GSK2 and GSK3 have the same ability to stop PCP-induced reversal learning deficits as lamotrigine, indicating that they may be used to treat cognitive symptoms of schizophrenia. Nonetheless, greater dosages than those needed for the medication's anticonvulsant efficacy are necessary for the reversal learning model to be active, indicating a narrow therapeutic window in comparison to the mechanism-dependent central adverse effects of this indication. The US Food and medicine Administration designated raxatrigine (GSK-1014802) as an orphan medicine in July 2013.

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The Nav1.7 channel represents a promising target for pain relief. In the recent decades, a number of Nav1.7 channel inhibitors have been developed. According to the effects on channel kinetics, these inhibitors could be divided into two major classes: reducing activation or enhancing inactivation. To date, however, only several inhibitors have moved forward into phase 2 clinical trials and most of them display a less than ideal analgesic efficacy, thus intensifying the controversy regarding if an ideal candidate should preferentially affect the activation or inactivation state. In the present study, we investigated the action mechanisms of a recently clinically confirmed inhibitor Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) using both electrophysiology and site-directed mutagenesis. We found that CNV1014802 inhibited Nav1.7 channels through stabilizing a nonconductive inactivated state. When the cells expressing Nav1.7 channels were hold at 70 mV or 120 mV, the half maximal inhibitory concentration (IC50) values (with 95% confidence limits) were 1.77 (1.20-2.33) and 71.66 (46.85-96.48) μmol/L, respectively. This drug caused dramatic hyperpolarizing shift of channel inactivation but did not affect activation. Moreover, CNV1014802 accelerated the onset of inactivation and delayed the recovery from inactivation. Notably, application of CNV1014802 (30 μmol/L) could rescue the Nav1.7 mutations expressed in CHO cells that cause paroxysmal extreme pain disorder (PEPD), thereby restoring the impaired inactivation to those of the wild-type channel. Our study demonstrates that CNV1014802 enhances the inactivation but does not reduce the activation of Nav1.7 channels, suggesting that identifying inhibitors that preferentially affect inactivation is a promising approach for developing drugs targeting Nav1.7 [2].

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Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) exerts a state-dependent inhibition on Nav1.7 channels. [2]
Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) does not affect steady-state activation. [2]
Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) causes a hyperpolarizing shift in the steady-state inactivation. [2]
Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) shows use-dependent inhibition of Nav1.7 channels. [2]
Influences of Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) on the development of inactivation. [2]
Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) slows the recovery from inactivation. [2]
Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) normalizes the functional effects of PEPD mutations. [2]

Sodium channel inhibition is a well precedented mechanism used to treat epilepsy and other hyperexcitability disorders. The established sodium channel blocker and broad-spectrum anticonvulsant lamotrigine is also effective in the treatment of bipolar disorder and has been evaluated in patients with schizophrenia. Double-blind placebo-controlled clinical trials found that the drug has potential to reduce cognitive symptoms of the disorder. However, because of compound-related side-effects and the need for dose titration, a conclusive evaluation of the drug's efficacy in patients with schizophrenia has not been possible. (5R)-5-(4-{[(2-Fluorophenyl)methyl]oxy}phenyl)-l-prolinamide (GSK2) and (2R,5R)-2-(4-{[(2-fluorophenyl)methyl]oxy}phenyl)-7-methyl-1,7-diazaspiro[4.4]nonan-6-one (GSK3) are two new structurally diverse sodium channel blockers with potent anticonvulsant activity. In this series of studies in the rat, we compared the efficacy of the two new molecules to prevent a cognitive deficit induced by the N-methyl-d-aspartic acid receptor antagonist phencyclidine (PCP) in the reversal-learning paradigm in the rat. We also explored the effects of the drugs to prevent brain activation and neurochemical effects of PCP. We found that, like lamotrigine, both GSK2 and GSK3 were able to prevent the deficit in reversal learning produced by PCP, thus confirming their potential in the treatment of cognitive symptoms of schizophrenia. However, higher doses than those required for anticonvulsant efficacy of the drugs were needed for activity in the reversal-learning model, suggesting a lower therapeutic window relative to mechanism-dependent central side effects for this indication. [1]

Electrophysiology [2]
Whole-cell patch-clamp recordings were conducted at room temperature using an Axopatch 200B patch clamp amplifier. Pipettes were pulled from borosilicate glass capillaries with an electrode resistance typically ranging from 1.5 to 4 MΩ. The recording pipette intracellular solution contained the following (in mmol/L): 140 CsF, 10 NaCl, 10 HEPES, 1.1 EGTA and 20 glucose (pH 7.3 adjusted by CsOH); the bath or extracellular solution contained the following (in mmol/L): 140 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES and 20 glucose (pH 7.3 adjusted by NaOH). During the recording, the bath solution was continuously perfused using a BPS perfusion system. Recording was performed after a 5-min equilibration period at −80 mV after breaking into the whole-cell configuration. Currents were acquired at a 50-kHz sampling frequency and filtered at 2 kHz. Series resistance compensation was used and set to 80%. P/N subtraction was never applied throughout the experiment. An unsaturated IC90 concentration (10 μmol/L) was applied throughout the whole study unless otherwise stated. If a change in activation was not observed at 10 μmol/L, a higher concentration of the drug (30 μmol/L) was further administered to confirm the lack of an effect. This higher concentration was also used in the parallel experiments, such as the inactivation recordings.

Cell culture and transfection [2]
Human embryonic kidney 293 (HEK293) cells stably expressing hNav1.7 were used. Cells were grown in high-glucose DMEM supplemented with 10% fetal bovine serum and were selected with 300 μg/mL of the antibiotic Hygromycin B under standard tissue culture conditions (5% CO2, 37 °C). For the functional expression of Nav1.7 mutants, Chinese hamster ovary (CHO) cells were used and cultured in 50/50 DMEM/F-12 supplemented with 10% fetal bovine serum. Two days prior to recording, the constructs were transfected into CHO cells with Lipofectamine reagent, according to the manufacture's protocol. A GFP construct was co-transfected to aid in the identification of transfected cells by fluorescence microscopy. Cells were seeded onto poly-L-lysine-coated glass coverslips before they were used for electrophysiology recording.

Single ascending dose study procedures[3]
This was a double‐blind crossover study conducted at a single clinical site from May 2007 to May 2008. Volunteers and all site personnel were blinded to study treatment allocation but sponsor personnel were unblinded to assist with appropriate dose selection decisions. Eligible volunteers were healthy men aged 18–65 years or healthy women with no childbearing potential aged 18–50 years. Volunteers were also required to be nonsmokers and have a body weight of > 50 kg and body mass index of 19–29.9 kg/m2 (± 10%). Exclusion criteria included significant abnormalities found on clinical examination, or clinical chemistry or hematology parameters. The sample size of 10 participants per cohort is a commonly used number in early studies. [3]

The steps recommended in the US Food and Drug Administration’s Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers were followed for the estimation of the starting dose. On normalizing the experimentally determined nontoxic dosage level for surface area, the most sensitive preclinical animal species examined was the dog. Using the conversion factor provided in the guidance, the nontoxic dosage level of 70 mg/kg/day in the dog translates to a human equivalent dose of 2,333 mg/day for a 60 kg human. Dividing this value by a conservatively estimated safety factor of 10, the maximum recommended starting dose of Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) was determined to be 233 mg/day; however, the results observed using Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) in pain models indicate that a pharmacologically active dosage of Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) is 1 mg/kg. This efficacious dose is expected to translate to a predicted clinical dose of 10 mg/day in a 60 kg human; thus, a starting dose of 10 mg q.d. was selected. [3]

Volunteers were recruited into 3 cohorts of 10 and treated with a starting dose of Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) 10 mg or placebo in cohort 1 (Figure S1 ). In each dosing session, 8 volunteers received vixotrigine and 2 volunteers received placebo, except cohort 2, dosing session 3 (vixotrigine, n = 4; placebo, n = 6). The highest vixotrigine dose tested in 1 cohort was the initial dose in the subsequent cohort (Figure S2 ). Vixotrigine doses were escalated up to 825 mg, until predefined safety or PK stopping limits were reached. Plasma samples were taken at baseline and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 72 hours after dosing. Each volunteer received a maximum of 4 vixotrigine doses and 1 placebo dose over 5 dosing sessions, with the exception of cohort 2 (2 vixotrigine doses and 1 placebo dose over 3 dosing sessions). Each session was separated by a ≥ 7‐day washout period. Volunteers attended a follow‐up visit ~ 7–14 days following the last dose of study medication. [3]

The primary endpoints of the SAD study were to (i) evaluate Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) safety and tolerability assessed through adverse events (AEs), vital signs (blood pressure, heart rate, and respiration rate), clinical laboratory evaluations (hematology, clinical chemistry, and urinalysis), and 12‐lead electrocardiograms (ECGs); and (ii) evaluate the following vixotrigine PK parameters: area under the concentration‐time curve from time 0 (predose) extrapolated to infinite time (AUC0–inf), AUC from predose to last time of quantifiable concentration (AUC0–t), maximum observed concentration (Cmax), and time to Cmax (Tmax). Dose proportionality of AUC0–inf, AUC0–t, and Cmax across doses was investigated by a power model fitted by restricted maximum likelihood method, with log(dose) fitted as covariate. The intercept for volunteers was fitted as a random effect. Estimated mean slope (β) and 90% confidence intervals were constructed for each parameter. [3]

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Multiple ascending dose study procedures[3]
This single‐blind study included 4 cohorts of parallel staggered doses. Eligible volunteers were healthy men or healthy women with no childbearing potential aged 18–55 years, and a body weight ≥ 50 kg and body mass index ≥ 19 kg/m2 and ≤ 29 kg/m2. No significant abnormalities on clinical examination or through evaluation of clinical chemistry or hematology parameters were permitted. [3]

Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) was supplied as 50, 100, or 200 mg film‐coated brownish yellow tablets. Placebo tablets visually matched the active tablets and all tablets were taken with 240 mL of water. Twelve volunteers in each of 4 parallel‐dose cohorts were randomized to vixotrigine or placebo in a 9:3 ratio. For all cohorts, a screening phase preceded study treatment and a follow‐up visit was conducted 7–14 days after the last dose. Cohort 1 received one 14‐day repeat‐dose phase (vixotrigine 150 mg q.d. or placebo). Cohort 2 received one 14‐day repeat‐dose phase (vixotrigine 400 mg q.d. or placebo). An additional dose of study drug was administered on day 15, within 30 minutes of consuming a high‐fat breakfast. Cohort 3 received an SD and 28‐day repeat‐dose of vixotrigine 300–400 mg b.i.d. (doses individually adjusted to keep the AUC below the originally defined PK limits) or placebo, with a morning dose given for the SD and on day 28 of the repeat‐dose period. Cohort 4 received an SD and 14‐day repeat‐dose of vixotrigine 350–450 mg b.i.d. (doses individually adjusted to keep below the PK limits for Cmax and AUC) or placebo, with a morning dose given for the SD and on day 15 of the repeat‐dose period. In addition, an assessment of exploratory endpoints (mechanical pain threshold, and pressure pain threshold and tolerance) was completed after day 1 and day 15 SD (see Supplementary Material Table S1 and Figure S3 ). Volunteers in cohorts 3 and 4 received the same treatment allocation (vixotrigine or placebo) in the SD and repeat‐dose periods, which were separated by ≥ 7 days. Predose blood samples were drawn and at prespecified time points to measure plasma vixotrigine levels (0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours; cohorts 1 and 4: days 1, 7, and 14; cohort 2: days 1, 7, 14, and 15; cohort 3: days 1, 7, 14, and 28). [3]

The primary endpoints of the repeat‐dose study were (i) to evaluate the safety and tolerability of Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) by monitoring AEs and concomitant medication, 12‐lead ECGs, lead II monitoring, 24‐hour Holter monitoring, vital signs, and laboratory parameters; and (ii) PK parameters estimated from plasma concentration‐time profiles for each analyte: Cmax, Tmax, and AUC from time 0 (predose) to 24 hours after dosing (AUC0–24; q.d. dose), AUC from time 0 (predose) to 12 hours after dosing (b.i.d. dose), and terminal half‐life (t 1/2). [3]

For both studies, plasma concentrations of Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) were determined using liquid chromatography‐tandem mass spectrometry after protein precipitation extraction, according to validated analytical methods at LGC. 17 The lower limit of quantification for vixotrigine was 10 ng/mL. PK parameters were derived using noncompartmental analyses with WinNonlin software version 5.0.1. Statistical analyses used SAS version 9.1.

Single ascending dose PK [3]
No quantifiable vixotrigine concentrations were reported in predose plasma samples, indicating no carryover between dosing periods. Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) was rapidly and extensively absorbed, with Cmax generally achieved at 1–2 hours postdose. Dose proportionality was approximate (Figure S4); although statistical significance was not confirmed for vixotrigine 10–825 mg, AUC0–inf showed no relevant deviation from dose proportionality (estimate of the slope from the power model, 1.088). The deviation from dose proportionality for Cmax was larger but still of limited importance (slope, 1.202). Following the maximal vixotrigine dose for this study (825 mg), Cmax and AUC0–inf were 6.53 μg/mL and 66.2 μg*h/mL, respectively (Figure 1). The estimated values for oral clearance and volume of distribution were 13.8 L/hr and 262 L, respectively. The concentration of vixotrigine increased with dose (Figure 2) and there were no dose‐dependent changes in total clearance of vixotrigine from plasma or volume of distribution, indicating linear kinetics. Vixotrigine appeared to have moderate plasma clearance and tissue distribution, with a t 1/2 of ~ 11 hours (Table 1).

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Multiple ascending dose PK [3]
Repeat‐dose PK parameters for Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) are summarized in Table 2. PK characteristics of single oral doses of vixotrigine 150–400 mg were in alignment with those reported in the SD study. Tmax was achieved in ~ 2 hours postdose and t 1/2 was 9–13 hours. Accumulation was observed following repeat vixotrigine doses; dose‐proportional increases in exposure, as measured by AUC0–24 and Cmax, were approximate (Figure S6). As expected, accumulation was higher after b.i.d. dosing by approximately twofold compared with q.d. dosing (Figure 3). Steady‐state of vixotrigine was generally achieved for all repeat‐dose regimens from day 5 onward. When administered a high‐fat meal, vixotrigine 400 mg q.d. AUC0–24 decreased by 3%, Cmax decreased by 15%, and Tmax was delayed by an average of 2.5 hours (Figure S5). Similar to SD PK, no dose‐dependent changes in oral clearance and volume of distribution were observed following repeat‐dose administration of vixotrigine.

Safety and tolerability [3]
Single ascending dose safety and tolerability [3]
In the SD study, Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) doses up to 825 mg were well‐tolerated in healthy volunteers (Table 3). Twenty‐three (77%) volunteers reported at least 1 AE. Dizziness was the most commonly reported AE (n = 11; 37%), with a higher incidence at higher vixotrigine doses (600 and 825 mg, reported by 4 of 10 (40%) and 5 of 7 (71%) volunteers, respectively). No other AEs appeared to increase with dose. Drug‐related AEs were reported by 14 (47%) volunteers (Table 3); dizziness was again the most commonly reported AE (n = 9; 30%). [3]
The majority of AEs following Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) administration were mild in nature, except for 8 events (4 for dizziness and 1 for each of somnolence, headache, diarrhea, and vasovagal syncope associated with sinus node pause) that were rated as moderate. No deaths, serious AEs, withdrawals due to an AE, drug‐related serious AEs, or clinically significant changes in ECG values or clinical laboratory evaluations were reported.

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Multiple ascending dose safety and tolerability [3]
Repeat Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) doses were well‐tolerated at all dose levels up to 450 mg in healthy volunteers (Table 4). An AE was reported by 11 (92%) placebo‐treated volunteers and by 9 (75%), 6 (67%), 9 (100%), and 7 (78%) volunteers treated with vixotrigine 150 mg q.d., 400 mg q.d., 300–400 mg b.i.d., and 350–450 mg b.i.d., respectively. Headache was the most commonly reported AE, with a similar incidence in vixotrigine‐treated and placebo‐treated volunteers. Any drug‐related AE was reported by 6 (50%), 3 (25%), 4 (44%), 8 (89%), and 6 (67%) volunteers across the placebo and vixotrigine 150 mg q.d., 400 mg q.d., 300–400 mg b.i.d., and 350–450 mg b.i.d. treatment groups, respectively. Dizziness was the most frequent drug‐related AE, reported by 1 (8%), 0, 2 (22%), 3 (33%), and 3 (33%) of the placebo and vixotrigine 150 mg q.d., 400 mg q.d., 300–400 mg b.i.d., and 350–450 mg b.i.d. treatment groups, respectively. [3]
All AEs were mild in nature, with the exception of 2 volunteers with vomiting of moderate intensity following placebo, and 2 volunteers with vomiting of moderate intensity following Raxatrigine (GSK1014802; Vixotrigine; CNV-1014802) (1 for each of the 400 mg q.d. and 300–400 mg b.i.d. groups) treatment. No deaths, severe AEs, serious drug‐related AEs, clinically significant abnormalities in ECG values or clinical laboratory evaluations, or withdrawals due to an AE were reported in the repeat‐dose study.

[1]. The efficacy of sodium channel blockers to prevent phencyclidine-induced cognitive dysfunction in the rat: potential for novel treatments for schizophrenia. J Pharmacol Exp Ther. 2011 Jul;338(1):100-13.

[2]. Enhancing inactivation rather than reducing activation of Nav1.7 channels by a clinically effective analgesic CNV1014802. Acta Pharmacol Sin . 2018 Apr;39(4):587-596.

[3]. Safety, Tolerability and Pharmacokinetics of Single and Repeat Doses of Vixotrigine in Healthy Volunteers. Clin Transl Sci. 2020 Dec 13;14(4):1272–1279.

Vixotrigine has been investigated for the treatment of Bipolar Disorder and Bipolar Depression.

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Neuropathic pain affects ~ 6.9–10% of the general population and leads to loss of function, anxiety, depression, sleep disturbance, and impaired cognition. Here, we report the safety, tolerability, and pharmacokinetics of a voltage‐dependent and use‐dependent sodium channel blocker, vixotrigine, currently under investigation for the treatment of neuropathic pain conditions. The randomized, placebo‐controlled, phase I clinical trials were split into single ascending dose (SAD) and multiple ascending dose (MAD) studies. Healthy volunteers received oral vixotrigine as either single doses followed by a ≥ 7‐day washout period for up to 5 dosing sessions (SAD, n = 30), or repeat doses (once or twice daily) for 14 and 28 days (MAD, n = 51). Adverse events (AEs), maximum observed vixotrigine plasma concentration (Cmax), area under the concentration‐time curve from predose to 24 hours postdose (AUC0–24), time to Cmax (Tmax), and terminal half‐life (t 1/2), among others, were assessed. Drug‐related AEs were reported in 47% and 53% of volunteers in the SAD and MAD studies, respectively, with dizziness as the most commonly reported drug‐related AE. SAD results showed that Cmax and AUC increased with dose, Tmax was 1–2 hours, and t 1/2 was ~ 11 hours. A twofold increase in accumulation was observed when vixotrigine was taken twice vs. once daily (MAD). Steady‐state was achieved from day 5 onward. These data indicate that oral vixotrigine is well‐tolerated when administered as single doses up to 825 mg and multiple doses up to 450 mg twice daily.[3]

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The Nav1.7 channel represents a promising target for pain relief. In the recent decades, a number of Nav1.7 channel inhibitors have been developed. According to the effects on channel kinetics, these inhibitors could be divided into two major classes: reducing activation or enhancing inactivation. To date, however, only several inhibitors have moved forward into phase 2 clinical trials and most of them display a less than ideal analgesic efficacy, thus intensifying the controversy regarding if an ideal candidate should preferentially affect the activation or inactivation state. In the present study, we investigated the action mechanisms of a recently clinically confirmed inhibitor CNV1014802 using both electrophysiology and site-directed mutagenesis. We found that CNV1014802 inhibited Nav1.7 channels through stabilizing a nonconductive inactivated state. When the cells expressing Nav1.7 channels were hold at 70 mV or 120 mV, the half maximal inhibitory concentration (IC50) values (with 95% confidence limits) were 1.77 (1.20-2.33) and 71.66 (46.85-96.48) μmol/L, respectively. This drug caused dramatic hyperpolarizing shift of channel inactivation but did not affect activation. Moreover, CNV1014802 accelerated the onset of inactivation and delayed the recovery from inactivation. Notably, application of CNV1014802 (30 μmol/L) could rescue the Nav1.7 mutations expressed in CHO cells that cause paroxysmal extreme pain disorder (PEPD), thereby restoring the impaired inactivation to those of the wild-type channel. Our study demonstrates that CNV1014802 enhances the inactivation but does not reduce the activation of Nav1.7 channels, suggesting that identifying inhibitors that preferentially affect inactivation is a promising approach for developing drugs targeting Nav1.7.[2]

C18H20CLFN2O2

350.8150

350.12

C, 61.63; H, 5.75; Cl, 10.10; F, 5.42; N, 7.99; O, 9.12

934240-31-0

Raxatrigine;934240-30-9; Raxatrigine hydrochloride;934240-31-0; 934240-35-4 (mesylate)

16046067

White to off-white solid powder

4.514

3

4

5

24

399

2

C1C[C@H](N[C@H]1C2=CC=C(C=C2)OCC3=CC=CC=C3F)C(=O)N.Cl

HEPUBAGOKRIZEO-PPPUBMIESA-N

InChI=1S/C18H19FN2O2.ClH/c19-15-4-2-1-3-13(15)11-23-14-7-5-12(6-8-14)16-9-10-17(21-16)18(20)22/h1-8,16-17,21H,9-11H2,(H2,20,22)1H/t16-,17+/m1./s1

(2S,5R)-5-(4-((2-fluorobenzyl)oxy)phenyl)pyrrolidine-2-carboxamide hydrochloride

GSK1014802; GSK 1014802; GSK-1014802; CNV1014802; CNV-1014802; CNV 1014802; Raxatrigine; Raxatrigine hydrochloride; Vixotrigine; 934240-31-0; Raxatrigine HCl; (2S,5R)-5-(4-((2-Fluorobenzyl)oxy)phenyl)pyrrolidine-2-carboxamide hydrochloride; CNV1014802 hydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ≥ 31 mg/mL (~88.36 mM)
H2O : ~14.29 mg/mL (~40.73 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.13 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.13 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.13 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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