H+/K+-ATPase (IC50 = 0.29~0.52 μM)

替格拉赞抑制猪、狗和人的 H+/K+-ATP 酶活性。 Tegoprazan 以可逆且钾竞争性的方式抑制 pH+/K+-ATP 酶。当替戈拉赞剂量高达 0.15 μM 时，86% 的 H+/K+-ATP 酶活性被 3 μM 的替戈拉赞抑制 [1]。

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Tegolazan[（S）-4-（（5,7-二氟色满-4-基）氧基）-N，N，2-三甲基-1H-苯并[d]咪唑-6-甲酰胺]是一种钾竞争性酸阻断剂（P-CAB），是一种新型强效、高选择性的胃H+/K+-ATP酶抑制剂。Tegolazan在体外抑制猪、犬和人的H+/K+-ATP酶，IC50值范围为0.29至0.52μM，而犬肾Na+/K+-APTP酶的IC50值超过100μM。动力学分析显示，替戈拉赞以钾竞争方式抑制H+/K+-ATP酶，这种结合是可逆的[3]。

在狗中，egoprazan（1.0 mg/kg，口服）可有效防止组胺引起的胃酸化。五肽胃泌素诱导酸化后，胃内 pH 值恢复到中性范围，剂量为 1.0–3.0 mg/kg，口服。在给予五肽胃泌素的狗中，依戈拉赞（3 mg/kg，面部）引起迁移性运动复合体立即胃 III 期收缩 [1]。

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炎症性肠病（IBD）是一种慢性免疫介导的疾病，其特征是胃肠道炎症持续时间延长。IBD可能是由肠道屏障功能障碍、肠道微生物群改变以及环境因素在遗传易感个体中诱导的肠道免疫异常引起的。质子泵抑制剂（PPI），如雷贝拉唑，经常用于抑制胃酸。然而，长期服用PPI会改变肠道微生物组的组成，可能会加重IBD的严重程度。本研究表明，替戈拉赞是一种钾竞争性酸阻断剂，可显著改善小鼠结肠炎，增强肠上皮屏障功能。Tegolazan缓解了肠道微生物群失调，促进了普通拟杆菌的生长。反过来，B.vulgatus通过抑制致病菌的上皮粘附来缓解肠道炎症。与雷贝拉唑不同，替戈拉赞不会引起肠道生态失调。我们的研究结果为替戈拉赞作为IBD的肠道保护剂和胃酸相关疾病的治疗剂的潜在作用提供了新的见解。[1]

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Tegolazan是一种新型的钾竞争性酸阻断剂（P-CAB），是下一代治疗药物，用于治疗胃食管反流病（GERD）和消化性溃疡等与酸相关的胃肠道疾病。在本研究中，我们比较了替戈拉赞与埃索美拉唑（一种代表性的质子泵抑制剂）的体外和体内药理学特性。使用从猪分离的含有胃H+/K+-ATP酶的离子泄漏囊泡进行体外酶测定。在胃食管反流病和消化性溃疡大鼠模型中评估了替戈拉赞的体内疗效。Tegolazan以可逆的方式抑制猪H+/K+-ATP酶的活性，IC50值为0.53μM，而埃索美拉唑显示出微弱且不可逆的抑制作用，IC50值是42.52μM。在GERD模型中，替戈拉赞在抑制食管损伤和胃酸分泌方面表现出剂量依赖性疗效，ED50为2.0mg/kg，比埃索美拉唑强15倍。在消化性溃疡模型中，与埃索美拉唑相比，替戈拉赞表现出更优的抗溃疡活性。在萘普生、乙醇和水浸泡束缚应激诱导的消化性溃疡模型中，替戈拉赞的ED50分别为0.1、1.4和0.1mg/kg。在醋酸诱导的消化性溃疡模型中，重复口服5天后，10mg/kg的替戈拉赞的治愈率高于30mg/kg的埃索美拉唑（分别为44.2%和32.7%）。因此，替戈拉赞是一种新型的P-CAB，对胃H+/K+-ATP酶具有强效和可逆的抑制作用，与之前的质子泵抑制剂相比，可能具有更强的疗效。[2]

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狗单次口服0.3至30mg/kg的替戈拉赞能很好地被吸收到血液中，并在胃组织/液体中的分布高于血浆。替戈拉赞能有效抑制组胺诱导的狗胃酸分泌，从给药后1小时开始，在1.0mg/kg的剂量下观察到完全抑制。此外，口服1和3mg/kg的替戈拉赞可将五肽胃泌素诱导的酸化胃pH值逆转至中性范围。有趣的是，在接受五肽胃泌素治疗的狗中，3 mg/kg的替戈拉赞立即诱发了迁移运动复合物的胃III期收缩，其他P-CAB，伏诺普拉赞也观察到了类似的效果。Tegolazan是一种新型的P-CAB，可能为胃酸相关和运动障碍疾病的治疗提供新的选择。[3]

H+/K+-ATP酶抑制的动力学分析。[2]
酶动力学研究基于之前描述的猪离子泄漏测定法进行，但载体量（1µg）除外，并用不同浓度的钾（终浓度为2.0、2.5、3.5、5.0和10 mM KCl）和替戈拉赞（终浓度分别为0.15、0.30、0.45和0.60µM）进行了测试。

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犬肾脏Na+/K+-ATP酶活性的测定。[2]
用250mM蔗糖溶液将犬肾Na+/K+-ATP酶稀释至7mg/ml。在96孔聚苯乙烯板中的60µl酶反应混合物中测量犬肾Na+/K+-ATP酶活性，该混合物含有11µg蛋白质、试验化合物、100 mM NaCl、2 mM KCl、3 mM MgSO4、3 mmol Na2ATP和40 mM Tris（37°C下pH 7.4）。对于0%和100%抑制对照，酶反应分别在1%二甲亚砜和100µM哇巴因存在下进行。通过加入Na2ATP开始反应，将混合物在37°C下孵育30分钟，然后加入30µl含10%SDS的消泡剂A以停止反应。采用与猪试验相同的方法进行比色分析。

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对药理学相关分子的结合/功能的抑制。[2]
在10µM替戈拉赞存在的情况下，测试了对药理学相关分子、受体、离子通道、转运蛋白和酶的结合或功能活性的抑制作用。关于Na+/K+-ATP酶，在30µM下测试了替戈拉赞。所有检测均由Eurofins Cerep在标准抑制剂作为检测对照的情况下，根据其验证的检测方法进行(http://www.cerep.fr/cerep/users/pages/catalog/assay/catalog.asp).

细胞培养和治疗[1]
人结肠癌细胞系HT-29 和Caco-2[HTB-37™，美国典型培养物保藏中心（ATCC）]在含有10%热灭活胎牛血清（FBS）和1%青霉素-链霉素溶液的RPMI 1640中，在含5%CO2的加湿培养箱中保持在37°C。Caco-2细胞在含有10%FBS和1%青霉素-链霉素溶液的Dulbecco改良Eagle培养基中培养。在显微镜下通过台盼蓝染色评估细胞存活率。Caco-2细胞用于评估肠屏障功能，用40 ng/mL肿瘤坏死因子（TNF）-α处理48小时可诱导屏障损伤。

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体外肠通透性测定[1]
接下来，为了评估肠上皮屏障功能，将Caco-2细胞植入Transwell系统的上腔（0.4μm孔）。通过分析上皮电阻（TEER）和FITC-葡聚糖（FD4）的细胞旁通量来评估上皮通透性。使用Millicell ERS仪器评估在Transwell室中培养的Caco-2细胞单层的电阻。将FITC-葡聚糖以1mg/ml的终浓度加入上室。加入FITC-葡聚糖两小时后，收集下室的培养基，并使用荧光微孔板读数器测量荧光强度。

Measurement of Plasma and Gastric Juice Concentration of tegoprazan in Dogs. [3]
Male beagle dogs at 8–11 months old were fasted overnight prior to dosing and dosed tegoprazan with 0.3, 3, and 30 mg/kg by mouth (n = 2) and blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, and 24 hours post dosing. Male Heidenhain pouch (HP) dogs were orally dosed tegoprazan with 1 or 3 mg/kg, and blood and gastric juice were collected at 5 or 16 hours after the drug dose. Plasma and gastric concentrations of tegoprazan were quantified using liquid chromatography–tandem mass spectrometry (API4000 Triple Quadrupole mass spectrometer) and calculated using Analyst Program version 1.4.1. Pharmacokinetic parameters were determined with noncompartmental analysis using WinNonlin version 5.2.1

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Measurement of Gastric Acid Secretion in the HP Dog Model. [3]
A gastric pouch was constructed according to the method introduced by Heidenhain (1879). Briefly, male beagle dogs (7–12 kg body weight) were anesthetized with midazolam (0.2 mg/kg, i.m.) and medetomidine (0.05 mg/kg, i.m.) and isoflurane inhalation. The abdominal cavity was opened after the injection of atropine sulfate (0.5 mg/ml, 1 ml, i.m.) and infusion of Lactec D (100 ml/h). After exposing the stomach in the surgical field, a portion of the greater curvature opposite the splenic hilum was converted into a pouch with an adequate blood supply from the intact gastroepiploic artery. The main body of the stomach was reconstituted, while the pouch drained into an implanted metal cannula. After closing the pouch, the cannula was brought out of the abdominal cavity through the left lateral abdominal wall. Animals were allowed to recover from surgery for at least at last 3 weeks. The experiment was started after overnight fasting with free access to water. Acid secretion was stimulated by continuous intravenous infusion of histamine (80 μg/mg per hour) and maintained throughout the experimental periods. Gastric juice samples were collected by gravity drainage every 15 minutes throughout the experiment. At 90 minutes after initiation of the histamine infusion, tegoprazan, omeprazole, or vehicle (0.5% methylcellulose) was administrated orally. In the 5-day repeated dosing study, test drug or vehicle was administered orally for 5 days and its inhibitory effect on histamine-stimulated acid secretion was examined on treatment days 1 and 5. The collected gastric juice samples were centrifuged and acidity in the supernatant was determined using an automatic titrator. Gastric acid secretion at intervals of every 15 minutes (acidity × volume of the supernatant) was expressed as milliequivalents per 15 minutes. All results were calculated by MS EXCEL software

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In Vivo Intestinal Permeability Assay [1]
Intestinal permeability was evaluated by measuring paracellular permeability to 4 kDa fluorescein isothiocyanate (FITC)-dextran on the day of sacrifice. Mice were orally administered 150 μL of 80 mg/mL FITC-dextran, and blood was collected 4 h after administration. Fluorescence intensity was measured using a fluorescent microplate reader

[1]. Novel Potassium-Competitive Acid Blocker, Tegoprazan, Protects Against Colitis by Improving Gut Barrier Function. Front Immunol. 2022 May 25;13:870817.

[2]. Effects of Tegoprazan, a Novel Potassium-Competitive Acid Blocker, on Rat Models of Gastric Acid-Related Disease. J Pharmacol Exp Ther. 2019 Jun;369(3):318-327.

[3]. Tegoprazan, a Novel Potassium-Competitive Acid Blocker to Control Gastric Acid Secretion and Motility. J Pharmacol Exp Ther. 2018 Feb;364(2):275-286.

Tegoprazan (also known as CJ-12420) is a novel therapeutic developed by CJ Healthcare Corp for treating acid-related gastrointestinal diseases. This drug is a potent and high-selective potassium-competitive acid blocker (P-CAB) with a fast onset of action and the ability to control gastric pH for a prolonged period of time. Tegoprazan’s strong and sustained effect is due to its ability to be slowly cleared from the gastric glands and exertion of effects independent of acid levels. It has also been observed to be efficacious independent of food intake.

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Mechanism of Action
Tegoprazan works as a potassium-competitive acid blocker that is potent and highly selective. Its mechanism of action is different from that of the proton-pump inhibitors as this drug does not require conversion into an active form and can directly inhibit H+/K+‐ATPase in a reversible and K+‐competitive way. This is because it is an acid-resistant weak base with the ability to remain in the highly acidic canaliculi of gastric parietal cells.

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In conclusion, tegoprazan, a novel P-CAB, exhibits stronger and more reversible gastric acid suppression than traditional PPIs. However, the effect of tegoprazan on intestinal inflammation remains unknown. Long-term PPI administration reportedly increases and exacerbates the severity of IBD. To our knowledge, this study is the first to demonstrate that tegoprazan potentially ameliorates intestinal inflammation by enhancing intestinal epithelial barrier integrity, increasing the levels of Tregs, and modulating the composition of the gut microbiota, as demonstrated by in vitro and in vivo experiments. Additionally, we determined that tegoprazan promotes the growth of a particular member of commensal bacteria, B. vulgatus, which crucially contributes to the suppression of pathogenic microorganisms. Specifically, our findings provide critical insights into the potential treatment strategies using PPIs and P-CABs in IBD, although detailed mechanisms underlying the changes described warrant further characterization. [1]

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P-CAB is a new class of drugs for acid-related gastrointestinal diseases and is positioned as the next generation of PPIs. Clinical evaluation of vonoprazan has demonstrated its potent and long-acting efficacy in patients with gastroesophageal reflux disease and peptic ulcers compared with that of the currently available PPIs. In vitro and in vivo animal pharmacology studies of tegoprazan in this study have demonstrated that tegoprazan has similar pharmacological properties to those of vonoprazan. Tegoprazan is a novel P-CAB that may provide a new option for therapy in gastric acid-related diseases as well as in gastrointestinal motility-impaired diseases in clinical use.[3]

C20H19F2N3O3

387.37997174263

387.139

C, 62.01; H, 4.94; F, 9.81; N, 10.85; O, 12.39

942195-55-3

Tegoprazan-d6

23582846

Off-white to light yellow solid powder

3.2

67.4

1

6

3

28

581

1

O([C@H]1CCOC2=CC(=CC(=C12)F)F)C1C=C(C(=O)N(C)C)C=C2N=C(NC=12)C

CLIQCDHNPDMGSL-HNNXBMFYSA-N

InChI=1S/C20H19F2N3O3/c1-10-23-14-6-11(20(26)25(2)3)7-17(19(14)24-10)28-15-4-5-27-16-9-12(21)8-13(22)18(15)16/h6-9,15H,4-5H2,1-3H3,(H,23,24)/t15-/m0/s1

7-[[(4S)-5,7-difluoro-3,4-dihydro-2H-chromen-4-yl]oxy]-N,N,2-trimethyl-3H-benzimidazole-5-carboxamide

Tegoprazan; 942195-55-3; Tegoprazan [INN]; CJ-12420; UNII-W017G7IF4S; W017G7IF4S; CJ 12420; CJ12420; RQ-00000004; RQ00000004; RQ 00000004;K-CAB; LXI-15028;

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)

DMSO : ~100 mg/mL (~258.14 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.45 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.45 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.45 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。