sphingosine 1-phosphate (S1P)(IC50 = 0.033 nM, in K562 and NK cells); PAK1

在与 NK 细胞一起存活之前，单核细胞衍生的未成熟树突状细胞 (iDC) 暴露于不同剂量的 S1P 中不同的时间。在这些培养皿中，自体或同种异体 iDC 暴露于 0.2-20 μM S1P 四个小时，可显着屏蔽 NK 细胞。对于自体 iDC 和同种异体 iDC，S1P 的 IC50 值分别确定为 160 nM 和 34 nM。随后，不同剂量的芬戈莫德或 SEW2871 能够抵消 S1P 的抑制作用，IC50 值分别为 173 或 15 nM [1]。据观察，真菌莫德通过阻止细胞中溶血磷脂酶的自动移动性而引起LPA的合成。轴突发育的重要组成部分，轴突 cAMP 浓度，与芬戈林治疗有很强的相关性。此外，芬戈莫德显着降低了神经中枢的 LPA 水平。髓磷脂厚度的改善与 PF-8380 治疗有关 [2]。

挤出后 14 天，芬戈莫德治疗导致野生型 C57BL/6 喷嘴中的神经显着增加。另一方面，用芬戈莫德治疗的 T 缺陷 B 缺失的 Foxn1-/-nozzles 表现出增强的神经再生。尽管用芬戈莫德治疗的 Foxn1-/- 喷嘴和对照 Foxn1-/- 喷嘴的神经生长平均速率表明 T 缺陷影响神经再生。尽管只有 Foxn1-/-治疗的小鼠的表现显着优于 C57BL/6，并且在功能检查中表现更好 [2]，但有可能发挥作用。离体放射性自动结合用于量化用芬戈莫德治疗动物 28 天后发生的 18F-GE180 示踪剂结合量。结果显示，芬戈莫德治疗后18F-GE180的结合电位显着降低（P<0.0001）。

利用ELISA阵列试剂盒检测细胞因子和趋化因子释放[1]
DC或NK细胞以1×106个细胞/ml的细胞浓度与培养基或与2μM S1P、10nM SEW2871、10nMFTY720或它们的组合孵育。DC也与1μg/ml LPS一起孵育。孵育24小时后，收获细胞，并将细胞悬浮液以1000×g离心10分钟，然后收集上清液。如制造商的用户手册所述，使用多分析ELISArray试剂盒检测各种细胞因子和趋化因子的水平。该试剂盒分析IL-1β、IL-4、IL-6、IL-10、IL-12、IL-17A、IFN-γ、TNF-α、TGF-β1、MCP-1、MIP-1α和MIP-1β的释放。试剂盒提供的阴性和阳性对照也包括在内[1]。

未成熟DC保持完整，或与2μM S1P、10nMFTY720、10nM SEW2871或S1P与这些药物的组合孵育4小时。作为对照，使用1μg/ml LPS。将细胞洗涤并在96孔板中孵育（v-bottom，每孔2×105个细胞），再次洗涤并重悬于含有0.1%叠氮化钠的PBS缓冲液中。用1μg/ml FITC缀合的小鼠抗人CD80、1μg/ml FITC缀合小鼠抗人CD43、1μg/ml FITC缀合小鼠抗人D86、1μmg/ml FITC偶联小鼠抗人HLA I类、1μg/ml FITC偶联鼠抗人HLA-DR、1μ/ml FITC偶联鼠标抗人HLA-E或1μg/ml FITC偶联小鼠IgG作为对照进行标记。将细胞洗涤两次，并在流式细胞仪中进行检查。根据同种型对照FITC缀合的小鼠IgG设置标记。[1]
为了用各种NK细胞激活受体的抗体对NK细胞进行染色，将其未经处理或与2μM S1P孵育4小时，用1μg/ml PE缀合的小鼠抗人NKp30（CD337）、1μg/ml聚乙烯缀合的鼠抗人NKp44（CD336）、1µg/ml PE缀合的鼠抗人类NKG2D（CD314）洗涤并染色，或作为对照，用1µg/ml聚乙烯缀合的小鼠IgG1在4°C下染色45分钟。NK细胞也用1μg/ml FITC偶联的抗杀伤抑制受体（KIR）/CD158抗体染色，该抗体识别KIR2DL2、KIR2DL3、KIR2DS2和KIR2DS4，并用FITC偶联小鼠IgG作为对照。将细胞洗涤两次，并在流式细胞仪中进行检查。根据同型对照PE缀合或FITC缀合的小鼠IgG设置标记。

Methods:[3]
Chronic focal experimental autoimmune encephalitis (EAE)-like lesions were induced in Lewis rats (n = 24) via stereotactic intrastriatal injection of heat-killed bacillus Calmette-Guérin (BCG) and subsequent activation using an intradermal injection of BCG in complete Freund adjuvant. This process resulted in a delayed-type hypersensitivity (DTH)-like EAE lesion. The extent of neuroinflammation surrounding the lesion was measured using (18)F-GE180 as a PET radioligand. The imaging was performed before and after treatment with fingolimod (0.3 mg/kg/d by mouth, 28 d) or vehicle as a control. In addition to imaging, autoradiography and immunohistochemistry experiments were performed to verify the in vivo results.
Results: [3]
The chronic DTH EAE lesion led to increased ligand binding in the ipsilateral, compared with contralateral, hemisphere when PET imaging was performed with the translocator protein-binding radioligand (18)F-GE180. Treatment with fingolimod led to a highly significant reduction in the binding potential, which could be demonstrated using both in vivo and ex vivo imaging (fingolimod vs. vehicle treatment, P < 0.0001). The area of increased (18)F-GE180 signal mapped closely to the area of activated microglial cells detected by immunohistochemistry.

Absorption, Distribution and Excretion
Fingolimod is slowly but efficiently absorbed in the gastrointestinal tract. AUC varies greatly, depending on the patient, and pharmacokinetic studies demonstrate a range of AUC values for fingolimod. The Tmax of fingolimod ranges between 12-16 hours and its bioavailability is 90-93%. Steady-state concentrations of fingolimod are achieved within 1-2 months after initiation when it is administered in a single daily dose.
About 81% of an oral dose of fingolimod is excreted in the urine in the form of inactive metabolites. Intact fingolimod and its active metabolite account for less than 2.5% of the dose, and can be found excreted in the feces.
The volume of distribution of fingolimod is about 1200±260 L. It is approximately 86% distributed in the red blood cells (RBC).
Fingolimod blood clearance is 6.3±2.3 L/h, according to prescribing information. Another resource mentions it ranges from 6-8 L/h.

---

Metabolism / Metabolites
Sphingosine kinase metabolizes fingolimod to an active metabolite, fingolimod phosphate. Fingolimod metabolism occurs via 3 major metabolic pathways: firstly, phosphorylation of the (S)-enantiomer of fingolimod-phosphate (pharmacologically active), secondly, oxidation by cytochrome P450 4F2 (CYP4F2), and thirdly, fatty acid-like metabolism to various inactive metabolites. The formation of inactive non-polar ceramide analogs of fingolimod also occurs during its metabolism.

---

Biological Half-Life
The half-life of fingolimod and its active metabolite ranges from 6-9 days.

Hepatotoxicity
In large randomized controlled trials of fingolimod in patients with multiple sclerosis, serum ALT elevations above 3 times ULN were reported in 8% to 14% of fingolimod compared to 2% to 3% of placebo recipients. The enzyme elevations were usually transient and not associated with symptoms or jaundice and required drug discontinuation in less than 1% of cases. No instances of acute hepatitis or clinically apparent liver injury were reported in the preregistration trials of fingolimod. Subsequent to its approval and more wide scale use, however, instances of clinically apparent liver injury attributed to fingolimod were reported including cases of acute liver failure requiring emergency liver transplantation. The onset of liver injury was often within days or weeks of starting treatment and the pattern of liver enzyme elevations was usually hepatocellular. In addition, at least one instance of reactivation of hepatitis B in an inactive HBsAg carrier has been reported. Thus, mild-to-moderate and transient serum enzyme elevations during therapy are not uncommon, and clinically apparent liver injury with jaundice due to fingolimod can occur.
Likelihood score: C (probable cause of clinically apparent liver injury as well as reactivation of hepatitis B in susceptible patients).

---

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Although fingolimod and its active metabolite are highly bound in maternal plasma and unlikely to reach the breastmilk in large amounts, it is potentially toxic to the breastfed infant. Because there is no published experience with fingolimod during breastfeeding, expert opinion generally recommends that it should be avoided during breastfeeding, especially while nursing a newborn or preterm infant. However, the manufacturer's labeling does not recommend against its use in breastfeeding.
◉ 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.

---

Protein Binding
The protein binding of fingolimod and its active metabolite exceeds 99.7%.

[1]. FTY720 and SEW2871 reverse the inhibitory effect of S1P on natural killer cell mediated lysis of K562 tumor cells and dendritic cells but not on cytokine release. Cancer Immunol Immunother. 2010, 59(4), 575-586.

[2]. Fingolimod promotes peripheral nerve regeneration via modulation of lysophospholipid signaling. J Neuroinflammation. 2016 Jun 10;13(1):143.

[3]. In vivo PET imaging demonstrates diminished microglial activation after fingolimod treatment in an animal model of multiple sclerosis. J Nucl Med. 2015 Feb;56(2):305-10.

[4]. Modification of lymphocyte migration to Peyer's patches by inhibition of sphingosine-1-phosphate lyase ameliorates murine colitis. J Gastroenterol Hepatol. 2018 Jan 15.

Pharmacodynamics
In multiple sclerosis, fingolimod binds to sphingosine receptors, reducing its associated neuroinflammation.In COVID-19, it may reduce lung inflammation and improve the clinical outcomes of patients with this disease. Fingolimod causes a transient reduction in heart rate and AV conduction during treatment initiation. It has the potential to prolong the QT interval.

C19H33NO2

307.478

307.251

C, 74.22; H, 10.82; N, 4.56; O, 10.41

162359-55-9

Fingolimod-d4;1346747-38-3;Fingolimod-d4 hydrochloride;1346604-90-7;Fingolimod hydrochloride;162359-56-0; 162359-55-9; 402615-91-2 (phosphate); 207113-62-0 (octanoic acid); 1242271-26-6 (palmitamide); 207113-64-2 (hexanoic acid)

107970

White to off-white solid powder

1.0±0.1 g/cm3

479.5±45.0 °C at 760 mmHg

102-107

243.8±28.7 °C

0.0±1.3 mmHg at 25°C

1.531

5.25

66.48

3

3

12

22

258

0

KKGQTZUTZRNORY-UHFFFAOYSA-N

InChI=1S/C19H33NO2/c1-2-3-4-5-6-7-8-17-9-11-18(12-10-17)13-14-19(20,15-21)16-22/h9-12,21-22H,2-8,13-16,20H2,1H3

2-Amino-2-[2-(4-octylphenyl)ethyl]-1,3-propandiol

FTY720 (free base); FTY720; 162359-55-9; 2-Amino-2-(4-octylphenethyl)propane-1,3-diol; 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol; Fingolimod [INN]; 2-Amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol; Fty-720; fingolimodum; Fingolimod HCl; FTY-720; FTY 720; Trade name: Gilenia and Gilenya.

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 : ~7.69 mg/mL (~25.01 mM)
DMSO : ~2 mg/mL (~6.50 mM)

配方 1 中的溶解度: ≥ 1 mg/mL (3.25 mM) (饱和度未知) in 10% EtOH + 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，将 100 μL 10.0 mg/mL 澄清乙醇储备液加入到 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。

---

配方 2 中的溶解度: ≥ 1 mg/mL (3.25 mM) (饱和度未知) in 10% EtOH + 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 10.0 mg/mL 澄清乙醇储备液加入 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。

---

View More

配方 3 中的溶解度: ≥ 1 mg/mL (3.25 mM) (饱和度未知) in 10% EtOH + 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 10.0 mg/mL 澄清 EtOH 储备液添加到 900 μL 玉米油中并充分混合。

---

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。