Anandamide membrane transporter (AMT)

Some synthetic agonists of the VR1 vanilloid (capsaicin) receptor also inhibit the facilitated transport into cells of the endogenous cannabinoid anandamide (arachidonoylethanolamide, AEA). Here we tested several AEA derivatives containing various derivatized phenyl groups or different alkyl chains as either inhibitors of the AEA membrane transporter (AMT) in intact cells or functional agonists of the VR1 vanilloid receptor in HEK cells transfected with the human VR1. We found that four known AMT inhibitors, AM404, arvanil, olvanil and linvanil, activate VR1 receptors at concentrations 400-10000-fold lower than those necessary to inhibit the AMT. However, we also found three novel AEA derivatives, named VDM11, VDM12 and VDM13, which inhibit the AMT as potently as AM404 but exhibit little or no agonist activity at hVR1. These compounds are weak inhibitors of AEA enzymatic hydrolysis and poor CB(1)/CB(2) receptor ligands. We show for the first time that, despite the overlap between the chemical moieties of AMT inhibitors and VR1 agonists, selective inhibitors of AEA uptake that do not activate VR1 (e.g. VDM11) can be developed [1].

There is some dispute concerning the extent to which the uptake inhibitor VDM11 (N-(4-hydroxy-2-methylphenyl) arachidonoyl amide) is capable of inhibiting the metabolism of the endocannabinoid anandamide (AEA) by fatty acid amide hydrolase (FAAH). In view of a recent study demonstrating that the closely related compound AM404 (N-(4-hydroxyphenyl)arachidonylamide) is a substrate for FAAH, we re-examined the interaction of VDM11 with FAAH.[1]
In the presence of fatty acid-free bovine serum albumin (BSA, 0.125% w v−1), both AM404 and VDM11 inhibited the metabolism of AEA by rat brain FAAH with similar potencies (IC50 values of 2.1 and 2.6 μM, respectively). The compounds were about 10-fold less potent as inhibitors of the metabolism of 2-oleoylglycerol (2-OG) by cytosolic monoacylglycerol lipase (MAGL).[1]
The potency of VDM11 towards FAAH was dependent upon the assay concentration of fatty acid-free bovine serum albumin (BSA). Thus, in the absence of fatty acid-free BSA, the IC50 value for inhibition of FAAH was reduced by a factor of about two (from 2.9 to 1.6 μM). A similar reduction in the IC50 value for the inhibition of membrane bound MAGL by both this compound (from 14 to 6 μM) and by arachidonoyl serinol (from 24 to 13 μM) was seen.[1]
An HPLC assay was set up to measure 4-amino-m-cresol, the hypothesised product of FAAH-catalysed VDM11 hydrolysis. 4-Amino-m-cresol was eluted with a retention time of ∼2.4 min, but showed a time-dependent degradation to compounds eluting at peaks of ∼5.6 and ∼8 min. Peaks with the same retention times were also found following incubation of the membranes with VDM11, but were not seen when the membranes were preincubated with the FAAH inhibitors URB597 (3′-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) and CAY10401 (1-oxazolo[4,5-b]pyridin-2-yl-9-octadecyn-1-one) prior to addition of VDM11. The rate of metabolism of VDM11 was estimated to be roughly 15–20% of that for anandamide.[1]
It is concluded that VDM11 is an inhibitor of FAAH under the assay conditions used here, and that the inhibition may at least in part be a consequence of the compound acting as an alternative substrate [2].

FAAH activity assays [1]
N18TG2 cells were cultured as described previously and references cited therein). The effect of compounds on the enzymatic hydrolysis of [14C]AEA (6 μM) was studied by using membranes prepared from N18TG2 cells incubated with increasing concentrations of compounds in 50 mM Tris–HCl, pH 9, for 30 min at 37°C. [14C]Ethanolamine produced from [14C]AEA hydrolysis was measured by scintillation counting of the aqueous phase after extraction of the incubation mixture with 2 volumes of CHCl3/CH3OH 2:1 (by vol).

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AEA transporter assays [1]
The effect of compounds on the uptake of AEA by RBL-2H3 cells was studied by a modification of the method described previously, and analogous to the protocol described except for the use of a higher concentration (4 μM) of [14C]AEA. Cells were incubated with [14C]AEA for 5 min at 37°C, in the presence or absence of varying concentrations of the inhibitors. Residual [14C]AEA in the incubation media after extraction with CHCl3/CH3OH 2:1 (by vol), determined by scintillation counting, was used as a measure of the AEA that was taken up by cells. We applied the same protocol also to C6 rat glioma cells, which also contain a membrane transporter for AEA. Data are expressed as the concentration exerting 50% inhibition of AEA uptake (IC50) calculated with GraphPad.

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CB1 and CB2 receptor binding assays [1]
Displacement assays for CB1 receptors were carried out by using [3H]SR141716A (0.4 nM, 55 Ci/mmol) as the high affinity ligand, and the filtration technique previously described, on membrane preparations (0.4 mg/tube) from frozen male CD rat brains, and in the presence of 100 μM PMSF. Specific binding was calculated with 1 μM SR141716A and was 84.0%. The spleen from CD rats were used to prepare membranes (0.4 mg/tube) to carry out CB2 binding assays by using [3H]WIN55,212-2 (0.8 nM, 50.8 Ci/mmol) as described previously, and again in the presence of 100 μM PMSF. Specific binding was calculated with 1 μM HU-348 and was 75.0%. In all cases, K i values were calculated by applying the Cheng–Prusoff equation to the IC50 values for the displacement of the bound radioligand by increasing concentrations of the test compounds.

Ca2+ influx assays [1]
The effect of the substances on the influx of Ca2+ into cells was determined by using Fluo-3, a selective intracellular fluorescent probe for Ca2+. Four days prior to experiments cells were transferred into six-well dishes coated with poly-L-lysine and grown in the culture medium mentioned above. The day of the experiment the cells (50–60 000 per well) were loaded for 2 h at 25°C with 4 μM Fluo-3 methylester in DMSO containing 0.04% Pluoronic. After the loading, cells were washed with Tyrode pH=7.4, and trypsinized to be suspended in the cuvette of the fluorescence detector under continuous mixing. Experiments were carried out by measuring cell fluorescence at 25°C (λ EX=488 nm, λ EM=540 nm) before and after the addition of the test compounds at various concentrations. Capsazepine (1–5 μM) or EGTA (4 mM) were added 30 or 10 min, respectively, before the test compounds. Data are expressed as the concentration exerting a half-maximal effect (EC50) calculated by using GraphPad software. The efficacy of the effect was determined by comparing it to the analogous effect observed with 4 μM ionomycin.

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

Colorless to light yellow liquid

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

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

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.)