SMN2

Risdiplam raises the amounts of SMN protein and controls the splicing of SMN2 pre-mRNA to generate full-length SMN2 mRNA. Risdiplam is a splicing modulator of SMN2 that can enhance the amount of full-length SMN2 protein, enhancing the functionality of SMN proteins. The most frequent genetic illness that kills infants is still (SMA). Due to doublets in the southeastern motor neuron 1 (SMN1) gene, low levels of early motor neuron protein (SMN) are the cause of this autosomal recessive neuropathy disorder, which is characterized by progressive movement and respiratory muscle weakening. Other sources of residual inactivation and gene loss [1].

To further explore risdiplam distribution, researchers assessed in vitro characteristics and in vivo drug levels and effect of risdiplam on SMN protein expression in different tissues in animal models. Total drug levels were similar in plasma, muscle, and brain of mice (n = 90), rats (n = 148), and monkeys (n = 24). As expected mechanistically based on its high passive permeability and not being a human multidrug resistance protein 1 substrate, risdiplam CSF levels reflected free compound concentration in plasma in monkeys. Tissue distribution remained unchanged when monkeys received risdiplam once daily for 39 weeks. A parallel dose-dependent increase in SMN protein levels was seen in CNS and peripheral tissues in two SMA mouse models dosed with risdiplam. These in vitro and in vivo preclinical data strongly suggest that functional SMN protein increases seen in patients' blood following risdiplam treatment should reflect similar increases in functional SMN protein in the CNS, muscle, and other peripheral tissues. [1]

In vitro transport assay [1]
Parent porcine kidney epithelial LLC PK1 (Lewis Lung Cancer Porcine Kidney 1) and canine kidney epithelial MDCKII cell lines were used. LLC‐PK1, MDCKII, L‐MDR1 (LLC‐PK1 cells transfected with human MDR1), L‐Mdr1a (LLC‐PK1 cells transfected with rodent Mdr1a), M‐BCRP (MDCKII cells transfected with human Breast Cancer Resistance Protein; BCRP), and M‐Bcrp1 (MDCKII cells transfected with rodent Bcrp1) cell lines were used under a license agreement. The rodent Mdr1a is a murine protein and shares 95% amino acid sequence identity with the rat Mdr1a, henceforth referred as “rodent” Mdr1a throughout the manuscript. The assays were conducted as previously described. Briefly, cells were cultured on semipermeable 96‐well inserts (surface area 0.11 cm2, pore size 0.4 mm; Millipore), and bidirectional transport measurements were performed either at Day 3 or 4 after seeding. The medium was removed from the apical (100 mL) and basolateral (240 mL) compartments and replaced on the receiver side by culture medium without phenol red and with or without inhibitor (zosuquidar, 1 μM for L‐MDR1 and L‐Mdr1a; Ko143, 1 μM for M‐BCRP and M‐Bcrp). Transcellular transport was initiated by the addition of media to the donor compartment containing test substrate (risdiplam or RG7800, tested at 1 μM) and 10 μM Lucifer yellow. Lucifer yellow was included to confirm monolayer integrity and reference substrates were incubated as controls to MDR1/Mdr1a or BCRP/Bcrp activity. Plates were incubated for 3.5 hours at 37°C and 5% CO2 under continuous shaking (100 rpm). Samples (triplicates for each condition) were taken from the donor and receiver compartments and analyzed by scintillation counting or high‐performance liquid chromatography with tandem mass spectrometry, as previously described for liquid chromatography, 10ADvp pump system coupled with a PAL HTS auto‐sampler was used, and for MS, API 4000 or QTrap4000 system equipped with a TurboIonspray source.

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.

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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).

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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.

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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.

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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.

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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.

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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.

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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

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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.

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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

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)