D1 Receptor (Ki = 1.18 nM); Sigma 1 Receptor; D5 Receptor (Ki = 7.56 nM); D2 Receptor (Ki = 920 nM); D3 Receptor (Ki = 399 nM)

PIP2 hydrolysis in membranes is stimulated by SKF83959 hydrobromide (10~250 μM). The EC50 value of SKF81297 is altered by SKF83959 Hydrobromic acid (0.1~10 μM; PC12 cells) from 0.5 nM in normal tissue to 31.6 nM, 251.2 nM, and 631.0 nM [2].

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Effects of SKF83959 on [35S]GTPγS binding to Gα proteins in FC [2]
We next attempted to define the guanine nucleotide-binding regulatory protein (G protein) that links the D1 DAR to the PI pathway by measuring receptor-stimulated binding of [35S]GTPγS to membrane Gα proteins. Incubation of rat FC membranes with 100 µm SKF83959 resulted in increases of 41% and 96% in [35S]GTPγS binding to Gαi and Gαq, respectively, whereas binding to Gαs and Gαo proteins was unchanged (Fig. 5). The results therefore suggest that SKF83959 stimulates PI hydrolysis by coupling to Gαq and/or Gαi proteins.

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The effect of SKF83959 on brain and D1A/PC12 cell membranes adenylyl cyclase activities [2]
The ability of SKF83959 to regulate adenylyl cyclase activity was tested in membranes obtained from striatum and from D1A/PC12 cells. SKF38393 and SKF81297 stimulated adenylyl cyclase activity in striatal and D1A/PC12 cell membranes, whereas SKF83959 did not activate the enzyme in the two membrane preparations (Figs 7a and b). In contrast, this benzazepine exhibited a clear antagonism of D1 DAR-stimulated adenylyl cyclase as demonstrated by the shift to the right of the SKF81297 dose–response curves. The EC50 value of SKF81297 was changed from 0.5 nm in control tissue to 31.6 nm, 251.2 nm and 631.0 nm at the three SKF83959 doses (0.1, 1.0, 10 µm) tested (Fig. 8).

In the Y-maze test and passive avoidance task, SKF83959 hydrobromide (0.5 and 1 mg/kg; i.p.; 1 hour) restores the cognitive impairment caused by scopolamine [1]. SKF83959 hydrobromide (1 mg/kg; i.p.; 30 minutes) induces memory enhancement, which is prevented when the brain-derived neurotrophic factor pathway is blocked [1]. In the mouse hippocampal region, SKF83959 hydrobromide exhibits anti-amnestic properties and can reestablish the BDNF signaling pathway that hyoscyamine had lowered [1].

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Patients with Alzheimer's disease (AD) always have cognitive impairments. In this study we investigated whether 6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF83959) has improvements on cognitive dysfunction. The scopolamine model of dementia was used to investigate the anti-amnesic activities of SKF83959, and then, Western blotting and pharmacological inhibitor were used to assay the anti-amnesic mechanisms of SKF83959. It was found that SKF83959 administration significantly improved the scopolamine-induced memory impairments in the passive avoidance task, Y-maze test, and Morris water maze task. Moreover, SKF83959 treatment significantly antagonized the down-regulating effects of scopolamine on brain-derived neurotrophic factor (BDNF) signaling cascade in the hippocampus, but not cortex. Importantly, the usage of K252a, a selective inhibitor of tyrosine kinase B (TrkB), significantly attenuated the protective effects of SKF83959 in the scopolamine model. Collectively, this study shows that SKF83959 has beneficial effects in the scopolamine model of dementia by modulation of hippocampal BDNF signaling, implying a novel and potential therapeutic agent for treating dementia in AD. [1]

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Characteristics of benzazepines-mediated phosphoinositide hydrolysis in brain [2]
Three benzazepine compounds, SKF83959, SKF81297 and SKF38393 were tested for their effects on the hydrolysis of PIP2 in membranes prepared from rat FC. As indicated in Fig. 1, all three compounds showed dose-related increases in PIP2 hydrolysis. Maximal stimulations (3.4, 1.3 or 2.1-fold) were reached at 250 µm for SKF83959 and SKF81297, and at 500 µm for SKF38393, respectively. Relative potencies (EC50) for SKF83959, SKF81297 and SKF38393, calculated by curve fitting were 34.8 ± 3.1, 17.8 ± 1.2 and 72.4 ± 4.6 µm, respectively.

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SKF83959-stimulated phosphoinositide hydrolysis is blocked by SCH23390 and cis-flupenthixol [2]
Seven receptor antagonists were tested for their abilities to block SKF83959-stimulated PIP2 hydrolysis (Fig. 2a). The selective D1 DAR antagonist, SCH23390, and the mixed D1 and D2 receptor antagonist, cis-flupenthixol, inhibited SKF83959-stimulated PIP2 hydrolysis, whereas the mucarinic acetylcholine receptor antagonist, atropine, α1-adrenoceptor antagonist, prazosin, the D2 DAR antagonists, spiperone and (+)butaclamol, and the serotonin 5-HT2A/2C receptor antagonist, mesulergine did not affect the SKF83959-stimulated effect. SCH23390 dose-dependently inhibited SKF83959-stimulated PIP2 hydrolysis with an estimated IC50 value of 110 µm (Fig. 2b), indicating that this compound stimulates PI hydrolysis via a D1-like DAR subtype. However, stimulation of membranes prepared from D1A DAR-expressing PC12 cells with either SKF38393 or SKF83959 did not produce significant activation of PIP2 hydrolysis (Fig. 3). These data therefore clearly indicate that stimulation of the D1A receptor is not responsible for benzazepine-mediated PIP2 hydrolysis in rat brain membrane preparation.

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SKF83959-induces PI hydrolysis in membranes prepared from distinct brain regions [2]
The ability of SKF83959 to elicit [3H]PIP2 hydrolysis in membranes prepared different brain regions was compared. The results summarized in Fig. 4 show that at the concentrations 10 µm, 50 µm and 250 µm, SKF83959 significantly and dose-dependently stimulated IP's production in FC, hippocampus, striatum and cerebellum. The results therefore indicate that the PI-linked D1-like receptor subtype is widely distributed in brain.

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The effect of SKF83959 administration on rat brain PI hydrolysis [2]
We have, thus far, demonstrated that SKF83959 activates PI hydrolysis in isolated brain membranes. We therefore tested if the drug is also able to stimulate the PI pathway in vivo. Rats injected intraperitoneally with 0.8 mg/kg SKF83959 were killed 8, 15 and 25 min thereafter and brain regional IP3 accumulations were assessed. As shown in Fig. 6, SKF83959 increased IP3 content 8–25 min post-injection in striatum and 8–15 min post-injection in hippocampus at a dose that was previously shown to elicit behavioral responses.

Adenylyl cyclase assay [2]
D1A/PC12 cells were harvested after washing with phosphate-buffered solution. The cells were homogenized in 10 volumes (w/v) of chilled buffer containing 50 mm Tris-HCl, pH 7.4, 2 mm EGTA and 10% sucrose. The homogenate was centrifuged at 800 g for 10 min and the supernatant centrifuged at 49 000 g for 20 min. The pellet was washed twice and suspended in 50 mm Tris-HCl buffer, pH 7.4.
Striatum was dissected from the rat brain and homogenized using a Teflon/glass homogenizer in 10 volumes (w/v) of pre-chilled buffer containing 50 mm Tris-HCl, 2 mm EGTA and 10% sucrose, pH 7.4. The homogenate was centrifuged at 8000 g for 10 min, and the supernatant was centrifuged at 49 000 g for 20 min. The pellet was washed twice and suspended in 50 mm Tris-HCl buffer, pH 7.4.
Membrane protein was determined using bovine serum album as standards. The adenylyl cyclase assay was measured by a modified method described by Salomon (1979). Each assay was performed in 250 µL of solution containing 50 mm Tris-HCl, pH 7.4, 2 mm MgCl2, 0.1 mm ATP, 10 mm phosphocreatine, 5 U of creatine phosphokinase, 0.2 mm EGTA, 100 µm 3-isobutyl-1-methylxanthine, 10 µm guanosine triphosphate (GTP), 1 mm dithiothreitol, 1 µCi of [α-32P]ATP and 50 µg of membrane protein. The reaction was carried out at 30°C for 20 min and terminated by the addition of 300 µL of a solution containing 2% sodium dodecyl sulfate, 25 mm ATP and 1.3 mm cAMP. [32P]cAMP was separated from [32P]ATP by Dowex and alumina chromatography. [3H]cAMP was added to each reaction mixture to allow calculation and correction for column recovery. Radioactivity in each sample was determined by liquid scintillation spectroscopy.

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[35S]GTPγS-binding to select G-proteins [2]
FC was homogenized in 10 volumes of ice-cold homogenization buffer containing 25 mm HEPES (pH 7.4), 100 mm sucrose, 1 mm EGTA, 0.2% 2-mercaptoethanol, protease-inhibitor mixture (50 µg/mL leupeptin, 25 µg/mL pepstatin A, 0.01 U/mL soybean trypsin inhibitor and 0.04 mm phenylmethylsulfonyl fluoride) using glass/glass homogenizer. The tissue homogenate was centrifuged for 10 min at 750 g (4°C). The resultant supernatant was then centrifuged for 10 min at 48 200 g (4°C). The pellet was resuspended in reaction buffer containing 20 mm HEPES (pH 7.4), 154 mm NaCl, 4.8 mm KCl, 1.2 mm KHPO4, 1.2 mm MgCl2 and protease-inhibitor mixture. The assay mixture (250 µL) contained 200 µg of membrane protein, 2 nm[35S]GTPγS in the absence or presence of 100 µm of SKF83959. Reaction was stopped after 10 min of incubation at 37°C with 750 µL of ice-cold reaction buffer containing 1 mm EDTA and 1 mm EGTA. It was then centrifuged at 16 000 g for 5 min. The pellet was solubilized in precipitation buffer and immunoprecipitated with specific anti-Gα antisera at 4°C overnight followed by addition of 15 µL protein-A conjugated agarose beads. The mixture was shaken gently for 2 h. The beads were recovered by centrifugation, washed two times with 1 mL of precipitation buffer as described previously (Friedman et al. 1993; Wang et al. 1995). The resulting pellet containing the antigen–antibody complex was briefly sonicated and radioactivity was measured by liquid scintillation spectrometry.

Cell culture and expression of rat D1A DAR [2]
PC12 cells were plated at a density of approximately 100–200/mm2 on collagen-coated dishes in culture medium RPMI media 1640 (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% horse serum, 5% fetal calf serum, 50 pg/mL streptomycin, 50 U/mL penicillin. The cells were cultured at 37°C in a water-saturated atmosphere of 95% air and 5% CO2 and used for transfection when they reached 40–60% confluence. The rat D1A DAR, cDNA was inserted into the polylinker region of the pTargeT vector (Promega, Madison, WI, USA) as described previously (Cai et al. 1999). For transfection of the D1A DAR, Lipofectin was mixed with an equal volume of plasmid DNA in buffer containing 10 mm Tris, 1 mm EDTA, pH 8.0, and allowed to stand for 20 min at room temperature. Cells were washed twice and incubated with 3 mL of OPTI-MEM, a reduced serum medium. The DNA–Lipofectin mixture was added to cultured cells and incubated for 24 h. The DNA-containing medium was removed and replaced with 3 mL of RPMI media 1640 supplemented with 20% fetal bovine serum and incubated for an additional 24 h. Stable expression clones were selected with 400 µm G418 (genestin), a selective agent in gene transfer experiments, and maintained in medium with 200 µm G418. The positive expression of D1A receptor in PC12 cells was confirmed by receptor binding assay using [3H]SCH23390 as ligand. The binding tests of current D1A-expressing PC12 (D1A/PC12) cells yielded Bmax of 4.8 ± 0.7 pmol/mg and Kd of 0.38 ± 0.09 nm.

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Measurement of PIP2 hydrolysis in rat brain and in D1A-expressing PC12 cell membranes [2]
D1A/PC12 cells were harvested with phosphate-buffered solution. The cells were homogenized in 10 volumes (w/v) of chilled buffer containing 50 mm Tris-HCl, pH 7.4, 2 mm EGTA and 10% sucrose. The homogenate was centrifuged at 800 g for 5 min and the supernatant centrifuged at 49 000 g for 20 min. The resultant pellet was washed twice and suspended in 25 mm Tris-maleate buffer (pH 6.8) with 3 mm EGTA. [2]
Rats were decapitated, the brains rapidly removed, and the brain regions – cerebellum, frontal cortex (FC), hippocampus and striatum, dissected on ice. Brain tissues were brought to room temperature in pH 7.4 Kreb's Ringer bicarbonate (KRB) buffer containing 20 mm HEPES, 118 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO4, 1.2 mm KH2PO4, 25 mm NaHCO3 and 10 mm glucose equilibrated with O2/CO2 (95 : 5). The tissues were chopped into 350 × 350 µm slices, washed twice with KRB at room temperature and incubated for 15 min in KRB at 37°C in a shaking water bath. The slices were then washed twice with pH 7.0 hypotonic buffer containing 20 mm Tris-HCl, 1 mm EGTA at 4°C and homogenized in this buffer using 10 hand strokes of a Teflon-glass homogenizer. Homogenates were centrifuged at 3300 g for 15 min, the supernatant was discarded and the pellets were resuspended in pH 7.0, Tris-HCl 20 mm, KCl 2 m and EGTA 1 mm buffer, and centrifuged. The resultant pellets were washed twice in hypotonic buffer and finally suspended in 25 mm Tris-maleate buffer (pH 6.8) with 3 mm EGTA as described previously (Gonzales and Crews 1984; Carter et al. 1990). [2]
To determine phosphoinositide hydrolysis, 100 µg membrane protein was added to assay tubes containing 25 mm Tris-maleate (pH 6.8), 1 µm GTPγS, 6 mm MgCl2, 8 mm LiCl, 1 mm sodium deoxycholate/[3H]PIP2 (0.01 µCi) mixture, 100 nm free Ca2+ adjusted with 3 mm EGTA, various concentrations of agonists and/or antagonists in a final volume of 250 µL. The assays were started by the addition of membrane suspension and incubated for 30 min at 37°C. The reactions were stopped by the addition of 1.5 mL of chloroform : methanol : 12 N HCl mixture (100 : 200 : 1, v/v/v) followed by the addition of 500 µL of chloroform and 750 µL of water. The tubes were vortexed for 1 min and centrifuged at 800 g for 5 min to enhance the separation of the phases. The [3H]inositol phosphate(s) in the aqueous phase was directly counted. [2]

Animal/Disease Models: Male ICR male mice (8 weeks) [1]
Doses: 1 mg/kg
Route of Administration: intraperitoneal (ip) injection; 30 minutes
Experimental Results: BDNF system blockade prevented the memory-enhancing effect.

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Measuring brain IP3 accumulation in situ in response to in vivo SKF83959 challenge [2]
Rats were injected intraperitoneally with 0.8 mg/kg SKF83959 or vehicle and decapitated 8, 15 or 25 min thereafter. Brains were removed and FC and hippocampus were dissected. Tissues were homogenized in 1.5 mL of 1 m trichloroacetic acid and placed on ice for 15 min before centrifugation at 13 000 g for 10 min. The pellets were washed three times with distilled water and digested in 1 m NaOH and protein content was determined by the Bradford metho. One milliliter of trichlorotrifluoroethane–trioctylamine (3 : 1) was added to 500 µL of the trichloroacetic acid tissue extract in a polypropylene Eppendorf tube, mixed vigorously for 15 s and centrifuged for 1 min at 10 000 g. Aliquots of upper phase were taken for the IP3 determination according to the instructions provided by the manufacturer of the kit. IP3 concentration is expressed as pmol/mg protein.

[1]. SKF83959 Has Protective Effects in the Scopolamine Model of Dementia. Biol Pharm Bull. 2018;41(3):427-434.

[2]. SKF83959 selectively regulates phosphatidylinositol-linked D1 dopamine receptors in rat brain. J Neurochem. 2003;85(2):378-386.

[3]. Receptor affinities of dopamine D1 receptor-selective novel phenylbenzazepines. Eur J Pharmacol. 2003;474(2-3):137-140.

[4]. SKF83959 is a potent allosteric modulator of sigma-1 receptor. Mol Pharmacol. 2013;83(3):577-586.

N-methyl-6-chloro-1-(3-methylphenyl)-2,3,4,5-tetrahydro-3-benzazepine-7,8-diol hydrobromide is a hydrobromide salt prepared from N-methyl-6-chloro-1-(3-methylphenyl)-2,3,4,5-tetrahydro-3-benzazepine-7,8-diol and one equivalent of hydrogen bromide. Dopamine D1-like receptor partial agonist (Ki values are 1.18, 7.56, 920 and 399 nM for rat D1, D5, D2 and D3 receptors respectively). May act as an antagonist in vivo, producing anti-Parkinsonian effects and antagonising the behavioral effects of cocaine. It has a role as a dopamine agonist and a prodrug. It contains a N-methyl-6-chloro-1-(3-methylphenyl)-2,3,4,5-tetrahydro-3-benzazepinium-7,8-diol(1+).

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Previously a distinct D1-like dopamine receptor (DAR) that selectively couples to phospholipase C/phosphatidylinositol (PLC/PI) was proposed. However, lack of a selective agonist has limited efforts aimed at characterizing this receptor. We characterized the in vitro and in vivo effects of SKF83959 in regulating PI metabolism. SKF83959 stimulates (EC50, 8 micro m) phosphatidylinositol 4,5-biphosphate hydrolysis in membranes of frontal cortex (FC) but not in membranes from PC12 cells expressing classical D1A DARs. Stimulation of FC PI metabolism was attenuated by the D1 antagonist, SCH23390, indicating that SKF83959 activates a D1-like DAR. The PI-linked DAR is located in hippocampus, cerebellum, striatum and FC. Most significantly, administration of SKF83959 induced accumulations of IP3 in striatum and hippocampus. In contrast to other D1 DAR agonists, SKF83959 did not increase cAMP production in brain or in D1A DAR-expressing PC12 cell membranes. However, SKF83959 inhibited cAMP elevation elicited by the D1A DAR agonist, SKF81297, indicating that the compound is an antagonist of the classical D1A DAR. Lastly, we demonstrated that SKF83959 enhances [35S]guanosine 5'-O-(3-thiotriphosphate) binding to membrane Galphaq and Galphai proteins, suggesting that PI stimulation is mediated by activation of these guanine nucleotide-binding regulatory proteins. Results indicate that SKF83959 is a selective agonist for the PI-linked D1-like DAR, providing a unique tool for investigating the functions of this brain D1 DAR subtype.[2]

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We prepared a series of 18 novel substituted phenylbenzazepine congeners of the dopamine D1/D5 receptor partial-agonist SKF-83959 (R,S-3-methyl-6-chloro-7,8-dihydroxy-1-[3'-methylphenyl]-2,3,4,5-tetrahydro-1H-benzazepine) and characterized their potency and selectivity in assays of dopamine, 5-HT and adrenoceptors in rat brain tissue or membranes of genetically transfected cells. The R-enantiomer of SKF-83959 (MCL-202) and three other novel racemic 1-phenyl-7,8-dihydroxybenzazepines (MCL-204, -203, and -207) showed very high dopamine D5 receptor affinity; MCL-209 displayed the greatest dopamine D5 receptor affinity. These five potent novel ligands also had >100-fold selectivity for dopamine D1 over dopamine D2, D3, serotonin 5-HT-2A receptors and alpha2-adrenoceptors. They require further functional testing to characterize their intrinsic activity, and for potential stimulant-antagonist actions, as observed with SKF-83959 and MCL-202.[3]

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SKF83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine), an atypical dopamine receptor-1 (D(1) receptor) agonist, has shown many D(1) receptor-independent effects, such as neuroprotection, blockade of Na(+) channel, and promotion of spontaneous glutamate release, which resemble the effects of the sigma-1 receptor activation. In the present work, we explored the potential modulation of SKF83959 on the sigma-1 receptor. The results indicated that SKF83959 dramatically promoted the binding of (3)H(+)-pentazocine (a selective sigma-1 receptor agonist) to the sigma-1 receptor in brain and liver tissues but produced no effect on (3)H-progesterone binding (a sigma-1 receptor antagonist). The saturation assay and the dissociation kinetics assay confirmed the allosteric effect. We further demonstrated that the SKF83959 analogs, such as SCH22390 [(R)-(1)-7-chloro-8- hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride] and SKF38393 [(+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrobromide], also showed the similar allosteric effect on the sigma-1 receptor in the liver tissue but not in the brain tissue. Moreover, all three tested chemicals elicited no significant effect on (3)H-1,3-di(2-tolyl)-guanidine ((3)H-DTG) binding to the sigma-2 receptor. The present data uncovered a new role of SKF83959 and its analogs on the sigma-1 receptor, which, in turn, may reveal the underlying mechanism for the D(1) receptor-independent effect of the drug. [4]

C18H21BRCLNO2

398.721843481064

397.044

C, 54.22; H, 5.31; Br, 20.04; Cl, 8.89; N, 3.51; O, 8.03

67287-95-0

SKF 83959;80751-85-5

11957685

White to off-white solid powder

4.575

3

3

1

23

380

0

CC1=CC(=CC=C1)C2CN(CCC3=C(C(=C(C=C23)O)O)Cl)C.Br

FHYWNBUFNGHNCP-UHFFFAOYSA-N

InChI=1S/C18H20ClNO2.BrH/c1-11-4-3-5-12(8-11)15-10-20(2)7-6-13-14(15)9-16(21)18(22)17(13)19;/h3-5,8-9,15,21-22H,6-7,10H2,1-2H3;1H

9-chloro-3-methyl-5-(3-methylphenyl)-1,2,4,5-tetrahydro-3-benzazepine-7,8-diol;hydrobromide

SKF 83959 hydrobromide; 67287-95-0; SKF-83959 hydrobromide; SKF 83959; ...; skf83959 hydrobromide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~20 mg/mL (~50.16 mM)
DMF : 20 mg/mL (~50.16 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.)