GLP-1 receptor

PF-06882961 可促进表达人类和猴子 GLP-1R 的 CHO 细胞中 cAMP 的积累，且 EC50 值相当。另一方面，PF-06882961 不会提高表达 GLP-1R 的小鼠、大鼠或兔细胞中的 cAMP 水平。 [2]

PF-06882961 可减少猴子的食物摄入量并增加胰岛素释放以响应葡萄糖刺激。[2]

Danuglipron口服生物可利用，可有效降低猴子的葡萄糖水平和食物摄入量。 静脉注射后，Danuglipron在大鼠（CLp=57.3 mL/min/kg）和猴子[CLp=13.8 mL/min/kg]中表现出中等至高的血浆清除率（CLp）值，大鼠和猴子的消除半衰期分别为1.1和1.9小时。三羟甲基纤维素盐形式的达那格列普仑在动物体内的口服生物利用度较低至中等，并以剂量依赖的方式增加，这足以研究在溶解在2%吐温80的标准0.5%甲基纤维素制剂中、口服灌胃递送的Danuglipron的临床前体内疗效和安全性。[3]

由于Danuglipron不会激活啮齿动物GLP-1R，因此在食蟹猴静脉注射和口服后，在静脉葡萄糖耐量试验（IVGTT）中检查了达那格列普仑对胰岛素和葡萄糖的治疗作用。使用猴子药代动力学（PK）研究的全身浓度预测输注速率和口服剂量，这些研究是实现受体占用率（RO）所必需的，包括根据利拉鲁肽临床有效剂量（1.8mg每日一次）报告的人体血浆暴露量估算的RO。在IVGTT期间静脉注射达那格列普仑导致胰岛素分泌率和葡萄糖消失率（K值）增加（图88B-D）。Danuglipron增强葡萄糖刺激的胰岛素分泌具有浓度依赖性，在口服给药和静脉输注途径后具有可比性（图88E）。与赋形剂处理的猴子相比，每天皮下注射达那格列普仑一次持续2天也抑制了食物摄入（图88F）。选择皮下给药途径是为了减少口服给药时注意到的全身暴露的可变性，比静脉注射更方便。

生物分析。Min6 Ca2+动员测定[3]
将MIN6-c4细胞以每孔5×104个细胞的密度接种到黑色96孔板中，并在37°C和5%CO2下培养20-24小时。对于细胞负载，吸取培养上清液，并根据制造商的说明将100μL的测定缓冲液（Krebs–Ringer缓冲液，10mM HEPES，0.1%BSA，2.5mM葡萄糖）和等体积的钙6染料（FLIPR钙6测定试剂盒，Molecular Devices，R8191）溶解在同一缓冲液中，添加到每个孔中。将细胞在37°C/5%CO2下孵育70分钟，并在黑暗中在室温下再平衡10分钟。为了评估受试化合物对葡萄糖介导的细胞内Ca2+增加的影响，在FLIPR Tetra仪器（分子装置）上检测期间，每个孔添加50μL的含有75 mM葡萄糖（最终浓度为15 mM葡萄糖）和受试化合物或DMSO的测定缓冲液。对于低血糖对照，添加50μL不含额外葡萄糖的饥饿缓冲液，以将最终葡萄糖浓度保持在2.5 mM。通过计算3 s至372 s的荧光读数曲线下的面积来量化钙通量。

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稳定表达人GLP-1R的PSC-HEK293细胞系中的cAMP刺激测定[3]
使用解冻和使用冷冻细胞在1536孔板中进行GLP-1R激动剂和阳性变构调节剂的体外细胞测定。使用前，将冷冻细胞在37°C下快速解冻，并用20mL细胞缓冲液（1×HBSS；20mM HEPES，0.1%BSA）洗涤（在900rpm下5分钟）。将细胞重悬于测定缓冲液（细胞缓冲液加2mM IBMX）中，并将其调节至100万个细胞/mL的细胞密度。向1536孔微量滴定板中加入2μL细胞（最终2000个细胞/孔）和2μL化合物，用于激动剂测定。对于PAM测定，应用了两种测定形式，即（a）用1μL不同剂量的化合物和1μL固定浓度（EC20）的GLP1（9–36）NH2进行增强子测定，以及（b）用1µL不同剂量GLP1（9-16）NH2和1μL 10μM和3μM化合物进行移位测定。将含有2μL每个细胞和化合物的混合物在室温下孵育30分钟
使用来自Cisbio Corp.的试剂盒（目录号62AM4PEC）基于HTRF（均匀时间分辨荧光）测定细胞的cAMP含量。在加入在裂解缓冲液（试剂盒组分）中稀释的HTRF试剂后，将板温育1小时，然后测量665/620nm的荧光比。使用内部软件计算剂量-反应结果Biost@t-Speed2.0版HTS，使用四参数逻辑模型

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人胰腺β细胞系1.1B4中的cAMP刺激测定[3]
使用人胰腺β细胞系1.1B4进行GLP-1（7-36）NH2、GLP1（9-36）NH2和测试化合物的体外细胞测定。在GLP-1R激活后，1.1B4细胞积聚细胞内的环磷酸腺苷（cAMP）。使用具有HTRF读数的商业免疫测定技术来测量环AMP的形成。在这些实验中，定量cAMP所需的所有试剂在试剂盒（目录号62AM4PEC，来自Cisbio Corp.，France）中提供，并根据供应商提供的方案应用。应用了两种测定形式，即（a）具有化合物浓度-反应曲线和固定浓度为10 nM的GLP1（9–36）NH2的增强子测定和（b）具有GLP1（9－36）NH2浓度-响应曲线和固定剂量为1μM的化合物的转移测定。20 000个细胞接种到96孔微量滴定板中。过夜培养后，将细胞洗涤两次，并将连续稀释的GLP-1R配体或试验化合物（含或不含各自固定浓度的试验化合物或GLP1（9–36）NH2）转移到细胞中。在与测试试剂孵育30分钟后，根据制造商的描述裂解细胞并制备用于cAMP测定。通过在665和620nm处的荧光测量、665/620nm比率的计算以及相对于阴性（0%）和阳性（100%）对照的效果百分比的表达来获得数据点。阴性对照为测定缓冲液（1×HBSS，0.1%BSA，1 mM IBMX），阳性对照为GLP-1（7-36）NH2。浓度-响应结果用内部软件计算Biost@t-Speed2.0版本LTS，使用四参数逻辑模型。使用SAS版本9.1.3中的Marquardt算法通过非线性回归获得调整。

稳定表达与绿色荧光蛋白 (GFP) 融合的 hGLP-1R（400,000 个细胞/孔）的 HEK293 细胞在 6 孔板上生长一整天，然后用 PF-06882961 刺激半分钟。在这些研究中，使用的激动剂浓度为 1 μM，已被证明可引起最大内化。为了测试内吞过程的可逆性，将细胞置于特定的孔中，用含有0.1% BSA的PBS冲洗3次，然后在37°C下再孵育2小时。用4％多聚甲醛在室温下固定细胞15分钟后，用含有0.1％BSA的PBS清洗细胞3次。

male cynomolgus monkeys
1 mg/kg, 5 mg/kg, 100 mg/kg
IV, Oral gavage

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Animal Pharmacokinetic Studies[3]
Jugular vein/carotid artery doubly cannulated male Wistar-Han rats (∼250 g), and male cynomolgus monkeys (∼7 kg) were used for these studies. Animals were fasted overnight and through the duration of the study (1.0 or 2.0 h), whereas access to water was provided ad libitum. Danuglipron was administered by slow iv bolus as a solution (1 mg/mL) in a 5:95 (v/v) polyethylene glycol 400/12% (w/v) sulfobutyl-β-cyclodextrin in water mixture or a 10:50:40 (v/v/v) DMSO/polyethylene glycol 400/water mixture via the tail vein in rats (n = 4) or the femoral vein in monkeys (n = 2) at a dose of 1.0 mg/kg in a dosing volume of 1 mL/kg. Serial blood samples were collected before dosing and 0.083, 0.25, 0.5, 1.0, 2.0, 4.0, 7.0, and 24 h after dose administration. The crystalline 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) salt form of Danuglipron was also administered by oral gavage to rats (5 and 100 mg/kg at 10 mL/kg) and monkeys [5 mg/kg (5.0 mL/kg) and 100 mg/kg (10 mL/kg)] as a homogeneous suspension in a 2:98 (v/v) Tween 80/0.5% (w/v) methylcellulose A4M in distilled water mixutre. Blood samples were taken prior to po administration, and then serial samples were collected 0.083, 0.25, 0.5, 1, 2, 4, 7, and 24 h after dosing. Blood samples from the pharmacokinetic studies were centrifuged to generate plasma. All plasma samples were kept frozen until analysis. For rat and monkey samples, aliquots of plasma (20–50 μL) were transferred to 96-well blocks, and acetonitrile (150–200 μL) containing verapamil (monkeys) or terfenadine (rat) as internal standard was added to each well. The supernatant was dried under nitrogen and reconstituted with 100 μL of water without evaporation. Following extraction, the samples were then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), and concentrations of Danuglipron in plasma were determined by interpolation from a standard curve as described in the Supporting Information.

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Determination of Preclinical Pharmacokinetic Parameters[3]
Pharmacokinetic parameters in animals were determined using noncompartmental analysis. Maximum plasma concentrations (Cmax) of Danuglipron in plasma after po dosing in rats and monkeys were determined directly from the experimental data, with Tmax defined as the time of first occurrence of Cmax. The area under the plasma concentration–time curve from time zero to infinity (AUC0–∞) was estimated using the linear trapezoidal rule. Systemic plasma clearance (CLp) was calculated as the intravenous dose divided by AUC0–∞iv. The terminal rate constant (kel) was calculated by a linear regression of the log-linear concentration–time curve, and the terminal elimination t1/2 was calculated as 0.693/kel. The apparent steady-state distribution volume (Vdss) in animals was determined as the iv or po dose divided by the product of AUC0–∞ and kel. The absolute bioavailability (F) of the po doses in animals was calculated using the equation F = AUC0–∞po/AUC0–∞iv × doseiv/dosepo. Detailed protocols used in animal pharmacology studies are provided in the Supplementary Methods.

[1]. Endocrinol Metab (Seoul). 2021 Feb;36(1):22-29

[2]. bioRxiv. 2020 Sep 30.

[3]. J Med Chem . 2022 Jun 23;65(12):8208-8226.

[4]. J Med Chem. 2020 Mar 12;63(5):2292-2307.

Glucagon-like peptide-1 (GLP-1) receptor agonists are efficacious glucose-lowering medications with salient benefits for body weight and cardiovascular events. This class of medications is now recommended as the top priority for patients with established cardiovascular disease or indicators of high risk. Until the advent of oral semaglutide, however, GLP-1 receptor agonists were available only in the form of subcutaneous injections. Aversion to needles, discomfort with self-injection, or skin problems at the injection site are commonly voiced problems in people with diabetes, and thus, attempts for non-invasive delivery strategies have continued. Herein, we review the evolution of GLP-1 therapy from its discovery and the development of currently approved drugs to the unprecedented endeavor to administer GLP-1 receptor agonists via the oral route. We focus on the pharmacokinetic and pharmacodynamic properties of the recently approved oral GLP-1 receptor agonist, oral semaglutide. Small molecule oral GLP-1 receptor agonists are currently in development, and we introduce how these chemicals have addressed the challenge posed by interactions with the large extracellular ligand binding domain of the GLP-1 receptor. We specifically discuss the structure and pharmacological properties of TT-OAD2, LY3502970, and PF-06882961, and envision an era where more patients could benefit from oral GLP-1 receptor agonist therapy.[1]

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Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) have revolutionized diabetes therapy, but their use has been limited because they require injection. Herein, we describe the discovery of the orally bioavailable, small-molecule, GLP-1R agonist PF-06882961 (danuglipron). A sensitized high-throughput screen was used to identify 5-fluoropyrimidine-based GLP-1R agonists that were optimized to promote endogenous GLP-1R signaling with nanomolar potency. Incorporation of a carboxylic acid moiety provided considerable GLP-1R potency gains with improved off-target pharmacology and reduced metabolic clearance, ultimately resulting in the identification of danuglipron. Danuglipron increased insulin levels in primates but not rodents, which was explained by receptor mutagensis studies and a cryogenic electron microscope structure that revealed a binding pocket requiring a primate-specific tryptophan 33 residue. Oral administration of danuglipron to healthy humans produced dose-proportional increases in systemic exposure (NCT03309241). This opens an opportunity for oral small-molecule therapies that target the well-validated GLP-1R for metabolic health.[3]

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The therapeutic success of peptidic GLP-1 receptor agonists for treatment of type 2 diabetes mellitus (T2DM) motivated our search for orally bioavailable small molecules that can activate the GLP-1 receptor (GLP-1R) as a well-validated target for T2DM. Here, the discovery and characterization of a potent and selective positive allosteric modulator (PAM) for GLP-1R based on a 3,4,5,6-tetrahydro-1H-1,5-epiminoazocino[4,5-b]indole scaffold is reported. Optimization of this series from HTS was supported by a GLP-1R ligand binding model. Biological in vitro testing revealed favorable ADME and pharmacological profiles for the best compound 19. Characterization by in vivo pharmacokinetic and pharmacological studies demonstrated that 19 activates GLP-1R as positive allosteric modulator (PAM) in the presence of the much less active endogenous degradation product GLP1(9-36)NH2 of the potent endogenous ligand GLP-1(7-36)NH2. While these data suggest the potential of small molecule GLP-1R PAMs for T2DM treatment, further optimization is still required towards a clinical candidate.[4]

C31H30FN5O4

555.599410533905

555.23

C, 67.01; H, 5.44; F, 3.42; N, 12.61; O, 11.52

2230198-02-2

2230198-02-2 (free acid); 2230198-03-3 (tris)

Off-white to light yellow solid powder

1.4

114Ų

C1CO[C@@H]1CN2C3=C(C=CC(=C3)C(=O)O)N=C2CN4CCC(CC4)C5=NC(=CC=C5)OCC6=C(C=C(C=C6)C#N)F

HYBAKUMPISVZQP-DEOSSOPVSA-N

InChI=1S/C31H30FN5O4/c32-25-14-20(16-33)4-5-23(25)19-41-30-3-1-2-26(35-30)21-8-11-36(12-9-21)18-29-34-27-7-6-22(31(38)39)15-28(27)37(29)17-24-10-13-40-24/h1-7,14-15,21,24H,8-13,17-19H2,(H,38,39)/t24-/m0/s1

2-[[4-[6-[(4-cyano-2-fluorophenyl)methoxy]pyridin-2-yl]piperidin-1-yl]methyl]-3-[[(2S)-oxetan-2-yl]methyl]benzimidazole-5-carboxylic acid

PF06882961; Danuglipron; PF 06882961; PF-06882961

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Ethanol: ~100 mg/mL
DMSO: ~10 mg/mL (~18 mM)