Monoamine oxidase B (MAO-B) (IC50 = 98 nM)

甲磺酸沙芬酰胺 (1-300 µM) 以浓度依赖性方式降低峰值钠电流的幅度。当电流从 -110 mV 的 Vh 驱动至 +10 mV 的 Vtest 时，IC50 值为 262 µM。由于 Safinamide mesylate 的抑制作用，大鼠皮质神经元的去极化保持电位为 -53 mV，其 IC50 值较低，为 8 µM。

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通过快速固相合成制备了Safinamide、（S）-N2-{4-[（3-氟苄基）氧基]苄基}丙氨酸酰胺甲磺酸盐（作为抗帕金森药物正在进行III期临床试验）和烷酰胺类似物库，并评估了它们的单胺氧化酶B（MAO-B）和单胺氧化酶a（MAO-a）抑制活性和选择性。（S） -3-氯苄氧基丙氨酸酰胺（8）和（S）-3-氯苄氧基丝氨酸酰胺（13）衍生物被证明是比Safinamide更有效的MAO-B抑制剂（IC50分别为33和43 nM，对98 nM），但MAO-B选择性较低（SI分别为3455和1967，对5918）。沙芬那胺的四氢异喹啉类似物（R）-21显示出最高的MAO-B抑制效力（IC50=17 nM）和良好的选择性（SI=2941）。结构亲和关系和对接模拟表明，苄氧基的α-氨基酰胺侧链和对位取代基具有强烈的负空间效应，间位取代基具有良好的疏水相互作用。许多R和Sα-氨基酰胺对映体（包括沙芬那胺的两种刚性类似物（21））的MAO-B亲和力差异很大，表明酶结合位点可能存在对映选择性相互作用。[1]

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大鼠皮质神经元钠通道抑制。[3]
+10 mV的电压脉冲从皮质神经元诱发快速向内钠电流，其幅度取决于条件脉冲的电压（见材料和方法）。诱发最大（静息状态，Vrest）和50%最大钠电流（半最大失活状态，Vlhalf）的调节电压分别为-110和-53 mV（图6A）。根据观察到的稳态失活曲线，在-110 mV（Vrest）和-53 mV（Vlhalf）的预处理电位下测试了Safinamide对钠电流和阻断电压/状态依赖性的影响。如图6B所示，Safinamide（1-300µM）以浓度依赖的方式降低了峰值钠电流（强直性阻滞）的振幅。当电流从-110 mV的Vh刺激到+10 mV的Vtest时，IC50值为262µM。沙芬那胺的抑制作用是电压依赖性的，因为当保持电位去极化至-53 mV时，IC50值显著降低（8µM）。洗脱导致抑制作用完全逆转。钠通道失活状态的亲和力常数（Ki）为4.1µM。

腹腔注射（90 mg/kg，每天一次，持续 14 天）时，甲磺酸沙芬酰胺可显着减少小鼠 MCAO 引起的脑梗塞体积，以及神经功能缺损、脑血屏障 (BBB) 破坏和ZO-1和紧密连接蛋白occludin的表达[3]。 GABA 和 Glu 的体内释放受到甲磺酸沙芬酰胺的剂量依赖性抑制（腹膜内注射；5 mg/kg、15 mg/kg 和 30 mg/kg）。当施用藜芦定时观察到这种效果。甲磺酸沙芬酰胺，剂量为 30 mg/kg，阻断藜芦定对 GABA 释放的影响（治疗 F1,8=4.04；时间 F8,64=3.76，时间×治疗相互作用 F8,64=2.83）和 Glu（治疗F1,8=1.31；时间×治疗相互作用F8,64=2.4）。在大鼠中，safinamide mesylate 在 5 和 15 mg/kg 剂量下完全抑制藜芦定刺激的 Glu 释放，而在 0.5 mg/kg 剂量下有轻微但不具有统计学意义的减少[3]。

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Safinamide最近被批准作为左旋多巴治疗帕金森病的附加药物。除了抑制B型单胺氧化酶外，它还在体外阻断钠通道并调节谷氨酸（Glu）的释放。由于这种特性可能有助于药物的治疗作用，我们进行了本研究，以调查沙芬酰胺是否也在体内抑制Glu的释放，以及这种作用是否在不同的脑区是一致的，并且对谷氨酸能神经元是选择性的。为此，使用体内微透析监测幼年清醒大鼠海马和基底神经节中自发和藜芦碱诱导的Glu和GABA释放。还测量了脑中沙芬那胺的水平。为了阐明沙芬那胺作用的机制，通过膜片钳记录大鼠皮质神经元的钠电流。沙芬那胺在15mg/kg时最大限度地抑制了藜芦碱诱导的海马中Glu和GABA的释放，达到1.89-1.37µM的游离脑浓度。该剂量减弱了藜芦碱刺激的丘脑底核、苍白球和网状黑质中Glu（但不是GABA）的释放，但纹状体中没有。Safinamide对自发神经递质释放无效。在体外，沙芬那胺抑制钠通道，在去极化（IC50=8µM）时比在静息（IC50=262µM）电位下显示出更大的亲和力。我们得出结论，沙芬酰胺抑制了受刺激神经末梢的体内Glu释放，可能是通过阻断具有特定放电模式的神经元亚群的钠通道。这些数据与沙芬那胺的抗惊厥和抗帕金森病作用一致，并为其作用的非多巴胺能机制提供了支持[3]。

体外酶活性测定。分别使用选择性底物14C-血清素（5-HT）和14C-苯乙胺（PEA）对MAO-a和MAO-B进行放射酶测定，评估酶活性。 将线粒体沉淀（500μg蛋白质）重新悬浮在200μL pH 7.40的0.1 M磷酸盐缓冲液中，并将其加入50μL抑制剂溶液（在向游离碱水溶液中加入化学计量量的0.01 M甲磺酸后转化为甲磺酸盐）或缓冲液溶液中，在37°C下孵育30分钟（预孵育）。然后加入50μL缓冲液中的底物（5μM 14C-5-HT或0.5μM 14C-PEA），将测定混合物在37°C下孵育30分钟（5-HT）或10分钟（PEA）。[1]
通过分别加入0.2mL HCl或高氯酸用于5-HT或PEA来停止反应。离心后，用3mL乙醚（用于5-HT）或甲苯（用于PEA）提取酸性放射性代谢物，并通过液体闪烁光谱法以90%的效率测量有机相的放射性。[1]
酶活性表示为每分钟每毫克蛋白质转化的底物纳摩尔数（nmol mg-1 min-1）。[1]
从5到8个不同浓度（10-10-10-10-5M）获得药物抑制曲线，每个浓度重复两次，使用非线性回归分析（GraphPad最佳拟合计算机程序）确定IC50。对于活性极低的抑制剂，在表1所示的浓度下，酶抑制百分比一式两份测定。

全细胞膜片钳记录。[3]
实验是在室温（25°C）下根据标准全细胞膜片钳记录技术（Hamill等人，1981）进行的。用含有（以毫摩尔计）NaCl（60）、氯化胆碱（60），CaCl2（1.3）、MgCl2（2）、CdCl2（0.4）、NiCl2（0.3）、TEACl（20）、葡萄糖（10）和HEPES（10）的细胞外溶液连续超灌注神经元细胞。使用Sutter P-87电极拔出器拔出贴片移液管，并填充由以下成分组成的内溶液（单位：毫摩尔）：CsF（65）、CsCl（65），NaCl（10）、CaCl2（1.3）、MgCl2（2）、EGTA（10），HEPES（10）和MgATP（1）。贴片电极的尖端电阻为2-3MΩ。使用Axopatch 200B放大器在5kHz下记录和过滤膜电流，并使用Axon Digidata 1322A对数据进行数字化。电压指令协议和数据采集使用Axon pClamp8软件进行控制。测量电极和参比电极均为AgCl-Ag电极。接入电阻范围为5至10 MΩ；使用P/4泄漏减法协议消除了线性泄漏和电容电流Safinamide（20mM蒸馏水储备溶液）在外部溶液中稀释，并施加2分钟以达到平衡反应。

Animal/Disease Models: Focal cerebral ischemia C57/BL6 male mouse Model[3]
Doses: 90 mg/kg
Route of Administration: intraperitoneal (ip)injection; one time/day; 14 days
Experimental Results: Dramatically diminished infarction volume in brain areas.

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Experimental Protocols and Design. [3]
Ninety-five rats were used for the microdialysis experiments, 84 for the study of veratridine-stimulated neurotransmitter release and 11 for the study of spontaneous release. The experimental protocols were approved by the Italian Ministry of Health (licenses 170/2013B and 714/2016-PR-B). As for the design of the experiments (Fig. 1, C and D and Fig. 2), each rat was randomized to saline/veratridine or Safinamide/veratridine (0.5, 5, or 15 mg/kg, Fig. 1, C and D; 5 or 15 mg/kg, Fig. 2) in the first and second microdialysis sessions, ensuring that no rat received the same treatment in the two sessions. Rats underwent two microdialysis sessions (i.e., at 24 and 48 hours after probe implantation), after which they were sacrificed with an isoflurane overdose, and placement of the probes was verified histologically. For study of veratridine-stimulated release (Fig. 1, A and B, 3, 4, and 5), each animal implanted with a single microdialysis probe was randomized to saline/veratridine or Safinamide/veratridine (30 mg/kg, Fig. 1, A and B; 15 mg/kg, Figs. 3–5) in the first microdialysis session, and treatments crossed in the second session. For the study on spontaneous Glu and GABA release, rats implanted with one probe in STN and another in the contralateral SNr were randomized to saline or veratridine 15 mg/kg in the first microdialysis session, and treatments crossed in the second session. Overall, seven animals were discarded for probe misplacement or probe clogs during microdialysis.

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In Vivo Microdialysis. [3]
Intracerebral microdialysis was performed as previously described (Morari et al., 1996; Paolone et al., 2015). One probe of concentric design was stereotaxically implanted under isoflurane anesthesia in five different brain regions according to the following coordinates (in millimeters) from the bregma and the dural surface (Paxinos and Watson, 1986): hippocampus (1-mm dialyzing membrane, antero-posterior (AP) −3.14, medio-lateral (ML) ± 1.8, dorso-ventral (DV) −4.2.), STN (1-mm dialyzing membrane, AP −3.7, ML ± 2.5, DV −8.6), SNr (1-mm dialyzing membrane, AP −5.5, ML ± 2.2, DV −8.3), DLS (3-mm dialyzing membrane, AP +1.0, ML ± 3.5, DV −6.0) and GP (2-mm dialyzing membrane, AP −1.3, ML ± 3.3, DV −6.5). When veratridine-stimulated neurotransmitter release was studied, each animal was implanted with one probe at the time. Conversely, when spontaneous neurotransmitter release was studied, each animal was implanted with two probes at the same time, one in the STN and another in the contralateral SNr. Probes were secured to the skull with dental cement. The wound was infiltrated with local anesthetic (lidocaine 2%) before surgery completion. Twenty-four hours after surgery, the probes were perfused with a modified Ringer’s solution (1.2 mM CaCl2, 2.7 mM KCl, 148 mM NaCl, and 0.85 mM MgCl2) at a flow rate of 3.0 μl/min, and sample collection (every 20 minutes) began after 6 hours of rinsing. At least four baseline samples were collected before systemic (i.p.) administration of saline or Safinamide. Thirty minutes later, veratridine (10 μM) was perfused for 30 minutes through the probe by reverse dialysis; at the end of veratridine perfusion, sample collection was continued for 80 minutes.

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Brain Pharmacokinetic Analysis. [3]
Twenty-seven rats were used for pharmacokinetic analysis.Safinamide was administered at three dose levels (5, 15, and 30 mg/kg, i.p.), and brains were removed 40, 60, and 80 minutes later to match the veratridine perfusion time in the microdialysis study. Brain samples were homogenized by sonication; after protein precipitation, the total Safinamide concentration was measured by HPLC-tandem mass spectrometry on a Sciex API4000 mass spectrometer (AB Sciex, Framingham, MA). Samples (5 µl) were injected using a CTC analytics HTS Pal autosampler (Zwingen, Switzerland) onto a Synergi MAX-RP 30 ×2.0 mm, 4-µm column at an eluent flow rate of 1.5 ml/min. Analytes were eluted using a high-pressure linear gradient program by an HP1100 binary HPLC system. To calculate the free brain concentration, the fraction of unbound Safinamide in the brain (fu,b) was determined by in vitro equilibrium dialysis (Summerfield et al., 2007). The fu,b percent was 3.27.

Absorption, Distribution and Excretion
Rapid with peak plasma concentrations ranging from 2 to 4 h, total bioavailability is 95%. Food prolonged the rate and did not affect the extent of absorption of safinamide.
76% renal, 1.5% faeces
1.8 litres/kg
total oral clearance of plasma , which accounts for parent safinamide as well as metabolites, was on average only 17.53 ± 2.71 ml/h × kg

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Metabolism / Metabolites
The principal step is mediated by amidases which have not been identified, and produces safinamide acid. It is also metabolized to O-debenzylated safinamide and N-delkylated amine. The N-dealkylated amine is then oxidized to a carboxylic acid and finally glucuronidated. Dealkylation reactions are mediated by cytochrome P450s (CYPs), especially CYP3A4. Safinamide acid binds to organic anion transporter 3 (OAT3), but no clinical relevance of this interaction has been determined. Safinamide also binds to ABCG2 transiently. No other transporter affinities have been found in preliminary studies.

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Biological Half-Life
22 h

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Safinamide Brain Levels. [3]
In a separate group of rats, the brain levels of safinamide were measured 40, 60, and 80 minutes after the administration of 5, 15, or 30 mg/kg safinamide. Free brain concentrations, derived by taking into account the brain-binding tissue of safinamide, correlated with doses, being highest for the 30 mg/kg dose and lowest for the 5 mg/kg dose at any time points examined (Table 1). In addition, for all doses, a gradual and linear decline was observed from the first through the last time point examined. During veratridine perfusion (100–120 minutes), safinamide-free brain levels for the 5, 15, and 30 mg/kg doses were in the 0.70–0.44, 1.89–1.70, and 4.77–3.04 µM concentration ranges, respectively.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of safinamide during breastfeeding. Because of liver toxicity in nursing rat pups, the manufacturer recommends that the drug not be used in nursing mothers. Alternate agents are preferred.
◉ 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. Hepatotoxicity
Safinamide has been reported to cause serum enzyme elevations in a small proportion of patients treated long term, although the abnormalities were usually mild and self-limiting and were usually no more frequent than with placebo or comparator agents. Safinamide has not been implicated in cases of acute liver injury, but such instances have been reported with nonspecific MAO inhibitors.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Protein Binding
88–90%

[1]. Solid-phase synthesis and insights into structure-activity relationships of safinamide analogues as potent and selective inhibitors of type B monoamine oxidase. J Med Chem, 2007, 50(20), 4909-4916.

[2]. Safinamide: from molecular targets to a new anti-Parkinson drug. Neurology. 2006 Oct 10;67(7 Suppl 2):S18-23.

[3]. Safinamide Differentially Modulates In Vivo Glutamate and GABA Release in the Rat Hippocampus and Basal Ganglia.J Pharmacol Exp Ther. 2018 Feb;364(2):198-206.

See also: Safinamide (has active moiety).

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Drug Indication
Xadago is indicated for the treatment of adult patients with idiopathic Parkinson's disease (PD) as add-on therapy to a stable dose of Levodopa (L-dopa) alone or in combination with other PD medicinal products in mid-to late-stage fluctuating patients.

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Safinamide is an amino acid amide.
Safinamide is for the treatment of parkinson's disease. It was approved in Europe in February 2015, and in the United States on March 21, 2017.
Safinamide is a Monoamine Oxidase Type B Inhibitor. The mechanism of action of safinamide is as a Monoamine Oxidase-B Inhibitor, and Breast Cancer Resistance Protein Inhibitor.
Safinamide is an inhibitor of monoamine oxidase used as adjunctive therapy in combination with levodopa and carbidopa in the management of Parkinson’s disease. Safinamide has been associated with a low rate of serum enzyme elevations during treatment, but has not been linked to instances of clinically apparent acute liver injury.
See also: Safinamide Mesylate (active moiety of).

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Drug Indication
Safinamide is indicated as an add-on treatment to levodopa with or without other medicines for Parkinson’s disease
Xadago is indicated for the treatment of adult patients with idiopathic Parkinson's disease (PD) as add-on therapy to a stable dose of Levodopa (L-dopa) alone or in combination with other PD medicinal products in mid-to late-stage fluctuating patients.

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Mechanism of Action
Safinamide is a unique molecule with multiple mechanisms of action and a very high therapeutic index. It combines potent, selective, and reversible inhibition of MAO-B with blockade of voltage-dependent Na+ and Ca2+ channels and inhibition of glutamate release. Safinamide has neuroprotective and neurorescuing effects in MPTP-treated mice, in the rat kainic acid, and in the gerbil ischemia model.

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Safinamide, (S)-N2-{4-[(3-fluorobenzyl)oxy]benzyl}alaninamide methanesulfonate, which is in phase III clinical trials as an anti-Parkinson drug, and a library of alkanamidic analogues were prepared through an expeditious solid-phase synthesis and evaluated for their monoamine oxidase B (MAO-B) and monoamine oxidase A (MAO-A) inhibitory activity and selectivity. (S)-3-Chlorobenzyloxyalaninamide (8) and (S)-3-chlorobenzyloxyserinamide (13) derivatives proved to be more potent MAO-B inhibitors than safinamide (IC50 = 33 and 43 nM, respectively, vs 98 nM) but with a lower MAO-B selectivity (SI = 3455 and 1967, respectively, vs 5918). The highest MAO-B inhibitory potency (IC50 = 17 nM) and a good selectivity (SI = 2941) were displayed by (R)-21, a tetrahydroisoquinoline analogue of safinamide. Structure-affinity relationships and docking simulations pointed out strong negative steric effects of alpha-aminoamide side chains and para substituents of the benzyloxy groups and favorable hydrophobic interactions of meta substituents. The significantly diverse MAO-B affinities of a number of R and S alpha-aminoamide enantiomers, including the two rigid analogues (21) of safinamide, indicated likely enantioselective interactions at the enzymatic binding sites.[1]

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Ideal treatment in Parkinson's disease (PD) aims at relieving symptoms and slowing disease progression. Of all remedies, levodopa remains the most effective for symptomatic relief, but the medical need for neuroprotectant drugs is still unfulfilled. Safinamide, currently in phase III clinical trials for the treatment of PD, is a unique molecule with multiple mechanisms of action and a very high therapeutic index. It combines potent, selective, and reversible inhibition of MAO-B with blockade of voltage-dependent Na+ and Ca2+ channels and inhibition of glutamate release. Safinamide has neuroprotective and neurorescuing effects in MPTP-treated mice, in the rat kainic acid, and in the gerbil ischemia model. Safinamide potentiates levodopa-mediated increase of DA levels in DA-depleted mice and reverses the waning motor response after prolonged levodopa treatment in 6-OHDA-lesioned rats. Safinamide has excellent bioavailability, linear kinetics, and is suitable for once-a-day administration. Therefore, safinamide may be used in PD to reduce l-dopa dosage and also represents a valuable therapeutic drug to test disease-modifying potential. [2]

C17H19FN2O2.CH4O3S

398.45

398.131

C, 54.26; H, 5.82; F, 4.77; N, 7.03; O, 20.08; S, 8.05

202825-46-5

Safinamide;133865-89-1

3038502

White to off-white solid powder

476.7ºC at 760 mmHg

210° (dec)

242.1ºC

2.98E-09mmHg at 25°C

4.044

127.1

3

7

7

27

438

1

C[C@@H](C(=O)N)NCC1=CC=C(C=C1)OCC2=CC(=CC=C2)F.CS(=O)(=O)O

YKOCHIUQOBQIAC-YDALLXLXSA-N

InChI=1S/C17H19FN2O2.CH4O3S/c1-12(17(19)21)20-10-13-5-7-16(8-6-13)22-11-14-3-2-4-15(18)9-14;1-5(2,3)4/h2-9,12,20H,10-11H2,1H3,(H2,19,21);1H3,(H,2,3,4)/t12-;/m0./s1

(S)-2-((4-((3-fluorobenzyl)oxy)benzyl)amino)propanamide methanesulfonate

PNU-151774E, FCE28073; Safinamide mesylate; Safinamide mesylate; 202825-46-5; (S)-2-((4-((3-Fluorobenzyl)oxy)benzyl)amino)propanamide methanesulfonate; Safinamide mesilate; PNU-151774E; NW-1015; safinamide methanesulfonate; Xadago; NW 1015; PNU 151774E; EMD 1195686; FCE-28073; Safinamide mesilate; FCE 28073; NW1015; NW-1015; EMD-1195686; EMD1195686; PNU-151774E;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

配方 1 中的溶解度: ≥ 2.08 mg/mL (5.22 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (5.22 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 20.8 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: ≥ 2.08 mg/mL (5.22 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 20.8 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: Saline: 30 mg/mL

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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。