TRPV4/transient receptor potential vanilloid 4

GSK1016790A (0.1-1000 nM) When added to mouse and human embryonic kidney (HEK) cells that express human TRPV4, it causes Ca2+ influx (EC50 values of 18 and 2.1 nM) and, at a dose of 1 nM, TRPV4 whole-cell currents. In a portion of neurons, treatment with (100 nM) results in a rapid rise in intracellular Ca2+ [2].

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The transient receptor potential (TRP) vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. In this study, we describe a small molecule TRPV4 channel activator, (N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), which we have used as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca2+ influx in mouse and human TRPV4-expressing human embryonic kidney (HEK) cells (EC50 values of 18 and 2.1 nM, respectively), and it evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast, the TRPV4 activator 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) was 300-fold less potent than GSK1016790A in activating TRPV4 currents. TRPV4 mRNA was detected in urinary bladder smooth muscle (UBSM) and urothelium of TRPV4+/+ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4-/- bladders. TRPV4 activation with GSK1016790A contracted TRPV4+/+ mouse bladders in vitro, both in the presence and absence of the urothelium, an effect that was undetected in TRPV4-/- bladders. Consistent with the effects on TRPV4 HEK whole-cell currents, 4alpha-PDD demonstrated a weak ability to contract bladder strips compared with GSK1016790A[1].

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TRPV4 located on myenteric neurons mediate the effect of GSK1016790A [2]
Studies in myenteric neurons isolated and cultured from the proximal and distal colon of WT and TRPV4−/− mice and HEK-293 cells expressing TRPV4 were then performed to further confirm that GSK1016790A selectively activates TRPV4 channels and to elucidate the role of extracellular and intracellular Ca2+ in GSK1016790A-induced effects.
GSK1016790A (100 nM) treatment elicited a rapid elevation of intracellular Ca2+ in a subset of neurons (48 ± 2 % or 1143/2289 neurons, n = 12) (Fig. 3a). Responses were predominantly sustained for the duration of the stimulation. Non-neuronal cells also responded to TRPV4 activators. These were small cells with multiple processes, suggesting functional expression of TRPV4 by interstitial cells of Cajal or enteric glial cells under these culture conditions. However, as the focus of the present study was neuronal TRPV4 expression, the identity of these cells was not characterized further.
Responses to GSK1016790A (100 nM) were effectively abolished following pretreatment with the TRPV4-selective antagonist HC067047 (10 μM, 30 min pretreatment). The neuronal responses to carbachol (10 μM) and KCl (50 mM) were retained after HC treatment, indicating that neuronal viability was not significantly affected (Fig. 3b). Similar block of responses to GSK1016790A occurred following pretreatment with the non-selective ion channel inhibitor RuR (1 μM, 30 min pretreatment). The functional expression of TRPV4 by myenteric neurons was confirmed by comparing wild-type to TRPV4−/− mice (Fig. 3b–d). GSK1016790A failed to increase intracellular Ca2+ levels in neurons cultured from TRPV4−/− mice. The responses to CCh and KCl were unaffected (Fig. 3d).
The specificity of GSK1016790A was examined in HEK cells stably expressing human TRPV4. GSK1016790A (10−10–10−6 M) elevated intracellular Ca2+ in a concentration-dependent manner (Supplementary Information, Fig. S2), and this effect was blocked by RN 1734 (5 × 10−6 M) or absent in cells in which TRPV4 expression was not induced. We also observed the absence of the stimulatory effect of GSK1016790A in Ca2+-free buffer containing EDTA, suggesting that the extracellular calcium is required for this action.
Depletion of the intracellular calcium stores in isolated smooth muscle strips from the mouse colon (for details, see Supplementary Information) abolished the GSK1016790A-induced relaxant effect on smooth muscle in the mouse colon (Supplementary Information, Fig. S3). We may thus hypothesize that the presence of Ca2+ ions in the intracellular stores is required for the GSK1016790A-induced effects.

In mice, GSK1016790A (0.001-0.1 mg/kg; intraperitoneal injection) inhibits the transcriptional transit time in a dose-dependent manner [2]. Significant inhibition occurs in a concentration-inhibitory manner with GSK1016790A (0.1-1000 nM; assuming 10 minutes) [2].

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In vivo, urodynamics in TRPV4+/+ and TRPV4-/- mice revealed an enhanced bladder capacity in the TRPV4-/- mice. Infusion of GSK1016790A into the bladders of TRPV4+/+ mice induced bladder overactivity with no effect in TRPV4-/- mice. Overall TRPV4 plays an important role in urinary bladder function that includes an ability to contract the bladder as a result of the expression of TRPV4 in the UBSM[1].

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GSK1016790A inhibits smooth muscle contractility in mouse colon [2]
Next, we characterized the effect of TRPV4 activation on smooth muscle contractility in the mouse ileum and colon segments in vitro using an organ bath. GSK1016790A significantly inhibited the EFS-induced twitch contractions in isolated mouse colon strips in a concentration-dependent manner (Fig. 2a). The effect of GSK1016790A in the mouse colon was blocked by RN 1734 (10−5 M) and RuR (10−6 M, Fig. 2a). To confirm that the effect of GSK1016790A was mediated by the TRPV4 channels, the experiments were repeated in the presence of a selective TRPV1 antagonist 4′-chloro-3-methoxycinnamanilide (SB 366791). SB 366791 (10−6 M) did not influence the inhibitory action of GSK1016790A on smooth muscle contractility in the mouse colon (Supplementary Information, Fig. S1). GSK1016790A in the concentrations used had no effect on basal tone, resting phasic activity, or smooth muscle pre-contracted with bethanechol (10−7–2 × 10−5 M, data not shown; n = 8), making a direct effect on smooth muscle unlikely.

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GSK1016790A inhibits gastrointestinal motility [2]
In order to determine whether the in vitro effects of TRPV4 activators on the intestine translated to the whole animal, the effects of GSK1016790A were studied in standardized tests of GI motility in mice.GSK1016790A (0.001–0.1 mg/kg, i.p.) produced a dose-dependent inhibitory effect on whole gut transit time in mice, which was reversed by RN 1734 (1 mg/kg, i.p.; Fig. 4a). The inhibitory effect of GSK1016790A (0.1 mg/kg, i.p.) was absent in TRPV4−/− mice. In addition, GSK1016790A produced a dose-dependent (Fig. 4b) and time-dependent (Fig. 4c) inhibitory effect on colonic expulsion after i.p. administration in mice, which was blocked by RN 1734 (1 mg/kg, i.p.). We observed no effect of GSK1016790A on colonic bead expulsion in TRPV4−/− animals (Fig. 4d).
The i.p. administration of GSK1016790A (0.1 mg/kg) did not produce any effect on upper GI transit time in mice (Supplementary Information, Fig. S4).
Consequently, two animal models mimicking pathophysiological conditions in humans, a reversible acetylcholinesterase inhibitor neostigmine- and stress-induced hypermotility, were used in the study. GSK1016790A (0.001–0.1 mg/kg, i.p.) administered in a dose-dependent manner decreased the GI hypermotility induced by neostigmine (2.5 μg/kg, i.p.) in mice (Fig. 4e). Furthermore, the i.p. administration of GSK1016790A (0.1 mg/kg, i.p.) reduced the fecal pellet output in mice exposed to mild stress (Fig. 4f). GSK1016790A had no effect in TRPV4−/− mice in this model (Fig. 4f).

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NOS is involved in the TRPV4-induced effect of GSK1016790A in the mouse colon through a soluble guanylate cyclase as downstream effector [2]
NO is a major inhibitory neurotransmitter produced by inhibitory motor neurons and descending interneurons. Here, we examined the involvement of NO in inhibitory effects of the TRPV4 agonist on colonic contractions and further determined the effectors downstream to TRPV4 using an established NO-imaging technique.
The experiments revealed that GSK1016790A (10−7 M) stimulated the release of NO in LMMP preparations of the mouse colon (Fig. 5a, b). The effect of GSK1016790A was blocked by RuR (10−6 M), and SMTC (10−5 M) and 1400W (10−5 M; Fig. 5b, c) in combination. Because NO is a gaseous neurotransmitter that readily diffuses across cell membranes, it is a significant challenge to determine the cellular source of NO within the myenteric plexus, as all cells are loaded with the NO-sensitive dye. The application of the extracellular NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO, 10−4 M) prevents the extracellular movement of NO, thereby permitting the localization of cells producing NO (versus responding to NO) based on cellular fluorescence. The percentage of neurons producing NO in response to GSK1016790A within the myenteric plexus was 40 ± 6 (n = 3). When SMTC and PTIO were applied in combination, the neuronal response to GSK1016790A was completely inhibited, while the neuronal response to GSK1016790A in the presence of 1400W and PTIO was unchanged compared to GSK alone (Fig. 5b).

In vitro experiments on isolated smooth muscle strips[2]
Contractility of isolated intestinal segments was examined as described (Supplementary Information). GSK1016790A ((N-((1S)-1-{[4-((2S)-2-{[(2,4-Dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide, 10−10–10−6 M) was added cumulatively to the organ baths and effects on the electrical field stimulation (EFS; 8 Hz)-induced contractions or relaxations were recorded. Each concentration was allowed to incubate for 10 min. Prior to addition of GSK1016790A, the mean amplitude of four successive twitch contractions or relaxations was used to generate an internal control. Changes in contraction or relaxation amplitudes were reported as the percentage of the internal control. The effect of the vehicle (DMSO) was tested in control experiments.[2]
In separate experiments, the TRPV4 antagonist 2,4-Dichloro-N-isopropyl-N-(2-isopropylaminoethyl)benzenesulfonamide (RN 1734, 10−5 M), the TRPV antagonist ruthenium red (RuR, 10−6 M), the NOS-1 blocker S-methyl-L-thiocitrulline (SMTC, 10−5 M), the NOS-2 blocker N-(3-aminomethyl) benzylacetamidine (1400W, 10−6 M), and the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10−5 M) were added to the organ baths 10 min prior to addition of GSK1016790A. The effects of antagonists and the control experiments were performed as paired assays, using eight organ baths in parallel.

Measurement of [Ca2+]i in myenteric neurons[2]
For measurement of intracellular calcium [Ca2+]i) in individual neurons, cells were loaded with Fura2-AM ester (2 μM, 30 min, 37 °C) in Hank’s balanced salt solution (HBSS) containing 20 mM HEPES and 0.1 % BSA. Fluorescence was measured at 37 °C using a Leica DMI-6000B imaging system with ×10 dry objective. Images were collected (5-s intervals) at 340 and 380 nm excitation and 510 nm emission wavelengths. Images were processed using ImageJ software with McMaster biophotonics Facility plugins (v1.46b; http://imagej.nih.gov/ij). Results were expressed as the 340/380 nm fluorescence emission ratio, as an indirect measurement of intracellular calcium ([Ca2+]i). Neurons were sequentially challenged with GSK1016790A (100 nM, TRPV4 agonist), carbachol (1 μM, muscarinic receptor agonist), and KCl (50 mM). In some experiments, cells were pretreated with HC067047 (10 μM, TRPV4 antagonist) or ruthenium red (10 μM) 30 min prior to addition of agonists. Cells that responded to GSK1016790A and KCl with a peak 340/380 nm ratio greater than 0.1 units above baseline were counted as TRPV4-positive neurons.

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In vitro experiments on isolated smooth muscle strips [2]
Contractility of isolated intestinal segments was examined as described (Supplementary Information). GSK1016790A ((N-((1S)-1-{[4-((2S)-2-{[(2,4-Dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide, 10−10–10−6 M) was added cumulatively to the organ baths and effects on the electrical field stimulation (EFS; 8 Hz)-induced contractions or relaxations were recorded. Each concentration was allowed to incubate for 10 min. Prior to addition of GSK1016790A, the mean amplitude of four successive twitch contractions or relaxations was used to generate an internal control. Changes in contraction or relaxation amplitudes were reported as the percentage of the internal control. The effect of the vehicle (DMSO) was tested in control experiments.
In separate experiments, the TRPV4 antagonist 2,4-Dichloro-N-isopropyl-N-(2-isopropylaminoethyl)benzenesulfonamide (RN 1734, 10−5 M), the TRPV antagonist ruthenium red (RuR, 10−6 M), the NOS-1 blocker S-methyl-L-thiocitrulline (SMTC, 10−5 MA), the NOS-2 blocker N-(3-aminomethyl) benzylacetamidine (1400W, 10−6 M), and the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were added to the organ baths 10 min prior to addition of GSK1016790A. The effects of antagonists and the control experiments were performed as paired assays, using eight organ baths in parallel.

Animal/Disease Models: S100β-GFP and TRPV4 knockout (TRPV4-/-) mice [2]
Doses: 0.001, 0.01, 0.1 mg/kg
Route of Administration: IP; Single dose
Experimental Results: Effect on whole intestinal transit time in mice Dose-dependent inhibition. Produces dose-dependent and time-dependent inhibition of colonic evacuation.

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Generation of TRPV4−/− Mice. [1]
Genomic fragments homologous to the TRPV4 locus were cloned by polymerase chain reaction (PCR) from the E14.1 embryonic stem (ES) cell line using the Expand long template PCR kit (Roche, Palo Alto, CA). Primers for the 5′ homology arm were VR4_5F (5′-TTC TTG TTG ACC CAC AAG AAG CGC CT-3′) and VR4_5R (5′-ATG GTG TCG TTG CGC CCG TTG CTT AGG TT-3′) spanning coding exons 3 to 4; for the 3′ arm, VR4_3F (5′-TTC TTC CAG CCC AAG GAT GAG GGA GGC T-3′) and VR4_3R (5′-AGA TGC CGG GTG TCC TCA TCT GTC ACC...

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In vivo whole gastrointestinal transit [2]
To evaluate the peristaltic motility in the mouse whole intestine, whole gut transit time was measured using the established protocol. Briefly, 0.15 ml of viscous liquid consisting of 5 % Evans blue (a non-absorbable colored marker) and 5 % gum arabic was administered intragastrically to mice, using an 18-gauge animal feeding tube. Fifteen minutes before marker administration, mice were treated (i.p.) with GSK1016790A or vehicle. In selected experiments, the TRPV4 antagonist RN 1734 was injected i.p. 15 min before agonist administration. Immediately after the administration of the marker, mice were returned to individual cages, which were placed on a white sheet in order to facilitate recognition of colored boluses. Time elapsed between intragastric administration of the marker and the excretion of the first colored fecal bolus was considered as time of whole gut transit.

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In vivo colonic expulsion test [2]
Distal colonic expulsion was measured using the following protocol: after an overnight fasting period, GSK1016790A or vehicle was injected i.p. and a pre-warmed (37 °C) glass bead (2 mm) was inserted 2 cm into the distal colon using a silicone pusher. After the bead insertion, mice were placed in individual cages and the time to bead expulsion was determined. Mice that did not expel the bead within 30 min were sacrificed to confirm the presence of the bead in the lumen of the intestine.

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In vivo fecal pellet output [2]
To test the effect of GSK1016790A on stress-induced defecation, non-fasted animals were injected i.p. with GSK1016790A (0.001–0.1 mg/kg) or vehicle 15 min prior to the start of the experiment. Mice were then placed on a metal grid in new clean cages or left undisturbed in their home cages and the number of fecal pellets excreted over a 60-min period was counted. Fecal pellet output was also measured in mice receiving an i.p. injection of neostigmine (2.5 μg/kg, i.p.), a reversible acetylcholinesterase inhibitor.

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Pharmacological treatments [2]
GSK1016790A (0.001–0.1 mg/kg) or vehicle was injected intraperitoneally (i.p.) 15–75 min prior to the start of the assay. In selected experiments, RN 1734 was injected i.p. 15 min before agonist administration.

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Nitric oxide imaging [2]
Nitric oxide (NO) imaging was performed as described (Supplementary Information). GSK1016790A (10−7 M) was bath applied for 30 s after a 5-min baseline period. Inhibitors were bath applied for the duration of the experiment and included RuR (10−6 M), SMTC (10−5 M), and 1400W (10−5 M). Additionally, the extracellular NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 10−4 M) was utilized.

[1]. N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I. J Pharmacol Exp Ther. 2008 Aug;326(2):432-42.

[2]. Transient receptor potential vanilloid 4 inhibits mouse colonic motility by activating NO-dependent enteric neurotransmission. J Mol Med (Berl). 2015 Dec;93(12):1297-309.

GSK1016790A is a tertiary carboxamide that is piperazine in which one of the amino groups has undergone condensation with the carboxy group of N-[(2,4-dichlorophenyl)sulfonyl]-L-serine, while the other has undergone condensation with the carboxy group of N-(1-benzothiophen-2-ylcarbonyl)-L-leucine. It is a cell-permeable, potent and selective agonist of the TRPV4 (transient receptor potential vanilloid 4) channel. It has a role as a TRPV4 agonist. It is a member of 1-benzothiophenes, a N-acylpiperazine, a sulfonamide, a dichlorobenzene, a tertiary carboxamide and an aromatic primary alcohol.

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Identification of the TRPV4 Activator GSK1016790A. [[1]
As part of a small molecule screening effort, GSK1016790A was identified as a novel TRPV4 channel activator (Fig. 1A). GSK1016790A potently induced Ca2+ influx in HEK cells expressing mouse TRPV4 channels with an EC50 value of 18 nM (Supplemental Fig. 2). GSK1016790A demonstrated a similar potency at human TRPV4 channels (EC50 = 2.1 nM). GSK1016790A was inactive against TRPV1 channels (see accompanying article, Willette et al., 2008), which, based on sequence homology, is the TRP superfamily..
GSK1016790A is a novel TRPV4 channel activator that is ∼300-fold more potent than the commonly used TRPV4 activator 4α-PDD. We have used molecular techniques, TRPV4−/− mice, in vitro contractility, and in vivo urodynamics in conjunction with GSK1016790A to support a role for TRPV4 in UBSM. TRPV4 mRNA was amplified by PCR, and protein expression was demonstrated by Western blot analysis and immunohistochemistry in UBSM and urothelium. GSK1016790A contracted bladder independent of the urothelium..

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In the present study, a TRPV4-selective agonist GSK1016790A served as a tool to evaluate the role of TRPV4 in GI motility. Our goal was to characterize the effect of GSK1016790A on smooth muscle contractility and relaxation in the mouse intestine in vitro and in animal models of GI transit. In order to elucidate the intracellular pathways implicated in TRPV4-dependent signaling, calcium- and NO-imaging techniques were used. In addition, studies of tissues from healthy human subjects and patients with motility disorders were performed. Our data indicate that TRPV4 could become a viable target in the treatment of motility-related disorders in humans. [2]

C28H32CL2N4O6S2

655.61

654.114

C, 51.30; H, 4.92; Cl, 10.81; N, 8.55; O, 14.64; S, 9.78

942206-85-1

23630211

White to off-white solid powder

5.285

3

8

10

42

1070

2

CC(C)C[C@@H](C(=O)N1CCN(CC1)C(=O)[C@H](CO)NS(=O)(=O)C2=C(C=C(C=C2)Cl)Cl)NC(=O)C3=CC4=CC=CC=C4S3

IVYQPSHHYIAUFO-VXKWHMMOSA-N

InChI=1S/C28H32Cl2N4O6S2/c1-17(2)13-21(31-26(36)24-14-18-5-3-4-6-23(18)41-24)27(37)33-9-11-34(12-10-33)28(38)22(16-35)32-42(39,40)25-8-7-19(29)15-20(25)30/h3-8,14-15,17,21-22,32,35H,9-13,16H2,1-2H3,(H,31,36)/t21-,22-/m0/s1

N-[(2S)-1-[4-[(2S)-2-[(2,4-dichlorophenyl)sulfonylamino]-3-hydroxypropanoyl]piperazin-1-yl]-4-methyl-1-oxopentan-2-yl]-1-benzothiophene-2-carboxamide

GSK1016790A; GSK-1016790A; GSK1016790A; 942206-85-1; GSK-1016790A; N-[(2S)-1-[4-[(2S)-2-[(2,4-dichlorophenyl)sulfonylamino]-3-hydroxypropanoyl]piperazin-1-yl]-4-methyl-1-oxopentan-2-yl]-1-benzothiophene-2-carboxamide; N-((S)-1-(4-((S)-2-(2,4-dichlorophenylsulfonaMido)-3-hydroxypropanoyl)piperazin-1-yl)-4-Methyl-1-oxopentan-2-yl)benzo[b]thiophene-2-carboxamide; CHEMBL4461515; (N-((1S)-1-{[4-((2S)-2-{[(2,4-Dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide; GSK 1016790A.

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)

DMSO : ≥ 33 mg/mL (~50.33 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.81 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (3.81 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.17 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)