Risdiplam (RG-7916, RO-7034067)   中文名称: 利司扑兰

SMN2

Risdiplam 提高 SMN 蛋白的量并控制 SMN2 前 mRNA 的剪接以生成全长 SMN2 mRNA。 Risdiplam 是 SMN2 的剪接调节剂，可以增加全长 SMN2 蛋白的量，从而增强 SMN 蛋白的功能。导致婴儿死亡的最常见遗传病仍然是SMA（SMA）。由于东南运动神经元 1 (SMN1) 基因中的双联体，早期运动神经元蛋白 (SMN) 水平低是导致这种常染色体隐性神经病的原因，其特征是进行性运动和呼吸肌减弱。残留失活和基因丢失的其他来源[1]。

为了进一步探索利斯地普兰的分布，研究人员在动物模型中评估了利斯地普兰的体外特征和体内药物水平，以及对不同组织中SMN蛋白表达的影响。小鼠（n = 90）、大鼠（n = 148）和猴子（n = 24）的血浆、肌肉和脑中的总药物水平相似。正如预期的那样，基于它的高被动渗透性和不是人类多药耐药蛋白1底物，里斯迪普兰CSF水平反映了猴子血浆中游离化合物的浓度。当猴子每天服用一次利西泮，连续39周，组织分布没有变化。在给药的两种SMA小鼠模型中，CNS和外周组织中SMN蛋白水平呈平行剂量依赖性增加。这些体外和体内的临床前数据有力地表明，利斯地普兰治疗后患者血液中功能性SMN蛋白的增加应该反映了CNS、肌肉和其他外周组织中功能性SMN蛋白的类似增加。[1]

体外转运试验[1]
使用亲本猪肾上皮LLC PK1 （Lewis肺癌猪肾1）和犬肾上皮MDCKII细胞系。LLC‐PK1， MDCKII, L‐MDR1 (LLC‐PK1细胞转染了人MDR1), L‐Mdr1a (LLC‐PK1细胞转染了啮齿动物Mdr1a), M‐BCRP (MDCKII细胞转染了人乳腺癌抵抗蛋白；BCRP)和M - Bcrp1（转染啮齿动物Bcrp1的MDCKII细胞）细胞系在许可协议下使用。啮齿动物Mdr1a是一种小鼠蛋白，其氨基酸序列与大鼠Mdr1a具有95%的同源性，因此本文将其称为“啮齿动物”Mdr1a。化验按前面所述进行。简单地说，将细胞培养在半透性的96孔植入物上(表面积0.11 cm2，孔径0.4 mm；在播种后第3天或第4天进行双向输运测量。将培养基从顶部（100 mL）和基底外侧（240 mL）室中取出，在受者一侧替换为不含酚红和含有或不含抑制剂（zosuquidar， L‐MDR1和L‐Mdr1a的抑制剂为1 μM）的培养基；Ko143, 1 μM M - BCRP和M - BCRP)。通过向含有试验底物（risdiplam或RG7800，在1 μM下测试）和10 μM路西弗黄的供体室中添加培养基，启动跨细胞运输。加入Lucifer黄以确认单层的完整性，对照底物孵育作为MDR1/Mdr1a或BCRP/ BCRP活性的对照。在37°C和5% CO2下连续摇瓶（100 rpm）孵育3.5小时。样品（每种条件下三份）从供体和受体区室中取出，并通过闪烁计数或串联质谱的高效液相色谱分析，如前所述的液相色谱，使用10ADvp泵系统与PAL HTS自动进样器耦合，对于MS， API 4000或QTrap4000系统配备TurboIonspray源。

Study design in rats (Studies 6, 7, and 8) [1]
Risdiplam was administered orally by gavage once daily as a solution in 10 mM ascorbic acid/0.01 mg/mL sodium thiosulfate pentahydrate, pH 3, and the dosing volume was 10 mL/kg (Study 6) or 4 mL/kg (Study 7, 8). For information on doses and length of dosing for each individual study, please see Table 1. Each animal was killed under isoflurane anesthesia. Animals were exsanguinated by the severing of major blood vessels. Terminal blood samples were taken from the jugular vein immediately prior to exsanguination and collected into tubes containing K3‐EDTA anticoagulant. The entire brain was collected into labeled 7 mL Precellys® homogenization tubes (CK14), snap‐frozen in liquid nitrogen and stored on dry ice. Tissues were homogenized by bead beating and/or diluted with blank tissue homogenate or blank rat plasma. The analyte was isolated from matrix (EDTA plasma or tissues homogenate) by protein precipitation with acetonitrile/ethanol containing the internal standard (13C, D2 stable isotope‐labeled risdiplam or RG7800) and separated from other constituents of the sample by narrow‐bore HPLC. Detection was accomplished utilizing heated electrospray (HESI) MS/MS positive‐ion selected reaction monitoring mode (SRM). CSF and tissue samples were quantified against rat plasma calibration curves diluted with the appropriate blank tissue homogenate or blank plasma. The LLOQ for risdiplam or RG7800 was 0.250 ng/mL in rat plasma using 20 μL aliquots, 0.500 or 1.00 ng/mL in CSF and 2 ng/g in tissues using 20 μL of tissue homogenate.

---

Study design in monkeys (Studies 9 and 10) [1]
Risdiplam or RG7800 was administered orally by gavage once daily as a solution in 10 mM ascorbic acid/0.01 mg/mL sodium thiosulfate pentahydrate, pH 3, and the dosing volume was 1.5 mL/kg (Study 9) or 5 mL/kg (Study 10). For information on doses and length of dosing for each individual study, please see Table 1. At the end of dosing, animals were killed, and terminal plasma and tissues were collected and stored frozen. In Study 10, brain stem and cortex samples (0.5 g) were separately collected. A sample of 0.5 mL of CSF was collected from all animals. Tissues were homogenized and diluted with blank cynomolgus monkey plasma. The analyte was isolated from matrix as described for Studies 6‐8. Detection was accomplished utilizing HESI MS/MS in positive ion SRM. The LLOQ in cynomolgus monkey plasma was 0.250 ng/mL (Study 9) or 0.500 ng/mL (Study 10) using 20 μL aliquots. All other samples were quantified against cynomolgus monkey plasma calibration curves. Due to sample dilution, the resulting LLOQs were 0.500 ng/mL in CSF (Study 9) and between 0.500 and 5000 ng/g in tissues (10.0 ng/g in brain). For Study 10, a dedicated, sensitive CSF method with LLOQ 0.100 ng/mL was used. Unbound (free) plasma concentrations were calculated by multiplying the measured total concentration in plasma by the measured free fraction in plasma (15% in adult cynomolgus monkeys).

---

Rat Quantitative Whole‐Body Autoradiography (QWBA) study design (Study 13) [1]
Wistar rats received either a single oral dose of 14C‐risdiplam or RG7800 (by gastric gavage), or a single intravenous dose of 14C‐risdiplam or RG7800 (by tail vein injection). Dose levels were 5 or 2 mg/kg, for oral and intravenous doses, respectively. For whole‐body autoradiography, following deep anesthesia under isoflurane, single animals were killed by cold shock (in a mixture consisting of an excess of dry‐ice in hexane) at the following times after dosing: 10 min for the IV‐dosed animals and 2, 24, 72, and 168 hours for the oral‐dosed animals. Once fully frozen, the carcasses were prepared for, and subjected to, whole‐body autoradiography procedures. Radioactivity concentration in tissues was quantified from the whole‐body autoradiograms, using a validated image analysis system. After exposure in a copper‐lined, lead exposure box for 7 days, the imaging plates were processed using a FUJI FLA 5000 or 5100 radioluminography system. The electronic images were analyzed using a validated PC‐based image analysis package. While under terminal anesthesia, blood (approximately 3 mL) was collected from each of the animals by cardiac puncture into tubes precoated with lithium heparin. Blood and plasma were assayed for total radioactivity.

---

Study design in mice (Studies 1, 2, 3, 4, 5, 11, and 12) [1]
Two different mouse models of SMA were utilized for these studies. For studies in adult mice, the C/C‐allele mouse model of mild SMA was utilized. C/C‐allele mice have a near‐normal life span but show decreased muscle function, reduced body weight gain, and peripheral necrosis in comparison to normal mice. Neonatal SMNΔ7 mice, a mouse model of severe SMA, were also used. These mice die approximately 2 weeks after birth. For oral dosing of adult mice, compounds were formulated as a suspension in 0.5% hydroxypropylmethyl cellulose with 0.1% Tween 80. For intraperitoneal (IP) dosing of juvenile mice, compounds were formulated in dimethyl sulfoxide and administered at a dosing volume of 2.5 mL/kg. For repeat administration, the compounds were administered once daily. For Study 5, mice were dosed IP from PND3 to PND23 and dosed by oral gavage from PND24 onward. For information on doses and length of dosing for each individual study, please see Table 1.

Absorption, Distribution and Excretion
The Tmax following oral administration is approximately 1-4 hours. Following once-daily administration with a morning meal (or after breastfeeding), risdiplam reaches steady-state in approximately 7-14 days. The pharmacokinetics of risdiplam were found to be approximately linear between all studied dosages in patients with SMA.
Following the oral administration of 18mg risdiplam, approximately 53% of the dose was excreted in the feces and 28% was excreted in the urine. Unchanged parent drug comprised 14% of the dose excreted in feces and 8% of the dose excreted in urine.
Following oral administration, risdiplam distributes well into the central nervous system and peripheral tissues. The apparent volume of distribution at steady-state is 6.3 L/kg.
For a 14.9kg patient, the apparent clearance of risdiplam is 6.3 L/kg.

---

Metabolism / Metabolites
The metabolism of risdiplam is mediated primarily by flavin monooxygenases 1 and 3 (FMO1 and FMO3), with some involvement of CYP1A1, CYP2J2, CYP3A4, and CYP3A7. Parent drug comprises approximately 83% of circulating drug material. A pharmacologically-inactive metabolite, M1, has been identified as the major circulating metabolite - this M1 metabolite has been observed _in vitro_ to inhibit MATE1 and MATE2-K transporters, similar to the parent drug.

---

Biological Half-Life
The terminal elimination half-life of risdiplam is approximately 50 hours in healthy adults.

Protein Binding
Risdiplam is approximately 89% protein-bound in plasma, primarily to serum albumin.

---

Hepatotoxicity
In preregistration clinical trials, there were no clinically significant changes in serum laboratory values with risdiplam therapy and no differences in serum ALT, AST, and bilirubin values between risdiplam vs placebo recipients. While safety results were based on only several hundred patients, there were no cases of suspected drug induced liver injury with jaundice. Furthermore, there have been no published cases of clinically apparent liver injury attributed to risdiplam since its approval in 2020.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

[1]. Risdiplam distributes and increases SMN protein in both the central nervous system and peripheral organs. Pharmacol Res Perspect. 2018 Nov 29;6(6):e00447.

Risdiplam is an orally bioavailable mRNA splicing modifier used for the treatment of spinal muscular atrophy (SMA). It increases systemic SMN protein concentrations by improving the efficiency of SMN2 gene transcription. This mechanism of action is similar to its predecessor [nusinersen], the biggest difference being their route of administration: nusinersen requires intrathecal administration, as does the one-time gene therapy [onasemnogene abeparvovec], whereas risdiplam offers the ease of oral bioavailability. Risdiplam was approved by the FDA in August 2020 for the treatment of spinal muscular atrophy (SMA). Set to be substantially cheaper than other available SMA therapies, risdiplam appears to provide a novel and relatively accessible treatment option for patients with SMA regardless of severity or type.
Risdiplam is a Survival of Motor Neuron 2 Splicing Modifier. The mechanism of action of risdiplam is as a Survival of Motor Neuron 2 Splicing Modifier, and Multidrug and Toxin Extrusion Transporter 1 Inhibitor, and Multidrug and Toxin Extrusion Transporter 2 K Inhibitor. The physiologic effect of risdiplam is by means of Increased Protein Synthesis.

---

Drug Indication
Risdiplam is indicated for the treatment of spinal muscular atrophy (SMA).
Evrysdi is indicated for the treatment of 5q spinal muscular atrophy (SMA) in patients with a clinical diagnosis of SMA Type 1, Type 2 or Type 3 or with one to four SMN2 copies. Â

---

Mechanism of Action
Spinal muscular atrophy (SMA) is a severe and progressive congenital neuromuscular disease resulting from mutations in the survival of motor neuron 1 (_SMN1_) gene responsible for making SMN proteins. Clinical features of SMA include degeneration of motor neurons in the spinal cord which ultimately leads to muscular atrophy and, in some cases, loss of physical strength. SMN proteins are expressed ubiquitously throughout the body and are thought to hold diverse intracellular roles in DNA repair, cell signaling, endocytosis, and autophagy. A secondary _SMN_ gene (_SMN2_) can also produce SMN proteins, but a small nucleotide substitution in its sequence results in the exclusion of exon 7 during splicing in approximately 85% of the transcripts - this means that only ~15% of the SMN proteins produced by _SMN2_ are functional, which is insufficient to compensate for the deficits caused by _SMN1_ mutations. Emerging evidence suggests that many cells and tissues are selectively vulnerable to reduced SMN concentrations, making this protein a desirable target in the treatment of SMA. Risdiplam is an mRNA splicing modifier for _SMN2_ that increases the inclusion of exon 7 during splicing, which ultimately increases the amount of functional SMN protein produced by _SMN2_. It does so by binding to two sites in _SMN2_ pre-mRNA: the 5' splice site (5'ss) of intron 7 and the exonic splicing enhancer 2 (ESE2) of exon 7.

---

Pharmacodynamics
Risdiplam helps to alleviate symptoms of spinal muscular atrophy by stimulating the production of a critical protein in which these patients are deficient. Early trials with risdiplam demonstrated up to a 2-fold increase in SMN protein concentration in SMA patients after 12 weeks of therapy.

C22H23N7O

401.464323282242

401.2

C, 65.82; H, 5.77; N, 24.42; O, 3.99

1825352-65-5

Risdiplam-d4

118513932

White to yellow solid powder

0.5

78.1

1

6

2

30

886

0

ASKZRYGFUPSJPN-UHFFFAOYSA-N

InChI=1S/C22H23N7O/c1-14-9-18(26-29-11-15(2)24-21(14)29)17-10-20(30)28-12-16(3-4-19(28)25-17)27-8-7-23-22(13-27)5-6-22/h3-4,9-12,23H,5-8,13H2,1-2H3

7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[1,2-b]pyridazin-6-yl)pyrido[1,2-a]pyrimidin-4-one

RG7916; RO703406; RG-7916; RO-7034067; Risdiplam; 1825352-65-5; Evrysdi; Risdiplam [INN]; RG 7916; RO 7034067; Evrysdi

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: ~1.7 mg/mL (~4.2 mM)
Ethanol: < 1 mg/mL
H2O: < 0.1 mg/mL

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

---

注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

View More

注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

---

口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

View More

口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

---

注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

---

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。