrected; n = 9, relative to starting [11C]CO2), radiochemical purity >99%, and molar activity of 58.8 ± 30.8 GBq/µmol (1.55 ± 0.83 Ci/µmol). Preliminary PET/magnetic resonance imaging with [11C]ibrutinib in experimental autoimmune encephalomyelitis (EAE) mice showed a 49% higher radioactivity accumulation in the spinal cord of mice with EAE scores of 2.5 vs. sham mice.
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Positron emission tomography (PET) is an important non-invasive tool to help guide the drug discovery and development process. Positron-emitting-radiolabeled drug candidates represent an important tool for drug hunters to gain insight into a drug's biodistribution and target engagement of exploratory biologic targets of interest. Recently, there have been several drug candidates that incorporate an acryloyl functional group due to their ability to form a covalent bond within the biological target of interest through Michael addition. Methods to incorporate a carbon-11 radionuclide into acrylamide derivatives remain challenging given the reactive nature of this moiety. Herein, we report the improved radiosynthesis of carbon-11-containing acrylamide drug candidates, [11C]ibrutinib, [11C]tolebrutinib, and [11C]evobrutinib, using [11C]CO and a novel "in-loop" 11 C-carbonylation reaction. [11C]Ibrutinib, [11C]tolebrutinib, and [11C]evobrutinib were reliably synthesized, generating 2.2-7.1 GBq of these radiopharmaceuticals in radiochemical yields ranging from 3.3 to 12.8% (non-decay corrected; relative to starting [11C]CO2) and molar activities of 281-500 GBq/µmol (7.5-13.5 Ci/µmol), respectively. This study highlights an improved method for incorporating carbon-11 into acrylamide drug candidates using [11C]CO within an HPLC loop suitable for clinical translation using simple modifications of standard automated synthesis modules used for cGMP manufacture of PET radioligands.
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The programmed death protein (PD-1) and its ligand (PD-L1) play critical roles in a checkpoint pathway cancer cells exploit to evade the immune system. A same-day PET imaging agent for measuring PD-L1 status in primary and metastatic lesions could be important for optimizing drug therapy. Herein, we have evaluated the tumor targeting of an anti-PD-L1 adnectin after 18F-fluorine labeling. Methods: An anti-PD-L1 adnectin was labeled with 18F in 2 steps. This synthesis featured fluorination of a novel prosthetic group, followed by a copper-free click conjugation to a modified adnectin to generate 18F-BMS-986192. 18F-BMS-986192 was evaluated in tumors using in vitro autoradiography and PET with mice bearing bilateral PD-L1-negative (PD-L1(-)) and PD-L1-positive (PD-L1(+)) subcutaneous tumors. 18F-BMS-986192 was evaluated for distribution, binding, and radiation dosimetry in a healthy cynomolgus monkey. Results:18F-BMS-986192 bound to human and cynomolgus PD-L1 with a dissociation constant of less than 35 pM, as measured by surface plasmon resonance. This adnectin was labeled with 18F to yield a PET radioligand for assessing PD-L1 expression in vivo. 18F-BMS-986192 bound to tumor tissues as a function of PD-L1 expression determined by immunohistochemistry. Radioligand binding was blocked in a dose-dependent manner. In vivo PET imaging clearly visualized PD-L1 expression in mice implanted with PD-L1(+), L2987 xenograft tumors. Two hours after dosing, a 3.5-fold-higher uptake (2.41 ± 0.29 vs. 0.82 ± 0.11 percentage injected dose per gram, P < 0.0001) was observed in L2987 than in control HT-29 (PD-L1(-)) tumors. Coadministration of 3 mg/kg ADX_5322_A02 anti-PD-L1 adnectin reduced tumor uptake at 2 h after injection by approximately 70%, whereas HT-29 uptake remained unchanged, demonstrating PD-L1-specific binding. Biodistribution in a nonhuman primate showed binding in the PD-L1-rich spleen, with rapid blood clearance through the kidneys and bladder. Binding in the PD-L1(+) spleen was reduced by coadministration of BMS-986192. Dosimetry estimates indicate that the kidney is the dose-limiting organ, with an estimated human absorbed dose of 2.20E-01 mSv/MBq. Conclusion:18F-BMS-986192 demonstrated the feasibility of noninvasively imaging the PD-L1 status of tumors by small-animal PET studies. Clinical studies with 18F-BMS-986192 are under way to measure PD-L1 expression in human tumors.
Assuntos
Antígeno B7-H1/metabolismo , Radioisótopos de Flúor , Neoplasias/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/síntese química , Animais , Feminino , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Marcação por Isótopo , Ligantes , Macaca fascicularis , Camundongos , Compostos Radiofarmacêuticos/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
Discovery of potent renin inhibitors which contain a simplified alkylamino Asp-binding group and exhibit improved selectivity for renin over Cyp3A4 is described. Structure-function results in this series are rationalized based on analysis of selected compounds bound to renin, and the contribution of each molecular feature leading to the reduced P450 inhibition is quantified.
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Ácido Aspártico/metabolismo , Citocromo P-450 CYP3A/metabolismo , Inibidores de Proteases/química , Renina/antagonistas & inibidores , Ácido Aspártico/química , Sítios de Ligação , Cristalografia por Raios X , Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Simulação de Dinâmica Molecular , Inibidores de Proteases/metabolismo , Ligação Proteica , Renina/metabolismo , Relação Estrutura-AtividadeRESUMO
The series of 4-(benzylaminomethylene)isoquinoline-1,3-(2H,4H)-dione and 4-[(pyridylmethyl)aminomethylene]isoquinoline-1,3-(2H,4H)-dione derivatives reported here represents a novel class of potential antitumor agents, which potently and selectively inhibit CDK4 over CDK2 and CDK1. In the benzylamino headpiece, a 3-OH substituent is required on the phenyl ring for CDK4 inhibitory activity, which is further enhanced when an iodo, aryl, heteroaryl, t-butyl, or cyclopentyl substituent is introduced at the C-6 position of the isoquinoline-1,3-dione core. To circumvent the metabolic liability associated with the phenolic OH group on the 4-substituted 3-OH phenyl headpiece, we take two approaches: first, introduce a nitrogen o- or p- to the 3-OH group in the phenyl ring; second, replace the phenyl headpiece with N-substituted 2-pyridones. We present here the synthesis, SAR data, metabolic stability data, and a CDK4 mimic model that explains the binding, potency, and selectivity of our CDK4 selective inhibitors.
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Antineoplásicos/síntese química , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Isoquinolinas/síntese química , Piridinas/síntese química , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligação de Hidrogênio , Técnicas In Vitro , Isoquinolinas/química , Isoquinolinas/farmacologia , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Fosforilação , Piridinas/química , Piridinas/farmacologia , Ratos , Proteína do Retinoblastoma/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The cyclin-dependent kinases (CDKs), as complexes with their respective partners, the cyclins, are critical regulators of cell cycle progression. Because aberrant regulations of CDK4/cyclin D1 lead to uncontrolled cell proliferation, a hallmark of cancer, small-molecule inhibitors of CDK4/cyclin D1 are attractive as prospective antitumor agents. The series of 4-(phenylaminomethylene)isoquinoline-1,3(2H,4H)-dione derivatives reported here represents a novel class of potent inhibitors that selectively inhibit CDK4 over CDK2 and CDK1 activities. In the headpiece of the 4-(phenylaminomethylene)isoquinoline-1,3(2H,4H)-dione, a basic amine substituent is required on the aniline ring for the CDK4 inhibitory activity. The inhibitory activity is further enhanced when an aryl or heteroaryl substituent is introduced at the C-6 position of the isoquinoline-1,3(2H,4H)-dione core. We present here SAR data and a CDK4 mimic model that explains the binding, potency, and selectivity of our CDK4 selective inhibitors.