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]
\nRisdiplam 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.\n

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\nStudy design in monkeys (Studies 9 and 10) [1]
\nRisdiplam 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.\n\nAt 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.\n\nTissues 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).\n

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\nRat Quantitative Whole‐Body Autoradiography (QWBA) study design (Study 13) [1]
\n\nWistar 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.\n

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\nStudy design in mice (Studies 1, 2, 3, 4, 5, 11, and 12) [1]
\nTwo 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.\n\nFor 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.\n

Absorption, Distribution and Excretion
The time to peak concentration (Tmax) after oral administration is approximately 1–4 hours. Administered once daily with breakfast (or after breastfeeding), lixitaplan reaches steady-state plasma concentrations in approximately 7–14 days. In SMA patients, the pharmacokinetics of lixitaplan were approximately linear at all study doses. After oral administration of 18 mg lixitaplan, approximately 53% of the dose is excreted in feces and 28% in urine. Unmetabolized parenteral drug accounts for 14% of the fecal excretion and 8% of the urinary excretion. Following oral administration, lixitaplan distributes well in the central nervous system and peripheral tissues. The steady-state apparent volume of distribution is 6.3 L/kg. For a patient weighing 14.9 kg, the apparent clearance of lixitaplan is 6.3 L/kg. Metabolism/Metabolites The metabolism of lisparin is primarily mediated by flavin monooxygenases 1 and 3 (FMO1 and FMO3), with CYP1A1, CYP2J2, CYP3A4, and CYP3A7 also involved. The parent drug comprises approximately 83% of the total circulating drug. A pharmacologically inactive metabolite, M1, has been identified as the major circulating metabolite—in vitro studies have shown that M1 inhibits the MATE1 and MATE2-K transporters, similar to the parent drug. Biological Half-Life The terminal elimination half-life of lisparin in healthy adults is approximately 50 hours.

Protein Binding
Rixitalin has a protein binding rate of approximately 89% in plasma, primarily binding to serum albumin. Hepatotoxicity
In pre-registration clinical trials, rixitalin treatment did not cause clinically significant changes in serum laboratory parameters; there were no differences in serum ALT, AST, and bilirubin levels between the rixitalin and placebo groups. Although safety results are based on only a few hundred patients, no cases of suspected drug-induced liver injury with jaundice have been reported. Furthermore, since rixitalin's approval in 2020, there have been no published cases of clinically significant liver injury. Probability Score: E (Unlikely to be the cause of clinically significant 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 modulator used to treat spinal muscular atrophy (SMA). It increases systemic SMN protein concentration by enhancing the transcriptional efficiency of the SMN2 gene. Its mechanism of action is similar to its prodrug, nusinersen, with the main difference being the route of administration: nusinersen requires intrathecal injection, as does the one-time gene therapy onasemnogene abeparvovec, while risdiplam offers the convenience of high oral bioavailability. Risdiplam received FDA approval in August 2020 for the treatment of SMA. Compared to other existing SMA treatments, risdiplam is expected to be significantly cheaper, potentially providing a novel and relatively accessible treatment option for patients with SMA of various severity and types. Risdiplam is a motor neuron survival 2 splicing modulator. Lisparan's mechanism of action is as a motor neuron survival 2 splicing regulator, a multidrug and toxin efflux transporter 1 (SMN1) inhibitor, and a MMN2K inhibitor. Lisparan's physiological effects are achieved by increasing protein synthesis. Indications: Lisparan is indicated for the treatment of spinal muscular atrophy (SMA). Everstrand is indicated for the treatment of 5q spinal muscular atrophy (SMA) in patients clinically diagnosed with SMA type 1, 2, or 3, or who carry 1 to 4 copies of the SMN2 gene. Mechanism of Action: Spinal muscular atrophy (SMA) is a severe, progressive, congenital neuromuscular disease caused by mutations in the motor neuron survival gene 1 (SMN1), which is responsible for the synthesis of SMN proteins. Clinical features of SMA include the degeneration of spinal motor neurons, ultimately leading to muscle atrophy and, in some cases, loss of muscle strength. SMN proteins are widely expressed throughout the body and are believed to play important roles in various intracellular processes, including DNA repair, cell signaling, endocytosis, and autophagy. A second SMN gene (SMN2) also produces SMN proteins, but a small nucleotide substitution in its sequence results in the deletion of exon 7 in approximately 85% of the transcripts during splicing—meaning that only about 15% of the SMN proteins produced by SMN2 are functional, insufficient to compensate for the defects caused by the SMN1 mutation. Increasing evidence suggests that many cells and tissues are selectively sensitive to reduced SMN concentrations, making SMN proteins an ideal target for the treatment of spinal muscular atrophy (SMA). Risdiplam is an SMN2 mRNA splicing modifier that increases the inclusion of exon 7 during splicing, ultimately increasing the amount of functional SMN protein produced by SMN2. It works by binding to two sites on the SMN2 precursor mRNA: the 5' splice site (5'ss) of intron 7 and exon splicing enhancer 2 (ESE2) of exon 7.

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Pharmacodynamics
Lixitabram helps alleviate the symptoms of spinal muscular atrophy (SMA) by stimulating the production of a key protein in the body. Early lixitabram trials showed that after 12 weeks of treatment, the concentration of SMN protein in SMA patients could increase by up to 2 times.

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.)