VDM11   中文名称: (5Z,8Z,11Z,14Z)-N-(4-羟基-2-甲基苯基)-5,8,11,14-二十碳四烯酰胺

Anandamide membrane transporter (AMT)

VR1香草酸（辣椒素）受体的一些合成激动剂也抑制内源性大麻素anandamide（花生四烯酸乙醇酰胺，AEA）向细胞内的促进转运。在这里，我们测试了几种含有各种衍生苯基或不同烷基链的AEA衍生物，作为完整细胞中AEA膜转运蛋白（AMT）的抑制剂，或转染人VR1的HEK细胞中VR1香草酸受体的功能激动剂。我们发现，四种已知的AMT抑制剂AM404、arvanil、olvanil和linvanil在比抑制AMT所需浓度低400-10000倍的浓度下激活VR1受体。然而，我们还发现了三种新的AEA衍生物，分别命名为VDM11、VDM12和VDM13，它们对AMT的抑制作用与AM404一样强，但在hVR1上几乎没有激动剂活性。这些化合物是AEA酶水解的弱抑制剂，CB（1）/CB（2）受体配体较差。我们首次表明，尽管AMT抑制剂和VR1激动剂的化学部分存在重叠，但可以开发出不激活VR1的选择性AEA摄取抑制剂（例如VDM11）[1]。

关于摄取抑制剂VDM11（N-（4-羟基-2-甲基苯基）花生四烯酸酰胺）在多大程度上能够通过脂肪酸酰胺水解酶（FAAH）抑制内源性大麻素anandamide（AEA）的代谢，存在一些争议。鉴于最近的一项研究表明，密切相关的化合物AM404（N-（4-羟基苯基）花生四酰亚胺）是FAAH的底物，我们重新研究了VDM11与FAAH的相互作用。[1]
在无脂肪酸的牛血清白蛋白（BSA，0.125%w v−1）存在的情况下，AM404和VDM11均以相似的效力抑制了大鼠脑FAAH对AEA的代谢（IC50值分别为2.1和2.6μM）。这些化合物作为细胞溶质单酰基甘油脂肪酶（MAGL）代谢2-油酰甘油（2-OG）的抑制剂的效力低约10倍。[1]
VDM11对FAAH的效力取决于无脂肪酸牛血清白蛋白（BSA）的测定浓度。因此，在没有不含脂肪酸的BSA的情况下，抑制FAAH的IC50值降低了约两倍（从2.9μM降至1.6μM）。该化合物（从14μM降至6μM）和花生四烯酸丝氨酸醇（从24μM降至13μM）抑制膜结合MAGL的IC50值也出现了类似的降低。[1]
建立了一种高效液相色谱法来测定4-氨基间甲酚，这是FAAH催化VDM11水解的假设产物。4-氨基间甲酚的洗脱保留时间约为2.4分钟，但对化合物的降解呈时间依赖性，洗脱峰分别为5.6和8 min。在用VDM11孵育膜后，也发现了具有相同保留时间的峰值，但在加入VDM11之前，用FAAH抑制剂URB597（3′-氨基甲酰基联苯-3-基环己基氨基甲酸酯）和CAY10401（1-恶唑并[4,5-b]吡啶-2-基-9-十八碳炔-1-酮）预孵育膜时，没有发现峰值。VDM11的代谢率估计约为anandamide的15-20%。[1]
结论是，在这里使用的测定条件下，VDM11是FAAH的抑制剂，这种抑制可能至少部分是该化合物作为替代底物的结果[2]。

FAAH活性测定[1]
如前所述和其中引用的参考文献培养N18TG2细胞）。通过使用由N18TG2细胞制备的膜，在37°C下用50 mM Tris-HCl（pH 9）中浓度递增的化合物孵育30分钟，研究了化合物对[14C]AEA（6μM）酶水解的影响。在用2体积的CHCl3/CH3OH 2:1（体积比）提取孵育混合物后，通过闪烁计数水相来测量由[14C]AEA水解产生的[14C]乙醇胺。

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AEA转运蛋白测定[1]
通过修改之前描述的方法，研究了化合物对RBL-2H3细胞摄取AEA的影响，除了使用更高浓度（4μM）的[14C]AEA外，与所述方案类似。在存在或不存在不同浓度抑制剂的情况下，细胞在37°C下与[14C]AEA一起孵育5分钟。用CHCl3/CH3OH 2:1（体积比）提取后，通过闪烁计数测定培养基中残留的[14C]AEA，用作细胞吸收AEA的指标。我们也将相同的方案应用于C6大鼠胶质瘤细胞，这些细胞也含有AEA的膜转运蛋白。数据表示为用GraphPad计算的对AEA摄取产生50%抑制的浓度（IC50）。

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CB1和CB2受体结合试验[1]
通过使用[3H]SR141716A（0.4 nM，55 Ci/mmol）作为高亲和力配体，并采用之前描述的过滤技术，在冷冻雄性CD大鼠脑的膜制剂（0.4 mg/管）上，在100μM PMSF的存在下，对CB1受体进行置换分析。用1μM SR141716A计算特异性结合率为84.0%。CD大鼠的脾脏用于制备膜（0.4 mg/管），以使用[3H]WIN55212-2（0.8 nM，50.8 Ci/mmol）进行CB2结合测定，如前所述，并再次在100μM PMSF的存在下进行。用1μM HU-348计算特异性结合率为75.0%。在所有情况下，通过将Cheng-Prusoff方程应用于通过增加测试化合物的浓度来置换结合的放射性配体的IC50值来计算K i值。

Ca2+内流测定[1]
通过使用Fluo-3（一种选择性细胞内Ca2+荧光探针）测定这些物质对Ca2+流入细胞的影响。在实验前四天，将细胞转移到涂有聚-L-赖氨酸的六孔培养皿中，并在上述培养基中生长。实验当天，细胞（每孔50-60 000个）在25°C下用含0.04%Pluoronic的DMSO中的4μM Fluo-3甲酯装载2小时。装载后，用Tyrode pH=7.4洗涤细胞，并在连续混合下将胰蛋白酶悬浮在荧光检测器的试管中。在添加不同浓度的测试化合物之前和之后，通过在25°C（λEX=488nm，λEM=540nm）下测量细胞荧光进行实验。在试验化合物前30或10分钟分别加入辣椒素（1-5μM）或EGTA（4 mM）。数据表示为使用GraphPad软件计算的产生半最大效应的浓度（EC50）。通过将其与4μM离子霉素观察到的类似效果进行比较来确定其疗效。

Metabolic stability of VDM11 [2]
The membrane fractions (100 μg of protein) from the cerebella of adult Sprague–Dawley rats were incubated at 37°C in Tris-HCl buffer (10 mM, pH 7.2) containing either ethanol, 50–100 μM 4-amino-m-cresol or 400 μM VDM11 and incubated at 37°C for the times shown in the text and figures. Reactions were stopped by adding 400 μl chloroform: methanol (1 : 1 v v−1). The phases were separated by centrifugation (10 min, 2500 r.p.m.). When FAAH inhibitors were used, they were preincubated with the membranes for 15 min at 37°C prior to addition of 4-amino-m-cresol or VDM11. Aliquots (20 μl) of the methanol/buffer phase were then injected to the high-performance liquid chromatography (HPLC) system. The system used comprised of a pump, and a UV absorbance detector (Waters 486, France), which was set at 250 nm (4-amino-m-cresol shows highest UV absorbance at 250 nm). Chromatographic separations were performed with the Chromolith Performance RP-18e 4.6 × 100 mm column. The mobile phase consisted of water/acetonitrile (95 : 5 v v−1) and the flow rate was 2.0 ml min−1. Injection volume was 20 μl. In these conditions, unchanged 4-amino-m-cresol is detected with a retention time of ∼2.4 min and a detection limit of 20 ng, corresponding to a concentration of ∼8 μM.

Metabolic stability of VDM11 [2] FAAH-catalyzed hydrolysis of VDM11 is expected to produce arachidonic acid and 4-amino-m-cresol (Figure 1). Therefore, we established an HPLC detection method to detect 4-amino-m-cresol. Under the detection conditions used, the retention time of 4-amino-m-cresol was approximately 2.4 min (detection limit 20 ng). The expected peak was obtained by adding 100 μM 4-amino-m-cresol to the membrane preparation (100 μg protein) and detecting it immediately, as well as minor peaks at retention times of approximately 5.6 min and approximately 11 min (Figure 3a). A retention time of approximately 11 min was also observed for samples with ethanol carrier instead of 4-amino-m-cresol (data not shown). The gain was reduced to quantify the early peak. It was found that the ratio of the two peaks changed with the extension of sample incubation time. Therefore, after membrane incubation for 0, 10, 30, 60, and 120 minutes, the AUC values of the peak at approximately 5.6 minutes were 8%, 13%, 19%, 30%, and 42% of the AUC values of the peak at approximately 2.4 minutes, respectively (data not shown). A peak at approximately 5.6 minutes was also observed when the 4-amino-m-cresol solution used in the experiment was left to stand overnight at room temperature and then analyzed by high-performance liquid chromatography (HPLC) (data not shown), suggesting that this peak represents the non-enzymatic oxidation product of the compound. At the 120-minute time point, another peak was observed at a retention time of approximately 8 minutes (data not shown). In two independent experiments, the membrane fraction (100 μg protein) was incubated with 4-amino-m-cresol for 24 hours. In both cases, the major peak appeared at approximately 8 minutes (see Figure 3b). This peak was not observed when 4-amino-m-cresol was incubated with distilled water at 37°C for 24 hours; however, it was observed when incubated with Tris buffer at pH 7.2, further indicating that this peak is a non-enzymatic oxidation product. No such peak was observed in membranes incubated with water, buffer, or ethanol alone (data not shown). Pre-incubation of the membrane with 3 μM URB597 for 15 minutes before adding 4-amino-m-cresol did not affect the main peak (Figure 3c). After adding VDM11 (400 μM) to the membrane and incubating, no other significant peaks were observed except for a peak at approximately 11 minutes after incubation at 0 or 10 minutes (data not shown). However, peaks were observed at a retention time of approximately 2.4 minutes at 30 and 60 minutes of incubation, and a small additional peak appeared at approximately 5.4 minutes at 120 minutes (Figure 3d). Three samples were incubated with 400 μM VDM11 for 120 min and 180 min, respectively. The peak at approximately 2.4 min was quantified and compared with the peaks at approximately 2.4 min and approximately 5.4 min obtained by incubating the same samples with 50 μM 4-amino-m-cresol for 0 min and then extracting and measuring them. Based on these data, the degradation rate of VDM11 was determined to be 160 ± 12 pmol min⁻¹ mg protein⁻¹ (data not shown). For comparison, the hydrolysis rate of the control sample with 2 μM [³H]AEA in Table 1 was 560 ± 46 pmol min⁻¹ mg protein⁻¹.
While incubation of 1–3 hours is sufficient to demonstrate the metabolism of VDM11, the peak values are too small to be used for VDM11 inhibitor sensitivity studies. Therefore, a longer incubation time (24 hours) was used. Under these conditions, a clear peak was observed at the same retention time as when 4-amino-m-cresol was added to the sample (Figure 3e). These peaks were not observed when VDM was incubated with water or buffer at 37°C for 24 hours (data not shown); more importantly, these peaks were also not observed when the membrane was pre-incubated with 3 μM URB597 for 15 minutes before adding VDM11, followed by 24 hours of incubation (Figure 3f). Preliminary experiments indicate that 1 μM URB597 completely blocks the metabolism of VDM11, while residual metabolic activity was observed at lower concentrations (10 and 100 nM) of URB597 (data not shown). Although URB597 exhibits much higher selectivity for FAAH than for cannabinoid receptors and MAGL (Kathuria et al., 2003), it has been reported to inhibit other serine hydrolases (Lichtman et al., 2004), suggesting the possible existence of FAAH-independent URB597-sensitive activity involved in the metabolism of VDM11. Therefore, we tested the ability of a series of FAAH inhibitors shown in Table 1 to inhibit VDM11 metabolism. Given the high assay concentration of VDM11 relative to its potency against FAAH (and the predicted Km value), the inhibitory sensitivity of these compounds to VDM11 should be compared with the data for high concentrations of [3H]AEA shown in Table 1. The results are consistent with these data. Thus, CAY10401 produced a concentration-dependent inhibition of VDM11 metabolism, with complete blockade observed at concentrations of 1 μM (Figure 4), 10 μM, 50 μM, and 100 μM (data not shown). OTMK (100 μM) also reduced the peak production of VDM11 metabolism, while no significant inhibitory effect on VDM11 metabolism was observed at 3 μM or 30 μM arachidonic acid serotonin, or at 1 μM or 3 μM OTMK (data not shown). At the highest concentration used, none of the compounds affected the peak production of 4-aminom-cresol (data not shown).

[1]. Overlap between the ligand recognition properties of the anandamide transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake with negligible capsaicin-like activity. FEBS Lett. 2000 Oct 13;483(1):52-6.

[2]. Vandevoorde S, Fowler CJ. Inhibition of fatty acid amide hydrolase and monoacylglycerol lipase by the anandamide uptake inhibitor VDM11: evidence that VDM11 acts as an FAAH substrate. Br J Pharmacol. 2005 Aug;145(7):885-93.

While polyunsaturated fatty acid chains are a necessary prerequisite for the effective activation of hVR1, they are not sufficient on their own to achieve effective activation. In fact, we found that another endogenous ligand of the cannabinoid receptor, 2-arachidonic acid glyceride, and another AEA analog, arachidonic acid glycine, have almost no activity against the hVR1 receptor (Table 1). Interestingly, these two compounds also showed weak or no activity as [14C]AEA uptake inhibitors, highlighting the similarity in ligand recognition properties between AMT and hVR1. However, by introducing a methyl or hydroxyl group into AM404, we synthesized two compounds, VDM11 and VDM12, which, while still exhibiting strong AMT inhibitory activity (Table 1), showed either no activity or significantly reduced efficacy against hVR1 (Figure 1b). VDM11, an ortho-methyl derivative of AM404, at concentrations up to 40 μM, had almost no significant effect on Ca2+ influx in hVR1-transfected cells, but its inhibitory IC50 value for AMT was approximately 10–11 μM. The ortho-hydroxy derivative of AM404, VDM12, only induced an effective agonist response in hVR1-transfected cells at a concentration of 40 μM, while its agonist effect on hVR1 was very weak when the concentration reached the half-maximal inhibitory concentration (IC50) for [14C]AEA transport to the cell (Table 1). VDM13 (arachidonicyl-5-methoxytryptamine) did not activate VR1 at all, even at high doses, but its AMT potency was comparable to VDM11 and VDM12. Under the same experimental conditions, AM404 was comparable in potency as an AMT inhibitor to VDM11, VDM12, and VDM13 (Table 1). Furthermore, none of these three new compounds antagonized the effect of capsaicin on the hVR1 receptor (1 μM, pre-incubation for 30 min, data not shown). Therefore, VDM11, VDM12, and VDM13 exhibited significantly higher selectivity for AEA-promoted transport than vanillin receptors compared to AM404, the most widely used AMT inhibitor. We also evaluated the affinity of these novel compounds for FAAH and CB1/CB2 cannabinoid receptors. VDM11, VDM12, and VDM13 showed weak inhibition of [14C]AEA hydrolysis on the N18TG2 cell membrane (IC50 > 50 or 25 ± 1.6 μM and 27 ± 0.9 μM, respectively). Under the same conditions, AM404 was slightly more potent, with an IC50 of 22 ± 0.7 μM. Finally, in endocannabinoid receptor binding assays using rat meninges or spleens, VDM11, VDM12, and VDM13 all showed ligand displacement rates below 50% at concentrations up to 5–10 μM (Ki > 5–10 μM in all cases). Therefore, in addition to being inactive against the CB2 receptor, these compounds also exhibited weaker ligand activity against the CB1 receptor than AM404, arvanil, or linvanil, whose Ki values against the CB1 receptor were 1.76, 0.5–2.6, and 3.4 μM, respectively [13, 14, 35]. In summary, these novel compounds (especially VDM11) demonstrated higher selectivity in all experiments than previously reported AMT inhibitors, making them effective pharmacological tools for investigating the role of AMT in the physiological termination of AEA. Consistent with the high selectivity of these compounds, we found that, unlike AM404, VDM11 and VDM12 did not dilate rat mesenteric arteries (a process mediated by VR1 and endocannabinoid receptors) or inhibit human breast cancer cell proliferation (a CB1-mediated effect) unless at very high doses (V. Di Marzo, D. Melck, Z. Járai, T. Bisogno, and G. Kunos, unpublished observations). In summary, we provide evidence of partial overlap in the recognition properties of hVR1 and AMT ligands. Based on these studies, we have identified chemical modifications that may enable AEA analogs to distinguish hVR1 and AMT, and developed novel selective inhibitors of the latter protein, which are expected to become essential tools for studying AEA inactivation in vivo. [1]

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This study has three conceptually simple objectives: to determine whether the differences in the sensitivity of FAAH to VDM11 observed in different laboratories are related to the presence of fatty acid-free BSA in the detection; whether VDM11 is a substrate of FAAH like AM404; and whether VDM11 inhibits MAGL. The data presented in this paper show that the presence of fatty acid-free BSA reduces the sensitivity of FAAH (and MAGL) to VDM11, which may be partly due to the avoidance of this highly viscous molecule binding to pipette tips, thereby increasing the concentration of AEA in the detection. However, this problem did not appear to occur with 2-OG, but the titer of MAGL showed the same change. Therefore, the change in the absolute concentration of the substrate may not be the sole reason for the influence of fatty acid-free BSA. Given the strong binding affinity of BSA to the arachidonic acid group (Bojesen & Bojesen, 1994; Bojesen & Hansen, 2003), it is reasonable to speculate that BSA can also bind to the arachidonic acid group of VDM11, thereby reducing its free concentration, although further experiments are needed to confirm this. In conclusion, since the detection methods with the highest sensitivity for inhibiting FAAH activity of VDM11 and AM404 reported in the literature all contain fatty acid-free BSA (see Introduction), the presence or absence of this reagent in the detection is clearly not the cause of the differences between the detection methods. Furthermore, the finding that the potency of VDM11 as a FAAH inhibitor decreases with increasing substrate concentration can also be ruled out as the main cause of the variation (although the competitive inhibition pattern is consistent with the role of VDM11 as a competitive substrate, see below), because the substrate concentration used is not significantly different from the Km values previously reported by the same laboratory (see Maurelli et al., 1995; Jonsson et al., 2001). Therefore, this variation remains an unexplained phenomenon. It is worth mentioning that this study provides data on the interaction of VDM11 and AM404 with MAGL and confirms that VDM11 is indeed a substrate for FAAH. Regarding the latter, this study shows that after VDM11 is incubated with the cerebellar meninges, the HPLC peak pattern (a) depends on the activity of FAAH, as URB597 and CAY10401 can inhibit the appearance of these peaks; (b) the retention times of these peaks are the same as the HPLC pattern of the presumed VDM11 decomposition product 4-aminom-cresol. It is well known that 4-amino-m-cresol is readily oxidized upon incubation with biological materials, producing a variety of metabolites (Eggenreich et al., 2004), so the presence of multiple peaks is not surprising. Some have suggested that longer incubation times and higher VDM11 concentrations limit the relevance of the data. However, these conditions are necessary given the relatively high detection limit of 4-amino-m-cresol, and VDM11 metabolism was observed within 60 minutes. Through incubation at 120 and 180 minutes, the degradation rate of 400 μM VDM11 was estimated to be 160 pmol mg protein⁻¹ min⁻¹. This value can be compared to the hydrolysis rate of 2 μM [³H]AEA observed in the preparation (560 pmol mg protein⁻¹ min⁻¹). Given that the Km value of AEA in our experiments was approximately 1 μM (see Jonsson et al., 2001), and assuming that the Vmax value measured at such a high VDM11 concentration (relative to its affinity for FAAH) is the Vmax value, the existing data suggest that the Vmax value of VDM11 metabolism is approximately 15-20% of that of AEA metabolism. In contrast, the rates of metabolism of myristamide, palmitamide, and oleamide (100 μM) by rat FAAH expressed in COS-7 cells were 5.8%, 9.9%, and 24% of the rate of metabolism of 100 μM AEA, respectively (Cravatt et al., 1996). This indicates that while the rate of FAAH metabolism of VDM11 is significantly lower than that of AEA, it is comparable in range to other alternative substrates of this enzyme.
In 2004, researchers at Fegley et al. used high-performance liquid chromatography/mass spectrometry (HPLC/MS) to determine the concentration of AM404. Their results showed that low concentrations of AM404 (0.1–1 nmol) were effectively removed after 30 minutes of incubation with wild-type mouse cell membranes, but not in FAAH-/− mouse cell membranes. Their study indicated that the substrate was lost in a FAAH-dependent manner, while our study suggests that the appearance of the hypothesized product is sensitive to both URB597 and CAY10401, with both being complementary. Of course, due to the different methods used, we cannot compare the relative rates of VDM11 and AM404 as FAAH substrates. However, regardless of their absolute kcat values, the mechanisms of action of these two compounds are consistent with their role as FAAH substrates, meaning they must interact with FAAH, thereby reducing AEA metabolism due to substrate competition. Whether this fully explains their inhibitory mechanisms remains to be elucidated. Regarding the interaction between AM404 and VDM11 with MAGL, Saario et al. (2004) recently reported that 1 mM AM404, measured at 25°C, did not inhibit the hydrolysis of 50 μM 2-AG (measured in the presence of 0.5% BSA) by rat cerebellar membrane components. Conversely, we found that after incubation with 100 μM AM404, cytoplasmic MAGL completely inhibited the metabolism of 2 μM 2-OG. Arachidonic acid trifluoromethyl ketone also showed similar differences in sensitivity. Saario et al. (2004) showed that this compound inhibited membrane-bound 2-AG metabolism with an IC50 value of 66 μM, while our study found it to exhibit stronger inhibitory activity in cytoplasmic 2-OG metabolism (IC50 value of 2.9 μM) (Ghafouri et al., 2004), and Dinh et al. (2002) also obtained similar results (IC50 value of 2.9 μM). This indicates that the difference in detection sensitivity for FAAH also exists in MAGL, emphasizing that comparisons between compounds must be performed in the same laboratory. The interactions of AM404 and VDM11 with MAGL are noteworthy. Their ability to interact does not imply that they are substrates, unlike the case of FAAH—in fact, in our experiments, AEA (not metabolized by MAGL, Dinh et al., 2002) inhibited the metabolism of 2-OG by soluble components of the rat cerebellum with an IC50 value of 60 μM (i.e., much lower than its affinity for FAAH), and a similar IC50 value was observed for arachidonic acid (Ghafouri et al., 2004). The situation for AM404 and VDM11 may be similar. Of course, these compounds may indirectly interfere with 2-AG reabsorption by reducing the metabolic rate of 2-AG, but the sensitivity of 2-AG absorption and/or 2-AG levels to these compounds (Bifulco et al., 2004; Hájos et al., 2004; Melis et al., 2004) is more likely to reflect the effect of these compounds on the absorption process itself.
Finally, there is a point regarding the relevance of the current data to the thorny issue of AEA absorption mechanisms. The purpose of this article is not to elucidate this issue, but simply to determine whether VDM11 interacts with endocannabinoid metabolic enzymes. It is clear that while FAAH plays an important role in absorption (Day et al., 2001; Deutsch et al., 2001), it is by no means the only mechanism involved, as the absorption of AEA and the in vivo effects of AEA absorption inhibitors have been demonstrated in FAAH−/− mice (Fegley et al., 2004; Ligresti et al., 2004; Ortega-Gutiérrez et al., 2004), and compounds such as UCM707 and OMDM-2, which have weak interactions with FAAH regardless of the detection method used (López-Rodríguez et al., 2003; Ortar et al., 2003; Fowler et al., 2004), enhance the in vivo effects of AEA (de Lago et al., 2002; 2004). Clearly, the debate about this elusive transporter will continue. [2]

C27H39NO2

409.60406

409.298079

313998-81-1

9887748

Typically exists as solids at room temperature

1.0±0.1 g/cm3

586.6±50.0 °C at 760 mmHg

308.5±30.1 °C

0.0±1.7 mmHg at 25°C

1.553

6.7

49.3 Ų

2

2

15

30

547

0

CCCCC/C=C\C/C=C\C/C=C\C/C=C\CCCC(=O)NC1=CC=C(C=C1C)O

WUZWFRWVRHLXHZ-ZKWNWVNESA-N

InChI=1S/C27H39NO2/c1-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19-20-27(30)28-26-22-21-25(29)23-24(26)2/h7-8,10-11,13-14,16-17,21-23,29H,3-6,9,12,15,18-20H2,1-2H3,(H,28,30)/b8-7-,11-10-,14-13-,17-16-

(5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)icosa-5,8,11,14-tetraenamide

313998-81-1; VDM 11; (5Z,8Z,11Z,14Z)-N-(4-HYDROXY-2-METHYLPHENYL)-5,8,11,14-EICOSATETRAENAMIDE; VDM-11; (5Z,8Z,11Z,14Z)-N-(4-Hydroxy-2-methylphenyl)icosa-5,8,11,14-tetraenamide; VDM11; VDM-11 (Solution in Ethanol); (5Z,8Z,11Z,14Z)-N-(4-Hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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。