5-HT_1A Receptor

Agonistic binding of Geissoschizine methyl ether/GM to 5-HT1A receptors [2]
The results of in vitro competitive binding assays of seven UH-derived alkaloids against [3H]8-OH-DPAT binding to 5-HT1A receptors are shown in Fig. 6. Geissoschizine methyl ether/GM (0.01–100 μM) strongly inhibited the binding of [3H]8-OH-DPAT to 5-HT1A receptors in a concentration-dependent manner compared with the other six alkaloids. The 50% inhibitory concentration (IC50) and inhibition constant (Ki) values of GM were estimated to be 0.904 μM and 0.517 μM, respectively.

To clarify the agonistic effect of Geissoschizine methyl ether/GM on the 5-HT1A receptor, [35S]GTPγS, an in vitro binding assay was performed (Fig. 7). The [35S]GTPγS binding was increased by GM (0.1–100 μM) or 5-HT, a full agonist (1–300 nM), in a concentration-dependent manner. However, the binding rate of GM was approximately 40% of that of 5-HT.

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GM/Geissoschizine methyl ether concentration in YKS [2]
Table 1 shows the concentrations of seven UH-derived alkaloids contained in YKS. GM comprised approximately 0.014% of the YKS extract. Thus, it was confirmed that approximately 140 μg of GM was contained in 1.0 g of YKS.

Effects of GM/Geissoschizine methyl ether on increased aggressive behavior and decreased social behavior in socially isolated mice [2]
A single administration of GM (75–300 μg/kg) did not alter increased aggressive behavior (Fig. 8A), whereas GM significantly ameliorated the decreased social behavior at 300 μg/kg (P<0.01) (Fig. 8B).

Repeated administration of GM/Geissoschizine methyl ether for 14 days significantly decreased aggressive behavior at 150 μg/kg (P<0.01) and 300 μg/kg (P<0.01) (Fig. 9A), and increased social behavior at 150 μg/kg (P<0.01) and 300 μg/kg (P<0.01) (Fig. 9B). These ameliorative effects by GM (300 μg/kg) administration for 14 days were counteracted by single administration of WAY-100635 (0.1 mg/kg) on the 14th day 30 min after GM administration (P<0.01) (Fig. 9A,B) as did YKS (Fig. 3) and UH (Fig. 5).

Coadministration effect of WAY-100635 on ameliorative effect of aggressive behavior and social behavior by repeated administration of Geissoschizine methyl ether/GM [2]
To examine the effect of repeated administration of WAY-100635 on ameliorative effects of the interactive behaviors by repeated administration of GM/Geissoschizine methyl ether, GM (300 μg/kg) was orally administered for 15 days, and WAY-100635 (0.1 mg/kg) was coadministered with GM for 14 days (Fig. 10). In the social interaction test on the 15th day, ameliorative effects of aggressive behavior (Fig. 10A) and social behavior (Fig. 10B) by GM administration for 15 days were counteracted by coadministrating WAY-100635 for 14 days (P<0.05).

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Sustention of ameliorative effects of aggressive behavior and social behavior by repeated administration of Geissoschizine methyl ether/GM [2]
GM/Geissoschizine methyl ether (300 μg/kg) was orally administered for 14 days to the isolated mice, and then the social interaction test was performed on the 15th day (Fig. 11). On the 15th day, that is, also in the condition that GM was not administered on the behavioral test day, ameliorative effects on the aggressive behavior (P<0.05) and social behavior (P<0.01) were observed (Fig. 11A,B).

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Effects of WAY-100635 alone on increased aggressive behavior and decreased social behavior in socially isolated mice [2]
As already shown in Fig. 3, Fig. 5, Fig. 9, Fig. 10, coadministration of WAY-100635 with YKS, UH, or Geissoschizine methyl ether/GM counteracted the ameliorative effects of aggressive and social behaviors by these test substances. Here, the effects of WAY-100635 alone on the isolation-induced aggressive and social behaviors were examined (Fig. 12). A single (0.1 and 1.0 mg/kg) or a repeated (0.1 mg/kg) administration of WAY-100635 for 14 days did not affect aggressive (Fig. 12A,D) and social behaviors (Fig. 12B,E) in the isolated mice. No significant differences were also observed in the motor activity of all groups (Fig. 12C,F).

Competitive binding assay to 5-HT1A receptors [2]
Various concentrations of rhynchophylline, isorhynchophylline, corynoxeine, isocorynoxeine, hirsuteine, hirsutine, and Geissoschizine methyl ether/GM were prepared by dissolving each in 0.5% dimethyl sulfoxide (DMSO). A competitive binding assay for 5-HT1A receptors was carried out using the method previously described (Terawaki et al., 2010). In brief, 5.25 μl of the test compound solution or vehicle was incubated in duplicate with 500 μl of membrane solution (60–92 μg protein/mL) of CHO-h5-HT1A cells and 20 μl of 40 nM [3H]8-OH-DPAT in 50 mM Tris–HCl buffer, pH 7.4, containing 0.1% ascorbic acid, 0.5 mM EDTA, and 10 mM MgSO4 for 60 min at 25 °C. Nonspecific binding was determined by adding 10 μM metergoline. After incubation, 5-HT1A receptor-ligand complexes were isolated by rapid filtration through a Whatman GF/B filter using a cell harvester. The trapped radioactive complexes were rinsed four times with 3 ml of ice-cold 50-mM Tris–HCl buffer and dried. Radioactivity (count per minute, cpm) trapped on the dried filter was measured using a liquid scintillation counter. The specific binding was determined by subtracting nonspecific binding from total binding, and expressed as the percentage inhibition using the following formula: Inhibition (%) = [1−(c−a)/(b−a)]×100, where “a” is the average cpm of nonspecific binding, “b” is the average cpm of total binding, and “c” is average cpm in the presence of the test substance.

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[35S]GTPγS binding assay to 5-HT1A receptors [2]
The effects of Geissoschizine methyl ether/GM on [35S]GTPγS binding to 5-HT1A receptors were examined using the method previously described (Terawaki et al., 2010). In brief, 0.42 μl of Geissoschizine methyl ether/GM solution dissolved in 50% DMSO or vehicle was preincubated for 20 min at 30 °C in duplicate with 50 μl of the CHO-h5-HT1A membrane solution (25–30 μg protein/mL) and 25 μl of 10 μM GDP in 20 mM HEPES buffer, pH 7.4, containing 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 1 mM EDTA, and further incubation was carried out for 60 min at the same temperature after adding wheat germ agglutinin-coated scintillation proximity assay beads. The binding reaction was initiated by the addition of 10 μl of 3.3 nM [35S]GTPγS to the mixture. After incubation for 30 min, the radioactivity was measured by using a liquid scintillation counter. Nonspecific binding was determined by adding 100 μM GTPγS. The rate of [35S]GTPγS binding induced by the test substance was compared with that induced by 300 nM of 5-HT, a full agonist of 5HT1A receptors.

Effects of a single administration of YKS, UH, and Geissoschizine methyl ether/GM on aggressive and social behaviors in socially isolated mice [2]
YKS (0.5 and 1.0 g/kg), UH (75 and 150 mg/kg), or Geissoschizine methyl ether/GM (75, 150, and 300 μg/kg) was orally administered in a single dose to the mice that had been isolated for 4 weeks. Distilled water (10.0 ml/kg) for the tests of YKS and UH, or 0.5% Tween-80 (10.0 ml/kg) for the test of GM was orally administered to the corresponding isolated control or group-housed mice by the same schedule. The social interaction test was performed 60 min after the drug administration.

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Effects of repeated administration of YKS, UH, and Geissoschizine methyl ether/GM on aggressive and social behaviors in socially isolated mice [2]
YKS (0.5 and 1.0 g/kg), YKS-UH (1.0 g/kg), UH (75 and 150 mg/kg), or Geissoschizine methyl ether/GM (150 and 300 μg/kg) was orally administered once a day for 14 days from the 4th week to 6th week to the isolated mice. Distilled water (10.0 ml/kg) for the tests of YKS and UH or 0.5% Tween-80 (10.0 ml/kg) for the test of GM was orally administered to the corresponding isolated control or group-housed mice by the same schedule. In this experiment, WAY-100635 (0.1 mg/kg) or saline (10.0 ml/kg) was administered i.p. once 30 min after administration of YKS (1.0 g/kg), UH (150 mg/kg), or GM (300 μg/kg) on the 14th day. The social interaction test was performed 60 min after the final administration of test substance on the 14th day.

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Coadministration effects of WAY-100635 on repeated administration effects of Geissoschizine methyl ether/GM [2]
GM/Geissoschizine methyl ether (300 μg/kg) was orally administered once a day for 15 days from the 4th to 6th week to the isolated mice, and WAY-100635 (0.1 mg/kg) or saline (10.0 ml/kg) was i.p. coadministered with GM once a day for 14 days; WAY-100635 was not administered on the 15th day. On the social interaction test day (the 15th day), GM was administered 60 min before the behavioral test.

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On sustention of repeated administration effects of Geissoschizine methyl ether/GM [2]
GM/Geissoschizine methyl ether (300 μg/kg) or 0.5% Tween-80 (10.0 ml/kg) was orally administered once a day for 14 days from the 4th to 6th week to the isolated mice, and then the social interaction test was performed on the 15th day; GM was not administered on the 15th day.

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Effects of WAY-100635 alone on increased aggressive behavior and decreased social behavior in socially isolated mice [2]
To evaluate single administration effect of WAY-100635 alone, WAY-100635 (0.1 and 1.0 mg/kg) or saline (10.0 ml/kg) was i.p. administered to mice isolated for 4 weeks. Social interaction test was performed 30 min after the administration of WAY-100635. To evaluate repeated administration effect of WAY-100635 alone, WAY-100635 (0.1 mg/kg) or saline (10.0 mg/kg) was i.p. administered once a day for 14 days from the 4th to 6th weeks to the isolated mice. On the 15th day of the next day, social interaction test was performed without administration of WAY-100635.

1. YKS (Yokan San) is a traditional Japanese herbal medicine, also known as Kampo medicine, used to treat symptoms such as neurasthenia, insomnia, night crying and restlessness in children. The main indole alkaloid in Uncaria rhynchophylla, rhynchophylline methyl ether (GM), has been confirmed as the main active ingredient of YKS, possessing psychoactive effects. Recent reports indicate that GM has a partial agonistic effect on 5-hydroxytryptamine 1A receptors. However, although several studies have reported the hemodynamics of GM in rats and humans, information on GM metabolism in humans remains limited. This study aims to investigate the metabolic pathways and enzymes of GM in humans. 2. Using recombinant human cytochrome P450 (CYP) isoenzymes and polyclonal antibodies against CYP isoenzymes, we found that GM is metabolized under the action of certain CYP isoenzymes into hydroxylated, dehydrogenated, hydroxylated + dehydrogenated, demethylated, and water adduct forms. 3. The relative activity factors of each CYP isoenzyme in human liver microsomes were calculated to determine the relative contribution of each CYP isoenzyme to GM metabolism in human liver microsomes (HLM). We identified CYP3A4 as the major CYP isoenzyme for GM metabolism in human liver microsomes. 4. These findings provide an important basis for understanding the pharmacokinetics and pharmacodynamics of GM and YKS. [1]

[1]. In vitro identification of human cytochrome P450 isoforms involved in the metabolism of Geissoschizine methyl ether, an active component of the traditional Japanese medicine Yokukansan. Xenobiotica. 2016;46(4):325-34.

[2]. Geissoschizine methyl ether, an alkaloid in Uncaria hook, is a potent serotonin ₁A receptor agonist and candidate for amelioration of aggressiveness and sociality by yokukansan. Neuroscience. 2012 Apr 5;207:124-36.

It has been reported that Uncaria sinensis and Uncaria rhynchophylla contain rhynchophylline methyl ether, and related data have been reported. Okugansan (YKS) is a traditional Japanese herbal medicine composed of seven dried herbs. Clinically, it is widely used to treat mental illnesses, such as aggressive behavior in dementia patients. We have previously demonstrated that Okugansan and its constituent herb Uncaria hook (UH) have partial agonistic effects on 5-HT(1A) receptors in vitro. However, whether this in vitro effect also exists in vivo, and what its active ingredient is, remains unclear. This study aims to identify the active ingredient of Okugansan and verify its in vivo effects. In this study, we first investigated the effects of Okugansan and Uncaria on aggression and social behavior in socially isolated mice. Both YKS and UH improved isolation-induced aggression and social isolation, and these improvements were offset by co-administration of the 5-HT(1A) receptor antagonist WAY-100635 or disappeared after removing UH from YKS. These results indicate that the effects of YKS are mainly attributed to UH, and its active ingredient is present in UH. To identify candidate components, we used Chinese hamster ovary cells artificially expressing the 5-HT(1A) receptor to conduct competitive binding experiments on seven major alkaloids in UH and [(35)S]guanosine 5'-O-(3-thiotriphosphate) (GTPγS) binding experiments. The results showed that only gemcicilline methyl ether (GM) among the seven alkaloids could effectively bind to the 5-HT(1A) receptor and exert a partial agonist effect. The effect of GM in vitro was further confirmed in socially isolated mice. Similar to YKS and UH, GM was able to improve isolation-induced aggression and socialization, while co-administration of WAY-100635 counteracted this effect. These results suggest that GM in UH is a potent 5-HT(1A) receptor agonist and may be a candidate component for the pharmacological effects of YKS in improving aggression and socialization in socially isolated mice. [1] Therefore, we screened components in UH to identify candidate components that bind to the 5-HT1A receptor. Specifically, we tested the in vitro competitive binding of seven UH-derived alkaloids. The binding assay results showed that only GM/Geissoschizine methyl ether had a strong binding affinity to the 5-HT1A receptor and its effect was similar to that of YKS and UH, being a partial agonist (Fig. 6, Fig. 7). This result is consistent with other previously reported in vitro findings: some guinea pig alkaloids, such as GM, cinnamidine, and dihydrocinnamidine, have been shown to have partial agonistic effects on 5-HT receptors in the guinea pig ileum, although the 5-HT receptor subtypes have not yet been determined (Kanatani et al., 1985). Pengsuparp et al. (2001) also confirmed GM as a 5-HT1A receptor agonist through in vitro binding experiments. However, these in vitro results have not been validated in vivo, and no one has confirmed the relative importance of GM in mental illness models. Therefore, in this study, we further evaluated the effects of GM on aggression and social behavior. As shown in Figure 9, we newly found that continuous administration of GM (150 and 300 μg/kg) for 14 days dose-dependently improved aggression and social behavior, similar to the effects of YKS and UH (Figures 3 and 5). Liquid chromatography analysis showed that the concentration of GM in the YKS extract was 0.014%, meaning that approximately 140 μg of GM was present in 1.0 g of YKS (Table 1). Therefore, the GM dose of 150 μg/kg is almost identical to the content in YKS. These results indicate that the ameliorative effect of YKS is primarily attributed to its UH-derived component, GM. Furthermore, the ameliorative effect of GM administration over 14 days could be offset by a single dose of the 5-HT1A receptor antagonist WAY-100635, along with YKS and UH. These results also suggest that the ameliorative effect of GM on aggression and social behavior is mediated at least through 5-HT1A receptors. While repeated administration of GM, as well as YKS and UH, showed ameliorative effects, these effects were not observed with a single dose. These results suggest the possible existence of neural adaptations, such as hypersensitivity or upregulation in response to repeated administration. In this study, we used the active compound GM (representing YKS, UH, and GM) to examine the role of 5-HT1A receptors in the ameliorative effect of repeated administration. As shown in Figure 10, continuous 15-day administration of GM improved aggression and social behavior, but this improvement was offset after 14 days of combined administration with WAY-100635. These results indicate that the neural adaptation to the 5-HT1A receptor induced by repeated administration of GM is involved in the improvement of aggressive and social behaviors. Furthermore, as shown in Figure 11, the improvement from 14 consecutive days of GM administration was observed even on the second day after GM administration was discontinued. Therefore, this sustained effect (likely attributed to neural adaptation) is a major factor in GM's improvement of aggression. Previous studies have shown that GM has various inhibitory effects on spontaneous movement (Sakakibara et al., 1999), seizures (Mimaki et al., 1997), and head-twitching behavior (Pengsuparp et al., 2001). However, these in vivo responses are induced by doses 100 to 1000 times higher (tens to hundreds of milligrams) than those contained in YKS. This paper reveals that the actual dose of GM contained in YKS can improve aggressive and social behaviors in socially isolated mice. This is the first time that GM in YKS has been found to improve aggression and social behavior. Furthermore, we recently demonstrated that GM was detectable in both the plasma and brain tissue of rats after oral administration of YKS, and that GM could cross the blood-brain barrier (BBB) in vitro (Imamura et al., 2011). These results indicate that GM from oral YKS is absorbed into the bloodstream, crosses the blood-brain barrier, and reaches the brain, and that GM's effects on YKS's aggression and social behavior are central. While further research is needed to confirm the details of the YKS-improving mechanism, our results suggest that YKS may have two distinct effects: an acute effect on social behavior and a chronic effect on both aggression and social behavior. A single administration of the test substances YKS, UH, and GM improved social contact deficits (Figures 2, 4, and 8), while improvements in aggression required long-term treatment (Figures 3, 5, and 9). Therefore, the ameliorative effects of YKS, UH, and GM on social behavior are thought to be primarily attributable to their direct partial agonistic effect on 5-HT1A receptors. Social behaviors between rats, such as sniffing, following, and touching, are often assessed as indicators of anxiety or anxiolytic effects, as benzodiazepines and serotonin (5-HT)-related anxiolytics increase these behaviors, while anxiolytics decrease them (File and Seth, 2003). Kuribara and Maruyama (1996) and Kamei et al. (2009) demonstrated the anxiolytic effects of YKS, and Jung et al. (2006) demonstrated the anxiolytic effects of UH aqueous extract. Therefore, our current data on social behaviors may suggest that YKS, UH, and GM have anxiolytic effects. Conversely, as previously mentioned, the long-term effects on aggression may be attributed to neural adaptations targeting the 5-HT1A receptor. Furthermore, future research needs to elucidate the detailed mechanisms by which the interaction of the 5-HT1A receptor with other receptors, such as the 5-HT2A receptor, induces neural adaptations. For example, Egashira et al. (2008) reported the involvement of 5-HT2A receptors in the effects of YKS: 14 consecutive days of YKS administration reduced head twitching responses induced by the 5-HT2A receptor agonist 2,5-dimethoxy-4-iodophenylamphetamine (DOI), while a single dose had no such effect. Therefore, repeated administration of YKS may lead to downregulation of 5-HT2A receptor proteins, which may be closely related to the ameliorative effect of YKS on aggressive behavior. Although the molecular and neural mechanisms by which YKS improves aggressive behavior require further investigation, this study suggests that GM may be a candidate component of YKS with psychoactive effects (such as improving aggression and sociability). Conclusion: YKS and its component UH significantly improved isolation-induced reduction in social behavior and increase in aggressive behavior. Screening of seven alkaloids in UH for binding to 5-HT1A receptors showed that GM could bind to 5-HT1A receptors and act as a partial agonist. Furthermore, GM could improve the abnormal behaviors associated with YKS and UH. In addition, co-administration with WAY-100635 weakened the effect of GM. These results suggest that GM may be the active ingredient in improving the effect of YKS and is mainly related to the 5-HT1A receptor. [1]

C22H26N2O3

366.4534

366.194

60314-89-8

6443046

White to off-white solid powder

1.2±0.1 g/cm3

Uncaria sinensis and Uncaria rhynchophylla

3.674

1

4

4

27

629

2

O(C([H])([H])[H])C(/C(=C(/[H])\OC([H])([H])[H])/[C@]1([H])/C(=C(/[H])\C([H])([H])[H])/C([H])([H])N2C([H])([H])C([H])([H])C3C4=C([H])C([H])=C([H])C([H])=C4N([H])C=3[C@]2([H])C1([H])[H])=O

VAMJZLUOKJRHOW-XEASWFAXSA-N

InChI=1S/C22H26N2O3/c1-4-14-12-24-10-9-16-15-7-5-6-8-19(15)23-21(16)20(24)11-17(14)18(13-26-2)22(25)27-3/h4-8,13,17,20,23H,9-12H2,1-3H3/b14-4-,18-13-/t17-,20-/m0/s1

methyl (Z)-2-[(2S,3E,12bS)-3-ethylidene-2,4,6,7,12,12b-hexahydro-1H-indolo[2,3-a]quinolizin-2-yl]-3-methoxyprop-2-enoate

Geissoschizine methyl ether; Geissoschizine methyl ether; 60314-89-8; methyl (Z)-2-[(2S,3E,12bS)-3-ethylidene-2,4,6,7,12,12b-hexahydro-1H-indolo[2,3-a]quinolizin-2-yl]-3-methoxyprop-2-enoate; TNN2THT2NX; (Z)-Methyl 2-((2S,12bS,E)-3-ethylidene-1,2,3,4,6,7,12,12b-octahydroindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate; methyl (Z)-2-((2S,3E,12bS)-3-ethylidene-2,4,6,7,12,12b-hexahydro-1H-indolo(2,3-a)quinolizin-2-yl)-3-methoxyprop-2-enoate; UNII-TNN2THT2NX; SCHEMBL22615910;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO: ~100 mg/mL (~272.9 mM)

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.)