Tyk2 JH2 (IC50 = 0.2 nM); JAK1 JH2 (IC50 = 1 nM)

小分子JAK抑制剂已成为治疗自身免疫性疾病的主要治疗进展。发现传统上靶向该激酶家族催化活性位点的异构体选择性JAK抑制剂是一个巨大的挑战。我们实现TYK2高选择性的策略依赖于靶向TYK2假激酶（JH2）结构域。在此，我们报道了后期的优化工作，包括结构导向设计和水置换策略，从而发现了BMS-986165（11）作为高亲和力JH2配体和TYK2的强效变构抑制剂。除了前所未有的JAK同种型和kinome选择性外，11还显示出优异的药代动力学特性，具有最小的分析责任，并在几种自身免疫性疾病的小鼠模型中有效。基于这些发现，11似乎与所有其他报道的JAK抑制剂不同，并已成为临床开发中第一种以假激酶为导向的治疗方法，作为自身免疫性疾病的口服治疗[1]。
药物化合物包括碳、氢和其他元素的稳定重同位素，在药物开发过程中主要作为定量示踪剂。由于氘化可能会影响药物的药代动力学和代谢特性，因此值得关注[1]。氘代化合物的潜在好处：生物体内的半衰期更长。氘代化合物可能能够增加化合物的药代动力学特性或体内半衰期。这可以促进给药并增强化合物的安全性、有效性和耐受性。第二，提高口服生物利用度。大量未代谢的药物能够达到其作用目标，因为氘化物质减少了肝脏和肠壁中不需要的代谢（首过代谢）量。低剂量下更好的耐受性和活性取决于高生物利用度。 (3)增强新陈代谢功能。氘代物质可以增强药物代谢并减少有害或反应性代谢物的产生。 （四）增强药品安全性。氘代化学物质是无害的，可以减轻或消除药物的不良副作用。 (5) 保持治疗效果。根据早期研究，氘化分子应保持与氢类似物相当的生物效力和选择性。

Tyk2-IN-4 治疗的 NZB/W 小鼠中狼疮样疾病得到强烈抑制。 Tyk2-IN-4 是安全的且总体耐受性良好。活性药物组（75%）和安慰剂组（76%）没有出现严重不良事件，且非严重不良事件发生的频率相似。口服后，Tyk2-IN-4 被迅速吸收，表观消除半衰期为 8-15 小时[1]。

使用均相时间分辨荧光（HTRF）测定法测定所有生物化学效价和选择性，其中化合物显示与荧光探针竞争结合人重组JAK1、JAK2、JAK3和TYK2 JH1结构域蛋白以及TYK2和JAK1 JH2蛋白结构域。生成剂量-反应曲线，以确定通过非线性回归分析得出的抑制50%HTRF信号（IC50）所需的浓度。使用稳定整合的STAT依赖性萤光素酶报告基因测定法测定T细胞中的细胞潜能和选择性，使用IFNα-刺激测定TYK2/JAK1依赖性信号传导，使用IL-23刺激测定TYK2/JAK1依存性信号传导。使用GM-CSF刺激在TF-1细胞中测量JAK2依赖性信号传导。生成剂量-反应曲线，以确定通过非线性回归分析得出的抑制50%细胞反应（IC50）所需的浓度。还使用特异性细胞因子刺激在人和小鼠全血中测量了JAK依赖性信号传导的效力和选择性，并通过细胞染色和流式细胞术测量了特异性STAT蛋白的磷酸化。先前已经报道了所有测定的实验细节。对所有在生物测定中具有活性的化合物进行电子过滤，以获得泛测定干扰化合物（PAINS）常见的结构属性，并发现其为阴性[1]。

使用T细胞中稳定整合的STAT依赖性萤光素酶报告子测定法测定细胞效力和选择性，使用IFNα刺激测量TYK2/JAK1依赖性信号传导，使用IL-23刺激测量TYK2/JAK1依存性信号传导。使用GM-CSF刺激在TF-1细胞中测量JAK2依赖性信号传导。生成剂量-反应曲线，以确定通过非线性回归分析得出的抑制50%细胞反应所需的浓度（IC50）。还使用特定的细胞因子刺激在人和小鼠全血中测量了JAK依赖性信号传导的效力和选择性，并通过细胞染色和流式细胞术测量了特定STAT蛋白的磷酸化。所有测定的实验细节之前都有报道。所有在生物测定中具有活性的化合物都经过了电子过滤，以获得泛测定干扰化合物（PAINS）共有的结构属性，结果为阴性[1]。

IL-23-Induced Acanthosis in Mice
Acanthosis was induced in 6–8-week-old C57BL/6 female mice (19–20 g average weight, Jackson Laboratories) by intradermal injection of dual chain, recombinant human IL-23 into the right ear. IL-23 injections were administered every other day from day 0 through day 9 of the study. Treatment groups consisted of eight mice per group. Compound 11 at 7.5, 15, and 30 mg/kg BID in vehicle (EtOH:TPGS:PEG300, 5:5:90) and vehicle alone dosed BID by oral gavage, with the first dose given the evening before the first IL-23 injection. An anti-IL-23 adnectin (3 mg/kg) and PBS control were administered subcutaneously approximately 1 h prior to the first IL-23 injection and then twice a week thereafter. Ear thickness was measured using a Mitutoyo (no. 2412F) dial caliper and calculated as the percent change in thickness from the baseline measurement taken on day 0 before initial IL-23 injections for each animal. At the end of the study, IL-23-injected ears as well as naïve control ears were collected from four animals per group for histological examination and gene expression analyses. Terminal blood samples collected via the retro-orbital sinus were used for PK determinations. Statistical analyses were performed using Student’s t tests or ANOVA with Dunnett’s post test. At the end of the study, ears were removed and fixed in 10% neutral-buffered formalin for 24–48 h. The fixed ears were then cut longitudinally, and two pieces were parallel embedded to make the paraffin blocks. The paraffin blocks were then sectioned and placed on microscope slides for H&E staining for histological evaluation. Severity of ear inflammation was scored using an objective scoring system based on the following parameters: extent of the lesion, severity of hyperkeratosis, number and size of pustules, height of epidermal hyperplasia (acanthosis, measured in interfollicular epidermis), and the amount of inflammatory infiltrate in the dermis and soft tissue. The latter two parameters, acanthosis and inflammatory infiltrate, were scored independently on a scale from 0 to 4: 0, none; 1, minimal; 2, mild; 3, moderate; 4, marked. The histological changes were blindly evaluated by a pathologist. Statistical analyses was performed using one-way ANOVA with Dunnett’s test for comparison of each treatment versus the vehicle control.

Absorption, Distribution and Excretion
Following oral administration, deucravacitinib plasma Cmax and AUC increased proportionally over a dose range from 3 mg to 36 mg (0.5 to 6 times the approved recommended dosage) in healthy subjects. The steady state Cmax and AUC24 of deucravacitinib following administration of 6 mg once daily were 45 ng/mL and 473 ng x hr/mL, respectively. The steady state Cmax and AUC24 of the active deucravacitinib metabolite, BMT-153261, following administration of 6 mg once daily were 5 ng/mL and 95 ng x hr/mL, respectively. The absolute oral bioavailability of deucravacitinib was 99% and the median Tmax ranged from two to three hours. A high-fat, high-calorie meal decreased Cmax and AUC of deucravacitinib by 24% and 11%, respectively, and prolonged Tmax by one hour; however, this has clinically significant effects on drug absorption and exposure.
After a single dose of radiolabeled deucravacitinib, approximately 13% and 26% of the dose was recovered as unchanged in urine and feces, respectively. Approximately 6% and 12% of the dose was detected as BMT-153261 in urine and feces, respectively.
The volume of distribution of deucravacitinib at steady state is 140 L.
The renal clearance of deucravacitinib ranged from 27 to 54 mL/minute.

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Metabolism / Metabolites
Deucravacitinib undergoes N-demethylation mediated by cytochrome P-450 (CYP) 1A2 to form major metabolite BMT-153261, which has a comparable pharmacological activity to the parent drug. However, the circulating exposure of BMT-153261 accounts for approximately 20% of the systemic exposure of the total drug-related components. Deucravacitinib is also metabolized by CYP2B6, CYP2D6, carboxylesterase (CES) 2, and uridine glucuronyl transferase (UGT) 1A9.

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Biological Half-Life
The terminal half-life of deucravacitinib was 10 hours.

Hepatotoxicity
In the preregistration clinical trials of deucravacitinib that included data on 1519 subjects, only 1.8% of patients had serum ALT or AST elevations above 5 times ULN, none of which were considered likely due to drug induced liver injury, with myositis accounting for many of the elevations, and underlying alcoholic or nonalcoholic fatty liver disease accounting for a few. Elevations of ALT levels above 3 times the ULN arose in 1.1% to 1.3% of recipients of deucravacitinib in a 24 week trial compared to 1.2% of placebo recipients. While there were no instances of reactivation of hepatitis B in patients receiving deucravacitinib, patients with preexisting HBsAg in serum were excluded from enrollment and most treatment courses were limited in duration. Since its approval and more widespread clinical use, there have been no further reports of clinically apparent liver injury attributed to deucravacitinib, but it has been available for a limited time only.
Likelihood score: E (suspected but unproven cause of clinically apparent liver injury including reactivation of hepatitis B).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of deucravacitinib during breastfeeding. Because it is more than 80% bound to plasma proteins, the amount in milk is likely to be low. However, it is well absorbed orally. If deucravacitinib is required by the mother of an older infant, it is not a reason to discontinue breastfeeding, but until more data become available, an alternate drug may be preferred, especially while nursing a newborn or preterm infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.
◈ What is deucravacitinib?
Deucravacitinib is a medication that has been approved for the treatment of moderate-to-severe plaque psoriasis. MotherToBaby has a fact sheet on psoriasis & psoriatic arthritis at: https://mothertobaby.org/fact-sheets/psoriasis-and-pregnancy/. A brand name for deucravacitinib is SOTYKTU™.Sometimes when people find out they are pregnant, they think about changing how they take their medication, or stopping their medication altogether. However, it is important to talk with your healthcare providers before making any changes to how you take this medication. Your healthcare providers can talk with you about the benefits of treating your condition and the risks of untreated illness during pregnancy.
◈ I take deucravacitinib. Can it make it harder for me to get pregnant?
Studies have not been done to see if deucravacitinib can make it harder to get pregnant.
◈ Does taking deucravacitinib increase the chance of miscarriage?
Miscarriage is common and can occur in any pregnancy for many different reasons. Studies have not been done to see if deucravacitinib increases the chance for miscarriage.
◈ Does taking deucravacitinib increase the chance of birth defects?
Every pregnancy starts out with a 3-5% chance of having a birth defect. This is called the background risk. Human pregnancy studies have not been with deucravacitinib. Experimental animal studies reported by the manufacturer did not find an increased chance of birth defects.
◈ Does taking deucravacitinib in pregnancy increase the chance of other pregnancy related problems?
Studies have not been done to see if deucravacitinib increases the chance for pregnancy-related problems such as preterm delivery (birth before week 37) or low birth weight (weighing less than 5 pounds, 8 ounces [2500 grams] at birth).
◈ Does taking deucravacitinib in pregnancy affect future behavior or learning for the child?
Studies have not been done to see if deucravacitinib can cause behavior or learning issues for the child.
◈ Breastfeeding while taking deucravacitinib:
Deucravacitinib has not been studied for use while breastfeeding. Be sure to talk to your healthcare provider about all of your breastfeeding questions.
◈ If a male takes deucravacitinib, could it affect fertility (ability to get partner pregnant) or increase the chance of birth defects in a partner’s pregnancy?
Studies have not been done to see if deucravacitinib could affect male fertility or increase the chance of birth defects in a partner’s pregnancy. In general, exposures that fathers or sperm donors have are unlikely to increase the risks to a pregnancy. For more information, please see the MotherToBaby fact sheet Paternal Exposures at https://mothertobaby.org/fact-sheets/paternal-exposures-pregnancy/.

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Protein Binding
Protein binding of deucravacitinib was 82 to 90% and the blood-to-plasma concentration ratio was 1.26.

[1]. Highly Selective Inhibition of Tyrosine Kinase 2 (TYK2) for the Treatment of Autoimmune Diseases: Discovery of the Allosteric Inhibitor BMS-986165. J Med Chem. 2019 Jul 18.

[2]. SAT0226 A first-in-human, study of BMS-986165, a selective, potent, allosteric small molecule inhibitor of tyrosine kinase 2. Annals of the Rheumatic Diseases 2017;76:859.

Pharmacodynamics
Deucravacitinib is a tyrosine kinase 2 (TYK2) inhibitor that works to suppress the immune signaling pathways in inflammatory disorders, such as plaque psoriasis. In clinical studies comprising patients with psoriasis, deucravacitinib reduced psoriasis-associated gene expression in psoriatic skin in a dose dependent manner, including reductions in IL-23-pathway and type I IFN pathway regulated genes. Following 16 weeks of once-daily treatment, deucravacitinib reduced inflammatory markers such as IL-17A, IL-19 and beta-defensin by 47 to 50%, 72%, and 81 to 84%, respectively. Deucravacitinib does not affect with JAK2-dependent hematopoietic functions.

C20H22N8O3

425.4590

425.2

C, 56.46; H, 5.92; N, 26.34; O, 11.28

1609392-27-9

1609392-28-0 (HCl);1609392-27-9;

134821691

Off-white to light yellow solid

1.2

136Ų

3

8

7

31

648

0

[2H]C([2H])([2H])NC(=O)C1=NN=C(C=C1NC2=CC=CC(=C2OC)C3=NN(C=N3)C)NC(=O)C4CC4

BZZKEPGENYLQSC-FIBGUPNXSA-N

InChI=1S/C20H22N8O3/c1-21-20(30)16-14(9-15(25-26-16)24-19(29)11-7-8-11)23-13-6-4-5-12(17(13)31-3)18-22-10-28(2)27-18/h4-6,9-11H,7-8H2,1-3H3,(H,21,30)(H2,23,24,25,29)/i1D3

6-(cyclopropanecarboxamido)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide

BMS 986165; Deucravacitinib; BMS-986165; Sotyktu; Tyk2-IN-4; Sotyktu; BMS986165; N0A21N6RAU; Deucravacitinib [USAN]; BMS986165

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 : ~33.33 mg/mL (~78.34 mM)

配方 1 中的溶解度: 3.83 mg/mL (9.00 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
*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 (5.88 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 3 中的溶解度: ≥ 2.5 mg/mL (5.88 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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配方 4 中的溶解度: 10 mg/mL (23.50 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.
*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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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。