Sigma 1/2 Receptor; vesicular acetylcholine transport

The blockage of acetylcholine transport into synaptic vesicles and the subsequent quantal release of acetylcholine are thought to be the pharmacological actions of (±)-Vesamicol hydrochloride [1].

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For the purpose of this discussion, the structure of Vesamicol has been divided into three major fragments:  A, B, and C (Chart 1). Although many vesamicol analogues have been synthesized in an effort to expand our understanding of the structure−activity relationships for binding to the vesicular acetylcholine transporter, the vast majority contains an unmodified B fragment, and the rest contains only single-point modifications of this fragment. Consequently, little is known about the interaction between this region of the molecule and the vesicular acetylcholine transporter. In previous studies,6,12,13 we and others utilized a strategy of conformational restriction (in fragment A and/or C) to develop selective high-affinity ligands for the vesicular acetylcholine transporter. To test the limits of this strategy, we chose to extend our studies to fragment B. [1]

In our first attempt, we replaced the piperidyl fragment B with a tropanyl moiety. Formally, tropane may be regarded as 2,6-ethanopiperidine. Since the ethylene bridge prevents the interconversion of piperidine conformers, the tropanyl fragment may be regarded as a conformationally restricted piperidyl residue. In a parallel effort, we also synthesized several derivatives of 1-methylspiro[1H-indoline-3,4‘-piperidine] (7) as conformationally restricted Vesamicol analogues. The latter study is an extension of our previous investigation of spirovesamicol analogues such 3−5. The target compounds were tested for binding to the vesicular acetylcholine transporter of Torpedo synaptic vesicles. Binding to σ receptors was also evaluated because Vesamicol and some of its analogues display moderate to high affinity for these sites.11 The compounds were also tested for binding to monoamine transporters because the 3β-phenyltropanyl moiety is found in many inhibitors of monoamine reuptake. Finally, the affinity of these compounds for dopamine D2 receptors was evaluated because of behavioral effects in rodents (data not shown). Since the reference ligand [125I]NCQ298 does not discriminate between dopamine D2 and D3 receptors, the data obtained with this radioligand reflect binding to both dopamine receptor subtypes. [1]

Although many of the tropane analogues displayed moderate affinity for the vesicular acetylcholine transporter, none of the new compounds was more potent than Vesamicol. In fact, compared to the corresponding piperidyl analogues, most of the new compounds fared rather poorly. Thus, while compound 10a displayed 4-fold lower affinity than Vesamicol, 10b−d were at least 2 orders of magnitude less potent than benzovesamicol (2), 25, and 26, respectively (Table 1). These disparities persisted even when the cyclohexyl moiety was replaced with a hydroxyethyl group (27 vs 10f−k) but disappeared when the N-alkyl substituent became more flexible (28 vs 10e). Taken together, the foregoing suggests that the two-carbon bridge (which converts the piperidyl into a tropanyl fragment) is profoundly detrimental to the ligand−receptor interaction. As the two-carbon bridge increases the steric volume of fragment B, one plausible explanation for the adverse effect of this bridge may be that fragment B fits into a narrow pocket within the binding site. [1]

Although spirovesamicols such as 3−5 display comparable or higher affinity for the vesicular acetylcholine transporter than Vesamicol, all analogues of 7 were significantly less potent than Vesamicol. Moreover, the new compounds generally displayed 1−2 orders of magnitude lower potency than the corresponding carbon analogues (compare 18a vs 4, 19a vs 19b, 20a vs 20b, and 21a vs 21b). While the disparity between the two series of compounds was obscured by the absence of a cyclohexyl group (compare 22a vs 22b and 23a vs 23b), it is clear that 7 is not a suitable replacement for the A−B fragment in Vesamicol.

(±)-Vesamicol hydrochloride (3 mg/kg; intraperitoneal; once; male Wistar rats) therapy increased brain levels of cytosolic acetylcholine (ACh) and decreased brain levels of vesicular ACh in all regions with the exception of the extrastriatal region [2].

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Vesamicol is known to inhibit the transport of acetylcholine (ACh) into synaptic vesicles in vitro, but much less is known about its effects in the brain in vivo. To assess the effect of vesamicol in vivo, we examined cholinergic parameters, such as the subcellular distribution of ACh, activities of enzymes, uptake of choline, and muscarinic receptor binding in the striatum, hippocampus, and cerebral cortex of rats 30 and 60 min after intraperitoneal injection of vesamicol (3 mg/kg) or of Vesamicol in combination with DDVP (5 mg/kg), which was administered 10 min before vasamicol. The levels of cytosolic ACh increased in all regions of the brain after injection of vesamicol, while those of vesicular ACh decreased in all regions except for the striatum. The increase in the levels of extracellular ACh and cytosolic ACh in the striatum induced by DDVP was generally enhanced after injection of vesamicol, Vesamicol did not reduce the level of vesicular ACh when DDVP had been injected previously. Vesamicol did not induce any significant changes in the activities of enzymes, choline uptake, or binding of [6H]quinuclidinyl benzilate to the muscarinic ACh receptors in the three regions. Changes in the cholinergic parameters caused by DDVP were not reversed by the combined administration of DDVP with vesamicol. The present results indicate that vesamicol can inhibit the transport of ACh into synaptic vesicles in the brain tissue in vivo, although it cannot reverse the effects of DDVP that has been injected prior to vesamicol. [2]

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Levels of Subcellular Acetylcholine [2]
As shown in Fig. 1, Vesamicol did not cause a significant change in the level of extracellular ACh in the three regions of the brain 30 and 60 min after its injection. The level of cytosolic ACh had increased in the striatum, hippocampus, and cortex 30 and/or 60 min after the injection of Vesamicol. The level of vesicular ACh, by constrast, had decreased in the hippocampus and cortex but not in the striatum both 30 and 60 min after the injection of Vesamicol. In the striatum, no significant changes were observed in the levels of vesicular ACh 30 and 60 min after the injection of vesamicol (Fig. 1).

Administration of DDVP increased the levels of extracellular ACh in the striatum, of cytosolic ACh in the striatum and hippocampus, and of vesicular ACh in the striatum at 40 and 70 min (Fig. 1). The level of vesicular ACh did not change in any region of the brain after injection of DDVP, although there was an insignificant increase in the striatum and hippocampus 40 min after the injection of DDVP. As shown in Fig. 1, combined administration of Vesamicol and DDVP caused a significant increase in the levels of striatal extracellular ACh and cytosolic ACh, as compared to those in both control and DDVP-treated rats. No significant differences were observed in the level of vesicular ACh between control rats and rats injected with DDVP and vesamicol 40 or 70 min after injection of DDVP in the three regions of the brain. 3.2. Activities of Acetylcholinesterase and Choline Acetyltransferase, the High-Affinity Uptake of Choline (HACU) and the Binding of Muscarinic Receptors [2]
Fig. 2 shows the activity of AChE in the three regions of the brain from rats injected with Vesamicol, DDVP, or DDVP plus vesamicol. Vesamicol alone did not cause any change in the activity in any brain regions 30 or 60 min after injection. Injection of DDVP caused a significant decrease in the activity of AChE in the three regions of the brain at 40 and 70 min. Administration of vesamicol did not have any effect on the decreased activity of AChE in the three regions of the brain of rats that had previously been injected with DDVP.

As shown in Fig. 4, there were no significant changes in the values of Km (Fig. 4A) and Vmax (Fig. 4B) for HACU in the three regions of the brain between the controls and rats examined 30 or 60 min after injection of Vesamicol. However, DDVP caused an increase in both parameters of HACU in the three regions of the brain 40 and 70 min after injection. The HACU of each region of the brain of rats that received both DDVP and vesamicol was approximately the same as that of rats injected with DDVP alone.

Fig. 5 shows the binding of [3H]QNB to mAChRs in the three regions of the brain of rats that had been injected with saline (controls), Vesamicol, DDVP, or DDVP plus vesamicol. There were no significant differences in the respective values of Kd (Fig. 5A) and Bmax (Fig. 5B) in all regions between the controls and the rats examined 30 and 60 min after the injection of vesamicol alone. Injection of DDVP generally decreased the values of Kd and Bmax. The values of these parameters in each region of the brain of rats that had received DDVP plus Vesamicol were approximately the same as those of rats that had been injected with DDVP alone.

Vesicular Acetylcholine Transporter Binding. [1]
Dissociation constants of novel compounds were determined by competition against the binding of [3H]Vesamicol to electric organ synaptic vesicles at 22 °C, after 24 h of incubation, by the method of Rogers et al.6 σ Receptor Binding. σ1 binding sites were labeled with the σ1- selective radioligand [3H]-(+)-pentazocine in guinea pig brain membranes according to published procedures. 33,34 σ-2 sites were assayed in rat liver membranes, a rich source of these sites, with [3H]DTG in the presence of (+)-pentazocine (100 nM).

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Membrane Preparation. [1]
The crude P2 membrane fraction was prepared from frozen guinea pig brains minus cerebellum. Brains were allowed to thaw slowly on ice before homogenization. The crude P2 membrane fraction was also prepared from the livers of male Sprague−Dawley rats (175−225 g). Animals were sacrificed by decapitation and the livers removed and minced before homogenization. Tissue homogenization was carried out at 4 °C in 10 mL/g of tissue weight 10 mM Tris-HCl/0.32 M sucrose, pH 7.4, using a Porter-Elvehjem tissue grinder. The crude homogenate was centrifuged for 10 min at 1000g and the supernatant saved on ice. The pellet was resuspended in 2 mL/g of tissue weight ice-cold 10 mM Tris-HCl/0.32 M sucrose, pH 7.4, by vortexing. After centrifugation at 1000g for 10 min, the pellet was discarded and the supernatants were combined and centrifuged at 31000g for 15 min. The pellet was resuspended in 3 mL/g 10 mM Tris-HCl, pH 7.4, by vortexing, and the suspension was allowed to incubate at 25 °C for 15 min. Following centrifugation at 31000g for 15 min, the pellet was resuspended by gentle homogenization to 1.53 mL/g in 10 mM Tris-HCl (pH 7.4), and aliquots were stored at −80 °C until used. The protein concentration of the suspension was determined by the method of Bradford and generally ranged from 6 to 11 mg of protein/mL.

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σ1 Binding Assay. [1]
Guinea pig membranes (100 μg of protein) were incubated with 3 nM [3H]-(+)-pentazocine (31.6 Ci/mmol) in 50 mM Tris-HCl, pH 8.0, at 25 °C for either 120 or 240 min. Test compounds were dissolved in ethanol and then diluted in buffer to give a total incubation volume of 0.5 mL. Assays were terminated by the addition of ice-cold 10 mM Tris-HCl, pH 8.0, followed by rapid filtration through Whatman GF/B glass filters (presoaked in 0.5% poly(ethylenimine)) using a Brandel harvester. Filters were washed twice with 5 mL of ice-cold buffer. Nonspecific binding was determined in the presence of 10 μM (+)-pentazocine. Liquid scintillation counting was carried out in Ecolite(+) using a Beckman LS 6000IC spectrometer with a counting efficiency of 50%. Typical counts were 70 dpm/μg of protein for total binding, 6 dpm/μg for nonspecific binding, and 64 dpm/μg for specific binding.

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σ2 Binding Assay. [1]
Rat liver membranes (35 μg of protein) or guinea pig brain membranes (360 μg) were incubated with 3 nM [3H]DTG (38.3 Ci/mmol) in the presence of 100 nM (+)-pentazocine to mask σ1 sites. Incubations were carried out in 50 mM Tris-HCl, pH 8.0, for 120 min at 25 °C in a total incubation volume of 0.5 mL. Assays were terminated by the addition of ice-cold 10 mM Tris-HCl, pH 8.0, followed by rapid filtration through Whatman GF/B glass filters (presoaked in 0.5% poly(ethylenimine)) using a Brandel harvester. Filters were then washed twice with 0.5 mL of ice-cold buffer. Nonspecific binding was determined in the presence of 5 μM DTG. Liquid scintillation counting was carried out in Ecolite(+) using a Beckman LS 6000IC spectrometer with a counting efficiency of 50%. Typical counts for rat liver were 297 dpm/μg of protein for total binding, 11 dpm/μg for nonspecific binding, and 286 dpm/μg for specific binding. Typical counts for guinea pig brain were 16 dpm/μg of protein for total binding, 2 dpm/μg for nonspecfic binding, and 14 dpm/μg for specific binding. Data Analysis. The IC50 values for σ sites were determined in triplicate by nonlinear regression analysis using JMP, with 5−10 concentrations of each acetylcholine compound. Ki values were calculated using the Cheng−Prusoff equation 35 and represent mean values ± SEM. All assays were done in triplicate unless otherwise noted. For the radioligands, the following previously33,34 reported Kd values were employed:  [3H]DTG, 17.9 nM (rat liver); [3H]-(+)-pentazocine, 4.8 nM (guinea pig brain). The Kd value for [3H]DTG in guinea pig brain was determined by a Scatchard analysis to be 21.6 nM.

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Dopamine D2/D3 Receptor Binding Assay. [1]
The frozen membrane preparations from Spodoptera frugiperda (Sf9) insect cells expressing rat dopamine D2 receptors were kindly provided by Dr. Perry Molinoff. Rat striatal homogenates were prepared as described previously. 36 Competition experiments were carried out with 0.2 nM [125I]NCQ298 (for Sf9/D2 receptors) or 0.05 nM [125I]NCQ298 (for D2/D3 receptors in striatal homogenates) as the radioligand and 8−10 concentrations (10-10−10-5 M) of competing drugs (serially diluted in Tris-HCl buffer containing 0.1% BSA) in these cell membranes as described previously. 37 Nonspecific binding was defined with 1 μM spiperone. Incubation was carried out at 37 °C for 30 min. The reaction was terminated by separation of bound from free radioligand by filtration through glass fiber filters presoaked with 1% poly(ethylenimine). The filters were then washed three times with 3 mL of ice-cold 20 mM Tris buffer and counted in a gamma counter with 70% efficiency. Competition experiments were analyzed using the iterative nonlinear least-squares curve-fitting program LIGAND.

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Dopamine and Serotonin Tranporter Binding Assay. [1]
Binding of [125I]IPT to dopamine and serotonin transporters was carried out in rat striatal and cortical homogenates, respectively, as previously described. 39 Competition experiments were performed in the buffer containing 50 mM Tris-HCl, pH 7.4, and 120 mM NaCl with 0.2 M [125I]IPT (for striatal homogenates) or 0.5 M [125I]IPT (for cortical homogenates) and 8−10 concentrations (10-10−10-5 M) of competing drugs. Nonspecific binding was defined in the presence of 40 μM (−)-cocaine. Competition experiments were analyzed using the iterative nonlinear least-squares curve-fitting program LIGAND.

Animal/Disease Models: Male Wistar rats (120-300 g)[2]
Doses: 3 mg/kg
Route of Administration: intraperitoneal (ip)injection; once
Experimental Results: The levels of cytosolic acetylcholine (ACh) increased in all regions of the brain, while those of vesicular ACh diminished in all regions except for the striatum.

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In the present experiments, cholinergic parameters were determined for regions of the brain of rats that had been treated with Vesamicol alone or combination with DDVP. Rats received a single dose (3 mg/5 ml saline/kg, IP) of Vesamicol [(±)-Vesamicol hydrochloride) and they were sacrificed 30 or 60 min later. Control rats were injected with 5 ml/kg of saline and killed 30 min later. Some animals received DDVP (5 mg/kg, SC; Kobayashi et al.) 10 min prior to Vesamicol or saline and were sacrificed 40 or 70 min later. A dose of Vesamicol of 3 mg/kg was chosen because a higher dose (5 mg/kg) killed two of five rats tested and the LD50 of this drug (administered IP) has been reported to be 4.2 mg/kg in mice [2].

Metabolism / Metabolites
Copper is mainly absorbed through the gastrointestinal tract, but it can also be inhalated and absorbed dermally. It passes through the basolateral membrane, possibly via regulatory copper transporters, and is transported to the liver and kidney bound to serum albumin. The liver is the critical organ for copper homoeostasis. In the liver and other tissues, copper is stored bound to metallothionein, amino acids, and in association with copper-dependent enzymes, then partitioned for excretion through the bile or incorporation into intra- and extracellular proteins. The transport of copper to the peripheral tissues is accomplished through the plasma attached to serum albumin, ceruloplasmin or low-molecular-weight complexes. Copper may induce the production of metallothionein and ceruloplasmin. The membrane-bound copper transporting adenosine triphosphatase (Cu-ATPase) transports copper ions into and out of cells. Physiologically normal levels of copper in the body are held constant by alterations in the rate and amount of copper absorption, compartmental distribution, and excretion. (L277, L279)

Toxicity Summary
Excess copper is sequestered within hepatocyte lysosomes, where it is complexed with metallothionein. Copper hepatotoxicity is believed to occur when the lysosomes become saturated and copper accumulates in the nucleus, causing nuclear damage. This damage is possibly a result of oxidative damage, including lipid peroxidation. Copper inhibits the sulfhydryl group enzymes such as glucose-6-phosphate 1-dehydrogenase, glutathione reductase, and paraoxonases, which protect the cell from free oxygen radicals. It also influences gene expression and is a co-factor for oxidative enzymes such as cytochrome C oxidase and lysyl oxidase. In addition, the oxidative stress induced by copper is thought to activate acid sphingomyelinase, which lead to the production of ceramide, an apoptotic signal, as well as cause hemolytic anemia. Copper-induced emesis results from stimulation of the vagus nerve. (L277, T49, A174, L280)

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212231 mouse LD50 oral 48931 ug/kg PERIPHERAL NERVE AND SENSATION: FLACCID PARALYSIS WITHOUT ANESTHESIA (USUALLY NEUROMUSCULAR BLOCKAGE) European Journal of Pharmacology., 8(93), 1969 [PMID:4243404]
212231 mouse LD50 intravenous 5874 ug/kg PERIPHERAL NERVE AND SENSATION: FLACCID PARALYSIS WITHOUT ANESTHESIA (USUALLY NEUROMUSCULAR BLOCKAGE) European Journal of Pharmacology., 8(93), 1969 [PMID:4243404]

[1]. N-hydroxyalkyl derivatives of 3 beta-phenyltropane and 1-methylspiro[1H-indoline-3,4'-piperidine]: vesamicol analogues with affinity for monoamine transporters. J Med Chem. 1997 Nov 21;40(24):3905-14.

[2]. Effects of systemic administration of 2-(4-phenyl-piperidino)-cyclohexanol (vesamicol) and an organophosphate DDVP on the cholinergic system in brain regions of rats. Brain Res Bull. 1997;43(1):17-23.

2-(4-phenyl-1-piperidinyl)-1-cyclohexanol is a member of piperidines.

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As part of our ongoing structure-activity studies of the vesicular acetylcholine transporter ligand 2-(4-phenylpiperidino)cyclohexanol (Vesamicol, 1), 22 N-hydroxy(phenyl)alkyl derivatives of 3 beta-phenyltropane, 6, and 1-methylspiro[1H-indoline-3,4'-piperidine], 7, were synthesized and tested for binding in vitro. Although a few compounds displayed moderately high affinity for the vesicular acetylcholine transporter, no compound was more potent than the prototypical vesicular acetylcholine transporter ligand Vesamicol. However, a few derivatives of 6 displayed higher affinity for the dopamine transporter than cocaine. We conclude that modification of the piperidyl fragment of 1 will not lead to more potent vesicular acetylcholine transporter ligands. [1]

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In summary, 3β-phenyltropanyl derivatives of Vesamicol exhibit lower affinity for the vesicular acetylcholine transporter than the parent compound. Consequently, the introduction of a two-carbon bridge across the C2 and C6 positions of the piperidyl moiety of Vesamicol is not a suitable strategy for enhancing affinity for the vesicular acetylcholine transporter or increasing selectivity for the vesicular acetylcholine transporter relative to σ receptors. The introduction of an aminomethyl bridge into the C fragment of Vesamicol (to yield analogues of 7) is also unsuitable for similar reasons. Since the results obtained with the tropanes suggest that fragment B binds to a narrow pocket within the binding site, we suggest that further modification of this fragment should be limited to single-point substitution. [1]

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The observation that the binding of QNB to mAChRs was unaffected 30 and 60 min after injection of Vesamicol indicates that this drug is unable to modify the mAChRs that have high affinity for ACh. A kinetic model has suggested that ACh has high affinity for the vesicular ACh transporter, through which ACh is transported and to which vesamicol binds at an allosteric site, referred to as the Vesamicol receptor. Kd and Bmax express the affinity for the ligand ACh and the density of mAChRs. Administration of DDVP caused an increase in Kd and a decrease in Bmax in the present experiments, suggesting that accumulation of ACh must have occurred around mAChRs. Vesamicol failed to reverse the effects of DDVP on the ability of QNB to bind to mAChRs.
In conclusion, the present study of cholinergic parameters in three regions of the rat brain demonstrated that Vesamicol, an inhibitor of the transport of ACh into synaptic vesicles in vitro and in situ caused reduced levels of vesicular ACh and increased levels of cytoplasmic ACh in vivo after systemic administration. Thus, vesamicol apears to inhibit the uptake of ACh in the cytoplasm into vesicles in vivo. Other parameters, such as HACU, the activity of ChAT and levels of mAChRs, appear not to be involved directly in the effects of vesamicol under the present experimental conditions.[2]

C17H25NO

259.39

259.194

C, 78.72; H, 9.71; N, 5.40; O, 6.17

22232-64-0

22232-64-0; 120447-62-3; 112709-59-8; 23965-53-9

5662

Typically exists as solid at room temperature

1.086g/cm3

393.5ºC at 760 mmHg

97ºC

6.73E-07mmHg at 25°C

1.57

3.107

1

2

2

19

266

0

C1(C2CCN(C3CCCCC3O)CC2)C=CC=CC=1

YSSBJODGIYRAMI-UHFFFAOYSA-N

InChI=1S/C17H25NO/c19-17-9-5-4-8-16(17)18-12-10-15(11-13-18)14-6-2-1-3-7-14/h1-3,6-7,15-17,19H,4-5,8-13H2

2-(4-phenylpiperidin-1-yl)cyclohexan-1-ol

AH-5183; vesamicol; 22232-64-0; 2-(4-phenylpiperidin-1-yl)cyclohexan-1-ol; CHEMBL20943; 2-(4-Phenyl-piperidin-1-yl)-cyclohexanol; MLS000554350; 2-(4-Phenyl-1-piperidinyl)cyclohexanol; Cyclohexanol, 2-(4-phenyl-1-piperidinyl)-; AH 5183; Vesamicol

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.)