Sodium channel Nav1.7

GSK2 和 GSK3 具有与拉莫三嗪相同的阻止 PCP 诱导的逆转性学习缺陷的能力，表明它们可用于治疗精神分裂症的认知症状。尽管如此，要使逆转学习模型活跃，需要比药物抗惊厥功效所需的剂量更大的剂量，这表明与该适应症的机制依赖性中心不良反应相比，治疗窗口较窄。美国食品药品监督管理局于 2013 年 7 月将Raxatrigin（GSK1014802；Vixotrigin；CNV-1014802）/雷沙三嗪 (GSK-1014802) 指定为孤儿药。

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Nav1.7通道是一个有前景的止痛靶点。近几十年来，已经开发了许多Nav1.7通道抑制剂。根据对通道动力学的影响，这些抑制剂可分为两大类：减少活化或增强失活。然而，迄今为止，只有几种抑制剂进入了2期临床试验，其中大多数表现出不太理想的镇痛效果，从而加剧了关于理想候选药物是否应优先影响激活或失活状态的争议。在本研究中，我们使用电生理学和定点突变研究了最近临床证实的抑制剂Raxatrigin（GSK1014802；Vixotrigin；CNV-1014802）的作用机制。我们发现CNV1014802通过稳定非导电失活状态抑制Nav1.7通道。当表达Nav1.7通道的细胞保持在70 mV或120 mV时，半数最大抑制浓度（IC50）值（95%置信区间）分别为1.77（1.20-2.33）和71.66（46.85-96.48）μmol/L。该药物引起通道失活的剧烈超极化转变，但不影响激活。此外，CNV1014802加速了失活的开始，并延迟了失活后的恢复。值得注意的是，应用CNV1014802（30μmol/L）可以挽救CHO细胞中表达的Nav1.7突变，这些突变会导致阵发性极度疼痛障碍（PEPD），从而将受损的失活恢复到野生型通道的失活状态。我们的研究表明，CNV1014802增强了Nav1.7通道的失活，但不会减少其激活，这表明鉴定优先影响失活的抑制剂是开发靶向Nav1.7药物的一种有前景的方法。[2]

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Raxatrigin（GSK1014802；Vixotrigin；CNV-1014802）对Nav1.7通道具有状态依赖性抑制作用。[2]
Raxatrigin（GSK1014802；Vixotrigin；CNV-1014802）不影响稳态激活。[2]
Raxatrigin（GSK1014802；Vixotrigin；CNV-1014802）导致稳态失活的超极化偏移。[2]
Raxatrigin（GSK1014802；Vixotrigin；CNV-1014802）显示出Nav1.7通道的使用依赖性抑制。[2]
Raxatrigin（GSK1014802；Vixotrigin；CNV-1014802）对失活发展的影响。[2]
Raxatrigin（GSK1014802；Vixotrigin；CNV-1014802）减缓失活的恢复。[2]
Raxatrigin（GSK1014802；Vixotrigin；CNV-1014802）使PEPD突变的功能效应正常化。[2]

钠通道抑制是一个很好的先例机制，用于治疗癫痫和其他高兴奋性障碍。已建立的钠通道阻滞剂和广谱抗惊厥药拉莫三嗪对治疗双相情感障碍也有效，并已在精神分裂症患者中进行了评估。双盲安慰剂对照临床试验发现，该药有可能减轻认知障碍的症状。然而，由于与化合物相关的副作用和剂量滴定的需要，无法对该药物对精神分裂症患者的疗效进行结论性评估。（5R）-5-（4-{（2-氟苯基）甲基]氧}苯基）-l-脯氨酸酰胺（GSK2）和(2R,5R)-2-（4-{（2-氟苯基）甲基]氧}苯基）-7-甲基-1,7-重氮斯匹罗[4.4]壬胺-6-酮（GSK3）是两种结构多样的新型钠通道阻滞剂，具有较强的抗惊厥活性。在这一系列的大鼠研究中，我们比较了两种新分子在大鼠逆向学习范式中预防n -甲基-d-天冬氨酸受体拮抗剂苯环利定（phencyclidine， PCP）诱导的认知缺陷的功效。我们还探讨了药物对PCP的脑活化和神经化学作用的影响。我们发现，像拉莫三嗪一样，GSK2和GSK3都能够预防PCP产生的逆转学习缺陷，从而证实了它们在治疗精神分裂症认知症状方面的潜力。然而，在反向学习模型中，需要比抗惊厥药物疗效所需的剂量更高的剂量，这表明相对于机制依赖的中枢副作用，该适应症的治疗窗口较低。[1]

电生理学[2]
使用Axopatch 200B膜片钳放大器在室温下进行全细胞膜片钳记录。移液管取自硼硅酸盐玻璃毛细管，电极电阻通常在1.5至4 MΩ之间。记录移液管细胞内溶液含有以下物质（单位为mmol/L）：140 CsF、10 NaCl、10 HEPES、1.1 EGTA和20葡萄糖（用CsOH调节pH 7.3）；浴或细胞外溶液含有以下物质（单位为mmol/L）：140 NaCl、3 KCl、1 MgCl2、1 CaCl2、10 HEPES和20葡萄糖（用NaOH调节pH 7.3）。在记录过程中，使用BPS灌注系统连续灌注浴液。在打破整个电池配置后，在-80 mV下平衡5分钟后进行记录。以50kHz的采样频率采集电流，并以2kHz的频率进行滤波。使用串联电阻补偿，并将其设置为80%。在整个实验过程中从未应用P/N减法。除非另有说明，否则在整个研究过程中应用不饱和IC90浓度（10μmol/L）。如果在10μmol/L时没有观察到活化变化，则进一步施用更高浓度的药物（30μmol/L）以确认没有效果。这种更高的浓度也用于平行实验，如灭活记录。

细胞培养和转染[2]
使用稳定表达hNav1.7的人胚胎肾293（HEK293）细胞。细胞在添加了10%胎牛血清的高糖DMEM中生长，并在标准组织培养条件下（5%CO2，37°C）用300μg/mL的抗生素潮霉素B进行选择。对于Nav1.7突变体的功能表达，使用中国仓鼠卵巢（CHO）细胞，并在添加了10%胎牛血清的50/50 DMEM/F-12中培养。根据制造商的方案，在记录前两天，用Lipofectamine试剂将构建体转染到CHO细胞中。共转染GFP构建体，以帮助通过荧光显微镜鉴定转染的细胞。在用于电生理记录之前，将细胞接种到聚-L-赖氨酸涂层玻璃盖玻片上。

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]

C18H19FN2O2

314.3541

314.143

C, 68.77; H, 6.09; F, 6.04; N, 8.91; O, 10.18

934240-30-9

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

16046068

White to off-white solid powder

4.161

65.34

2

4

5

23

399

2

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

JESCETIFNOFKEU-SJORKVTESA-N

InChI=1S/C18H19FN2O2/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)/t16-,17+/m1/s1

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

CNV1014802; CNV-1014802; CNV 1014802; 934240-30-9; Vixotrigine; GSK1014802; BIIB074; GSK-1014802; GSK1014802; GSK 1014802; GSK-1014802; Raxatrigine.

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 : ~83 mg/mL (~264.04 mM)

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。

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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。

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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)

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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。

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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

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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。

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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。