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. Yokukansan (YKS) is a traditional Japanese medicine also called kampo, which has been used to treat neurosis, insomnia, and night crying and peevishness in children. Geissoschizine methyl ether (GM), a major indole alkaloid found in Uncaria hook, has been identified as a major active component of YKS with psychotropic effects. Recently, GM was reported to have a partial agonistic effect on serotonin 5-HT1A receptors. However, there is little published information on GM metabolism in humans, although several studies reported the blood kinetics of GM in rats and humans. In this study, we investigated the GM metabolic pathways and metabolizing enzymes in humans. 2. Using recombinant human cytochrome P450 (CYP) isoforms and polyclonal antibodies to CYP isoforms, we found that GM was metabolized into hydroxylated, dehydrogenated, hydroxylated+dehydrogenated, demethylated and water adduct forms by some CYP isoforms. 3. The relative activity factors in human liver microsomes were calculated to determine the relative contributions of individual CYP isoforms to GM metabolism in human liver microsomes (HLMs). We identified CYP3A4 as the CYP isoform primarily responsible 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.

Geissoschizine methyl ether has been reported in Uncaria sinensis and Uncaria rhynchophylla with data available.

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okukansan (YKS), a traditional Japanese medicine, is composed of seven kinds of dried herbs. It is widely prescribed in clinical situation for treating psychiatric disorders such as aggressiveness in patients with dementia. We previously demonstrated that YKS and Uncaria hook (UH), which is a constituent herb of YKS, had a partial agonistic effect to 5-HT(1A) receptors in vitro. However, it has still been unclear whether this in vitro effect is reflected in in vivo, and what the active ingredients are. The purpose of the present study is to find the active ingredient in YKS and to demonstrate the effect in in vivo. In the present study, we first studied the effect of YKS and UH on aggressiveness and sociality in socially isolated mice. YKS and UH ameliorated the isolation-induced increased aggressiveness and decreased sociality, and these ameliorative effects were counteracted by coadministration of 5-HT(1A) receptor antagonist WAY-100635, or disappeared by eliminating UH from YKS. These results suggest that the effect of YKS is mainly attributed to UH, and the active ingredient is contained in UH. To find the candidate ingredients, we examined competitive binding assay and [(35)S] guanosine 5'-O-(3-thiotriphosphate) (GTPγS) binding assay of seven major alkaloids in UH using Chinese hamster ovary cells expressing 5-HT(1A) receptors artificially. Only Geissoschizine methyl ether(GM) among seven alkaloids potently bound to 5-HT(1A) receptors and acted as a partial agonist. This in vitro result on GM was further demonstrated in the socially isolated mice. As did YKS and UH, GM ameliorated the isolation-induced increased aggressiveness and decreased sociality, and the effect was counteracted by coadministration of WAY-100635. These lines of results suggest that GM in UH is potent 5-HT(1A) receptor agonist and a candidate for pharmacological effect of YKS on aggressiveness and sociality in socially isolated mice.[1]

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Therefore, we screened the ingredients in UH to determine the candidate binding to 5-HT1A receptors. That is, we examined the in vitro competitive binding of seven UH-derived alkaloids. The result of the binding assay showed that only GM/Geissoschizine methyl ether strongly bound to 5-HT1A receptors and that it acted as a partial agonist as well as YKS and UH (Fig. 6, Fig. 7). This result is supported by other in vitro results previously reported: several UH-derived alkaloids, such as GM, corynantheine, and dihydrocorynantheine, have been demonstrated to possess partial agonistic effects on 5-HT receptors in guinea-pig ileum, although the subtype of 5-HT receptors was not determined (Kanatani et al., 1985). Pengsuparp et al. (2001) also demonstrated that GM is a 5-HT1A receptor agonist by an in vitro binding assay. However, these in vitro results were still not verified in vivo, and no one has demonstrated the relative importance of GM in psychiatric disorder model. Thus, in this study, we further evaluated the effects of GM on aggressive and social behaviors.

As shown in Fig. 9, we newly demonstrated that repeated administration of GM (150 and 300 μg/kg) for 14 days ameliorated aggressiveness and sociality in a dose-dependent manner, as did YKS and UH (Fig. 3, Fig. 5). A liquid chromatographic assay indicated that the concentration of GM in YKS extract was 0.014%, that is, 1.0 g of YKS is considered to contain approximately 140 μg of GM (Table 1). Therefore, the dosage of 150 μg/kg of GM is almost the same as the amount included in YKS. These results suggest that the ameliorating effect of YKS is mainly attributed to UH-derived ingredient GM. In addition, the ameliorating effects by GM administration for 14 days were counteracted by single coadministration of a 5-HT1A receptor antagonist WAY-100635 as well as YKS and UH. These results also suggest that the ameliorating effects of aggressive and social behaviors by GM, at least, are mediated by 5-HT1A receptors. Although the ameliorating effects of GM as well as YKS and UH were observed by repeated administration, their effects were not observed by a single administration. These results suggest the possibility of neuroadaptation such as hypersensitivity or upregulation to the repeated administration effect. In the present study, the involvement of 5-HT1A receptors on the ameliorative effect by repeated administration was examined by using active compound GM, on behalf of YKS, UH, and GM. As shown in Fig. 10, the ameliorative effects of aggressive and social behaviors by GM administration for 15 days were counteracted by coadministrating WAY-100635 for 14 days. This result suggests that neuroadaptation against 5-HT1A receptors by repeated administration of GM is involved in the amelioration of aggressive and social behaviors. In addition, the ameliorating effect by repeated administration of GM for 14 days was observed even on the next day that GM was not administered, as shown in Fig. 11. Therefore, such sustained effect presumably attributed to neuroadaptation mainly contributed to the amelioration of aggressiveness by GM.

It has been demonstrated that GM has several inhibitory effects on spontaneous motor activity (Sakakibara et al., 1999), convulsion (Mimaki et al., 1997), and head-twitching behavior (Pengsuparp et al., 2001). However, these in vivo responses are induced by doses 100- or 1000-fold higher (several dozen mg to several hundred mg) than the amount contained in YKS. Here, we revealed that real dose of GM contained in YKS ameliorated aggressive and social behavior in socially isolated mice. This is a first finding that GM in YKS ameliorates aggressiveness and sociality. In addition, we recently demonstrated that GM was detected in the plasma and brain of rats after oral administration of YKS and also demonstrated that GM was able to cross BBB in the in vitro BBB assay (Imamura et al., 2011). These results suggest that GM in YKS administered orally is absorbed into the blood and then reaches the brain through BBB, and effects of GM on aggressive and social behaviors in YKS is central action.

Although we need further studies to confirm the details of the amelioration mechanism of YKS, our results show the possibility that YKS has two different effects: an acute effect on sociality and a chronic effect on aggressiveness and sociality. Deficits in social contacts were ameliorated by a single treatment with the test substances, YKS, UH, and GM (Fig. 2, Fig. 4, Fig. 8), whereas chronic treatment was needed to ameliorate aggressiveness (Fig. 3, Fig. 5, Fig. 9). Thus, the ameliorating effects on social behavior by YKS, UH, and GM are thought to be mainly due to its direct partial agonistic effect on 5-HT1A receptors. Social behaviors such as the sniffing, following, and contacting observed between two rats have been often evaluated as an index of anxiety or anxiolytic effects because benzodiazepine- and 5-HT-related anxiolytic drugs increase social behaviors, whereas anxiogenic agents decrease them (File and Seth, 2003). Kuribara and Maruyama (1996) and Kamei et al. (2009) demonstrated the anxiolytic effect of YKS, and Jung et al. (2006) demonstrated anxiolytic effects of the aqueous extract of UH. Therefore, our present data regarding social behavior might suggest that YKS, UH, and GM have an anxiolytic effect. In contrast, chronic effect on aggressiveness may be thought to be due to neuroadaptation against 5-HT1A receptors, as already described. Furthermore, it will be necessary to clarify the detailed mechanisms for the interaction between 5-HT1A receptors and other receptors such as 5-HT2A receptors to induce the neuroadaptation, in future studies. For example, Egashira et al. (2008) reported the involvement of 5-HT2A receptors in the effect of YKS: a 5-HT2A receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI)-induced head-twitch response was reduced by 14 days administration of YKS but not by a single administration. Thus, it is possible that downregulation of the 5-HT2A receptor protein by repeated administration of YKS is closely related to the ameliorative effect on aggressive behavior.

Although the molecular and neuronal mechanism for amelioration of YKS will be performed in the future, the present study demonstrated that GM is candidate of active ingredient for the psychotropic effects such as aggressiveness and sociality of YKS.

Conclusion
YKS and its component UH significantly ameliorated the isolation-induced decrease in social behavior and increase in aggressive behavior. Screening of seven alkaloids in UH for binding to 5-HT1A receptors revealed that GM bound to 5-HT1A receptors and acted as a partial agonist. Furthermore, GM ameliorated both abnormal behaviors as well as YKS and UH. In addition, the effects of GM were attenuated by coadministration of WAY-100635. These results suggested the possibility that GM is the active ingredient responsible for amelioration by YKS, and mainly 5-HT1A receptors are associated with the ability.[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.)