PPARα (EC50 = 0.63 μM); PPARγ (EC50 = 32 μM)

As an agonist of PPARα, Pirinixic Acid (Wy-14643) has EC50s of 0.63 μM and 32 μM for PPARα and PPARγ in mice, and 5.0 μM, 60 μM, 35 μM, and PPARδ for PPARα and PPARγ in humans [1]. In synovial fibroblasts, pirinixic acid (Wy-14643; 0, 10, 100 μM) increases the expression of the PPAR-α protein. LPS-stimulated synovial fibroblasts' generation of NO and PGE2 is inhibited by pirinixic acid (0, 10, 100 μM). Additionally, pirinixic acid efficiently inhibits LPS-induced NF-kB activation, IkB phosphorylation, and TF in synovial fibroblasts as well as downregulating the production of inflammatory mediators in these cells, including VCAM-1, ICAM-1, ET-1, and TF. PPAR-α silenced cells, whereas pirinixic acid had little effect on NF-kB nuclear translocation [2].

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Osteoarthritis (OA), the most prevalent form of arthritis that results from breakdown of joint cartilage and underlying bone, has been viewed as a chronic condition manifested by persistence of inflammatory responses and infiltration of lymphocytes. Regulation of the inflammatory responses in synovial fibroblasts might be useful to prevent the development and deterioration of osteoarthritis. Pirinixic Acid/WY-14643, a potent peroxisome proliferator activator receptor-α (PPAR-α) agonist, has been described to beneficially regulate inflammation in many mammalian cells. Here, we investigate the potential anti-inflammatory role of WY-14643 in lipopolysaccharide (LPS)-induced synovial fibroblasts. WY-14643 greatly inhibited the production of NO and PGE2 induced by LPS. In addition, the mRNA expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelin-1 (ET-1), and tissue factor (TF) was significantly suppressed by Pirinixic Acid/WY-14643, as well as the secretion of pro-inflammatory cytokines including interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α (TNF-α), and monocyte chemotactic protein-1 (MCP-1). Furthermore, the transcription activity and nuclear translocation of NF-kB were found to be markedly decreased by WY-14643, while the phosphorylation of IkB was enhanced, indicating that the anti-inflammatory role of WY-14643 was meditated by NF-kB-dependent pathway. The application of WY-14643 failed to carry out its anti-inflammatory function in PPAR-α silenced cells, suggesting the role of PPAR-α. These findings may facilitate further studies investigating the translation of pharmacological PPAR-α activation into clinical therapy of OA [2].

In obese rats, pirinixic acid (Wy-14643; 10 mg/kg, IV) lowers MDA levels and liver damage. In the Sham and ischemia-reperfusion (IR) groups, pirinixic acid likewise increased SIRT1 activity, but it had no effect on the production of SIRT3 protein. In rats, pirinixic acid can prevent endoplasmic reticulum stress (ERS) and raise NAD+ and ATP levels [3].

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WY-14643 Administration Decreased Hepatic Injury and MDA Levels in Obese Rats [3]
First of all, we aimed to investigate the effect of WY-14643 pretreatment on hepatic injury in obese rats. As shown in Table 1, IR group was associated with increased ALT levels, which was prevented after treatment with WY-14643 (Table 1). In addition, pretreatment with the PPARα agonist decreased the release of lipid peroxidation products as observed for the low MDA levels (Table 2).

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WY-14643 Treatment Increased SIRT1 Activity, While No Effects Were Found on SIRT1 and SIRT3 Protein Expression [3]
It is known that hepatic deletion of SIRT1 alters PPARα signaling, but we then explored whether PPARα activation could affect the protein expression of SIRT1 and SIRT3. No changes on SIRT3 protein expression were observed among all the experimental groups (Figure 1(b)). By contrast, although SIRT1 protein expression increased during ischemia-reperfusion, its levels were not significantly different between IR and WY-14643 pretreated rats (Figure 1(a)). In addition, WY-14643 treatment resulted in enhanced SIRT1 activity in comparison to both Sham and IR group (Figure 1(c)).

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WY-14643 Administration Enhanced NAD+ Levels [3]
Due to the fact that SIRT1 depends on NAD+ levels, we determined the NAD+/NADH levels and the protein expression of nicotinamide phosphoribosyltransferase (NAMPT), a well-known mediator of NAD+ biosynthetic pathways. As evidenced in Figure 2(a), both IR and WY-14643 + IR groups showed augmented NAMPT levels when compared to Sham group. Moreover, obese rats submitted to IR presented significant decreases of NAD+/NADH levels in contrast to untreated animals, but WY-14643 contributed to more elevated NAD+ levels than IR group (Figure 2(b)).

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WY-14643 Pretreatment Augmented ATP Levels [3]
As PPARα induces fatty acid oxidation which is a source of ATP production, we then measured ATP levels. We observed that IR significantly decreased ATP levels when compared to Sham group, whereas WY-14643 administration previous to IR provoked an overwhelming increase in ATP levels (Figure 3).

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PPARα Enhancement Decreased ERS [3]
Excessive lipid accumulation in the tissues has been associated with ERS induction. Thus, possible alterations in protein expression of ERS parameters were evaluated. As shown in Figure 4, expression of IRE1α, p-eIF2, caspase 12, and CHOP was exacerbated by IR and restored by pretreatment with the PPARα agonist WY-14643.

Transaminases Assay [3]
Hepatic injury was assessed in terms of transaminases levels with a commercial kit from RAL. Briefly, blood samples were centrifuged at 4°C for 10 min at 3000 rpm and then were kept at −20°C. In order to assay transaminase activity, 200 μL of the supernatant was added to the substrate provided by the commercial kit. ALT levels were determined at 365 nm with an UV spectrometer and calculated following the supplier instructions.

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Lipid Peroxidation Assay [3]
Lipid peroxidation in liver was used as an indirect measurement of the oxidative injury induced by ROS. Lipid peroxidation was determined by measuring the formation of malondialdehyde (MDA) with the thiobarbiturate reaction. MDA in combination with thiobarbituric acid (TBA) forms a pink chromogen compound whose absorbance at 540 nm was measured. The result was expressed as nmols/mg protein.

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SIRT1 Activity Assay [3]
SIRT1 activity was determined according to the method described by Becatti et al. with some modifications. Protein extracts were obtained using a mild lysis buffer (50 mM Tris-HCl pH 8, 125 mM NaCl, 1 mM DTT, 5 mM MgCl2, 1 mM EDTA, 10% glycerol, and 0.1% NP40). SIRT1 activity was measured using a deacetylase fluorometric assay kit, following the manufacturer's protocol. A total of 25 μL of assay buffer containing the same quantity of protein extracts (10 μg/μL) was added to all wells, and the fluorescence intensity was monitored every 2 min for 1 h using the fluorescence plate reader Spectramax Gemini, applying an excitation wavelength of 355 nm and an emission wavelength of 460 nm. The results are expressed as the rate of reaction for the first 30 min, when there was a linear correlation between the fluorescence and this period of time.

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TP Quantification [3]
Tissue samples (20 mg) were pulverized in liquid N2 and homogenized in ice-cold 25 μL of KOH buffer (KOH 2.5 M, K2HPO4 1.5 M). Homogenates were then vortexed and centrifuged at 14,000 ×g at 4°C for 2 min. The supernatants were collected and dissolved in 100 μL of K2HPO4 1 M. Following this, pH was adjusted to 7 and samples were frozen at −80°C for posterior use. Finally, adenosine nucleotides were quantified with an ATP bioluminescent assay kit on a Victor 3 plate reader.

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NAD+/NADH Determination [3]
Hepatic NAD+/NADH levels were quantified with a commercially available kit according to the manufacturer's instructions.

Determine of NO Production Synovial fibroblasts were treated with LPS (100 μg/mL) in the presence or absence of GW7647. PPAR-α siRNA-transfected cells were also treated with LPS (100 μg/mL) together with WY-14643. After stimulation, the production of NO was determined using Griess reagents. The procedure was performed in accordance with the manufacturer’s instructions. Briefly, 300 μL of supernatant was mixed with 100 μL of Griess reagent and 2.6 mL of deionized water. The mixture was incubated for 30 min at room temperature, and the absorbance at 548 nm was measured. The concentrations of NO in the supernatants were calculated from a standard curve. [2]

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WY-14643, a potent peroxisome proliferator activator receptor-α (PPAR-α) agonist, has been described to beneficially regulate inflammation in many mammalian cells. Here, we investigate the potential anti-inflammatory role of WY-14643 in lipopolysaccharide (LPS)-induced synovial fibroblasts. WY-14643 greatly inhibited the production of NO and PGE2 induced by LPS. In addition, the mRNA expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelin-1 (ET-1), and tissue factor (TF) was significantly suppressed by WY-14643, as well as the secretion of pro-inflammatory cytokines including interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α (TNF-α), and monocyte chemotactic protein-1 (MCP-1). Furthermore, the transcription activity and nuclear translocation of NF-kB were found to be markedly decreased by WY-14643, while the phosphorylation of IkB was enhanced, indicating that the anti-inflammatory role of WY-14643 was meditated by NF-kB-dependent pathway. The application of WY-14643 failed to carry out its anti-inflammatory function in PPAR-α silenced cells, suggesting the role of PPAR-α. These findings may facilitate further studies investigating the translation of pharmacological PPAR-α activation into clinical therapy of OA.[2]

Rats were randomly divided into three experimental groups: (1) Sham, n = 6; (2) ischemia-reperfusion (IR), n = 6; and (3) WY-14643 + IR, n = 6. A model of partial (~70%) hepatic warm ischemia was applied. Briefly, a midline laparotomy was performed and the portal triad was dissected free of surrounding tissue. Then, an atraumatic clip was placed across the portal vein and hepatic artery to interrupt the blood supply to the left lateral and median lobes of the liver. After 60 min of partial hepatic ischemia, the clip was removed to recover hepatic reperfusion for 24 hours. Sham control rats underwent the same protocol without vascular occlusion. In the group of WY-14643 + IR, rats were treated with WY-14643 (10 mg/kg intravenously) 1 hour before the induction of IR. After 24 h of reperfusion, rats were sacrificed; blood samples were drawn from aorta and ischemic lobes were collected and stored at −80°C until assayed.[3]

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1 mg/kg i.v. bolus

Rats

Interactions
Inflammatory mediators orchestrate the host immune and metabolic response to acute bacterial infections and mediate the events leading to septic shock. Tumor necrosis factor (TNF) has long been identified as one of the proximal mediators of endotoxin action. Recent studies have implicated peroxisome proliferator-activated receptor alpha (PPARalpha) as a potential target to modulate regulation of the immune response. Since PPARalpha activators, which are hypolipidemic drugs, are being prescribed for a significant population of older patients, it is important to determine the impact of these drugs on the host response to acute inflammation. Therefore, we examined the role of PPARalpha activators on the regulation of TNF expression in a mouse model of endotoxemia. CD-1 mice treated with dietary fenofibrate or Wy-14,643 had fivefold-higher lipopolysaccharide (LPS)-induced TNF plasma levels than LPS-treated control-fed animals. Higher LPS-induced TNF levels in drug-fed animals were reflected physiologically in significantly lower glucose levels in plasma and a significantly lower 50% lethal dose than those in LPS-treated control-fed animals. Utilizing PPARalpha wild-type (WT) and knockout (KO) mice, we showed that the effect of fenofibrate on LPS-induced TNF expression was indeed mediated by PPARalpha. PPARalpha WT mice fed fenofibrate also had a fivefold increase in LPS-induced TNF levels in plasma compared to control-fed animals. However, LPS-induced TNF levels were significantly decreased and glucose levels in plasma were significantly increased in PPARalpha KO mice fed fenofibrate compared to those in control-fed animals. Data from peritoneal macrophage studies indicate that Wy-14,643 modestly decreased TNF expression in vitro. Similarly, overexpression of PPARalpha in 293T cells decreased activity of a human TNF promoter-luciferase construct. The results from these studies suggest that any anti-inflammatory activity of PPARalpha in vivo can be masked by other systemic effects of PPARalpha activators.

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Non-Human Toxicity Values
LD50 Rat oral 4150 mg/kg
LD50 Mouse oral 1600 mg/kg

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5694 rat LD50 oral 1050 mg/kg Journal of Medicinal Chemistry., 27(1621), 1984
5694 mouse LD50 oral 1600 mg/kg Atherosclerosis, 30(45), 1978 [PMID:209796]

[1]. The PPARs: from orphan receptors to drug discovery. J Med Chem. 2000 Feb 24;43(4):527-50.

[2]. PPAR-α Agonist WY-14643 Inhibits LPS-Induced Inflammation in Synovial Fibroblasts via NF-kB Pathway. J Mol Neurosci. 2016 Aug;59(4):544-53.

[3]. PPARα Agonist WY-14643 Induces SIRT1 Activity in Rat Fatty Liver Ischemia-Reperfusion Injury. Biomed Res Int. 2015;2015:894679.

Pirinixic acid is a member of pyrimidines, an organochlorine compound and an aryl sulfide. It is functionally related to an acetic acid.
Pirinixic Acid is a synthetic thiacetic acid derivative used in biomedical research, carcinogenic Pirinixic acid is a peroxisome proliferator that activates specific peroxisome proliferator-activated receptors (PPAR). PPARs play an important role in diverse cellular functions, including lipid metabolism, cell proliferation, differentiation, adipogenesis, and inflammatory signaling. (NCI04)

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Mechanism of Action
Effects of several classes of peroxisomal proliferators on peroxisomal functions, hepatomegaly, hepatocarcinogenesis and lipid metabolism have been extensively investigated in rodents. Less is known about influences of these agents, some used as hypolipidemic drugs, on various metabolic parameters in humans. We examined effects of clofibrate, di(2-ethyl-hexyl)phthalate (DEHP) and pirinixic acid (WY-14,643) on phospholipid metabolism in human fibroblasts in culture. Clofibrate inhibited incorporation of [1-(14)C]hexadecanol and [1-(14)C]linolenic acid into ethanolamine phosphoglycerides in a time- and concentration-dependent manner; labeling of plasmalogens and non-plasmalogen ethanolamine phosphoglycerides was reduced by 40-80% compared to a generalized 10-30% inhibition of labeling of other phospholipids, including phosphatidylcholine. In pulse and pulse-chase experiments, selective inhibition of incorporation of [1,2-(14)C]ethanolamine, compared to [methyl-(3)H]choline, confirmed relative specificity of inhibition of ethanolamine phosphoglycerides. Similar concentration dependence and specificity for inhibition of phospholipid turnover was observed for DEHP and WY-14,643, in both control and mutant (Zellweger and adrenoleukodystrophy) fibroblasts, in the absence of major effects on peroxisomal markers. These observations that peroxisomal proliferators specifically inhibit ethanolamine phosphoglyceride turnover in human fibroblasts should be considered when assessing the efficacy and safety of such agents as hypolipidemic drugs or when evaluating mechanisms of proliferator action at the cellular level.
Pirinixic acid (Wy-14,643) is an agonist of the peroxisome proliferator-activated receptor (PPAR) subtype alpha exhibiting beneficial effects in various inflammation-related processes in a slow, long-termed fashion. We recently showed that alpha-substituted pirinixic acid derivatives are agonists of PPAR alpha and act as dual inhibitors of 5-lipoxygenase (5-LO, EC 1.13.11.34) and the microsomal prostaglandin E(2) synthase-1 (EC 5.3.99.3). Here, we explored short-term effects of alpha-substituted pirinixic acid derivatives on typical neutrophil functions evoked by the agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP) including leukotriene formation, generation of reactive oxygen species, and release of human leukocyte elastase (EC 3.4.21.37), and we investigated the modulation of related signalling pathways. Pirinixic acid derivatives that are substituted with alkyl residues in alpha-position of the carboxylic group and with a 6-aminoquinoline residue at the pyrimidine moiety cause inhibition of leukotriene formation, reactive oxygen species formation, and leukocyte elastase release in response to fMLP. In parallel, Ca(2+) mobilisation and the phosphorylation (activation) of p38 mitogen-activated protein kinase was significantly reduced, whereas phosphorylation of the extracellular signal-regulated kinase-2 was unaffected. Pirinixic acid itself was not or only marginally active in all these assays. Conclusively, targeted structural modification of pirinixic acid leads to bioactive compounds that display immediate anti-inflammatory properties in human neutrophils with potential therapeutic value.
Normal function of the peroxisome proliferator-activated receptor alpha (PPARalpha) is crucial for the regulation of hepatic fatty acid metabolism. Fatty acids serve as ligands for PPARalpha, and when fatty acid levels increase, activation of PPARalpha induces a battery of fatty acid-metabolizing enzymes to restore fatty acid levels to normal. Hepatic fatty acid levels are increased during ethanol consumption. However, results of in vitro work showed that ethanol metabolism inhibited the ability of PPARalpha to bind DNA and activate reporter genes. This observation has been further studied in mice. Four weeks of ethanol feeding of C57BL/6J mice also impairs fatty acid catabolism in liver by blocking PPARalpha-mediated responses. Ethanol feeding decreased the level of retinoid X receptor alpha (RXRalpha) as well as the ability of PPARalpha/RXR in liver nuclear extracts to bind its consensus sequence, and the levels of mRNAs for several PPARalpha-regulated genes were reduced [long-chain acyl coenzyme A (acyl-CoA) dehydrogenase and medium-chain acyl-CoA dehydrogenase] or failed to be induced (acyl-CoA dehydrogenase, liver carnitine palmitoyl-CoA transferase I, very long-chain acyl-CoA synthetase, very long-chain acyl-CoA dehydrogenase) in livers of the ethanol-fed animals. Consistent with this finding, ethanol feeding did not induce the rate of fatty acid beta-oxidation, as assayed in liver homogenates. Inclusion of WY14,643, a PPARalpha agonist, in the diet restored the DNA-binding activity of PPARalpha/RXR, induced mRNA levels of several PPARalpha target genes, stimulated the rate of fatty acid beta-oxidation in liver homogenates, and prevented fatty liver in ethanol-fed animals. Blockade of PPARalpha function during ethanol consumption contributes to the development of alcoholic fatty liver, which can be overcome by WY14,643.
Endothelium injury is a primary event in atherogenesis, which is followed by monocyte infiltration, macrophage differentiation, and smooth muscle cell migration. Peroxisome proliferator-activated receptors (PPARs) are transcription factors now recognized as important mediators in the inflammatory response. The aim of this study was to develop a human endothelial model to evaluate anti-inflammatory properties of PPAR activators. PPAR proteins (alpha, delta and gamma) are expressed in EAhy926 endothelial cells (ECs). Pirinixic acid (Wy-14643), fenofibrate, fenofibric acid, the Merck ligand PPARdelta activator L-165041, 15-deoxy-Delta(12,14)-prostaglandin J2, but not rosiglitazone (BRL-49653) inhibited the induced expression of vascular cell adhesion molecule-1 (VCAM-1), as measured by enzyme linked immunosorbent assay (ELISA), and monocyte binding to activated-EAhy926 cells. The PPARdelta activator L-165041 had the greatest potency to reduce cytokine-induced monocyte chemotactic protein-1 (MCP-1) secretion. All PPAR activators tested which impaired VCAM-1 expression reduced significantly nuclear p65 amount. These results show that EAhy926 endothelial cells are an adequate tool to substantiate and characterize inflammatory impacts of PPAR activators.
For more Mechanism of Action (Complete) data for Pirinixic acid (10 total), please visit the HSDB record page.

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Ischemia-reperfusion injury (IRI) remains a frequent complication in surgery, especially in case of steatotic livers that present decreased tolerance towards IRI. Apart from its major role in metabolism, activation of peroxisome proliferator-activated receptor α (PPARα) has been related with positive effects on IRI. In addition, the deacetylase enzyme sirtuin 1 (SIRT1) has recently emerged as a promising target for preventing IRI, through its interaction with stress-related mechanisms, such as endoplasmic reticulum stress (ERS). Taking this into account, this study aims to explore whether PPARα agonist WY-14643 could protect steatotic livers against IRI through sirtuins and ERS signaling pathway. Obese Zucker rats were pretreated or not pretreated with WY-14643 (10 mg/kg intravenously) and then submitted to partial (70%) hepatic ischemia (1 hour) followed by 24 hours of reperfusion. Liver injury (ALT levels), lipid peroxidation (MDA), SIRT1 activity, and the protein expression of SIRT1 and SIRT3 and ERS parameters (IRE1α, peIF2, caspase 12, and CHOP) were evaluated. Treatment with WY-14643 reduced liver injury in fatty livers, enhanced SIRT1 activity, and prevented ERS. Together, our results indicated that PPARα agonist WY-14643 may exert its protective effect in fatty livers, at least in part, via SIRT1 induction and ERS prevention.[3]

C14H14CLN3O2S

323.8

323.049

C, 51.93; H, 4.36; Cl, 10.95; N, 12.98; O, 9.88; S, 9.90

50892-23-4

5694

Typically exists as White to off-white solids at room temperature

1.4±0.1 g/cm3

514.4±50.0 °C at 760 mmHg

155°C

264.9±30.1 °C

0.0±1.4 mmHg at 25°C

1.658

4.92

2

6

5

21

361

0

ClC1C([H])=C(N=C(N=1)SC([H])([H])C(=O)O[H])N([H])C1=C([H])C([H])=C([H])C(C([H])([H])[H])=C1C([H])([H])[H]

SZRPDCCEHVWOJX-UHFFFAOYSA-N

InChI=1S/C14H14ClN3O2S/c1-8-4-3-5-10(9(8)2)16-12-6-11(15)17-14(18-12)21-7-13(19)20/h3-6H,7H2,1-2H3,(H,19,20)(H,16,17,18)

[[4-chloro-6-[(2,3-dimethylphenyl)amino]-2-pyrimidinyl]thio]-acetic acid

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.42 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 20.8 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: ≥ 2.08 mg/mL (6.42 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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 (6.42 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.)